ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
121 Ocular Fibrosis: Myofibroblast to Therapeutics Minisymposium
Sunday, May 05, 2013 10:30 AM-12:15 PM
TCC 303 Minisymposium
Program #/Board # Range: 388-392
Organizing Section: Cornea
Contributing Section(s): Anatomy/Pathology, Glaucoma, Lens,
Retina
Program Number: 388
Presentation Time: 10:30 AM - 10:50 AM
Fibrosis - Keeping an Eye on the Myofibroblast
Boris Hinz. University of Toronto, Toronto, ON, Canada.
Commercial Relationships: Boris Hinz, None
Program Number: 389
Presentation Time: 10:50 AM - 11:05 AM
Fibrosis in Glaucoma: Role in Pathologenesis and Surgical
Failure
Colm J. O'Brien. Ophthalmology, Mater Misericordiae Univ
Hospital, Dublin, Ireland.
Commercial Relationships: Colm J. O'Brien, None
Program Number: 390
Presentation Time: 11:05 AM - 11:20 AM
Prevention of Keratocyte-Myofibroblast Conversion by
Targeting the Matrix-Cytokine System
Shizuya Saika. Ophthalmology, Wakayama Medical University,
Wakayama, Japan.
Commercial Relationships: Shizuya Saika, None
Program Number: 391
Presentation Time: 11:20 AM - 11:35 AM
Fibrosis in the lens: Regulation of TGFß-induced lens Epithelial
to Mesenchymal Transition (EMT) leading to cataract
Frank J. Lovicu. Anatomy & Histology - F13, University of Sydney,
Sydney, NSW, Australia.
Commercial Relationships: Frank J. Lovicu, None
Program Number: 392
Presentation Time: 11:35 AM - 11:50 AM
Regulation of Retinal and Subretinal Fibrosis
David R. Hinton. Pathology, Keck School of Medicine USC, Los
Angeles, CA.
Commercial Relationships: David R. Hinton, RPT (I), RPT (P)
126 Contact Lens I
Sunday, May 05, 2013 10:30 AM-12:15 PM
Exhibit Hall Poster Session
Program #/Board # Range: 487-523/B0124-B0160
Organizing Section: Cornea
Program Number: 487 Poster Board Number: B0124
Presentation Time: 10:30 AM - 12:15 PM
Evaluation of Surface Water Characteristics of Novel Daily
Disposable Contact Lenses Using Refractive Index Shifts after
Wear
Jeffery Schafer, Robert B. Steffen. Bausch & Lomb, Rochester, NY.
Purpose: Several novel daily disposable contact lenses have been
introduced with unique water characteristics. Nesofilcon A lenses are
described as having 78% water, the same water content as the cornea
throughout the lens matrix. Delefilcon A lenses are described as
having a surface water content of 80% and a bulk water content of
33%. The delefilcon A high water content at the surface is reported to
be the result of a surface modification using a copolymer of
polyamidoamine and poly(acrylamide-acrylic acid) which is highly
anionic (negative charge). During wear, these high water materials
are exposed to air and tear components that may change the
properties of the lenses. The objective of this study was to investigate
surface water characteristics using refractive index shifts after wear
with delfilcon A lenses compared to nesofilcon A and etafilcon A
lenses.
Methods: Twenty subjects wore each of the three lens types in a
randomly determined order for 15 minutes. The worn lenses for each
subject were measured for surface refractive index on the Metricon
M-2010 Prism Coupler. To establish baseline refractive index values,
unworn lenses of each type were also measured for refractive index
directly from the package.
Results: The mean changes in refractive index (unworn - worn) were
0.006 for nesofilcon A lenses, 0.012 for etafilcon A lenses and 0.093
for delefilcon A lenses. With the highly accurate measurement
capability of the Metricon instrument, (routine refractive index
accuracy of ± 0.00053 and standard deviation from 0.0008 to
0.0046), the difference between unworn and worn average values
were statistically significant for each lens, p<0.0001.
Conclusions: Lenses with higher surface water content have a
surface refractive index closer to the refractive index of water (1.33),
while lenses with lower surface water content will have a higher
refractive index. The refractive index is typically 1.46-1.48 for a 20%
water lens and 1.37-1.38 for a 75% water lens. The results of this
study show a change in mean surface refractive index for the
delefilcon A lenses from 1.34, typical of >80% water, to 1.43, typical
of a 33% water, following just 15 minutes of wear due to a change in
the water content at the surface. There was no change in refractive
index at the surface following lens wear for either the 78% water
nesofilcon A lenses or the 58% water etafilcon A lenses.
Commercial Relationships: Jeffery Schafer, Bausch & Lomb (E);
Robert B. Steffen, Bausch + Lomb (E)
Program Number: 488 Poster Board Number: B0125
Presentation Time: 10:30 AM - 12:15 PM
Effect of Protein Adsorption on Dewetting and Corneal cell
adhesion on Contact Lenses
Saad Bhamla, Claire M. Elkins, David Bergsman, Gerald G. Fuller.
Chemical Engineering, Stanford University, Stanford, CA.
Purpose: We discuss the influence of lysozyme adsorption on
contact lenses using two different experimental approaches dewetting on lenses and adhesion of corneal cells to a contact lens
substrate.
Methods: Dewetting is studied by stretching a contact lens flat on an
elevation stage built on a miniature Langmuir trough. By raising the
stage, the lens surface captures a sessile droplet coated with DPPC
(1,2-dipalmitoyl-sn-glycero-3-phosphocholine) at surface pressure of
the eye (20-25 mN/m). Liquid is slowly drained and dewetting
dynamics captured using a CCD camera.
Corneal cell adhesion is studied using an apparatus developed in the
Fuller lab. A contact lens is descended upon a cultured monolayer of
live epithelial cells. After a waiting period, the contact lens is sheared
laterally relative to the bottom plate, subjecting the cells to a
controlled strain while a force transducer measures the applied stress.
Our rheometer-based design allows precise control over the strain
down to 0.001 strain units, as well as highly sensitive measurement
of the applied stress. The entire apparatus is mounted on a DIC
microscope, allowing live cell imaging.
Results: Two commercial lenses are tested for both experimental
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
approaches. In the absence of DPPC, clean contact lenses were highly
hydrophilic and resisted dewetting, whereas when soaked overnight
in 1 mg/ml lysozyme showed substantial dewetting. Standard
deviation across 4 samples was about 10%. In the presence of DPPC,
dewetting is stabilized on the lysozyme adsorbed contact lens surface.
For clean lenses, a 200% step-strain produces a low force response
indicating no attachment of cells to the lens substrate. Whereas for
contact lens soaked overnight in 1 mg/ml lysozyme solution, a higher
maximum force is exhibited, indicating relatively greater cell
adhesion to the contact lens. This stress relaxes quickly to a new
equilibrium value, likely due to the re-organization and relaxation of
the adhered corneal cells.
Conclusions: Our findings show that adsorbed lysozyme decreases
the hydrophilicity of the lens surface leading to dewetting. Deposition
of lysozyme also makes the lens surface more attractive to cell
adhesion.
Fig1: Normalized wet area as a function of time with standard
deviation of 4 samples, for pure and lysozyme adsorbed contact
lenses.
Fig2: Step-strain data for uncoated and lysozyme coated contact lens
with corneal epithelial cells.
Commercial Relationships: Saad Bhamla, None; Claire M.
Elkins, None; David Bergsman, None; Gerald G. Fuller, None
Program Number: 489 Poster Board Number: B0126
Presentation Time: 10:30 AM - 12:15 PM
Secretory Phospholipase A2 Type IIA Levels across the Wear
Cycle: Response to Lens Age or Indication of CL Intolerance
Walter L. Nash, Alan Landers, Manal M. Gabriel, Jennifer D. Lane,
Michael S. Foster. Vision Care, Alcon, Duluth, GA.
Purpose: To evaluate the difference in day one versus day twentyeight levels of secretory phospholipase A2 Type IIA (sPLA2IIA) in
the tear envelope of symptomatic (decline in comfort over wear
cycle) and asymptomatic subjects.
Methods: Lenses were collected from subjects aseptically and stored
at below -20 degrees C. Subjects wore the same non-ionic silicone
hydrogel lens material and used the same lens care regimen. Lenses
were equilibrated to ambient temperature and rinsed in 2 mL of
phosphate buffered saline for 5 minutes with agitation. The rinsate
was subjected to a sandwich ELISA using a monoclonal antibody
specific for sPLA2IIA. The plate was washed and an
Acetylcholinesterase (ACHe); Fab’conjugate was added to the plate
which selectively binds to a different epitope on the sPLA2IIA
molecule. Finally, the enzymatic activity of ACHe was measured by
adding a reagent containing the ACHe substrate and reading the plate
at a wavelength of 420 nm by spectrophotometry.
Results: Symptomatic and asymptomatic subjects exhibited similar
levels of sPLA2IIA at day 1 as on day 28; with symptomatic subjects
exhibiting 17.2±6.2 and 17.8±8.9 ng/lens sPLA2IIA, respectively,
and asymptomatic subjects exhibiting 21.7±5.6 and 24.6±9.2 ng/lens
sPLA2IIA, respectively. Comparing the amount sPLA2IIA in the tear
envelope between symptomatic and asymptomatic subjects on both
day 1 and day 28 showed no statistical differences using nonparametric Mann-Whitney analysis.
Conclusions: sPLA2IIA hydrolyzes fatty acids generating free
arachidonic acid and lysophospholipids, the precursors of proinflammatory lipid mediators. Studies have reported elevated levels
of sPLA2IIA in the tears of intolerant contact lens wearers. This
study demonstrates no changes in the level of sPLA2IIA in the tear
envelope over the course of the wear cycle in symptomatic or
asymptomatic lens wearers. Our findings, suggest that the mechanism
driving comfort at the time of lens replacement may not be the same
as the mechanism driving contact lens intolerance as described in the
literature.
Commercial Relationships: Walter L. Nash, ALCON, a Novartis
Company (E); Alan Landers, Alcon (E); Manal M. Gabriel, Alcon,
A Novartis company (E); Jennifer D. Lane, Alcon Laboratories (E),
Novartis (E); Michael S. Foster, Alcon, a Novartis Company (E)
Program Number: 490 Poster Board Number: B0127
Presentation Time: 10:30 AM - 12:15 PM
Characterization of contact lenses through oxygen permeability,
equilibrium water content, and silicone content
Scott Curtin, Michelle Seitz, Meng Ouyang, Katarina Tomic,
Meredith Wiseman, Hanny Vanwersch. DSM, Berkeley, CA.
Purpose: In an effort to contribute to the growing knowledge base of
materials properties of silicone hydrogel (“SiHy”) contact lenses and
improve material design for formulation, DSM has evaluated three
principal material parameters that affect oxygen permeability (Dk);
amount of silicone component present, intrinsic permeability of
silicone component, and connectivity of silicone-containing phase
Methods: The coulometric oxygen permeability (Dk) and
equilibrium water content (EWC) of commercial SiHy contact lenses
were measured at DSM using methods described in ISO 183694:2006 and ANSI Z80.20-2010. A modified Mocon MH 2/21 was
used to measure the oxygen permeability for 5 contact lenses using
CIBA Vision O2 Optix for the Q value. The silicon and fluorine
contents of dried commercial lenses were measured by X-ray
fluorescence to approximately ± 0.5 wt%.
Results: CIBA Vision Air Optix 108±3.3 (110), CIBA Vision Dailies
Total One 131±4.3 (140), Acuvue Oasys 105±1.6 (105), Acuvue
Trueye 114±2.1 (110), Coopervision Biofinity 128±6.1(128) with
manufacturer-claimed values in parentheses and all lenses besides
CIBA Vision lenses were reported using polarographic method . A
wet blot gravimetric method was used to measure EWC for six lenses
with measured values closely matching reported values. CIBA Vision
Air Optix 32.7±0.5% (33%), CIBA Vision Dailies Total One
29.9±0.8% (33%), Acuvue Oasys 37.3±0% (38%), Acuvue Trueye
44.5±0.4% (46%), Coopervision Biofinity 46.3±0.8% (48%), and
CIBA Vision O2 Optix 30.5±0.5% (33%). Additionally, the atomic
silicon and fluorine contents of the dried lenses were measured with
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
X-ray Fluorescence.
Conclusions: Dk of SiHy lenses can be understood in terms of three
principal material parameters: the amount of silicone component
present, the intrinsic permeability of silicone component, and the
connectivity of silicone-containing phase. The volume fraction of the
silicone phase is a more meaningful parameter than EWC in
understanding Dk and it can be estimated using the EWC and atomic
percentages of Si and F. A simple morphologically based
permeability model can describe the Dk of commercial lenses. This
gives a framework for how to achieve sufficient Dk while minimizing
use of silicone materials by optimizing connectivity and intrinsic
permeability of silicone phase.
©2012 DSM. All rights reserved.
Commercial Relationships: Scott Curtin, None; Michelle Seitz,
None; Meng Ouyang, None; Katarina Tomic, None; Meredith
Wiseman, None; Hanny Vanwersch, None
Program Number: 491 Poster Board Number: B0128
Presentation Time: 10:30 AM - 12:15 PM
Effect of lens care system on silicone hydrogel contact lens
wettability
Cecile A. Maissa1, Michel Guillon1, Stephanie Wong1, Anna Lane1,
Trisha Patel1, Renee J. Garofalo2. 1OTG Research & Consultancy,
London, United Kingdom; 2Alcon, Fort Worth, TX.
Purpose: The purpose of this study was to compare the effect of the
repeated usage of two different care systems (one hydrogen peroxide
cleaning and disinfecting system and one PHMB containing
multipurpose system) with silicone hydrogel lenses worn on a daily
wear basis for a three month period. A specific aspect of interest was
the study of effects of the care systems on contact lens wettability.
Methods: Seventy-four symptomatic contact lens wearers, currently
wearing either ACUVUE® OASYS® (n=37) or PureVision (n=37),
constituted the study population. The investigation was a two-arm
prospective, bilateral study of three month duration to evaluate the
potential beneficial effects of CLEAR CARE® compared with renu®
freshTM. The subjects were randomized to use one of the two care
systems. The effects on contact lens wettability were assessed with
the Tearscope and reported in terms of Pre-lens Tear Film Non
Invasive Break Up Time. Baseline assessments were carried out at
the dispensing visit with new contact lenses. At the two and three
month follow-up visits the contact lenses had been worn for at least
six hours prior to the visits, and were at least 11 days old for
ACUVUE® OASYS® and 25 days old for PureVision.
Results: The results obtained showed that:
i. Whereas no difference in contact lens wettability was observed at
dispensing between the two lens care groups (Mean Median NIBUT:
4.5 vs. 4.2s, p=0.518) a significantly more stable pre-lens tear film
was observed with CLEAR CARE® than with renu® freshTM at
both the two (Mean Median NIBUT: 4.6 vs. 3.7s p=0.005) and three
(Mean Median NIBUT: 5.8 vs. 4.2s p=0.028) month visits.
ii. With CLEAR CARE® a significant improvement in contact lens
wettability was recorded compared with their habitual contact lenses
and care system at three months (Mean Median NIBUT 5.8 vs. 4.0s
p<0.001). Further, a significant increase in wettability was observed
after three months of use compared with dispensing (Mean Median
NIBUT 5.8 vs. 4.5s p=0.022).
iii. With renu® freshTM, no significant differences were observed at
the end of three months of use compared with either the subjects’
own care system or the new contact lenses at dispensing (Mean
Median NIBUT: 3M 4.2 vs. Disp 4.2 (p=0.420) vs. Enrol 5.1s
(p=0.734)).
Conclusions: Significantly better contact lens wettability was
achieved for ACUVUE® OASYS® and PureVision with CLEAR
CARE® than with renu® freshTM at the end of three months of use.
Commercial Relationships: Cecile A. Maissa, Alcon (F),
Optometric Technology Group (E), Alcon (C), Allergan (C), JJVCI
(C), Member of CLDW Tear film subcommittee (Tear Film & Ocular
Surface Society) (S), Wipes/Optometric Technology Group Ltd (P);
Michel Guillon, Johnson & Johnson Vision Care (C), Alcon (C),
Allergan (C), OTG Research & Consultancy (F), Wipes / Optometric
technology Group Ltd (P), Contact Lens Anterior Eye (S), Eye and
Contact lens (S), International society for Contact Lens Research (S),
Tear Film and Ocular Surface (S), Alcon (F); Stephanie Wong,
Optometric Technology Group (E), Alcon (F); Anna Lane,
Optometric Technology Group (E), Alcon (F); Trisha Patel,
Optometric Technology Group (E), Alcon (F); Renee J. Garofalo,
Alcon Research, Ltd. (E)
Support: Financial Support received from Alcon
Clinical Trial: NCT01494818
Program Number: 492 Poster Board Number: B0129
Presentation Time: 10:30 AM - 12:15 PM
Effect of care solutions on contact lens in-vivo wettability at
insertion and end of day
Alan Tomlinson, Raied Fagehi, Velitchko Manahilov. Department of
Life Sciences, Glasgow Caledonian University, Glasgow, United
Kingdom.
Purpose: To investigate the wetting of a soft contact lens (SCL) after
storage in various multipurpose solutions (MPS) (compared to
wetting with the pack solution), to assess the sustained benefits after
8 hours of wear.
Methods: Ten SCL wearers (age range 26.6± 6.3 yrs) were included
in this study. A Doane interferometer captured images of the pre-lens
tear film on a single type of SCL, ACUVUE OASYS (Johnson &
Johnson). The SCL wettability was measured after storage in the
pack solution or one of five MPS: Opti-Free EverMoist and Express
(Alcon), COMPLETE MPS (AMO), ReNu and Biotrue (Bausch &
Lomb). The wettability was measured after 15 min (time A) and 8
hours (time B) of wear of the lens by each subject. Four previously
described parameters of wettability were assessed: onset latency
(OL), the time to first break-up; drying duration (DD), the duration of
drying after first break-up; the maximum speed of drying (MS); and
the time to reach this maximum speed (PL).* The OSDI
questionnaire was completed by the subjects, at each visit, to evaluate
the subjective response to the solutions.
Results: There was no significant difference between CL wetting
measured after storage in the MPS compared to the pack solution for
all four wetting variables (Friedman test, P>0.05). However, storage
of the lens in some MPSs showed more sustained wetting after 8
hours wear, with similar values at (A) and (B); for OL, ReNu and
Complete; for DD and MS Biotrue, Express and Complete; and for
PL ReNu, Express and Complete. The OSDI questionnaire showed
less discomfort after 8 hours wear (at B) with storage in all the MPSs
compared to the pack solution (p< 0.02).
Conclusions: The effect of all MPS on the initial wetting of a SCL
(compared to that with the pack solution) was limited. However, the
ability to sustain wetting of a SCL over 8 hours of wear was better
with Biotrue, Opti-Free Express and Complete. The wetting
parameter of drying duration gave the closest objective measure to
subjective responses to lens wear (Figure 1and 2).
*Fagehi R, Tomlinson A, Manahilov V. ARVO 2011 E-abstract:
6524, 2011.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
did not substantially change over the course of the 720 minutes of
mechanical wear. Optical microscopy confirmed that the lenses still
maintained a surface gel layer.
Conclusions: A simple laboratory method for wearing contact lenses
through mechanical agitation was developed and characterized. Even
after 720 minutes of simulated wear, the lubricity and surface gel
layers were maintained on the delefilcon A lenses.
Brennan, AAO 2009.
Dunn, et al., Tribology Letters, 2013.
Commercial Relationships: Alan Tomlinson, Allergan (E),
Allergan (R), Bausch and Lomb (C), TearLab (C), TearLab (I),
Alcon, CibaVision (C), Pfizer (R), Pfizer (C); Raied Fagehi, None;
Velitchko Manahilov, None
Program Number: 493 Poster Board Number: B0130
Presentation Time: 10:30 AM - 12:15 PM
Laboratory Model for Wear of Contact Lenses and Effects on
Lens Lubricity of Surface Gel Layers
W G. Sawyer1, Juan M. Uruena1, Thomas E. Angelini1, Alison C.
Dunn1, John Pruitt2. 1Mechanical and Aerospace Eng, University of
Florida, Gainesville, FL; 2Biocompatibility Projects, Alcon Vision
Care Research, Johns Creek, GA.
Purpose: Lubricity of contact lenses is recognized as an important
contributor of comfort (Brennan 2009). Experiments aimed at
quantifying the lubricity of lenses are traditionally measured on new
lenses that have been removed from the package and either gently
rinsed in a saline solution or tested in the packing solution directly
(Dunn 2013). The purpose of this research is to establish a simple and
scalable method to mechanically exercise contact lenses and simulate
wear in a controlled laboratory environment, and then to perform
lubricity measurements on the front curve surface of the lenses at
varying time points of wear.
Methods: Poly(HEMA) (60% pHEMA) hydrogels were molded
inside of transparent acrylic containers to a thickness of
approximately 3 mm. A series of poly(HEMA) spheres of 6 mm
diameter were also molded and added to the hydrogel lined cup to a
packing fraction of approximately 25%. A single contact lens
(delefilcon A) was placed within the ensemble of hydrogel spheres
and the container was filled with Phosphate Buffered Saline. The
entire collection was shaken at 31 Hz for various time points. Image
analysis was used to track the hydrogel spheres and to quantify the
collision frequencies. Based on this analysis a mathematical model of
mechanical energy and tribological severity was developed and
compared to the tribological severity of 1,000 blinks per hour of
wear. Prior to friction testing lenses were examined for evidence of
wear and damage was quantified using scanning optical microscopy.
Results: This delefilcon A lenses showed a characteristic friction
coefficient of mu=0.02, with a standard deviation for each
measurement of approximately mu = 0.005. The friction coefficients
Experimental Setup for Laboratory Wear Studies.
Plot of Friction Coefficient versus Simulated Wear Time.
Commercial Relationships: W G. Sawyer, Alcon (F); Juan M.
Uruena, Alcon (F); Thomas E. Angelini, alcon (F); Alison C.
Dunn, Alcon (F); John Pruitt, Alcon (E)
Program Number: 494 Poster Board Number: B0131
Presentation Time: 10:30 AM - 12:15 PM
Assessment of the relationship between contact lens coefficient of
friction and subject lens comfort
Jami R. Kern1, Joseph M. Rappon2, Erich Bauman2, Ben Vaughn3.
1
Global Medical Affairs, R&D, Alcon, Fort Worth, TX; 2Vision Care
R&D, Alcon, Fort Worth, TX; 3Biostatistics, Rho, Chapel Hill, NC.
Purpose: To examine the relationship between subjective comfort
and contact lens coefficient of friction among multiple soft contact
lens materials.
Methods: A meta-analysis of clinical data (n=157) exploring the
association between comfort and contact lens lubricity was conducted
on 5 soft contact lens materials (delefilcon A, lotrafilcon B,
balafilcon A , balafilcon A2, etafilcon A+). Subjective data for
insertion comfort, overall comfort and end of day comfort were
obtained from a database of clinical trials that included studies
conducted between 2004 and 2011. Trials were included in the
analysis unless they met exclusion criteria, including; extended or
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
continuous lens wear, exclusively acute measures, multi-purpose lens
care utilized, contra-lateral eye study design, small sample size (n<
30). The last on-eye treatment value for a lens was used and the
association between reported comfort and the coefficient of friction,
measured using the inclined plane method (Tucker et al), was
assessed. Each comfort assessment (Insertion, Overall, End of Day)
was analyzed separately in a mixed effect model with a fixed effect
for the coefficient of friction and random effects for clinical trial and
subject (for crossover studies). A serial gate-keeping approach was
used to adjust for multiple comparisons.
Results: There were 117 females included in the analysis (74.5%)
and the average age of subjects was 32 years. An inverse relationship
was found between coefficient of friction and comfort. Coefficient of
friction was highly predictive of each measure of comfort (all
p<0.001), with higher coefficient of friction associated with lower
comfort. Each of the outcomes showed a highly significant
association between the respective comfort measure and coefficient
of friction; the estimates were very similar for all 3 comfort outcomes
assessed, with approximately a 0.025 reduction in coefficient of
friction associated with a 1-unit improvement in comfort on a 10point scale.
Conclusions: Using a database of clinical studies, we were able to
demonstrate a strong relationship between lens coefficient of friction
and subjective comfort. This association was not only statistically
significant, but given the range of soft contact lens coefficients of
friction, also clinically relevant. The lubricity of a lens should be
taken into consideration when optimizing lens wear, especially as it
relates to lens comfort.
Commercial Relationships: Jami R. Kern, Alcon (E); Joseph M.
Rappon, Alcon (E); Erich Bauman, Alcon Laboratories Inc. (E);
Ben Vaughn, None
Support: Sponsored by Alcon
Program Number: 495 Poster Board Number: B0132
Presentation Time: 10:30 AM - 12:15 PM
Surface Elastic Modulus of a Unique Hydrogel Material
Measured with Colloidal Probe AFM
Yuchen Huo1, Alexander Rudy1, Scott S. Perry1, John Pruitt2.
1
Materials Sicence and Engineering, University of Florida,
Gainesville, FL; 2Alcon Vision Care Research, Johns Creek, GA.
Purpose: This study reports the compressive elastic modulus of a
unique water gradient contact lens material, formally known as
delefilcon A, which consists of a low water content silicone hydrogel
core material that transitions through a gradient to an ultra-soft, high
water content surface gel layer via a proprietary chemical anchoring
process. Through comparison with two other lens materials,
balafilcon A and senofilcon A, the influential role of the surface gel
layer is highlighted.
Methods: The lenses employed in this study were commercially
fabricated and measured approximately 100 μm in thickness at the
center. Prior to each experiment, the samples were soaked in saline
(Unisol®4, Alcon, Fort Worth, TX) for 24 hours in order to remove
the blister pack solutions. Elastic modulus was measured in saline by
indenting a colloidal probe into the surface in a controlled manner
(i.e. fixed approach speed and maximum applied force), such that the
maximum indentation depth was restricted to the nanometer scale.
All modulus values were obtained from the anterior surface of the
sample. Calibrated cantilevers modified with 5-µm (diameter) silica
colloidal probes provided access to quantitative modulus values
measured for physiologically relevant contact pressures. A modulus
value for each material was determined via a Hertzian analysis of
force versus indentation behavior measured in the near surface
region.
Results: The lens materials examined exhibited large differences in
compressive surface modulus. Balafilcon A, which receives a post
production surface plasma oxidation treatment, exhibited the highest
elastic modulus, 2000 kPa, followed by senofilcon A, 700 kPa, and
the novel delefilcon A, 14 kPa. The large difference in surface
mechanical response is correlated to the structure of the near surface
region , with delefilcon A achieving the very low modulus through
low polymer content, low crosslinking density, and water content
greater than 80% by volume in this region.
Conclusions: The surface chemistry of silicone hydrogel contact lens
materials is seen to influence the elastic modulus of the lens surface,
as measured on the nanometer scale. Specifically, the incorporation
of a high water content surface gel (delefilcon A) is seen to produce
an exceptionally low modulus.
Commercial Relationships: Yuchen Huo, Alcon Vision Care
Research (F); Alexander Rudy, Alcon Vision Care Research (F);
Scott S. Perry, Alcon Research (F); John Pruitt, Alcon (E)
Program Number: 496 Poster Board Number: B0133
Presentation Time: 10:30 AM - 12:15 PM
Prosthetic Replacement of the Ocular Surface Ecosystem
(PROSE) causes corneal epithelial thinning without altering
overall corneal thickness or hysteresis
Peter G. Coombs, Ryan M. St Clair, Yvonne Wang, Michelle N. Lee,
Christopher E. Starr, Jessica Ciralsky, Kimberly C. Sippel, Priyanka
Sood, Mark Rosenblatt, Ana G. Alzaga Fernandez. Ophthalmology,
Weill Cornell Medical College, New York, NY.
Purpose: To assess the effect of the Prosthetic Replacement of the
Ocular Surface Ecosystem (PROSE) wear on corneal epithelial
thickness, total corneal thickness, and corneal hysteresis.
Methods: This was a prospective study of all patients referred for
PROSE treatment at Weill Cornell Ophthalmology. 32 eyes (16
patients) treated with extended PROSE wear, defined as more than 6
hours per day for longer than 30 days, were included in the analysis.
Each eye had corneal hysteresis measurements taken with the Ocular
Response Analyzer (Reichert, Inc, Buffalo, NY) and Visante anterior
segment OCT (Carl Zeiss International, Oberkochen, Germany) at 90
and 180 degrees during the initial visit before PROSE wear and after
extended PROSE wear. Epithelial and central corneal thickness were
determined using Neurolucida software (MBF Bioscience, Williston,
VT) with the quick measure line tool. Corneal hysteresis, central
corneal thickness, and epithelial thickness before PROSE wear and
after 30 days of more than 6 hours of daily PROSE wear were
compared using a paired t-test for means.
Results: Corneal epithelium was significantly thinner after PROSE
wear (32.11 +/- 4.24 μm) than before (35.11 +/- 5.5 μm) extended
PROSE wear (p=0.13). Central corneal thickness was unaltered by
extended PROSE wear (492.36 +/-22.6 μm vs. 488.93 +/- 27.7 μm,
p=0.385). Corneal hysteresis, a test of corneal biomechanics, was
also not significantly different before and after extended PROSE
wear (9.05 +/- 2.6 mmHg vs. 9.74 +/- 2.79 mmHg, p=0.249).
Conclusions: Patients had thinner corneal epithelium after a period
of extended PROSE wear. This thinning may represent morphologic
changes to the corneal epithelium after extended exposure to the
PROSE fluid reservoir and lack of exposure to air. However, the lack
of air exposure did not effect other corneal physical properties,
including central corneal thickness, and corneal hysteresis.
Commercial Relationships: Peter G. Coombs, None; Ryan M. St
Clair, None; Yvonne Wang, None; Michelle N. Lee, None;
Christopher E. Starr, None; Jessica Ciralsky, None; Kimberly C.
Sippel, None; Priyanka Sood, None; Mark Rosenblatt, None; Ana
G. Alzaga Fernandez, None
Support: Research to Prevent Blindness
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Program Number: 497 Poster Board Number: B0134
Presentation Time: 10:30 AM - 12:15 PM
Selenium covalently incorporated into the polymer of contact lens
case material inhibits bacterial biofilm formation
Ted W. Reid1, 2, Phat L. Tran1, Thomas Mosley2, Courtney Jarvis1,
Daniel Webster4, Robert E. Hanes3, Abdul Hamood5. 1Ophthal &
Visual Science, Texas Tech Univ Health Sciences Ctr, Lubbock, TX;
2
Selenium Ltd., Lubbock, TX; 3Selenium Ltd., Austin, TX; 4Cell
Biology, Texas Tech University Health Sciences Ctr, Lubbock, TX;
5
Microbiolgy, Texas Tech University Health Sciences Ctr, Lubbock,
TX.
Purpose: Contact lens case bacterial biofilm formation has become a
major cause of contact lens contamination. This is a serious problem
since it has been found that bacteria grow even in the presence of
contact lens cleaning solution. Silver as an antimicrobial has been
incorporated into contact lens cases, however, silver has several
drawbacks. Any patient with silver or metal allergies cannot use these
cases and silver has minimal effects against Staphylococcus aureus
(S. aureus), Stenotrophomonas maltophilia (S. maltophilia), and
different fungi. In addition, silver is expensive and has to leach out of
the case to be active. In contrast, selenium does not have to leach out
of the material to be active since it kills by the catalytic formation of
superoxide radicals and it is much less expensive. Thus, this project
was carried out to test the ability of selenium, covalently incorporated
into the polymer of contact lens case material, to inhibit biofilm
formation by different bacteria.
Methods: A polymer of polypropylene was produced that
incorporated organo-selenium monomers into the final polymer. This
material was then injection molded. The resulting material was tested
for its ability to inhibit biofilm formation. with silver or metal
allergies cannot use these cases and silver has minimal effects against
S. aureus and S. maltophilia were tested since these bacteria are
resistant to killing with silver. Pseudomonas aeruginosa and Serracia
marcessans were also tested. The bacteria were allowed to grow in
the presence of the polypropylene (with or without selenium) for 24
hours. The bacteria were then removed by vortexing and assayed.
The bacterial concentration was determined by a colony forming unit
assay (plating on agar). The biofilm was also imaged by confocal
laser scanning spectroscopy and was then quantitated by COMSTAT
analysis.
Results: Selenium containing polypropylene showed over 7 logs
(complete) inhibition against S. aureus, S. maltophilia and P.
aeruginosa, and also was fully active after soaking in PBS for the
equivalent of 8 weeks. S. marcessans showed 4 logs of killing.
Conclusions: The results showed that selenium covalently
incorporated into a polypropylene polymer could be injection molded
yet showed total inhibition of S. aureus S. maltophilia and P.
aeruginosa biofilm and was stable to soaking for 8 weeks.
Commercial Relationships: Ted W. Reid, Selenium Ltd. (I),
Selenium Ltd. (P); Phat L. Tran, None; Thomas Mosley, Selenium,
Ltd. (E); Courtney Jarvis, None; Daniel Webster, None; Robert E.
Hanes, Selenium, Ltd. (F), Selenium, Ltd. (I), Selenium, Ltd. (E),
Selenium, Ltd. (P); Abdul Hamood, None
Program Number: 498 Poster Board Number: B0135
Presentation Time: 10:30 AM - 12:15 PM
Effect of Oxidation on Binding of Fatty Acids to PureVision and
Acuvue Contact Lenses
Thomas J. Millar, Burkhardt S. Schuett. School of Science and
Health, Univ of Western Sydney, Penrith, NSW, Australia.
Purpose: Contamination reduces the longevity of contact lenses.
Although lipids contaminate contact lenses, it is not known if this is
preferentially by oxidised lipids. Such knowledge can be useful in
developing cleaning solutions.
Methods: A Fenton reaction was optimized and used to prepare
oxidized oleic acid and linolenic acid. The degree of oxidation was
quantified by measuring peroxides, malonyldialdehyde reactive
species and build-up of polymerized aldehydes. Based on these
measurements, 14C fatty acids were oxidised to different degrees.
PureVision and Acuvue contact lenses were loaded with these at
35°C and lipid binding was determined by measuring the ratio of
bound to unbound radioactivity.
Results: The degree of oxidation using the Fenton reaction depended
on the amount of desaturation. With time, only peroxides were
formed from oleic acid whereas linolenic acid was eventually broken
down completely. It was determined that 20h incubation with
50µg/mL of lipids gave optimal binding. Acuvue lenses bound ~50%
more non-oxidised lipids than PureVision lenses. There was no
increase in binding of oxidised lipids compared with non-oxidised
lipids in Acuvue lenses. There was an increase of ~50% in binding of
mildly oxidised lipids in PureVision lenses. If the lipids were
strongly oxidised then they bound less than non-oxidised lipids in
both contact lens types.
Conclusions: There are differences in the ability of different contact
lenses to bind oxidised lipids. It appears that mildly oxidised lipids,
such as might occur in vivo can bind more strongly to some types of
contact lenses than others. This experimental procedure provides a
platform for testing the ability of multi-purpose cleaning solutions to
remove oxidised lipids.
Commercial Relationships: Thomas J. Millar, Alcon (F), Allergan
(F); Burkhardt S. Schuett, Alcon (F), Allergan (S)
Support: Alcon
Program Number: 499 Poster Board Number: B0136
Presentation Time: 10:30 AM - 12:15 PM
In-Vivo Wettability of Contact Lenses Worn in a Low Humidity
Environmental Exposure Chamber (LH-EEC) Show Comparable
Changes to Traditional Field Trials
Fiona Soong1, Jalaiah P. Varikooty2, Nancy J. Keir2, Lyndon W.
Jones2, Piyush Patel1. 1R & D, Inflamax Ressearch, Toronto, ON,
Canada; 2CCLR, University of Waterloo, Waterloo, ON, Canada.
Purpose: The LH-EEC is a natural provocation research model,
which tightly controls environmental variables (humidity;
temperature; air flow) and is a valuable tool to study dry eye, with
little information on its utility to evaluate contact lens (CL) wear. The
purpose of this study was to use the LH-EEC model to observe invivo CL wettability changes and to compare these results with those
reported in typical CL field studies.
Methods: Ten symptomatic CL wearers were randomized and fit
with 1-day Acuvue® Moist® (etafilcon A): CLA in one eye and 1Day Acuvue® TruEye™ (narafilcon A): CLB in the contralateral
eye. They were exposed to LH-EEC for 180 mins with instruction to
watch a movie screen. Following CL insertion, 3 consecutive
measures of tear osmolarity were taken with TearLab® prior to LHEEC entry and exit. Dryness symptoms were rated from 0 (no
discomfort) to 4 (constant discomfort), and were collected at
specified intervals throughout the chamber visit as were observations
of blink rate (#blinks/min). In-vivo wettability was graded using a 0
(excellent) to 4 (extremely poor) scale with 0.25 steps.
Results: After only 180 mins in the LH-EEC, there was trend of
increasing tear osmolarity for both CLA (7.4±3.6mOsmol) and CLB
(4.80±3.23mOsmol), but this was not significant (p>0.05). Dryness
symptom scores showed non-significant increase from pre to post
chamber for CLA (+1.10±0.53) but a significant (p=0.001) increase
for CLB (+1.40±0.31). Blink rate significantly increased (p<0.003)
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
from pre-EEC rates of 42.0±4.8 blinks/min to average maximum of
61.2±4.3 blinks/min. Lens wettability worsened significantly over
time for both CLA and CLB 0.58±0.18 (p=0.01) and 0.65±0.25
(p=0.03) respectively. These values are comparable to changes in
wettability seen after 8 hrs of wear with both study materials
(Luensmann et al; Keir et al.)
Conclusions: The LH-EEC exacerbates ocular symptoms and signs
after 180 mins with CL wear. Significant changes in lens wettability
were seen during the exposure and yielded values comparable to
results shown in traditional trials with 8 hrs of lens wear. The LHEEC provides a way to accelerate extended day CL wear and
provides noteworthy research options for a controlled provocation
study of CL and dry eye signs and symptoms in a shorter time course.
Commercial Relationships: Fiona Soong, Inflamax Research (C);
Jalaiah P. Varikooty, Alcon (F); Nancy J. Keir, TearScience (F),
Alcon (F), Alcon (R), Allergan (F), Johnson & Johnson (F),
CooperVision (F), Visioneering, Inc. (F); Lyndon W. Jones, Alcon
(F), Alcon (R), Allergan (F), Abbott Medical Optics (R), Bausch &
Lomb (R), Ciba Vision (F), Ciba Vision (R), CooperVision (F),
Johnson & Johnson (F), Johnson & Johnson (R); Piyush Patel,
Inflamax Research (E)
Program Number: 500 Poster Board Number: B0137
Presentation Time: 10:30 AM - 12:15 PM
Viscoelasticity and mesh-size at the surface of hydrogels
characterized with microrheology
Thomas E. Angelini, Ryan M. Nixon, Alison C. Dunn, Juan M.
Uruena, John Pruitt, W G. Sawyer. MAE, University of Florida,
Gainesville, FL.
Purpose: Delefilcon A contact lenses contain a water gradient
structure that transitions from a low water content silicone hydrogel
core to a high water content surface gel. This work explores the
properties of the high water content surface gel, designed to mimic
the physical properties of the corneal surface, improving contact lens
comfort. The ~6 µm thick gels have an average water content that
exceeds 80%, yet the properties of the outer layer that makes direct
contact with the eye are not known. This study examines the elastic
and viscous moduli at the outermost surface of these soft gels to
understand the physical interactions between contact lenses and the
eye.
Methods: Microrheological tests were performed on the surfaces of a
standard silicone hydrogel (balafilcon A) and on water gradient
contact lenses (delefilcon A). Microspheres (0.5 micron radius) were
sandwiched between 3mm sections of lens material and kept in
deionized water. Video microscopy was performed at 90x
magnification, and the beads were tracked using digital image
analysis software.
Results: The mean-squared-displacement (MSD) was computed for
each particle, and averaged (N=32 for nelfilcon A; N=80 for
balafilcon A). Beads embedded in the surface gel layer of delefilcon
A exhibited significant motion, displacing between 50 to 100nm over
timescales below two seconds. By contrast, no detectable bead
motion above the noise threshold was observed in the balafilcon A
system; beads moved only 10nm to 16nm over the same two second
period. We compute the frequency-dependent elastic and viscous
moduli, G’ and G’’ from the MSD measurement. We find moduli
with very weak frequency dependence between 10 and 100 radians
per second, and an elastic modulus that varies between 0.3 and 1 Pa.
Thus, from elasticity theory of cross-linked flexible polymers, we
estimate that the water content at the outermost region of the
delefilcon A surface gel layer is above 99%.
Conclusions: Surface gel layers on contact lenses possess a
frequency dependence similar to low concentration polymer gels.
Remarkably, the modulus at the outermost surface is over 1000 times
lower than the mean elastic modulus of the entire surface gel layer.
This large modulus change, however, only requires roughly 15%
reduction in polymer concentration. This suggests that the polymer
concentration marginally drops near the surface as polymer chains
and crosslinks become sparse.
Commercial Relationships: Thomas E. Angelini, alcon (F); Ryan
M. Nixon, Alcon (F); Alison C. Dunn, Alcon (F); Juan M. Uruena,
Alcon (F); John Pruitt, Alcon (E); W G. Sawyer, Alcon (F)
Program Number: 501 Poster Board Number: B0138
Presentation Time: 10:30 AM - 12:15 PM
In vitro Uptake and Release of Natamycin Dex-b-PLA
Nanoparticles from Silicone Hydrogel Contact Lens Materials
Chau-Minh Phan1, Lakshman N. Subbaraman1, Lyndon W. Jones1,
Shengyan Liu2, Frank Gu2. 1Centre for Contact Lens Research,
School of Optometry and Vision Science, University of Waterloo,
Waterloo, ON, Canada; 2Department of Chemical Engineering,
University of Waterloo, Waterloo, ON, Canada.
Purpose: To evaluate the uptake and release of the antifungal agent
natamycin encapsulated within poly(D,L-lactide)-dextran
nanoparticles (Dex-b-PLA NPs) from model silicone hydrogel
contact lens materials.
Methods: Six model contact lens materials (gel A:
poly(hydroxyethyl methacrylate, pHEMA); gel B: 85% pHEMA:
15% [Tris(trimethylsiloxy)silyl]-propyl methacrylate (TRIS); gel C:
75% pHEMA: 25% TRIS; gel D: 85% N,N dimethylacrylamide
(DMAA): 15% TRIS; gel E: 75% DMAA: 25% TRIS; gel F:
DMAA) were prepared using photoinitiation. The resulting lens
materials were incubated in two conditions: (1) natamycin dissolved
in deionized (DI) water, and (2) natamycin encapsulated within Dexb-PLA NPs in 9.1 % dimethylsulfoxide (DMSO)/DI water for 7 days
(d) at 25±3 oC. The release of natamycin from these materials in 2
mL of unpreserved saline solution, pH 7.4 at 32±2 oC was monitored
using UV-Visible spectrophotometry at 304 nm over 7 d. The release
solution was replenished every 24 hours (h).
Results: The uptake of natamycin by all model lens materials
increased between 1 and 7 d (p<0.001). There were no differences in
drug uptake between lens materials containing pHEMA and DMAA.
However, the uptake of natamycin encapsulated with NPs was higher
than the uptake of the drug dissolved in DI water (p<0.05). The
release of the drug was observed to be higher in materials containing
DMAA than pHEMA (p<0.05). Additionally, all gels loaded with
natamycin NPs also released more drug compared to gels soaked with
natamycin in DI water (p<0.001). The drug release by all materials
reached equilibrium within 12 h. After replenishing the release
solution every 24 h, gels loaded with natamycin NPs continued to
release drugs for up to 4 d (p<0.05).
Conclusions: Model contact lens materials loaded with drug-Dex-bPLA NPs provides a drug delivery vehicle capable of releasing
natamycin for up to 12 h. In addition, materials containing DMAATRIS may be more suitable for drug delivery of natamycin due to the
higher drug release observed with these materials.
Commercial Relationships: Chau-Minh Phan, None; Lakshman
N. Subbaraman, None; Lyndon W. Jones, Alcon (F), Alcon (R),
Allergan (F), Abbott Medical Optics (R), Bausch & Lomb (R), Ciba
Vision (F), Ciba Vision (R), CooperVision (F), Johnson & Johnson
(F), Johnson & Johnson (R); Shengyan Liu, None; Frank Gu, None
Support: NSERC 20/20 Network for the Development of Advanced
Ophthalmic Materials
Program Number: 502 Poster Board Number: B0139
Presentation Time: 10:30 AM - 12:15 PM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Effects of Silicone Hydrogel Contact Lenses Applied Immediately
After Sub-Bowman’s Keratomileusis
Xingwu Zhong, Shaohui Gao. State Key Laboratory of
Ophthalmology, Zhongshan Ophthalmic Center, Guangzhou, China.
Purpose: To evaluate the effects of silicone hydrogel contact lenses
applied immediately after Sub-bowman keratomileusis.
Methods: The study was based on 46 myopic patients (92
consecutive eyes) who underwent bilateral Sub-Bowman
Keratomileusis (SBK). Each patient wore a PureVision contact lens
in one randomly-selected eye and nothing in the fellow eye
immediately after SBK. The contact lens was removed the next day.
Fluorescein staining(FL), tear film break up time(TBUT), schirmer I
test(SIT), central corneal thickness(CCT), ocular surface disease
index(OSDI), corneal hysteresis(CH), corneal resistance factor (CRF)
and corneal flap-related complications (postoperatively) were
assessed preoperatively and 1 day (except for CH and CRF), 1 week,
1 month and 3 months postoperatively.
Results: FL and TBUT of treated eye were improved at 1 day and 1
week postoperatively in contrast with the controlled, but significantly
aggravations were found at 1 day and 1 week after SBK compared
with preoperative level. There was no significantly difference in SIT
between treated and controlled eye postoperatively, but both eyes
showed the same reduction contrasted with preoperation except for 1
day. CCT of treated eye was thinner than controlled at 1 day after
surgery and both decreased obviously compared with preoperatively.
OSDI of treated eye was alleviated more at 1 day after SBK than the
controlled, but a significant severity existed at any visit for both in
contrast with that before surgery. There were no differences in CH
and CRF between treated and controlled eye after surgery, but both
eyes showed significantly decrease at any follow-up time. No
obviously differences were found in complications related to the
corneal flap between treated and controlled eye postoperatively.
Conclusions: Silicone hydrogel contact lenses applied immediately
after SBK can effectively reduce corneal epithelium staining ,
increase tear film stability , relieve corneal edema and alleviate
discomfort. However, it can’t shorten the duration of ocular surface
changes and symptoms revalant to SBK.
Commercial Relationships: Xingwu Zhong, None; Shaohui Gao,
None
Support: National Natural Science Foundation of China
GrantNO.81070754 and the Fundamental Research Funds of State
Key Lab of Ophthalmology Grant NO.2010C07.
Clinical Trial: under check
Program Number: 503 Poster Board Number: B0140
Presentation Time: 10:30 AM - 12:15 PM
High levels of sIgA and exudated serum albumin in tears of
contact lens related Dry Eye patients three months after
discontinuation of lens use
Piera Versura1, Alberto Bavelloni2, Chiara Coslovi1, William
Blalock3, Marco Grillini1, giuseppe giannaccare1, Emilio C.
Campos1. 1Ophthalmology Unit, DIMES Department, Alma Mater
Studiorum University of Bologna and S.Orsola-Malpighi Teaching
Hospital, Bologna, Italy; 2Laboratory of Musculoskeletal Cell
Biology, Istituto Ortopedico Rizzoli, Bologna, Italy; 3Institute of
Molecular Genetics, CNR-National Research Council of Italy,
Bologna, Italy.
Purpose: To evaluate discontinued contact lens (CL) wearers with
particular reference to secretory IgA (sIgA) and exudated serum
albumin in tears as markers of local inflammation.
Methods: 45 CL wearers diagnosed as having dry eye (DE)
according to DEWS guidelines (DEWS grade 1-3) and 25 matched
normal non-CL wearer volunteers were included in this study.
Patients had discontinued the use of various types of CLs (12 RGP,
33 soft disposable or frequent replacement CL-average time from last
use 60±30 days) because of associated discomfort. OSDI
questionnaire score, Schirmer test I, Break Up Time (BUT),
Lissamine green vital staining, corneal esthesiometry (CochetBonnet), conjunctival scraping and imprint cytology were performed.
Total tear protein content (TP), Lysozyme-C (LYS-C), Lactoferrin
(LACTO) and exudated serum albumin (ALB) were evaluated
(mg/ml) with the 2100 Bioanalyzer (Agilent Technology, CA, USA,
P230 Lab-chip kit). Validation of kDa range for heavy chain IgA
bands in each lane was carried out as previously described (Versura P
et al, Mol Vis 2012). The sIgA/LYS-C ratio was calculated as an
index of the increased activity of the IgA-producing cells in the
lachrymal gland. Data were statistically evaluated and correlated with
wear parameters (Pearson’s r) (significance p<0,05).
Results: A statistically significant decrease was found in CL-related
DE patients vs controls (media±S.D.) for BUT (respectively
8.6±4.9vs12.4±3.6 sec), corneal esthesiometry (50.2±2.3vs55.5±3.2
mm/lenght), TP (7.5±2.9vs9.3±4.3 mg/ml), LYS-C (2.08±0.9vs
2.9±0.9), LACTO (1.7±0.9vs 2.1±0.9), while an increase was found
for OSDI score (22.4±14,4vs5.5±2.2), scraping cytology score
(4.3±1.5vs2.1±0.3), imprint cytology score (1.8±0.8vs0.5±0.2), ALB
(1.1±1.3 vs 0.3±0.5) heavy chain sIgA (0.51±0.41vs0.38±0.33, band
validated in the range 48.0-51.0 kDa) and sIgA/LYS-C ratio
(0.21±0.1vs0.12±0.1). sIgA/LYS-C was found to be positively
correlated to DE severity grade, scraping cytology score and OSDI
(r=0,52, 0,42 and 0,45 respectively, p<0.05). No significant
correlation was found with CL type, wear regimen, discontinuation
time.
Conclusions: Ocular surface parameters and tear proteins were
significantly altered in CL-related DE patients, especially as regards
local inflammation indexes. Data suggest that recovery of ocular
surface homeostasis exceeds a three month CL use stop.
Commercial Relationships: Piera Versura, None; Alberto
Bavelloni, None; Chiara Coslovi, None; William Blalock, None;
Marco Grillini, None; giuseppe giannaccare, None; Emilio C.
Campos, None
Support: This work was partially supported by a grant from
Fondazione Cassa di Risparmio di Bologna and RFO ex-60% funds
from University of Bologna to ECC
Program Number: 504 Poster Board Number: B0141
Presentation Time: 10:30 AM - 12:15 PM
Comparative Cleaning Ability of Rigid Gas Permeable Lens Care
Systems
Kimberly A. Millard, Suzanne F. Groemminger. Formulation
Development, Bausch & Lomb, Rochester, NY.
Purpose: This study compared the cleaning efficacy of Rigid Gas
Permeable (RGP) lens care systems.
Methods: The care systems tested included two single bottle
multipurpose products; Boston Simplus and Unique pH, a two bottle
system; Boston Advance Cleaner and Conditioning Solution and a 3
bottle system; Lobob Optimum. Efficacy testing was completed using
Boston IV (itafocon B) RGP lenses.
Ten lenses were deposited in vitro for each product evaluation. The
deposition solution contained a combination of lipids and proteins.
The lipids included cholesterol and a palmitic acid ester while the
proteins consisted of lysozyme, lactoferrin, mucin, and albumin.
These were intended to simulate lens exposure to the tear film.
Squalene was also added to the mixture to mimic the oils from lens
handling often seen as “finger prints” on RGP lens surfaces. One
deposition cycle consisted of exposure to the simulated tear solution
followed by drying. After the completion of 10 deposition cycles, the
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
level of deposition was determined by computer enhanced image
analysis. An image of each lens was converted to mean grey scale,
which is a numerical representation of the degree of deposition. This
value was used as the baseline for calculation for each individual
lens. Each lens was re-imaged after a single cleaning regimen and the
% deposition removal was calculated.
Results: The image analysis demonstrated that three of the care
systems, Simplus, Advance, and Optimum removed significantly
greater amounts of lipid/protein deposits (p-value<0.05) than Unique
pH. The mean % removal was 95% (Simplus), 91% (Advance), and
95% (Optimum) compared to 83% (Unique pH).
Conclusions: RGP lens care products demonstrated varying degrees
of effectiveness to clean lipid and protein deposits. The Boston
Advance system, Lobob Optimum, and Boston Simplus provided the
best overall cleaning performance. Unique pH was not as effective in
lipid or protein removal. Deposit removal may have implications for
contact lens wearing comfort and vision.
Commercial Relationships: Kimberly A. Millard, Bausch & Lomb
(E); Suzanne F. Groemminger, Bausch & Lomb Inc (E)
Program Number: 505 Poster Board Number: B0142
Presentation Time: 10:30 AM - 12:15 PM
Corneal Uptake of Oxygen In Vivo During Soft Contact Lens
Wear
Percy Lazon De La Jara1, 2, Sho C. Takatori3, Klaus Ehrmann1,
Arthur Ho1, 2, Brien A. Holden1, 2, Clayton J. Radke3. 1Brien Holden
Vision Institute, Sydney, NSW, Australia; 2School of Optometry and
Vision Science, University of New South Wales, Sydney, NSW,
Australia; 3Department of Chemical and Biomolecular Engineering,
University of California, Berkeley, CA.
Purpose: Critical oxygen need of the human cornea during soft
contact lens (SCL) wear has received decades of attention with
polarograpy the primary in-vivo experiment. Unfortunately, classical
analysis of the polarographic oxygen sensor (POS) misinterprets
electrode behavior. We develop a new method to assess in-situ
corneal oxygen uptake during SCL wear using a micro-polarographic
Clark electrode.
Methods: After steady SCL wear and subsequent lens removal, a
membrane-covered POS is immediately placed onto the cornea and
transient oxygen tension recorded. A semi-logarithmic graph of
oxygen tension versus time at long time is created to give corneal
oxygen uptake, Jo(0). We apply our procedure to polarographic data
for ten human subjects with 12 different commercial SCL during
open eye.
Results: The average corneal oxygen uptake rates for ten subjects
with 12 different commercial SCLs vary from 2 - 10 μL(STP)/cm2 /h
at open eye. In Fig. 1, filled circles give oxygen uptake into the
cornea with lens wear, Jo(0), relative to that without lens wear,
Jo*(0), as a function of SCL transmissibility. The solid line
corresponds to a theoretical model, which corroborates well with the
experimental data. Lenses with oxygen transmissibilities around Dk/t
~ 150 hBarrer/cm have uptake rates of ~ 10 μL(STP)/cm2/h, in close
agreement with our previously obtained no-lens human uptake rates
of 9 - 13 μL(STP)/cm2/h at open eye (Takatori et al. IOVS. 2012; 53:
6331-6337).
Conclusions: Application of the classical procedure to our
experimental data gives corneal-uptake results that are about three to
five times smaller those classically reported. According to our
corrected procedure, full oxygenation of the human eye, 11.3 μL/cm2
/h for the 10 subjects studied, is reached only asymptotically (see Fig.
1). That is, very high lens transmissibility is required before the lens
no longer impedes oxygen transport. Conversely, 95 % anterior
corneal oxygenation is achieved with a lens transmissibility of 150
hBarrer/cm.
Fig 1. Reduction of oxygen uptake into the human cornea by SCL
wear at open eye, as a function of lens transmissibility.
Commercial Relationships: Percy Lazon De La Jara, AMO (F),
Allergan (F); Sho C. Takatori, None; Klaus Ehrmann, None;
Arthur Ho, None; Brien A. Holden, Allergan (F), AMO (I);
Clayton J. Radke, novartis corporation (F)
Program Number: 506 Poster Board Number: B0143
Presentation Time: 10:30 AM - 12:15 PM
Adhesion of Stenotrophomonas maltophilia, Delftia acidovorans
and Achromobacter xylosoxidans to contact lenses
Ajay Kumar Vijay, Mark D. Willcox. School of Optometry & Vision
Science, Sydney, NSW, Australia.
Purpose: Contact lens cases become contaminated with microbes
during use with certain MPDS containing Polyquaternium/Aldox
associated with high levels of Gram-negatives in cases, and the
degree of contamination is dependent on the type of multipurpose
disinfecting solution (MPDS) that is used. Furthermore, the MPDS
solution that has high levels of case contamination is also associated
with higher levels of infiltrates during lens wear. Thus, we wished to
establish whether microbes isolated from these MPDS/lens cases
were able to adhere to contact lenses, as this is hypothesized to be the
likely mechanism for transfer to the eye leading to the production of
infiltrates.
Methods: Strains of D. acidovorans, S. maltophilia, A. xylosoxidans
and P. aeruginosa isolated from contact lens cases (or contact lenses
at time of infiltrative response, P. aeruginosa only) were used.
Initially S. maltophilia was grown on agar plate overnight, cells
collected and washed once in PBS and re-suspended in 1:100
TSB:PBS at 1.0 x 105 to 1.0 x 109 cfu/ml and allowed to adhere to
lenses (senofilcon A or etafilcon A) for 24 hours. Subsequently, all
strains of bacteria were allowed to adhere to lenses for 24 hours at
starting inoculum of 1.0 x 107 cfu/ml. The numbers of bacteria
adherent to each lens type was estimated by culture.
Results: The adhesion of S.maltophilia to lenses was not affected by
initial inoculum size, with adhesion reaching 6.3 ± 0.2 log10cfu/lens
after 24 hours. Using initial inocula of 1.0 x 10 7cfu/ml, adhesion to
etafilcon A ranged from 5.0 ± 0.1 log10cfu/lens for A. xylosoxidans to
6.2 ± 0.0 log10cfu/lens for S.maltophilia. Adhesion to senofilcon A
ranged from 5.1 ± 0.3 log10cfu/lens for D. acidovorans to 6.2 ± 0.3
log10cfu/lens for P. aeruginosa. Adhesion to etafilcon A was
significantly higher (p<0.05) for P. aeruginosa and S. maltophilia
compared to D. acidovorans or A. xylosoxidans; adhesion to
senofilcon A was significantly higher (p<0.05) for P.aeruginosa
only.
Conclusions: This study has found that bacteria that are commonly
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
found in contact lens cases of Polyquaternium/Aldox care systems
can adhere to contact lenses in relatively high numbers. This may
facilitate their transfer into the eye and the production of corneal
infiltrates.
Commercial Relationships: Ajay Kumar Vijay, Bausch+Lomb
(F); Mark D. Willcox, Allergan Inc (C), Allergan Inc (R), Brien
Holden Vision Institute (P), Bausch + Lomb (C), Basuch + Lomb (R)
Program Number: 507 Poster Board Number: B0144
Presentation Time: 10:30 AM - 12:15 PM
Antimicrobial Activity of Melimine or Cathelicidin Bound to
Contact Lenses
Debarun Dutta1, 2, Mark D. Willcox2. 1Brien Holden Vision Institute,
Sydney, NSW, Australia; 2School of Optometry and Vision Science,
University of New South Wales, Sydney, NSW, Australia.
Purpose: Development of antimicrobial contact lens could have the
capacity to reduce the rate of contact lens related adverse events. The
purpose of this study was to evaluate two cationic peptides coated on
contact lenses for their activity against P. aeruginosa and S. aureus.
Methods: Minimal inhibitory concentration (MIC) of two peptides,
Melimine (a synthetic peptide) and Cathelicidin (LL37) was
measured against strains of P. aeruginosa and S. aureus. Increasing
concentrations of peptides were covalently bound to contact lenses.
Antimicrobial activity against the bacteria was evaluated by
measuring the amount of cell death compared to control lenses with
no melimine or LL37.
Results: MIC of LL37 against both P. aeruginosa and S. aureus was
3.9µg ml-1, whereas for Melimine against the same bacteria it was
500 µg ml-1 and 250 μg ml-1 respectively. Contact lenses covalently
reacted with 1mg ml-1 Melimine showed 0.8 log and 2.6 log
inhibition against P. aeruginosa and S. aureus respectively whereas
no inhibition was detected with LL37 at that concentration. Contact
lenses prepared with 3mg ml-1 melimine and LL37 showed 3.1 log
and 3.3 log inhibition against P. aeruginosa , 3.9 log and -0.2 log
inhibition against S. aureus respectively.
Conclusions: Whilst Melimine on contact lenses had activity against
both bacterial types; covalently bound LL37 was not active against S.
aureus. These differences suggest different mechanisms of action
against Gram-negative or Gram-positive bacteria by these two
cationic peptides.
Commercial Relationships: Debarun Dutta, None; Mark D.
Willcox, Allergan Inc (C), Allergan Inc (R), Brien Holden Vision
Institute (P), Bausch + Lomb (C), Basuch + Lomb (R)
Program Number: 508 Poster Board Number: B0145
Presentation Time: 10:30 AM - 12:15 PM
Establishing a standard method for evaluating efficacy against
Acanthamoeba
Monica Crary, Rhonda Walters, John Bartell, Manal M. Gabriel,
Jagath Kadurugamuwa, Bradley J. Catalone. Alcon Laboratories,
Fort Worth, TX.
Purpose: To establish a standard method for Multipurpose Contact
Lens Solution (MPS) efficacy testing against Acanthamoeba by
identifying and reducing variables.
Methods: Bacterized and axenic cultures of trophozoites were used
to determine if culture methods affected MPS efficacy. Cysts that
were prepared by starvation with or without Escherichia coli were
tested to determine variability in susceptibility to MPS following
bacterization. Additionally, the effect of heterogeneous cultures on
MPS efficacy was determined by examining the increasing ratios of
cysts to trophozoites in axenic cultures that were harvested between
3-7 days of age. Most Probable Number was used for enumeration at
different post-test time points to determine the earliest appropriate
read date for both cell types. Other variables such as inoculum
concentrations and MPS volumes were also examined.
Results: Current recommendations suggest that bacterization of
Acanthamoeba is the preferred culture method when evaluating MPS
efficacy. However, residual E. coli decreases the available biocide,
thereby resulting in variability correlated with bacterial
concentration. Current culture methods are limited in their ability to
generate homogeneous populations of trophozoites. Axenic
trophozoite cultures can contain cysts which contribute to the
observed variability of MPS efficacy. Harvesting of trophozoites at 3
days results in a more homogenous population of trophozoites as
compared to harvesting at 5 or 7 days. Another variable that
contributes to conflicting results in the literature is the duration of
incubation prior to enumeration. These studies clearly demonstrate
that the minimum incubation period for trophozoites plates is 7 days,
whereas for cysts it is 14 days. Earlier time points result in incorrect
enumeration. Other potential sources of variability include surface to
volume ratio of organisms and MPS.
Conclusions: The maximization of a homogenous population of
trophozoites or cysts is necessary to obtain reproducible results when
testing MPS efficacy. Variability of test results is affected by the
purity of the target cell type. For maximum accuracy, enumeration of
cells should not be performed prior to the minimum incubation
period. These identified variables have a significant effect on the
reproducibility and the performance of MPS against Acanthamoeba.
Commercial Relationships: Monica Crary, Alcon Labs (E);
Rhonda Walters, Alcon Laboratories (E); John Bartell, Alcon Labs
(E); Manal M. Gabriel, Alcon, A Novartis company (E); Jagath
Kadurugamuwa, Alcon Labs (E); Bradley J. Catalone, Alcon, a
Novartis Company (E)
Program Number: 509 Poster Board Number: B0146
Presentation Time: 10:30 AM - 12:15 PM
Risk Factors for Contact Lens Related Microbial Keratitis in
Singapore
Chris Lim1, Nicole A. Carnt1, 2, Mohamed Farook3, Janice Lam3,
Jodhbir S. Mehta3, Donald T. Tan3, Fiona Stapleton1. 1School of
Optometry and Vision Science, University of New South Wales,
Sydney, NSW, Australia; 2Moorfields Eye Hospital Trust, London,
United Kingdom; 3Singapore National Eye Centre, Singapore,
Singapore.
Purpose: Patterns of contact lens prescribing, wearer behavior and
environmental microbiota vary across different cultures and climates,
which may impact risks for microbial keratitis. This study
investigates independent risk factors for microbial keratitis in contact
lens wearers in Singapore.
Methods: Cases were contact lens wearers presenting to Singapore
National Eye Centre with microbial keratitis between 2009-2010.
Contact lens wearers attending for routine aftercare at a nearby
University Clinic over the same time period were identified as
controls. All wearers completed a previously validated questionnaire
describing contact lens wear history, hygiene and compliance habits
and demographics. Risk factors significant in univariate analysis
(p<0.2) were evaluated using multiple binary logistic regression.
Results: Fifty eight cases of microbial keratitis and 152
contemporaneous controls were identified. When controlling for
other variables, showering in lenses was associated with a 3x higher
risk (95% CI 1.1-6.6). Washing and drying hands prior to handling
was associated with an 8x lower risk (95% CI 1.3-47.6). Complete
lens care solution (AMO, CA) had a higher risk compared to
hydrogen peroxide and other multipurpose lens care solutions (OR
27.8 95% CI 2.5-333.3; OR 8.9, 95% CI 3.1-22.2, respectively).
Chinese ethnicity had a 7x lower risk compared to other races (95%
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
CI 2.3-18.7).
Conclusions: Consistent with previous findings, independent risk
factors for contact lens related microbial keratitis included poor hand
hygiene. Showering in lenses and type of lens care solution was also
associated with increased risk in this population. A case control study
of fungal keratitis in Singapore similarly found a lower risk for
Chinese compared to Malays, similar to findings in this study. While
this may be associated with socioeconomic factors, other behavioural
and innate factors warrant further investigation.
Commercial Relationships: Chris Lim, None; Nicole A. Carnt,
Vistakon (C), Alcon (C); Mohamed Farook, None; Janice Lam,
None; Jodhbir S. Mehta, None; Donald T. Tan, Network Medical
Products (P), Carl Zeiss Meditec (F), Alcon Labs (F), Bausch &
Lomb (F), Allergan (F), Santen (F); Fiona Stapleton, None
Program Number: 510 Poster Board Number: B0147
Presentation Time: 10:30 AM - 12:15 PM
The Antimicrobial Efficacy of Human Tear Proteins after
Repeated Exposure to Contact Lens Solutions
Bianca L. Price1, Philip B. Morgan2, Carole Maldonado-Codina2,
Curtis B. Dobson1. 1Faculty of Life Sciences, University of
Manchester, Manchester, United Kingdom; 2Eurolens research,
Faculty of Life Sciences, University of Manchester, Manchester,
United Kingdom.
Purpose: We have previously shown that tear proteins extracted
from soft contact lenses after one-day of wear possess potent
antimicrobial activity, which functions synergistically with certain
MPS. This effect is demonstrable during exposure to MPS and can
add to the antimicrobial activity of the solution. Here we investigated
whether this activity is detectable during conditions that better reflect
typical lens disinfection procedures (i.e. incubation at room
temperature for four hours in MPS). Additionally, we investigated
whether ex vivo protein extracts are still active after one week of
wear. In this study we examined whether the MPS used to treat lenses
daily during a week of wear affected the activity of the tear proteins.
Methods: Clinical samples of PureVision lenses worn for a week
were collected from four subjects and tear proteins were extracted
with ACN/TFA. For each pair of lenses, one was treated with Biotrue
each night, and one with Evermoist. Bacterial overnight cultures were
prepared and normalized to an OD600 of 1.0 in PBS. Suspensions of
Pseudomonas aeruginosa (ATCC 9027) or Staphylococcus aureus
(ATCC 6538) were challenged with extracts from worn or unworn
lenses in the presence of PBS+EDTA for four hours at room
temperature.
Results: Incubation of S. aureus and P. aeruginosa with tear proteins
extracted from worn lenses after treatment with full formulation MPS
showed readily observable antibacterial effects after four hours
incubation at room temperature. Tear proteins repeatedly exposed to
Biotrue (which has been previously shown to stabilize tear proteins)
showed greater antimicrobial activity than those exposed to
Evermoist.
Conclusions: These data show that tear proteins absorbed to soft
contact lenses possess substantial antimicrobial activity, and that this
activity can be modulated by ex vivo treatments of worn lens with
fully formulated Biotrue MPS under conditions likely to be
encountered during contact lens disinfection. Additionally, the
antimicrobial activities of tear proteins extracted from worn lenses
remain observable after multiple days of MPS treatments.
Commercial Relationships: Bianca L. Price, Bausch and Lomb (F);
Philip B. Morgan, Bausch & Lomb (F); Carole MaldonadoCodina, Bausch + Lomb (F), CooperVision Inc. (F), Johnson &
Johnson Vision Care (F), Sauflon Pharmaceuticals (F), Alcon Inc.
(F); Curtis B. Dobson, Bausch + Lomb (F)
Support: BA06951
Program Number: 511 Poster Board Number: B0148
Presentation Time: 10:30 AM - 12:15 PM
Traveler's Contact Lens Associated Keratitis (TCLAK):
Establishing Preventive and Treatment Guidelines To Close A
Gap in Ophthalmic Care
Fallon Ukpe1, Stephanie Youlios2, 4, Bela Parekh4, Jai G. Parekh3.
1
Duke University School of Medicine, Durham, NC; 2University of
Maryland, Baltimore, MD; 3New York Eye & Ear Infirmary, New
York, NY; 4Brar-Parekh Eye Associates, Edison, NJ.
Purpose: To evaluate preventive care and education provided to
patients with contact lenses in order to prevent infectious keratitis.
The authors propose the categorization of contact lens related
infectious keratitis acquired during travel as Traveler's Contact Lens
Associated Keratitis (TCLAK) and recommend specific preventive
and treatment measures for contact lens wearing patients. While the
incidence of contact lens associated keratitis is low, there are
increasing numbers of cases we have defined as TCLAK due to
higher contact lens usage, longer duration of lens wear, and
increasing international travel. This is relevant as TCLAK presents
higher morbidity risks since there is often decreased access to
ophthalmic care abroad, thereby leading to a delay in treatment and
poorer prognosis and outcomes.
Methods: Conducted a review of guidelines for safe contact lens use
and care from the American Association of Ophthalmology (AAO),
the Contact Lens Association of Ophthalmologists (CLAO), the
Centers for Disease Control (CDC), and the Food and Drug
Administration (FDA). More specifically, the review searched for
preventive and educational care guidelines for contact lens wearers
prior to travel.
Results: While guidelines for contact lens use and care caution
patients in order to prevent complications through appropriate lens
cleaning and disinfection as well as discarding lenses after the
prescribed duration of wear, there are no specific recommendations
regarding precautions to take when traveling internationally;
furthermore, there are no warnings of the potentially higher risks of
keratitis due to conditions abroad and the prevalence of certain
infectious microbes in particular areas.
Conclusions: The lack of preventive patient education and provider
care guidelines specifically aimed at decreasing the incidence of
TCLAK is a gap that requires attention. As patients with TCLAK
have a higher risk for devastating outcomes such as loss of sight or
even enucleation, the specific recognition of TCLAK infections as a
group as well as the development of preventive guidelines for
patients and clinical care guidelines for providers is essential. The
authors have designed a set of guidelines to specifically address this
gap and recommend dissemination of these guidelines for patients
traveling with contact lenses and for providers caring for contact lens
wearing patients.
Commercial Relationships: Fallon Ukpe, None; Stephanie
Youlios, None; Bela Parekh, None; Jai G. Parekh, None
Program Number: 512 Poster Board Number: B0149
Presentation Time: 10:30 AM - 12:15 PM
Lipid Adherence to Model Contact Lens Materials
Holly I. Lorentz1, Giuliano Guidi1, Lyndon W. Jones2, Heather
Sheardown1. 1Chemical Engineering, McMaster University,
Hamilton, ON, Canada; 2CCLR - School of Optometry & Vision
Science, University of Waterloo, Waterloo, ON, Canada.
Purpose: The purpose of this study was to analyze the impact of
incorporating novel hydrophilic substances into silicone materials
and their impact on cholesterol (CH) and phosphatidylcholine (PC)
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
deposition using radiochemical analysis.
Methods: Thirteen model lens materials were created based on
combinations of dimethylacrylamide (DMAA), hydroxyethyl
methacrylate (HEMA) and
methacryloxypropyltris(trimethylsiloxy)silane (TRIS). The materials
were prepared with and without the incorporation of a hydrophilic
novel substance such as hyaluronic acid, alginate, and a silicone
surfactant. The model materials were hydrated and advancing contact
angles were measured using the sessile drop technique. All materials
were incubated in a complex artificial tear solution (ATS) containing
protein, lipid, mucin and a trace amount of either 14C-CH or 14C-PC
for 3 and 14 days. All materials were then extracted, processed and
masses of deposition were quantified (ng/disc) using standard
calibration curves.
Results: For the HEMA-based materials tested, only the materials
that incorporated the silicone surfactant and alginate showed a
statistically significant increase in wettability (p<0.05) over the
controls by decreasing the contact angle from 90.8° for the HEMATRIS control down to 12.7° for the surfactant HEMA-TRIS material
and 60.9° the alginate HEMA-TRIS material. For the DMAA-TRIS
materials only the silicone surfactant material showed an
improvement in material wettability (p<0.001) with a contact change
from 97.1 to 48.2°. For lipid deposition, the incubation time in the
ATS had a significant impact on deposition (p ≤0.001) with the 14
day incubations depositing up to 3.4x more lipid than the 3 day
incubations. No statistically significant decreases in deposition were
seen for any material which incorporated the hydrophilic substance
when compared with the controls. However, there were some
statistically significant increases in CH and PC deposition (p<0.05)
when compared to the corresponding controls, especially for the
HEMA-TRIS surfactant containing materials.
Conclusions: Despite the incorporation of hydrophilic substances,
such as hyaluronic acid, alginate, and silicone surfactants, into
HEMA-TRIS and DMAA-TRIS materials, no reductions in CH and
PC deposition were found.
Commercial Relationships: Holly I. Lorentz, None; Giuliano
Guidi, None; Lyndon W. Jones, Alcon (F), Alcon (R), Allergan (F),
Abbott Medical Optics (R), Bausch & Lomb (R), Ciba Vision (F),
Ciba Vision (R), CooperVision (F), Johnson & Johnson (F), Johnson
& Johnson (R); Heather Sheardown, Alcon (F), Alimera Sciences
(F)
Support: NSERC 20/20 Ophthalmic Network
Program Number: 513 Poster Board Number: B0150
Presentation Time: 10:30 AM - 12:15 PM
Quorum Sensing Molecules in the Preferential Selection of
Pseudomonas aeruginosa from Contaminated Contact lens Cases
Darlene Miller, Katyayini Aribindi, Galina Dvoriantchikova, Dmitry
V. Ivanov, Sanjoy K. Bhattacharya, Jorge Maestre, Eduardo C.
Alfonso. Bascom Palmer Eye Institute, Univ of Miami Miller Sch of
Med, Miami, FL.
Purpose: To document and correlate the presence of quorum sensing
(QS) proteins/genes and their role in the selection of Pseudomonas
aeruginosa as a preferential corneal pathogen from contaminated
contact lens cases.
Methods: We used a combination of Proteomics, RT-PCR and plate
biosensor bioassays to detect the presence of Pseudomonas
aeruginosa quorum sensing proteins (lasI/lasR, rhlI/rhlR and rpoD)
and signaling molecules (acyl homoserine lactones) in 15 randomly
selected contaminated contact lens cases recovered from patients with
keratitis (N=26, Jan2011-June 2012). Results were correlated with
cultures and number of microbial communities members. Contact
lens cases were retrieved from storage at 4 C, refreshed with 2 ml
trypticase soy broth and incubated at room temperature for 24-48
hours. 1 ml aliquots were retrieved and used for the study.
Results: Pseudomonas aeruginosa emerged as the cornea pathogen
from 76.9% (20/26, N=83 species) of all matched ctl/cornea cultures.
There was an average of 3.8 species per well.
P. aeruginosa quorum sensing and or metabolic proteins including
protease IV, elastase, and elastase B were recovered in 10/22 (45.4%)
of the proteins identified by mass spectrometer in sample 1 of three
samples. No proteins were recovered that correlated with any of the
other community members (Acanthamoeba species, Klebsiella
oxytoxca, Mycobacteria chelonae). Proteins specific for S.
marcescens (#2) was documented in 5/44 (11.4%). Neither proteins
from P. aeruginosa or S. marcescens were recovered in the 37
proteins from sample #3. No proteins were recovered from the tsb
control.
Both quorum sensing systems (las and rhl) were expressed in aliquots
from at least one well in 8 of the 15 evaluated contact lens cases.
Pseudomonas aeruginosa was recovered in 5/8 (62.5%) in the
presence of at least 2 other community members (N=21).
Acyl homoserine lactones production from case aliquots was evident
in 3/15 (20%) of C. violaceum screening vs 6/15 (40%) for A.
tumefaciens.
Conclusions: The production and expression of quorum sensing
genes and signaling molecules in contact lens case ecosystems may
allow for the preferentially selection of P. aeruginosa as a corneal
pathogen. Deciphering this mechanism can lead to solutions to reduce
and or neutralize the advantage.
Commercial Relationships: Darlene Miller, None; Katyayini
Aribindi, None; Galina Dvoriantchikova, None; Dmitry V.
Ivanov, None; Sanjoy K. Bhattacharya, None; Jorge Maestre,
None; Eduardo C. Alfonso, Bio Tissue (C)
Support: unrestricted grant from Research to Prevent Blindlness,
Core grant, NIH
Program Number: 514 Poster Board Number: B0151
Presentation Time: 10:30 AM - 12:15 PM
Selenium Contact Lens Hydrogel Polymer: Inhibition of
Bacterial Biofilm Formation
Phat L. Tran1, Abdul Hamood2, Daniel Webster3, Courtney Jarvis1,
Robert E. Hanes5, Ted W. Reid1, 4. 1Ophthalmology and Visual
Science, Texas Tech University Health Sciences Ctr, Lubbock, TX;
2
Microbiology, Texas Tech University Health Sciences Ctr, Lubbock,
TX; 3Cell Biology, Texas Tech University Health Sciences Ctr,
Lubbock, TX; 4Selenium Ltd., Lubbock, TX; 5Selenium Ltd., Austin,
TX.
Purpose: Biofilm formation on contact lenses has been cited as a
possible cause of corneal infection and acute red eye. A contact lens
that blocks biofilm formation should reduce the frequency of these
clinically significant problems. Selenium compounds have the ability
to catalyze the formation of superoxide radicals in the tear film,
which are cytotoxic to bacteria. Thus, this study investigated the
effectiveness of a covalent organo-selenium polymerized into a
hydrogel against bacterial biofilm formation.
Methods: Organo-selenium compounds were polymerized directly
into a hydrogel. The inhibition of biofilm formation with the organoselenium hydrogel was investigated by incubating organo-selenium
hydrogels and selenium free hydrogel in a nutrient broth containing
Staphylococcus aureus for 24 hours at 37 degrees C. Biofilms were
quantified by determining the CFU (colony forming units) per lens.
To determine the CFU/lens, each lens was gently rinsed with sterile
distilled water, and placed into a microcentrifuge tube containing 1
ml phosphate buffered saline (PBS), and then vigorously vortexed
three times for 1 min vortex to detach the cells. Suspended cells were
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
serially diluted 10-fold in PBS and 10-μl aliquots of each dilution
were spotted on LB agar plates. The plates were incubated at 37
degrees C for 24 hours and the CFU were counted. For confocal laser
scanning microscopy (CLSM), we used the S. aureus strain AH1333
which carries the gene that encodes the green fluorescent protein.
Results: Colony forming unit assays showed total inhibition,
representing over 6 logs of Staphylococcus aureus killing on organoselenium polymerized hydrogels. Confocal laser scanning
microscopy confirmed these results.
Conclusions: The organo-selenium hydrogel polymer successfully
blocked the formation of a bacterial biofilm on the polymer by
Staphylococcus aureus in vitro.
Commercial Relationships: Phat L. Tran, None; Abdul Hamood,
None; Daniel Webster, None; Courtney Jarvis, None; Robert E.
Hanes, Selenium, Ltd. (F), Selenium, Ltd. (I), Selenium, Ltd. (E),
Selenium, Ltd. (P); Ted W. Reid, Selenium Ltd. (I), Selenium Ltd.
(P)
Program Number: 515 Poster Board Number: B0152
Presentation Time: 10:30 AM - 12:15 PM
Contact Lens Disinfecting Solution vs. Blister Pack: a Subjective
Evaluation
Scott Schatz1, Balamurali Vasudevan2, Sara N. Gaib3, Kimbal
Cooper4. 1Appalachian College of Optometry, Grundy, VA; 2College
of Optometry, Midwestern University, Glendale, AZ; 3College of
Optometry, Midwestern University, Glendale, AZ; 4College of Health
Sciences, Midwestern University, Glendale, AZ.
Purpose: To study subjectively the effect of contact lens disinfecting
solution on the ocular surface in the presence of either a hydrogel or a
silicone hydrogel soft contact lens.
Methods: Twenty young adult subjects were examined for this study
over 2 visits. Subjects were fit (randomized) with either a hydrogel
lens or silicone hydrogel lens in either eye. The lenses were presoaked overnight in either Puremoist or Revitalens contact lens
disinfecting solution or were obtained from the blister pack in a
randomized process. Subjective feedback on discomfort, burning,
dryness and irritation on a scale of 1 to 5 was obtained at baseline,
prior to lens insertion, and after 8 hours of wear. Chi-square analysis
was performed.
Results: There was a statistically significant increase in dryness after
lens insertion for Proclear and Purevision lenses taken from the
blister pack , relative to baseline (p = 0.01). Following 8 hours of lens
wear, there was a statistically significant increase in burning
sensation relative to baseline for all Proclear lenses. This held true
whether they were obtained from the blister pack or pre-soaked in
Revitalens MPDS or Puremoist MPDS (p=0.01).
Conclusions: Subjectively, lenses from the blister pack produced
more subjective symptoms of dryness upon initial insertion than
those pre-soaked in either solution. Proclear lenses produced more
subjective symptoms of burning after 8 hours of wear relative to
baseline irrespective of whether they were obtained from the blister
pack, or pre-soaked in either solution.
Commercial Relationships: Scott Schatz, Abbot Medical Optics
Inc (F); Balamurali Vasudevan, None; Sara N. Gaib,
Bausch+Lomb (C), Alcon (R), Vistakon (R), Coopervision (R);
Kimbal Cooper, None
Support: Study was supported by a grant from Abbott Medical
Optics
Program Number: 516 Poster Board Number: B0153
Presentation Time: 10:30 AM - 12:15 PM
Antimicrobial Efficacy of New Investigational Multipurpose
Disinfecting Solution and Comparison to Commercially
Available Multipurpose Disinfecting Solutions
Marina Milenkovic, Nancy Brady, Anthony Lam. Corneal R&D
Microbiology, Abbott Medical Optics, Santa Ana, CA.
Purpose: To evaluate antimicrobial efficacy of investigational
multipurpose formulation against bacteria, fungi and Acanthamoeba
spp. and compare it to commercially available multipurpose
disinfecting solutions (MPS). Study was done according to ISO
14729:2001/A.2010 standard.
Methods: The multipurpose disinfecting solutions studied were Investigational MPS-1: polyhexamethylene biguanide (PHMB) +
poloxamer (PLX) and currently marketed Japan products MPS-2:
polyquaternium (PQ1) + tetronic 1304, MPS-3: PHMB + poloxamine
(PLA) and MPS-4: PHMB +PLX.
Test organisms were: Staphylococcus aureus (ATCC 6538),
Pseudomonas aeruginosa (ATCC 9027), Serratia marcescens (ATCC
13880), Candida albicans (ATCC 10231), Fusarium solani (ATCC
36031) and Gram-negative clinical isolates. Test solutions were
evaluated at the minimum recommended disinfection time of 4 hours.
Disinfectant efficacy of MPS was also evaluated against
Acanthamoeba trophozoites.
Results: After 4 hours exposure, Investigational MPS-1 and MPS-3
showed >3 log kill of ISO bacteria and fungi. MPS-2 and MPS-4
failed to meet stand-alone criteria for ISO bacteria and fungi. MPS-1,
MPS-3 and MPS-4 showed >3 log kill against clinical isolates, while
MPS-2 showed <1 log kill. MPS-1 and MPS-3 achieved >3 log
reduction for Acanthamoeba trophozoites, while MPS-4 achieved <2
log reduction and MPS-2 achieved <1 log reduction.
Conclusions: New Investigational MPS-1 exhibited broad
antimicrobial activity against ISO panel organisms and showed
additional robustness against clinical isolates and Acanthamoeba
trophozoites. MPS-1 showed similar activity as MPS-3, while MPS-2
and MPS-4 failed to meet stand-alone criteria against ISO organisms
and clinical isolates.
Commercial Relationships: Marina Milenkovic, Abbott Medical
Optics (E); Nancy Brady, Abbott Medical Optics (E); Anthony
Lam, Abbott Medical Optics (E)
Program Number: 517 Poster Board Number: B0154
Presentation Time: 10:30 AM - 12:15 PM
Efficacy of multi-purpose solutions in removing cholesterol
deposits from silicone hydrogel contact lenses
Hendrik Walther, Lakshman N. Subbaraman, Lyndon W. Jones.
Centre for Contact Lens Research, School of Optometry and Vision
Science, University of Waterloo, Waterloo, ON, Canada.
Purpose: To determine the efficacy of saline and multi-purpose
solutions (MPS) on the removal of cholesterol deposits from silicone
hydrogel (SH) contact lens materials using an in vitro model.
Methods: Six SH lens materials: senofilcon A (Acuvue Oasys),
comfilcon A (Biofinity), balafilcon A (Pure Vision2), lotrafilcon A
(Air Optix® Night and Day Aqua), lotrafilcon B (Air Optix® Aqua)
and lotrafilcon B toric (Air Optix® for Astigmatism) were removed
from the blister pack (n=4 for each lens type), incubated for 7 days at
37°C in an artificial tear solution (ATS) containing 14-C radiolabeled
cholesterol. Thereafter, lenses were cleaned with an unpreserved
saline solution (Sensitive Eyes) or one of five MPS (Opti-Free®
PureMoist, renu fresh, RevitaLens, Biotrue, SoloCare Aqua) using a
rub and rinse technique, according to the manufacturer
recommendations, and stored in the MPS for a minimum of six hours.
Lenses were then extracted with 2:1 chloroform:methanol, analyzed
in a beta counter and µg/lens of cholesterol was determined.
Results: Balafilcon A and senofilcon A showed the highest amounts
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
of accumulated cholesterol (0.93±0.02µg/lens, 0.95±0.01µg/lens
respectively), while lotrafilcon A and lotrafilcon B deposited the
lowest amounts (0.37±0.03; 0.47±0.12). OptiFree PureMoist
removed more cholesterol than the other solutions for all lens
materials; however, the amount of cholesterol cleaned was
statistically significant for balafilcon A and senofilcon A lens
materials (p=0.006 and p=0.042). Sensitive Eyes and the other MPS
evaluated showed no significant effect on lipid removal (p>0.05).
Conclusions: Lipid-removal efficacy varies depending on the
combination of lens material and solution. Only one MPS showed a
significant reduction of lipids for any of the lenses tested. It will be
valuable to conduct further work to determine the efficacy of MPS in
removing lipid deposits on worn lenses and how these deposits may
impact subjective comfort.
Commercial Relationships: Hendrik Walther, None; Lakshman
N. Subbaraman, None; Lyndon W. Jones, Alcon (F), Alcon (R),
Allergan (F), Abbott Medical Optics (R), Bausch & Lomb (R), Ciba
Vision (F), Ciba Vision (R), CooperVision (F), Johnson & Johnson
(F), Johnson & Johnson (R)
Support: Alcon USA
Program Number: 518 Poster Board Number: B0155
Presentation Time: 10:30 AM - 12:15 PM
Antimicrobial Efficacy of Multipurpose Disinfecting Solutions
against Clinical Isolates after Prolonged Storage
Anthony Lam, Nancy Brady, Marina Milenkovic. Corneal R&D
Microbiology, Abbott Medical Optics, Santa Ana, CA.
Purpose: To compare antimicrobial efficacy of commercially
available multipurpose disinfecting solutions (MPS) against Gramnegative clinical isolates following prolonged storage in lens case in
the presence of a lens.
Methods: The multipurpose disinfecting solutions studied were MPS-1: polyquaternium (PQ1) + alexidine dihydrochloride (ALX),
MPS-2: PQ1 + polyhexamethylene biguanide (PHMB), MPS-3: PQ1
+ myristamidopropyl dimethylamine (ALDOX) and MPS-4: PQ1 +
ALDOX + nonanoyl ethylenediaminetriacetic acid (EDTA).
Test solutions were inoculated with Gram-negative clinical isolates in
the appropriate lens case, in the presence of Acuvue2 (etafilcon A)
lens. Test solution efficacy was evaluated at minimum recommended
manufacturer disinfection time of 6 hours and following prolonged
storage.
Test solutions were also inoculated with Gram-negative clinical
isolates in the test tube and tested according to ISO 14729.
Results: After 6 hours exposure, MPS-1 and MPS-2 showed >3 log
kill against clinical isolates in a test tube, while MPS-3 and MPS-4
failed stand-alone criteria. MPS-1 and MPS-2 maintained efficacy in
the lens case in the presence of Acuvue2 lens and achieved >2.5 log
kill. MPS-3 and MPS-4 showed <1 log kill or organism regrowth at 6
hours exposure.
Following 7 days storage in a lens case, MPS-1 achieved >3 log kill
for all organisms tested. MPS-2 and MPS-3 failed to achieve 3 log
kill for one organism tested. MPS-4 failed to achieve 3 log kill for
two organisms tested.
Following 30 days storage in a lens case, MPS-1, MPS-2 and MPS-3
achieved >3 log kill for all organisms tested, while MPS-4 failed to
achieve 3 log kill for two organisms tested.
Conclusions: Gram-negative clinical isolates are resistant to MPS-3
and MPS-4. MPS-1 and MPS-2 showed ability to reduce microbial
load under worst case conditions, tested in a lens case with Acuvue2
lens present.
Commercial Relationships: Anthony Lam, Abbott Medical Optics
(E); Nancy Brady, Abbott Medical Optics (E); Marina Milenkovic,
Abbott Medical Optics (E)
Program Number: 519 Poster Board Number: B0156
Presentation Time: 10:30 AM - 12:15 PM
Disinfection Efficacy of Rigid Gas Permeable Lens Care Systems
Suzanne F. Groemminger, Denise Callahan. Bausch & Lomb Inc,
Rochester, NY.
Purpose: This study compared the disinfecting efficacy of Rigid Gas
Permeable (RGP) lens care products.
Methods: The primary disinfecting solution from each lens care
system was evaluated using the ISO/FDA Stand Alone Procedure for
Disinfecting Products. Three distinct lots of each product were tested
using the regimen time listed on the patient instructions. The systems
tested using a 4 hour disinfection time included two-single bottle
multipurpose products: Boston Simplus and Unique pH, a two bottle
system: Boston Advance Cleaner and Conditioning Solution. The
three 3 bottle system: Lobob Optimum, was tested using a 6 hour
disinfection time.
Products were challenged with 3 bacterial species, Staphylococcus
aureus (Sa), Pseudomonas aeruginosa (Pa) and Serratia
marcescens(Sm) a yeast, Candida albicans (Ca) and a mold, Fusarium
solani (Fs).
The primary acceptance criteria states that the number of bacteria
recovered per mL shall be reduced by a mean value of not less than
3.0 logs within the minimum recommended disinfection period. The
mold and yeast recovered per mL shall be reduced by a mean value
of not less than 1.0 log within the minimum disinfection time with no
increase at not less than 4 times the disinfection time.
There are secondary acceptance criteria which require that there is a
combined log reduction for the mean values of all three bacteria of
not less than 5.0 logs within the recommended disinfection time. The
minimum acceptable mean log reduction for any single bacteria type
is 1.0 log. Stasis for the yeast and mold must be observed for the
recommended disinfection time.
Results: Disinfection efficacy results demonstrated Simplus,
Advance and Unique pH passed the primary criteria, with >4.7 log
reduction for all three bacterial species and >4.2 log reduction of the
mold. Both single bottle systems gave >4.2log reduction for the
yeast, while Advance demonstrated a 1.4 log reduction. Optimum
failed to pass the primary criteria for 2 of 3 of the bacterial species
(Sa-2.7 logs and Sm-0.6 logs) and also failed the secondary criteria
for yeast and mold.
Conclusions: RGP care products demonstrated varying degrees of
effectiveness to in their ability to disinfect RGP contact lenses.
Boston Simplus, Boston Advance, and Unique pH all passed the
primary acceptance criteria for disinfection efficacy set forth by ISO
and the FDA. Lobob Optimum had very weak activity, and failed to
meet even the secondary stand alone criteria.
Commercial Relationships: Suzanne F. Groemminger, Bausch &
Lomb Inc (E); Denise Callahan, Bausch and Lomb, Inc. (E)
Program Number: 520 Poster Board Number: B0157
Presentation Time: 10:30 AM - 12:15 PM
Effect of Contact Lenses and Lens Cases on Disinfection Efficacy
of Four Multipurpose Disinfection Solutions
Manal M. Gabriel, Cynthia McAnally, John Bartell, Rhonda Walters,
Jebree Spencer, Linda Clark, Bradley J. Catalone. Vision Care,
Alcon, Fort Worth, TX.
Purpose: To evaluate the effect of different contact lens materials
and cases on the disinfection efficacy of Multipurpose Disinfection
Solutions (MPDS) following the November 12, 2008 draft standard,
Antimicrobial Efficacy Endpoint Methodology to Determine
Compatibility of Contact Lens Solutions, Lens Cases and Hydrogel
Lenses for Disinfection (AEEMC).
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Methods: AEEMC testing was conducted in manufacturer supplied
lens cases. Two silicone hydrogel lenses (balafilcon A and senofilcon
A) and one conventional lens (etafilcon A) were inoculated with a
test organism (P. aeruginosa, S. marcescens, S. aureus, C. albicans,
or F. solani) in the presence of organic soil. After an exposure time
of 3-to10-minutes, MPDS was dispensed into each well. Aliquots of
the inoculated solutions were removed at the disinfection time (4 or 6
hrs as stated on the product label) and 24 hours for microbial
enumeration. Lens cases without lenses inoculated with organisms
were included as controls.
Results: Reduction in efficacy associated with contact lens was most
pronounced with C. albicans as a challenge organism at both
disinfection time and 24 hrs for all MPDS. Only one solution
consistently met > 1 log reduction for all three lenses at disinfection
time. Challenges with Fusarium also resulted in reduced efficacy in
the presence of contact lens for most of the MPDS at disinfection
time, although OPTI-FREE® PureMoist® showed no reduction in
efficacy. A reduction in efficacy against S. marcescens was also
observed at disinfection time with 2 out of 4 solutions. All MPDS
exhibited similar efficacy against P. aeruginosa and S. aureus.
Conclusions: The AEEMC studies are designed to more closely
simulate the conditions during actual use and evaluate the effect of
contact lenses in a lens case on the antimicrobial efficacy of MPDS.
Reduced efficacy of several MPDS were observed with C. albicans,
F. solani, and S. marcescens when tested following AEEMC
protocols in conjunction with balafilcon A, senofilcon A and
etafilcon A lenses. The current ISO standard 14729 does not address
the reduction in MPDS antimicrobial efficacy in the presence of
contact lenses. OPTI-FREE® PureMoist® demonstrated a high level
of efficacy under these simulated in-use conditions relative to other
MPDS.
Commercial Relationships: Manal M. Gabriel, Alcon, A Novartis
company (E); Cynthia McAnally, Alcon Research, Ltd. (E); John
Bartell, Alcon Labs (E); Rhonda Walters, Alcon Laboratories (E);
Jebree Spencer, Alcon Laboratories Inc. (E); Linda Clark, Alcon
Laboratories (E); Bradley J. Catalone, Alcon, a Novartis Company
(E)
Program Number: 521 Poster Board Number: B0158
Presentation Time: 10:30 AM - 12:15 PM
The Evaluation of the Biocidal Efficacy of Multi-Purpose
Solutions Against Mixed Cultures of Pseudomonas aeruginosa
with a Variety of Individual Fungal Organisms
Jessica M. Burger, Deborah McGrath, Brien C. David.
Microbiology, Bausch & Lomb, Inc, Rochester, NY.
Purpose: This study was performed to evaluate the biocidal activity
of Multi-Purpose Solutions (MPS) against Pseudomonas aeruginosa
mixed with fungal organisms. This testing was performed to
understand how effective MPS are against a multi-organism
challenge, which may better simulate polymicrobial contamination
reported for contact lens storage cases.
Methods: Stand-alone biocidal testing was used with modifications
to ISO Standard 14729. Organisms used in this study were not the
standard 5 ISO 14729 organisms, but rather mixtures of 6 separate
preparations that included P. aeruginosa and one of 6 different fungal
organisms. The organisms mixed with P. aeruginosa were: Candida
albicans, Candida tropicalis, Fusarium solani, Fusarium oxysporum,
Aspergillus brasiliensis, and Aspergillus fumigatus. The mixed
challenge inoculum was prepared at the concentration of ~5.0X10 5
and resuspended in DPBST with the incorporation of 10% organic
soil to provide a greater challenge. The mixed inoculum were then
used to challenge the MPS. The time points evaluated were 4 and 6
hours for each of the solutions. Each was plated out with Trypticase
Soy Agar, and results were recorded in log reductions.
Results: The results varied for the MPS tested. Fungal organisms
were recovered more than P. aeruginosa, as expected. Each solution
was assessed at the 4 and 6 hour time points. The recovery for the 4
hour time points ranged from 0.2 log reduction to >4.6 log reduction
(no microbial recovery observed), and the 6 hour time point ranged
from 0.0 log reduction to >4.6 log reductions (no microbial recovery
observed).
Conclusions: The results show that MPS have a broad range of in
vitro antimicrobial activity against the various P. aeruginosa and
fungal mixtures. Since environmentally sourced contaminants are
frequently mixtures of microorganisms, these results could
demonstrate MPS performance that reflects actual use conditions.
Further study is warranted to assess the microbiological and clinical
significance.
Commercial Relationships: Jessica M. Burger, Bausch & Lomb,
Inc (E); Deborah McGrath, Bausch&Lomb (E); Brien C. David,
Bausch + Lomb (E)
Program Number: 522 Poster Board Number: B0159
Presentation Time: 10:30 AM - 12:15 PM
Evaluation of Biocidal Efficacy of Multipurpose Solutions
Against Gram Negative Organisms in a Lens Case
Brien C. David, Denise Callahan, Julie Bair, Susan E. Norton. R&D
Microbiology, Bausch & Lomb, Rochester, NY.
Purpose: Evaluating the biocidal efficacy of multipurpose solutions
(MPS) against Gram - organisms associated with corneal infiltrative
events (CIEs), Achromobacter xylosoxidans, Stenotrophomonas
maltophilia, and Delftia acidovirans, in a lens case. Time points from
4 hrs to 7 days of exposure.
Methods: ISO Standard 14729 was used as a guideline to evaluate
biocidal efficacy of 6 MPS: Biotrue, ReNu Fresh, RevitaLens,
OptiFree Express,OptiFree Puremoist, OptiFree Replenish which
included the preservatives PHMB/PQ; PHMB; PQ/alexidine; and 3
different PQ/aldox based MPS; to challenges of Gram - organisms,
A.xylosoxidans, S. maltophilia, and D. acidovirans. Testing was
performed in lens cases appropriate to each Manufacturer's solution.
Each lens case was filled with 3 ml of MPS. Biocidal testing was
performed on 3 lots of each MPS on 3 separate days, in the lens case,
using ISO Standard 14729. Inoculum was prepared at ~5 x 10E5
colony forming units/mL. Biocidal testing was performed at 4, 6 and
24 hrs, and Day 7 after inoculation. .
Results: Test results varied depending on organism and solution. 3
solutions, Biotrue, ReNu Fresh, and ReVitalens showed >3 log
reduction for each organism at all time points. For OptiFree Express
and OptiFree Puremoist, there was increasing efficacy at each time
point, showing no microbial recovery by Day 7. However, OptiFree
Replenish did not achieve a 1 log reduction, even after 7 days
exposure. Overall, log reductions against these organisms ranged
from >5.0 log reduction (no microbial recovery) to -0.9 log reduction
(regrowth).
Conclusions: The ISO 14729 Stand Alone test is performed against
"ISO organisms" in test tubes. The current testing was performed in
contact lens cases using organisms associated with CIEs. In this
study, the three MPS that contained PQ/aldox as their preservatives
showed limited activity (<3 log reduction) against the three
organisms when tested at recommended soak times. Although two of
these solutions did show improvement with greater time of exposure,
one PQ/aldox solution (OptiFree Replenish) did not improve with
time of exposure. These studies show that ISO 14729 methodology
can be adapted to distinguish differences in antimicrobial effects
between various solutions. Further studies are needed to determine
the significance of these in-vitro results.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Commercial Relationships: Brien C. David, Bausch + Lomb (E);
Denise Callahan, Bausch and Lomb, Inc. (E); Julie Bair, Bausch &
Lomb, Inc. (E); Susan E. Norton, Bausch and Lomb, Inc. (E)
Program Number: 523 Poster Board Number: B0160
Presentation Time: 10:30 AM - 12:15 PM
Efficacy Of Contact Lens Solutions Against Achromobacter
xylosoxidans Biofilms Using Confocal Microscopy
David J. McCanna, Jaclyn M. Chang, Lakshman N. Subbaraman,
Lyndon W. Jones. Centre for Contact Lens Research, School of
Optometry and Vision Science, Waterloo, ON, Canada.
Purpose: Biofilms of Achromobacter xylosoxidans (Ax) can develop
in contact lens cases. These microorganisms can attach to the contact
lens and cause microbial keratitis. This study evaluated the
antimicrobial efficacy of contact lens solutions against Ax biofilms
by measuring the damage to cell membranes of Ax using confocal
microscopy.
Methods: Ax biofilms were formed by incubating the bacteria
overnight on glass coverslips. The biofilms were then exposed to
contact lens solutions for four hours. Commercial contact lens
solutions evaluated contained the antimicrobials polyhexamethylene
biguanide (PHMB), polyquaternium-1 (PQ1) and alexidine (ALX),
and PQ1 and Aldox (AD). After exposure, the bacteria were stained
with SYTO 9 and propidium iodide (PI). Using a confocal
microscope with a 488nm laser and the appropriate emission filters
the number of cells with damaged cell membranes was determined.
In addition to evaluating contact lens solutions, four concentrations of
benzalkonium chloride (BAK) 0.05%, 0.01%, 0.005% and 0.001% in
phosphate buffer saline were also evaluated to demonstrate dose
related effects at exposure times as short as 5 minutes.
Results: The contact lens solution that caused the greatest damage to
the Ax cell membranes was the formulation based on PQ1-ALX. The
other formulations tested based on PHMB and PQ1 with AD caused
some of the bacteria to lose membrane integrity but did not cause as
much damage to the bacteria cell membranes (p < 0.05) as the PQALX formulation. Dose effects of the preservative BAK could be
seen at 5 minutes of exposure time. BAK at 0.005% and 0.01%
caused an increase in the number of cells that were permeable to PI
compared to the phosphate buffered control (48% and 62%
respectively, p < 0.05). All of the Ax bacteria were permeable to PI
after exposure to 0.05% BAK.
Conclusions: One of the five lens care systems tested caused a
substantial number of Ax bacteria to lose membrane integrity. Also,
this method was able to detect the effect different concentrations of
BAK have on the membrane integrity of the Ax biofilm bacteria.
Understanding the ability of antimicrobials to damage bacteria cell
membranes could help in the development of lens care solutions that
can reduce and/or eliminate Ax biofilms from lens cases.
Commercial Relationships: David J. McCanna, None; Jaclyn M.
Chang, None; Lakshman N. Subbaraman, None; Lyndon W.
Jones, Alcon (F), Alcon (R), Allergan (F), Abbott Medical Optics
(R), Bausch & Lomb (R), Ciba Vision (F), Ciba Vision (R),
CooperVision (F), Johnson & Johnson (F), Johnson & Johnson (R)
127 Corneal Epithelium and Imaging I
Sunday, May 05, 2013 10:30 AM-12:15 PM
Exhibit Hall Poster Session
Program #/Board # Range: 524-567/B0161-B0204
Organizing Section: Cornea
Program Number: 524 Poster Board Number: B0161
Presentation Time: 10:30 AM - 12:15 PM
Body mass index, peripheral corneal thickness and anterior
chamber depth in young European adults - A pilot study
Sven Jonuscheit1, 2, Michael J. Doughty1, Raul Martin3, Ana del Rio
San Cristóbal3, Lisa J. Mackintosh1, David MacTaggart1, Michael
Hiscock1. 1Vision Sciences, Dept. of Life Sciences, Glasgow
Caledonian University, Glasgow, United Kingdom; 2Diabetes
Research Group, Institute for Applied Health Research, Glasgow
Caledonian University, Glasgow, United Kingdom; 3Optometry
Research Group, Instituto Universitario de Oftalmobiología Aplicada
(IOBA), Universidad de Valladolid, Valladolid, Spain.
Purpose: To assess the relationship of body mass index (BMI) and
the corneal thickness profile in normal white European individuals.
Methods: For this pilot study, 63 eyes of 63 healthy subjects were
assessed. Following completion of an ocular and general health
questionnaire body height and weight were measured and the BMI
calculated. Ocular assessments included habitual visual acuity, slitlamp biomicroscopy, and optical coherence tomography. Non-contact
specular microscopy was performed to rule out corneal
endotheliopathy. Scheimpflug photography (Pentacam) was used to
assess central, mid-peripheral and peripheral corneal thickness as
well as anterior chamber depth (ACD) at eleven locations nominally
1 mm apart along the horizontal meridian. Two consecutive
Scheimpflug scans were performed and the mean value used for
analyses. Descriptive statistics were generated. The association
between BMI, corneal thickness and ACD at central as well as offcenter locations was assessed using univariate regression analyses.
The coefficient of correlation (r) was calculated.
Results: The mean [SD] age was 27 [7] years. Mean body height and
body weight were 1.69 [0.08] meters and 64.6 [11.4] kilograms
respectively, the mean BMI was 22.7 [3.1]. Mean central corneal
thickness was 558 [33] micrometers (µm). Corneal thickness
increased progressively and asymmetrically from the corneal center
to the periphery with a significantly greater thickness at all nasal
locations as compared to the respective temporal sites (P<0.001,
related samples t-test). Temporal corneal thickness 5 mm away from
the center was 772 [50] µm and the corresponding nasal thickness
was about 6 % greater (823 [53] µm). While central corneal thickness
was independent of BMI (P = 0.241; r = -0.15), temporal corneal
thickness at 4 mm (P = 0.026; r = -0.28) and 5 mm (P = 0.012; r = 0.31) was inversely associated with BMI. Anterior chamber depth
averaged 3.03 [0.35] mm and was independent of BMI P>0.05).
Conclusions: For this cohort of young, healthy white European
adults with normal-weight average BMI, peripheral corneal thickness
was inversely related to BMI. The findings suggest the possibility of
a different corneal thickness profile in individuals with aboveaverage BMI. Further studies on the relationship between BMI,
obesity and corneal parameters are indicated.
Commercial Relationships: Sven Jonuscheit, Santander UK plc
(F); Michael J. Doughty, None; Raul Martin, None; Ana del Rio
San Cristóbal, None; Lisa J. Mackintosh, Santander UK plc (F);
David MacTaggart, Santander UK PLC (F); Michael Hiscock,
None
Support: Santander UK plc Grant
Program Number: 525 Poster Board Number: B0162
Presentation Time: 10:30 AM - 12:15 PM
Objective Estimation for Uncertainty of Restoring Corneal
Topography Surface
Anatoly Fabrikant. R&D, Abbott Medical Optics, Fremont, CA.
Purpose: Corneal topography (CT) field can be restored from
measurements data by decomposing available data into Zernike
polynomials and mapping the CT field in the desired area. The
accuracy of such restoration depends on the measurement noise and
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
number of available data. Here we present an algorithm for CT data
assimilation, which yields both CT map and an objective estimation
of the restoration accuracy.
Methods: The Kalman-Bucy technique is used to combine measured
CT heights with a priori mean and covariance of Zernike coefficients,
estimated for the general population (CT data for 308 virgin eyes).
This algorithm yields a statistically optimal estimate of Zernike
coefficients for the measured field and also their covariance matrix,
which provides a measure of the measurement uncertainty. The
efficiency of the proposed method is demonstrated using archived
corneal topography data from previous clinical studies.
Results: For any CT measurement the proposed technique yields an
estimate of Zernike amplitudes and their covariance matrix, which
result in a reconstructed map of CT heights with no gaps within the
area (Fig.1A). The covariance matrix gives the uncertainty (std) of
CT height restoration (Fig.1B). The uncertainty is higher at the area
edges, because the restoration is based mainly on the data from
internal area. Restored field in the measurement gaps has the highest
uncertainty, close to the a priori variance of the general population.
Conclusions: The proposed algorithm assimilates measurement data
together with a priori information, derived from statistics of general
population, which protects the results from measurement outliers. It
restores the CT field in the entire area and provides an objective
estimate of measurement uncertainty, based on the measurement
noise level and the number of available data. The uncertainty map
displays the areas where the map is less reliable and to what extent.
A B Fig. 1. A - CT height measured with Atlas topographer and
restored within 6mm diameter circle. B - Estimated uncertainty of
measured and restored CT height (Hyperopia Sph=2.25D,
Cyl=0.25D)
Commercial Relationships: Anatoly Fabrikant, Abbott Medical
Optics (E)
Program Number: 526 Poster Board Number: B0163
Presentation Time: 10:30 AM - 12:15 PM
Corneal nerve plexus condition in type 2 Diabetes Mellitus
patients assessed by Confocal Microscopy
Juan Antonio De la Campa, Oscar Baca, Alejandro Babayan, Regina
Velasco, Cristina Pacheco-Del-Valle. Cornea and Refractive
Surgery, Fundacion Hospital Nuestra Señora de la Luz, Mexico,
Mexico.
Purpose: To correlate the metabolic status of patients with type 2
Diabetes Mellitus and its relationship with corneal nerve plexus
alterations assessed by Confocal Microscopy along with clinical
questionnaires to detect peripherial diabetic neuropathy.
Methods: Patients with type 2 Diabetes Mellitus within the first year
of diagnosis were included. All patients were asked for glycosylated
hemoglobin (HbA1C). Inclusion criteria included values of
glycosylated hemoglobin ≥7%. We excluded patients with diagnosis
of central or peripheral neuropathy, ocular disease not related to
diabetes or history of previous refractive surgery. A control group of
healthy patients were also included. All patients were screened for
peripherial neuropathy using the MNSI questionnaire and classified
depending on the score obtained as without, mild-moderate or severe
peripherial neuropathy. Confocal microscopy was performed to
obtain corneal analysis from endothelium to epithelium and four
corneal subepithelial nerve plexus parameters were evaluated:
Number of fibers, tortuosity of fibers, number of beading and
branching grade. Statistical analysis was made using Pearson and tStudent tests, a p ≤ 0.05 was considered statistically significant.
Results: We included 14 patients in each group. In the study group
the mean value of HbA1C was 9.5%. In the study group, 28.5% of
the patients were classified as without peripheral neuropathy, 28.5%
as mild-moderate peripheral neuropathy and 42.8% as severe
peripheral neuropathy. The number of fibers, beadings and the
branching grade was decreased in diabetic patients compared with the
control group (p = 0.004, p ≤ 0.001 and p = 0.028 respectively). The
grade of tortuosity was higher in patients with diabetes compared
with healthy subjects with a p ≤ 0.001. There was a tendency of
progression of corneal neuropathy with higher levels of HbA1C.
Conclusions: The assessment of the corneal nerve plexus is a useful
tool in the global study of the diabetic patient by establishing the
absence or presence of early neuropathic damage, even before
clinical manifestations appear.
Commercial Relationships: Juan Antonio De la Campa, None;
Oscar Baca, None; Alejandro Babayan, None; Regina Velasco,
None; Cristina Pacheco-Del-Valle, None
Program Number: 527 Poster Board Number: B0164
Presentation Time: 10:30 AM - 12:15 PM
Non-invasive objective metrics of Bulbar Hyperemia for Clinical
Trials Endpoints
Neha Gadaria-Rathod, Kyu-In Lee, Benyamin Y. Ebrahim, Penny A.
Asbell. Ophthalmology, Mount Sinai School of Med, New York, NY.
Purpose: Bulbar hyperemia is a prominent feature of ocular irritation
associated with dry eye disease (DED), infection and allergy. The
aims of this study were to evaluate a modified topographer
(OCULUS Keratograph 5M) as a non-invasive objective metric for
evaluation of DED and ocular surface inflammation, and to evaluate
its precision in grading bulbar redness and its correlation with
clinician and subject grading.
Methods: An IRB approved prospective study of patients presenting
with either normal eyes or external ocular disease was conducted. 39
eyes of 20 patients were evaluated for bulbar redness independently
by 2 ophthalmologists (CLS1 and CLS2), by patients’ selfassessment (SS) and by the keratograph using the R-scan software.
Standardized CCLRU grading scales were used to score the bulbar
redness on a scale of 0-4. To establish precision of the keratograph, 2
pictures (KR1 and KR2) of the same eye were taken by the same
clinician, 2 minutes apart. The repeatability of measurements was
then analyzed using ANOVA and the repeatability index ‘r’.
Bivariate correlation analysis was done to get the Spearman’s
correlation coefficient.
Results: The keratograph grading showed high repeatability; r=0.90,
p<0.001. Both KR1 and KR2 showed high correlation with each
other (r=0.93; p<0.01) and with subject scores (r1=0.63, r2=0.70,
p<0.01), but variable correlation with clinical scores; clinician 1
scores (r1=0.59, r2=0.61, p<0.01); clinician 2 scores (r1=0.22,
p1=0.1; r2=0.31, p=0.05).(Table 1) The clinician scores
(mean=1.58+/-0.6) and subject scores(mean=2.3+/-0.7) were
significantly higher as compared to the keratograph
scores(mean=1.0+/-0.5) (p<0.01).
Conclusions: The keratograph showed high precision in measuring
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
bulbar redness. Being an objective measure, it reduces the inherent
variability of subjective assessments. Subjective assessment may
overestimate redness due to normally present large blood vessels on
the conjunctiva that are not indicative of inflammation. The
keratograph takes into account proportion of bulbar area occupied by
vessels, number of vessels and the proportion of area occupied by
thin vessels and thus reduces overestimation of hyperemia. Thus this
novel instrument may prove to be a non-invasive, objective
biomarker of ocular surface inflammation and thus serve as an
important endpoint in clinical trials of ocular surface disease.
Table 1
Commercial Relationships: Neha Gadaria-Rathod, None; Kyu-In
Lee, None; Benyamin Y. Ebrahim, None; Penny A. Asbell, RPS
(F)
Support: This study was supported in part by Research to Prevent
Blindness (RPB) Foundation and the Martin and Toni Sosnoff
Foundation.
Program Number: 528 Poster Board Number: B0165
Presentation Time: 10:30 AM - 12:15 PM
Clinical Validation of the Cassini Color LED Corneal
Topography (CLCT) in post penetrating keratoplasty (PKP)
Ronald Ensing1, Fleur de Lange2, Harry de Vries1, Michel Zaal2,
Victor A. Sicam1. 1i-Optics B.V., The Hague, Netherlands; 2OMC
Zaandam, Zaandam, Netherlands.
Purpose: To report results of a clinical investigation involving
comparison of corneal aberration measurements of CLCT with
conventional Placido based and Scheimpflug based topographers.
Methods: 23 PKP eyes from 16 subjects (age: 58 years ± 14 years,
ranging from 35 to 81 years, 9 OD and 14 OS) were measured by
three instruments: Cassini (i-Optics BV, The Hague, The
Netherlands), OPD Scan (Nidek, Gamagori, Japan) and Pentacam
(Oculus, Wetzlar, Germany). Corneal aberrations (Zernike
Convention at 6 mm corneal zone) were compared. An artificial toric
surface with gold standard measurement of 2.22 Diopter was also
measured to assess the accuracy of the instruments. The standard
deviation of three trials for every eye measurement was used to
characterize precision of the instrument. The measurement with
median defocus was used for inter-instrument comparison. The
paired student’s t-test was used on the median data to find
statistically significant differences.
Results: Nidek OPD measures astigmatism of the toric surface with
2.7% error while CLCT measures the toric surface with with 0.5%
error. The Pentacam has a precision reaching a mean of 0.25 μm for
astigmatism measurements. Both the OPD and CLCT do not exceed a
mean of 0.096 μm of precision in the measurement of corneal
aberrations. Statistically significant differences have been found
between the Cassini and the OPD for astigmatism (p = 0.0292) and
quadrafoil (p = 0.0281) aberrations. The spherical aberration
measured with Pentacam was significantly different from both
Cassini and OPD (p = 0.0114 and p = 0.0194 respectively).
Conclusions: The lower accuracy of the OPD in measuring
rotationally non-symmetric aberrations such as astigmatism and
quadrafoil, can be explained by the fact that rings are used in the
measurement. The use of rings does not measure the irregular
features of the cornea accurately. The poorer repeatability of the
Pentacam is a result of motion artifacts during acquisition. The
Cassini and the OPD have comparable repeatability because it takes
instantaneous measurements and is therefore not affected by motion
artifacts. Among the three instruments, the Cassini measures corneal
aberrations both accurately and precisely.
Commercial Relationships: Ronald Ensing, i-Optics B.V. (E);
Fleur de Lange, None; Harry de Vries, i-Optics (E); Michel Zaal,
i-Optics (F); Victor A. Sicam, i-Optics BV, The Hague, The
Netherlands (E), Patent/i-Optics BV, The Hague, The Netherlands
(P), Patent/VU University Medical Center, Amsterdam, The
Netherlands (P)
Support: The research is supported by i-Optics BV, The Hague, The
Netherlands.
Program Number: 529 Poster Board Number: B0166
Presentation Time: 10:30 AM - 12:15 PM
Topographically Guided Corneal Cross-Linking
David B. Usher, Radha Pertaub, Marc D. Friedman, Ronald F.
Scharf, David Muller. Avedro Inc, Waltham, MA.
Purpose: To determine the feasibility of a topographically guided
corneal cross-linking device.
Methods: A proprietary corneal cross-linking device was developed.
A UVA LED source illuminates a digital micromirror device (DMD).
Light reflected from the DMD is projected on to a subject’s eye. The
system controls the configuration of the DMD’s mirrors such that an
arbitrary UVA pattern can be projected on to the eye and modulated
at video rates. A digital camera is used to record views of the
subject’s eye. A Graphical User Interface allows an operator to
import topographies from a third party corneal topographer and
define UVA irradiation patterns based on the imported topography
data set. A common reference frame between the cross-linking device
and the topographer is established via a registration algorithm.
Images of the subject’s iris exported by the topographer are compared
to images recorded by the cross-linking device’s digital camera. Iris
textures and limbus boundaries visible within each dataset are used to
calculate a geometrical relationship between the two camera views.
Eye motion during the application of the UVA light is accounted for
by tracking the location of the eye in the cross-linking device’s
digital camera. This detected eye motion forms feedback for
modulating the DMD mirrors such that the incident UVA
illumination tracks relative to the eye. Eye motion, in terms of pupil
displacements, was calculated for 20 eyes from 10 subjects each over
30 second periods.
Results: An average frame-to-frame (60 Hz) eye motion of 23.9 um
(Range: 16.6 - 38.0) was recorded across subjects. Rotations of 2.1°
and 2.8° were measured for two subjects where images were recorded
by both the cross-linking device and the corneal topographer.
Conclusions: The proposed UVA treatment system demonstrates
unique features that will be able to advance the science of corneal
cross-linking. Each individual mirror of the DMD can be controlled
to correct beam uniformity through irradiance calibrations. Its
flexibility allows a surgeon complete freedom when configuring the
UVA dose across different areas of the cornea to a high degree of
accuracy. The eye tracking preserves this accuracy by accounting for
eye motion. The integration of the topography data allow a surgeon to
use variables such as corneal elevations, power maps, k readings,
corneal thickness maps, and epithelial thickness maps when creating
patent specific cross-linking pretreatment plans and procedures.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Commercial Relationships: David B. Usher, Avedro Inc (E);
Radha Pertaub, Avedro Inc (E); Marc D. Friedman, Avedro Inc
(E); Ronald F. Scharf, Avedro, Inc. (E); David Muller, Avedro Inc
(E)
Program Number: 530 Poster Board Number: B0167
Presentation Time: 10:30 AM - 12:15 PM
Corneal nerve morphology: a non-invasive surrogate of nerve
fibre damage and repair in chemotherapy-associated peripheral
neuropathy
Bernardo F. Sanchez Dalmau1, 2, Marisol Lopez Moreno1, 2, Ruben
Torres1, 2, Santiago Ortiz-Perez1, 2, Dolores Vela3, Elena Fraga2,
Pablo Villoslada2. 1Institut Clinic d'Oftalmologia, Hospital
Clinic.Seu Maternitat, Barcelona, Spain; 2NeuroImmunology Group,
IDIBAPS, Barcelona, Spain; 3Hematology Unit, Hospital General de
Granollers, Granollers, Spain.
Purpose: Chemotherapy-associated neuropathy is a cause of
disability in patients with cancer, for diagnosis invasive techniques
such as skin or sural nerve biopsy are used. Corneal confocal
microscopy is a noninvasive technique that allows to assess “in-vivo”
all structures of the cornea, including the corneal nerves. The purpose
of the current study is to determine the ability of this technique to
quantify the degeneration and regeneration of corneal nerves in
patients with peripheral neuropathy secondary to chemotherapy.
Methods: Twelve patients with chemotherapy-associated neuropathy
(study group) and 12 healthy volunteers were included. Laser in vivo
confocal microscopy (IVCM) of the cornea was performed in one eye
of each participant using the Heidelberg Retina Tomograph with the
Rostock Cornea module. By a combination of clinical assessment and
symptomatic neuropathy score an overall neuropathy score was
obtained.
Results: Median age was 59 year in the study group and 57.2 years in
the control group. The average time between evaluation and
completion of chemotherapy was 11,8 month (2-38 months). The
severity of the neuropathy was mild in one patient (8.3%), moderate
in 8 (66.6%) and severe in 3 patients (25%).The IVCM revealed a
reduction in nerve density and number of branching in 7 patients
(58.3%) compared with 1 (8.3%) in the control group (p=0.0272).
There was not a significant correlation between the sub-basal nerve
findings and the severity of the neuropathy and with the time of
chemotherapy ending.
Conclusions: Correlation between the chemotherapy-associated
neuropathy and sub-basal nerve morphology has been found. Given
that IVCM is a non invasive and painless technique which allows
serial assessment of the sub-basal nerve morphology we suggest that
IVCM may be an important tool to assess nerve degeneration and
regeneration and therefore become a surrogate marker in the
monitoring of chemotherapy associated neuropathy.
Commercial Relationships: Bernardo F. Sanchez Dalmau, None;
Marisol Lopez Moreno, None; Ruben Torres, None; Santiago
Ortiz-Perez, None; Dolores Vela, None; Elena Fraga, None; Pablo
Villoslada, Heidelberg Engineering (C), Novartis (F), Novartis (F),
Roche (C), Bionure (I), Bionure (P)
Program Number: 531 Poster Board Number: B0168
Presentation Time: 10:30 AM - 12:15 PM
Laser In Vivo Confocal Microscopy Demonstrates a Lower
Density of Peripheral Corneal Nerve Fibers Compared to the
Central Cornea in Normal Subjects
Jae Young You1, Bernardo M. Cavalcanti1, Susan Cheng1, Monique
L. Trinidad1, Douglas Critser3, Amy Watts2, Charles D. Leahy2,
Christine W. Sindt3, Pedram Hamrah1. 1Cornea and Refractive
Surgery, Massachusetts Eye and Ear Infirmary, Boston, MA;
2
Contact Lens, Massachusetts Eye and Ear Infirmary, Boston, MA;
Ophthalmology, University of Iowa, Iowa City, IA.
Purpose: To quantify subbasal corneal nerve density by in vivo
confocal microscopy in the central and four peripheral quadrants of
normal subjects.
Methods: In vivo confocal microscopy (IVCM; HRT3/RCM) of the
central cornea and 4 peripheral quadrants (superior, inferior, nasal,
and temporal) was performed in 37 subjects with normal clinical slitlamp examination. Three representative images were chosen for each
area and quantified using NeuronJ, a plugin for ImageJ (NIH). Two
masked observers measured the number and density of total nerves,
main trunks and branches. Statistical analysis was performed with
ANOVA with Bonferroni correction to compare the differences
between the 5 areas. A linear regression model was applied to assess
the changes for gender and age.
Results: The average age of normal subjects was 30 years, ranging
from 19 to 69 years. The central cornea had a significantly higher
number and density of total nerves (16.2 n/frame [14.8-17.6] 95%CI;
and 20067.7 µm/mm2 [18776.6-21358.8] 95%CI), main trunks (3.3
[2.9-3.6]; and 8848.4 [7915.3-9781.4]), and branches (12.8 [11.414.2]; and 11219.3 [10152.2-12286.4]) in comparison to all
peripheral areas (p<0.001). All peripheral areas demonstrated similar
distribution of the subbasal nerve plexus for all parameters (p>0.05),
including the number and density of total nerves for superior (9.2
[7.8-10.6]; and 11638.1 [9834.7-13441.5]), inferior (9.2 [7.5-11.0];
and 11169.3 [9080.2-13258.4]), temporal (8.9 [7.5-10.3]; and 9535.3
[8452.7-10617.9]), and nasal (7.8, [6.2-9.4] and 9621.0, [7832.311409.8]). An inverse correlation to age (R=-0.20; p=0.017), but not
for gender (p=0.781) was shown for all nerve parameters.
Conclusions: In normal eyes, IVCM shows a higher number and
density for subbasal nerve fibers for the corneal center in comparison
to the periphery. An inverse correlation is observed for nerve
parameters and age. A standardized approach is demonstrated to
measure the subbasal plexus for future clinical studies.
Commercial Relationships: Jae Young You, Alcon Research LTD
(F); Bernardo M. Cavalcanti, None; Susan Cheng, None; Monique
L. Trinidad, None; Douglas Critser, None; Amy Watts, None;
Charles D. Leahy, Vista Scientific LLC (I), Vista Scientific LLC
(P); Christine W. Sindt, alcon (F); Pedram Hamrah, None
Support: NIH K08-EY020575, New England Corneal Transplant
Research Fund, Falk Medical Research Trust, Alcon Research LTD
3
Program Number: 532 Poster Board Number: B0169
Presentation Time: 10:30 AM - 12:15 PM
Riboflavin Dosimetry in the Cornea using a KXL-II and the
Scheimpflug Principle
Marc D. Friedman, David B. Usher, Radha Pertaub, David Muller.
Avedro, Waltham, MA.
Purpose: To determine the feasibility of a new device (KXL-II) that
combines custom UVA illumination patterns with a Scheimpflug
imaging system for measuring riboflavin (RF) diffusion rates in the
cornea.
Methods: A proprietary corneal imaging device was developed. A
UVA LED source illuminates a digital micromirror device (DMD)
and is in turn projected on to a subject’s eye. Two digital cameras are
mounted ±45° to the apex of the cornea. The mirrors on the DMD are
configured such that UVA illumination forms a series of slits. As the
UVA passes through the cornea the resultant fluorescence profiles
change with the cross-sectional distribution of RF. Profiles of
multiple slits observed simultaneously map the distribution of
riboflavin in the radial direction (Figure 1). The two off axis cameras
employ a Scheimpflug principle whereby their focal planes are tilted
in order to optimize the system’s optical design in terms of focal
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
depth (Figure 2). Whole porcine eyes were placed at the device’s
focal plane. Images from the side cameras were recorded at regular
intervals after the application of RF solution.
Results: Sequences of images recorded at regular intervals during
and after the application of RF solution showed increased
fluorescence at increased depths within the cornea. A focal plane
angle of 18.2° to the apex of the eye was found to increase the
useable depth of field of the system by a factor of over 2.5.
Conclusions: An advanced riboflavin dosimetry device is presented.
The KXL-II system combines the use of UVA projection via a DMD
with side cameras that monitor riboflavin fluorescence in the cornea.
The camera’s tilted focal plane relaxes the constraints on the systems
depth of field. The DMD allows for rapid alternation between
illumination profiles intended for cross-linking treatment and slit
configurations intended for fluorescent dosimetry. With this device it
may be possible to respond and compensate to live dosimetry
readings as riboflavin is consumed and measured at various depths
within the cornea. Real time dosimetry measurements will play a
significant role in determining the underlying mechanisms of corneal
cross-linking and assist with the development of additional riboflavin
formulations.
Figure 1 Schematic of slit UVA illumination projected on to the
Cornea.
Figure 2 Schematic showing side camera’s use of Sceimpflug
principle. Focal plane intersects with measurement zone in cornea.
Commercial Relationships: Marc D. Friedman, Avedro Inc (E);
David B. Usher, Avedro Inc (E); Radha Pertaub, Avedro Inc (E);
David Muller, Avedro Inc (E)
Program Number: 533 Poster Board Number: B0170
Presentation Time: 10:30 AM - 12:15 PM
The clinical utility of the Eye Surface Profiler
D Robert Iskander. Institute of Biomedical Engineering and
Instrumentation, Wroclaw University of Technology, Wroclaw,
Poland.
Purpose: To evaluate the effectiveness of the Eye Surface Profiler in
measuring the topography of the anterior eye surface.
Methods: Eye Surface Profiler (ESP) is a newly developed cornea
and sclera topographer that can measure up to 20mm of the anterior
surface of the eye. It is an evolution of a wide field height eye
topographer [Jongsma et al. OVS; 1998]. Three tasks were set. Since
ESP requires instillation of fluorescein, the optimal combination of
the fluorescein strips and the eye physiological solution that results in
the best quality of recorded images was assessed. Further, the
repeatability of the instrument in measuring artificial and real eye
surfaces was studied. Finally, the new topographer was tested in
clinical settings against traditional Placido disk topographer (E300,
Medmont) for a range of normal, astigmatic, and highly aberrated
eyes (post corneal grafts).
Results: The best results in terms of highly resolved ESP images
have been achieved with BIO GLO sterile strips (1 mg fluorescein
sodium) with Hial eye drops (0.4 mg/ml hialuronian sodium). For
artificial surfaces, the repeatability of instrument in a dynamic case,
when operator manually focused the instrument, was very high while
the accuracy of the instrument in terms of the RMS error was less
than 10um but depended on the instrument-surface distance. The
working distance was estimated at +/-250um from the best focal
plane. For real eyes, the coverage area was routinely greater than
16mm and often reached up to 20mm diameter. Single instillation of
fluorescein allowed acquisition from 3 to 20 measurements (taken in
less than 30 second intervals). Repeatability, evaluated with
refractive spherical component over an 8 mm corneal diameter, was
high (differences less than 0.125 D). When tested against the E300
topographer, ESP showed excellent repeatability for spherical power
(in an 8 mm corneal diameter) but seemed to overestimate the
astigmatic component, which seemed to depend on the instrument’s
working distance. For highly aberrated eyes, in situations where E300
could not perform a valid measurement, the ESP was still performing
well.
Conclusions: ESP has high potential clinical utility. It could
substitute the currently used videokeratoscopes and provide a new
diagnostic quality in those applications in which information on
corneoscleral topography is of essence.
Commercial Relationships: D Robert Iskander, Eaglet Eye (F),
Eaglet Eye (I)
Support: Eaglet Eye - research grant
Program Number: 534 Poster Board Number: B0171
Presentation Time: 10:30 AM - 12:15 PM
Subclinical Keratoconus Detection Based On Pentacam
Scheimpflug Tomography Indices
Pablo R. Ruisenor Vazquez1, 2, Marianella Delrivo2, Fernando
Fuentes Bonthoux1, 2, Tomás Pfortner2, Pablo Chiaradia1, Jeremias
G. Galletti1, 2. 1Ophthalmology Division, Hospital de Clínicas,
University of Buenos Aires, Buenos Aires, Argentina; 2ECOS
(Clinical Ocular Studies) Laboratoy, Buenos Aires, Argentina.
Purpose: To evaluate the diagnostic performance of corneal indices
provided by the Pentacam tomograph for detecting subclinical
keratoconus.
Methods: Observational case series of 136 eyes from 136 healthy
subjects in group 1 and 42 topographically-unremarkable eyes from
42 keratoconic patients in group 2, evaluated with corneal
topography, aberrometry, and Scheimpflug tomography for BelinAmbrosio D indices. For data analysis, Student's t test was used to
compare means and receiver operating characteristic (ROC) curves
were used to analyze the diagnostic performance of D indices for
keratoconus detection. The statistical significance criteria used was p
<0.05.
Results: Gender distribution (group 1 vs group 2, 42% vs 67% male,
p <0.01), but not age (31.0 ± 8.4 vs 32.5 ± 11.4 years), was different
between groups. Keratoconic corneae were centrally thinner (535 ±
30 vs 512 ± 77 µm, p <0.01), had similar average corneal power
(44.26 ± 1.50 vs 44.40 ± 1.96 D) and more higher-order aberrations
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
(0.24 ± 0.07 vs 0.66 ± 0.35 µm, p <0.0001) than control corneae. The
area under the ROC curve, the sensitivity and specificity of BelinAmbrosio D indices were: Df = 0.80, 57.1% and 89.0%, Db= 0.83,
52.4% and 99.3%, Dp= 0.92, 64.3% and 97.8%, Dt= 0.79, 42.9% and
94.9%, Dy= 0.80, 54.8% and 90.4%, D= 0.93, 73.8% and 98.5%.
Comparing the global D index’s false negative (n = 11) and true
positive cases (n = 31), there were no differences in age (28.0 ± 6.8
vs 34.0 ± 12.4 years), in corneal thickness (509 ± 40 vs 514 ± 88 µm)
and corneal power (44.34 ± 1.44 vs 44.42 ± 2.13 D), but higher-order
aberrations were increased in the latter (0.35 ± 0.19 vs 0.76 ± 0.33
µm, p <0.001).
Conclusions: Back corneal surface index (Db) was not clinically
superior to the anterior surface index (Df) in sensitivity but provided
greater specificity, unlike the pachymetric progression index (Dp) or
the global D index that showed higher sensitivity and specificity. The
false negative cases of the best index had lower corneal aberrations in
the anterior surface than the true positive, suggesting that the first
represent earlier stages of keratoconus progression. These data
probably underestimate the diagnostic capability of the device due to
the strict inclusion criteria used. Although the detection rate is good,
there is significant margin for combining this technology with other
methods and thus improve performance.
Commercial Relationships: Pablo R. Ruisenor Vazquez, None;
Marianella Delrivo, None; Fernando Fuentes Bonthoux, None;
Tomás Pfortner, None; Pablo Chiaradia, None; Jeremias G.
Galletti, None
Program Number: 535 Poster Board Number: B0172
Presentation Time: 10:30 AM - 12:15 PM
Change of corneal irregular astigmatism after pterygium excision
surgery
Keiichiro Minami, Ayami Masuda, Yosai Mori, Ryohei Nejima,
Kazunori Miyata. Miyata Eye Hospital, Miyakonojo, Japan.
Purpose: Advanced pterygium degrades the visual acuity due to an
irregular astigmatism on the cornea. Surgical excision of the
pterygium healed or reduced the irregular astigmatism while the
corneal disorder may persist after the surgery. There were assessment
of the postoperative disorder irregularity, but the number of subjects
was not enough. This retrospective survey was to investigate change
of irregular astigmatism after the pterygium excision.
Methods: Eyes that had undergone excision surgeries of pterygium
on the nasal side at Miyata Eye Hospital from August 1999 to
December 2008 were reviewed. Size of the pterygium was graded by
the position of the end of pterygium: G1 was until one-third of
corneal diameter, and G2-4 corresponded to until, within, and over
the pupil, respectively. After excision of the pterygium and
intraoperative mitomycin C, the bare sclera was covered by sliding
adjacent superior conjunctiva. Corneal topography was measured at
pre-op and 1, 3, 6, and 12 months after the surgery. Changes of the
topographic index SRI were examined postoperatively for each grade
by the paired-ANOVA with Bonferroni multiple comparison.
Difference between the grades was also assessed by one-way
ANOVA. Effect of an advanced ratio that was ratio of length of
pterygium to the corneal diameter, on the SRI at 1 month was also
evaluated.
Results: 562 eyes were included, consisting of 119, 338, and105 eyes
of G1, G2, and G3, respectively. The mean age at the surgeries was
66.4 years (SD: 8.9). Mean SRI in G1 was 0.75 at 1month and
decreased to the level before the surgery in 3 months. The SRI of G2
was 0.85 at 1 month and significantly decreased (P<0.001) up to 3
months (0.70). There was no significant difference between G1 and
G2. In the SRI in G3, there was a significant decrease from 1.02 at 1
month to 0.91and 0.72 at 3 and 12 months, and G3 was also higher
than both G1 and G2 until 6 months. The advance ratio was
significantly and weekly correlated with the SRI (P<0.001).
Conclusions: The irregular astigmatism after the excision surgery
and the restoration period were associated with the size of pterygium.
Since topographic index SRI is associates with visual acuity, it was
demonstrated that cases in G3 would require 6 months to restore the
normal visual acuity.
Commercial Relationships: Keiichiro Minami, None; Ayami
Masuda, None; Yosai Mori, None; Ryohei Nejima, None;
Kazunori Miyata, None
Program Number: 536 Poster Board Number: B0173
Presentation Time: 10:30 AM - 12:15 PM
Measuring Corneal Epithelial Thickness Change after LASIK
with Optical Coherence Tomography
Maolong Tang, Yan Li, David Huang. Casey Eye Institute, Oregon
Health and Science University, Portland, OR.
Purpose: The purpose of this study is to measure corneal epithelial
thickness changes after LASIK with optical coherence tomography
(OCT).
Methods: A Fourier-domain OCT system with 26,000 axialscans/second scan speed and 5µm axial resolution was used. A
pachymetry scan pattern (8 radials, 1024 axial-scans each, 6mm
diameter) centered at the pupil center was used to image the cornea.
A computer algorithm was developed to generate the epithelial
thickness (tear film included) map automatically. Central corneal
epithelial thickness was measured before and 3 month after LASIK.
Pre- and post-operative central and peripheral epithelial thickness
was compared using paired t-test.
Results: Twelve eyes from 7 subjects were included in the study. All
subjects had myopic LASIK ranging from -1.33D to -6.31D. The
optical zone diameter was either 8.0mm or 8.5mm. The average
central epithelial thickness was measured to be 53.5 ± 2.0 μm before
LASIK and 56.7 ± 3.0 μm after LASIK (p = 0.02). The central
epithelial thickness change is correlated with LASIK dioptric
correction (R = 0.80, p = 0.056)
Conclusions: Corneal epithelial thickness increases after LASIK. It
would be interesting to measure epithelial thickness change at the
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
edge of the optical zone (8-9mm diameter) with future wide-field
OCT.
Commercial Relationships: Maolong Tang, Optovue Inc. (F); Yan
Li, Optovue, Inc. (F), Optovue, Inc. (P), Carl Zeiss Meditec, Inc. (P);
David Huang, Optovue (F), Optovue (I), Optovue (P), Optovue (R),
Carl Zeiss Meditec (P)
Support: NIH grant EY018184
Program Number: 537 Poster Board Number: B0174
Presentation Time: 10:30 AM - 12:15 PM
Normal Variants of Human Limbus Detected by In Vivo Laser
Scanning Confocal Microscopy
Elfren R. Baclagon1, Siamak Zarei-Ghanavati2, Sophie X. Deng1.
1
Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA; 2Cornea
Department and Eye Research Center, Mashhas University of
Medical Sciences, Mashhad, Islamic Republic of Iran.
Purpose: To report normal variations of the human limbal structures
using in vivo laser scanning confocal microscopy (LSCM).
Methods: Fourteen eyes from 13 healthy individuals between the
ages of 22 to 94 years were included in this study. Confocal imaging
of the cornea and limbus was performed using Heidelberg Retina
Tomograph III Rostock Corneal Module.
Results: The typical structure of the palisade of Vogt (POV) was
detected in 57% of eyes. Four additional structures and patterns were
found in the normal limbus that had not been reported previously in
eyes that did not have the POV. Whorl-like distribution of limbal
epithelial cells was present in two eyes. Mixed corneal and
conjunctival epithelial cells in a mosaic pattern were found at the
posterior limbus in two eyes. Bright dots pattern within the normal
limbal epithelial cells was observed in the wing and the basal layer in
five eyes. The forth structure, “limbal lacuna” was detected in two of
the subjects. It consisted of deep stromal lacuna filled with limbal
epithelial cells.
Conclusions: There are several structures variants in the normal
limbus. POV may not be the only structure that harbors limbal
stem/progenitor cells.
Commercial Relationships: Elfren R. Baclagon, None; Siamak
Zarei-Ghanavati, None; Sophie X. Deng, None
Support: CIRM TR2-01768 and NEI 5T32EY007026-35
Program Number: 538 Poster Board Number: B0175
Presentation Time: 10:30 AM - 12:15 PM
Imaging of epithelial downgrowth following penetrating
keratoplasty by confocal microscopy and high-resolution optical
coherence tomography
Vivian Lien1, Michael Chen1, Dennis E. Cortes1, 2, Jennifer Li1, Mark
J. Mannis1. 1Ophthalmology & Vision Science, University of
California Davis Eye Center, Sacramento, CA; 2Ophthalmology,
Pontificia Universidad Católica de Chile, Santiago, Chile.
Purpose: To report the characteristics of epithelial downgrowth
following penetrating keratoplasty using in vivo confocal microscopy
(IVCM) and high-resolution anterior segment optical coherence
tomography (AS-OCT).
Methods: A retrospective case review was performed of 3 eyes of 3
patients that developed epithelial downgrowth after multiple
penetrating keratoplasties. IVCM images were obtained at various
time points using the Heidelberg Retina Tomograph III Rostock
Cornea Module (Heidelberg Engineering, Germany) and AS-OCT
images were obtained using a high-resolution spectral-domain OCT
(Heidelberg Engineering, Germany). In two cases, the diagnosis was
confirmed by histopathologic evaluation.
Results: Three cases developed epithelial downgrowth. In case one, a
40 year-old male with a history of a corneal laceration complicated
by fungal keratitis was diagnosed with epithelial downgrowth after
undergoing 3 penetrating keratoplasties and placement of a glaucoma
drainage device over a 3-year period. In case two, a 48 year-old male
with a history of acanthamoeba keratitis developed epithelial
downgrowth after undergoing two therapeutic keratoplasties over a
one-year period. In case three, a 40 year-old female with a history of
perforating fungal keratitis developed epithelial downgrowth after 2
therapeutic keratoplasties over a three-month period. In all three
cases, IVCM revealed hyper-reflective nuclei characteristic of
epithelium and AS-OCT identified an epithelial layer at the level of
the endothelium.
Conclusions: This report provides useful images of epithelial
downgrowth from both IVCM and AS-OCT. These noninvasive
imaging modalities may potentially be more sensitive in identifying
and monitoring epithelial downgrowth than routine light
biomicroscopy.
Figure 1: A) Slit lamp photo showing diffuse epithelial downgrowth.
B) IVCM showing hyper-reflective nuclei of epithelial cells at the
level of the endothelium. C and D) AS-OCT with a hyper-reflective
epithelial layer posteriorly and growing over the iris. The angle is
completely sealed.
Figure 2: A) Slit lamp photo of diffuse epithelial downgrowth with
unaffected area centrally. B) IVCM showing hyper-reflective nuclei
of epithelial cells at the level of the endothelium. C) AS-OCT with
hyper-reflective layer of epithelial downgrowth posteriorly with
corresponding uncompromised area centrally.
Commercial Relationships: Vivian Lien, None; Michael Chen,
None; Dennis E. Cortes, None; Jennifer Li, None; Mark J.
Mannis, None
Program Number: 539 Poster Board Number: B0176
Presentation Time: 10:30 AM - 12:15 PM
Role of glutathione peroxidase 4 in maintaining homeostasis of
corneal epithelial cells
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Osamu Sakai1, 2, Takashi Ueta1, Yasuo Yanagi1, Shiro Amano1.
1
Opthalmology, The University of Tokyo, School of Medicine,
Tokyo, Japan; 2Research Laboratory, Senju Pharmaceutical Co Ltd,
Kobe, Japan.
Purpose: Antioxidant enzymes are involved in the homeostasis of
corneal epithelial cells. Glutathione peroxidase 4 (GPx4) is one of the
antioxidant enzymes that can directly reduce lipid peroxidation
caused by oxidative stress. The purpose of the present study was to
investigate the role of GPx4 in maintaining homeostasis of corneal
epithelial cells.
Methods: Simian virus 40 immortalized human corneal epithelial
(HCE) cells were used. HCE cells were transfected with GPx4
siRNA, and we tested for lipid oxidation, wound healing, and
cytotoxicity. Lipid oxidation was confirmed by immunostaining of 4hydroxy-2-nonenal (4-HNE). In the wound healing model, hole
defects were formed in confluent HCE cells, and wound areas were
evaluated 2 days after defect formation. Evaluation of cell
cytotoxicity was conducted by assay of LDH activity, staining of
annexin V/PI, immunostaining of apoptosis inducing factor (AIF),
and western blot of caspase 3. In oxidative stress study, cultured HCE
cells were transfected with GPx4 overexpressing plasmid and GPx4
siRNA, and then were treated with hydrogen peroxide. Cytotoxicity
was evaluated by assay of LDH activity 24 hours after the treatment.
Results: GPx4 knockdown caused 2.3-fold increase in the levels of
lipid oxidation (P<0.01 vs control). The mean wound area of GPx4
knockdown group was larger than that of control group (18.9 ± 1.2 %
and 9.3 ± 3.2% as a ratio of the initial damaged area, respectively;
P<0.01), showing that GPx4 knockdown delayed wound healing. The
levels of LDH activity were increased 4.6-fold by GPx4 knockdown
(P<0.01 vs control). Moreover, cell death in Gpx4 siRNA treated
cells was characterized by positive staining for annexin V, and
mediated by AIF, but not caspase. In oxidation stress study, GPx4
overexpression prevented hydrogen peroxide induced-cytotoxicity by
31% (P<0.01 vs control). Conversely, GPx4 siRNA knockdown
enhanced cytotoxicity by 6.3 fold (P<0.01 vs control).
Conclusions: GPx4 knockdown induced delay of wound healing and
cytotoxicity in corneal epithelial cells, and GPx4 overexpression
prevent cytotoxicity by oxidative stress. These results suggest that
GPx4 is involved in migration and survival of HCE cells.
Commercial Relationships: Osamu Sakai, None; Takashi Ueta,
None; Yasuo Yanagi, None; Shiro Amano, Topcon (P)
Program Number: 540 Poster Board Number: B0177
Presentation Time: 10:30 AM - 12:15 PM
A New Method for the Efficient and Less-Stress Dissociation of
Corneal Epithelial Cells
Katsuhiko Shinomiya1, Satoshi Kawasaki1, Keita Aoi2, 1, Koji
Kitazawa1, Shigeru Kinoshita1. 1Department of Ophthalmology,
Kyoto Prefectural University of Medicine, Kyoto, Japan; 2Faculty of
Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
Purpose: For the primary culture of corneal epithelial cells (CECs)
from human and animal ocular tissue, the cells should be
enzymatically separated and dissociated from the tissue via the use of
proteases such as dispase or trypsin. However, it is well known that
protease treatments have some cytotoxic effects and may cause cell
damage. In this present study, we report a new technique for the
enzymatic dissociation of CECs which allows for lower cytotoxicity
and a higher yield of cells.
Methods: The central region (8.75-mm diameter) of the cornea was
trephined from the eye of a Japanese white rabbit and treated with
dispase for 1 hour at 37°C (Method A) or overnight at 4°C (Method
B). While being observed under a microscope, the corneal epithelium
was then carefully separated from the underlying stroma. The
separated corneal epithelium was further dissociated by the use of a
trypsin-like protease. The histology of the separated corneal epithelial
sheet was assessed by hematoxylin-eosin staining. The dissociated
cells were counted and observed under a phase contrast light
microscope for the assessment of their cell morphology. For the
assessment of apoptotic status, the dissociated cells were subjected to
a commercial chemiluminescence-based apoptosis assay.
Results: The average number of CECs obtained by the Method B
was 1.38±0.15×106 cells per corneal button, which was significantly
larger than that obtained by the Method A (6.75±0.46×10 5 cells per
corneal button). Compared to the Method A, the Method B resulted
in a significantly lower number of apoptotic cells. A significantly
larger number of large cell aggregates were observed in the cell
suspension of the Method A compared with that of the Method B.
Significantly severe histological damage was found in the corneal
epithelial sheets obtained by the Method A compared to those
obtained by the Method B.
Conclusions: The findings of this study show that a low-temperature
dispase treatment method produced a higher yield of dissociated
CECs with lower cytotoxicity. This method should prove useful in
research involving CEC cultures, as well as corneal epitheliumrelated regenerative medicine.
Commercial Relationships: Katsuhiko Shinomiya, None; Satoshi
Kawasaki, None; Keita Aoi, None; Koji Kitazawa, None; Shigeru
Kinoshita, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co
(P), Otsuka Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)
Support: Grant-in-Aid for Scientific Research (C) #24592675 from
Japan Society for the Promotion of Science
Program Number: 541 Poster Board Number: B0178
Presentation Time: 10:30 AM - 12:15 PM
Characterization of cultured corneal epithelial cells on the
genipin-crosslinking Descemet membrane
Lung-Kun Yeh, Shih-Chun Huang. Ophthalmology, Chang Gung
Memorial Hospital -Linko, Taipei, Taiwan.
Purpose: Purpose: The Descement membrane is a good alternative
culture carrier for cell culture. However, the Descement membrane is
easily teared and rolled while peeling off from corneal tissue.
Genipin, a nature crosslinking material extracted from the fruits of
Gardenia jasminoides, has been widely used in many biological
applications. In this study, we try to study the phenotypes of cultured
epithelial cells on the genipin-crosslinker Descement membrane.
Methods: Methods: The crosslinking process between Descement
membrane and genipin was performed before cell plating. The
crosslinking characteristics of the genipin-fixed Descemet membrane
was recorded. The mechanical strength and resistance were compared
between the genipin-fixed and non-genipin-fixed Descemet
membrane. The proliferation rate were study by MTT assay. The
phenotype and morphological analysis of cultured cells were
photographed and observed by scanning EM. The
immunohistochemistry study was performed to characterize the
phenotype of cultured bovine corneal epithelial cells.
Results: Results: The MTT assay showed the proliferation rate was
increased in the group of genipin-fixed Descemet membrane. The
mechanical strength and resistance were increased in the group of the
genipin-fixed Descemet membrane. The genipin-fixed Descemet
membrane was easy handled, more smooth surface, and not easy
teared. The SEM analysis showed the normal epithelial cells
phenotype between the two groups. The immunohistochemistry
staining showed the Keratin-3 positive , Cx-43 positive, and more
P63 and Ki-67-positive cells on the genipin-fixed Descemet
membrane.
Conclusions: Conclusions: The results demonstrated that genipin can
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
form stable crosslinked products and is an effective crosslinking
agent for Descemet membrane fixation. Therefore, these results
indicated that the genipin-fixed Descemet membrane may be applied
as a excellent culture carrier. The non-toxic nature crosslinker
genipin may be useful for the application on the reconstruction of the
ocular surface.
Commercial Relationships: Lung-Kun Yeh, None; Shih-Chun
Huang, None
Support: Chang Medical Research Project G CMRPG3A1291 ;
National Science Council Grants (Taiwan) 1012314B182A056MY3
Program Number: 542 Poster Board Number: B0179
Presentation Time: 10:30 AM - 12:15 PM
The Role of Nrf2-Mediated Defense System in Corneal Epithelial
Wound Healing
Ryuhei Hayashi1, Noriko Himori2, Keiko Taguchi3, Yuki Ishikawa1,
Kohji Uesugi1, Motokazu Tsujikawa1, Toru Nakazawa2, Masayuki
Yamamoto3, Kohji Nishida1. 1Ophthalmology, Osaka University
Medical School, Suita, Japan; 2Ophthalmology, Tohoku university
school of medicine, Sendai, Japan; 3Department of Medical
Biochemistry, Tohoku university school of medicine, Sendai, Japan.
Purpose: The Nrf2-mediated defense system plays a central role in
protecting cells by activating genes against these types of stress. In
the present study, we investigated the role of the Nrf2-mediated
defense system in corneal epithelial wound healing by using Nrf2knockout (KO) mice.
Methods: The corneal epithelium of wild type (WT) and Nrf2 KO
mice were removed by treatment of n-heptanol for 1 minute under
anesthesia. The epithelial defect was stained with 1% fluorescein
solution and photographed at 0, 6, 12, 18, 24, 30, 36, 48, 60, and 72 h
after epithelial debridement. Injured corneas healed at various time
points were subjected to immunohistochemistry with Ki-67and Nrf2
antibody. Telomerase-immortalized corneal epithelial cell line
(C/TERT) was used for cell migration assay and siRNA experiment.
Results: Nrf2 was expressed in the corneal epithelium of WT mice,
but not in KO mice. Observation of wounds after 24 h of healing
revealed that healing of the corneal epithelium was significantly
delayed in the Nrf2 KO mice, while Nrf2 was activated in the corneal
epithelium of WT mice. Ki-67-staining revealed that the number of
Ki-67-positive proliferation cells was significantly lower in the Nrf2
KO mice than in the WT mice at 24-36 h after injury; however, these
numbers were approximately equivalent by 48 h. To clarify the role
of Nrf2 during wound healing, we performed in vitro experiments of
siRNA for Nrf2. The result showed that Nrf2 knock-down
significantly delayed corneal epithelial cell migration.
Conclusions: This study provides evidence that the Nrf2-mediated
defense system plays a crucial role in corneal epithelial wound
healing, by regulating the cell-migration activities of corneal
epithelial cells.
Commercial Relationships: Ryuhei Hayashi, None; Noriko
Himori, None; Keiko Taguchi, None; Yuki Ishikawa, None; Kohji
Uesugi, None; Motokazu Tsujikawa, Shionogi & Co. (C), Daiichi
Sankyo Co. (F), Daiichi Sankyo Co. (R), Santen Co. (R), AMO Co.
(R); Toru Nakazawa, Kowa Company Ltd. (F), Kowa Company
Ltd. (C); Masayuki Yamamoto, None; Kohji Nishida, Alcon (C),
Alcon (F), HOYA (F), Senju (F), Pfizer (F), Santen (F), Osaka
University (P)
Support: Grants-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology in Japan
Program Number: 543 Poster Board Number: B0180
Presentation Time: 10:30 AM - 12:15 PM
Increased fragility and acceleration of migration of corneal
epithelial cells in an epiplakin deficiency
Masahide Kokado1, Yuka Okada1, Kazushi Ishikawa2, Hiromitsu
Shimada2, Sakuhei Fujiwara2, Masayasu Miyajima3, Shizuya Saika1.
1
Department of Ophthalmology, Wakayama Medical University,
Wakayama, Japan; 2Department of Dermatology, Oita University,
Oita, Japan; 3Laboratory animal center, Wakayama Medical
University, Wakayama, Japan.
Purpose: Epiplakin is one of intermediate filament-related
components. To examine fragility and mechanism of migration of
corneal epithelial cells in an epiplakin deficiency. We previously
reported that the loss of epiplakin facilitates healing of a defect in
corneal epithelium in mice (ARVO 2009), and epiplakin knockdown
by Short-interfering RNA (siRNA) was accelerated the migration of
HCEC and suppressed TGFb1 activation of p38 and JNK signals
(ARVO2012).
Methods: (1) We used Epiplakin KO (KO) (n=4) and wild type
(WT) (n=4). The corneal surface was gently brushed with a surgical
micro-sponge. Then ultrathin sections were cut and observed under
transmission electron microscopy. (2) We ran real-time RT-PCR for
cell-cell connection-related components in RNA samples obtained
from KO (n=12)and WT (n=12). (3)Effect of siRNA knockdown of
EPPK on E-cadherin expression was also studied in AV40immortalized corneal epithelial cells by using western blotting.
Results: (1)Transmission electron microscopy showed that each
layer of the epithelial cells was well maintained following brushing in
a WT epithelium although partial separation between cells of the
superficial layer. In a KO epithelium one or two layer(s) of basal or
supra-basal epithelial cells were found to be in the original position
following the treatment and upper layer cells were found to be
removed. (2)Real-time RT-PCR also showed that mRNA expression
of E-cadherin was suppressed by the loss of epiplakin, while that of
desmoglein-1 or desmoplakin-1 was not affected by epiplakin gene
knockout in a mouse cornea. (3)EPPK knockdown suppressed Ecadherin expression in the cells.
Conclusions: The loss of epiplakin affects the corneal epithelium
integrity. The mechanism of acceleration of cell migration in the KO
corneal epithelium is to be further investigated, although suppression
of expression of E-cadherin in the absence of EPPK might be
included. The possibility of the potential corneal epithelium
disturbance in the patient who has abnormalities to EPPK was
suggested.
Commercial Relationships: Masahide Kokado, None; Yuka
Okada, None; Kazushi Ishikawa, None; Hiromitsu Shimada,
None; Sakuhei Fujiwara, None; Masayasu Miyajima, None;
Shizuya Saika, None
Program Number: 544 Poster Board Number: B0181
Presentation Time: 10:30 AM - 12:15 PM
Transfer of mucosal epithelial cells and extracellular matrix by a
gelatin matrix technique in vitro
Allen Ho1, Li-Fang Wang2. 1Jianguo School, Taipei, Taiwan;
2
National Taiwan University, Taipei, Taiwan.
Purpose: Poor connection and adhesion of epithelial cells to
underlying connective tissues is the basic defect of blister disease of
conjunctiva, skin, and mouth mucosal membrane such as ocular
cicatricial pemphigoid. Transplantation of extracellular matrix
(ECM) along with the autologous buccal mucosal epithelial cells may
modulate the survival and subsequent proliferation of transplanted
epithelial cells. Thus, we have developed a technique to transfer
native ECM with human mucosal epithelial cells in vitro.
Methods: Confluent monolayer human mucosal epithelial cells in the
culture plate was pretreated with 0.25% edetic acid for 15 minutes
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
and coated with a 800 μ layer of 20% gelatin. Patches of the mucosal
epithelial cells were harvested and transferred to another culture
plate. Cell viability and the ability of the transferred cells to
proliferate in culture were determined. The ECM transferred along
with the cells was characterized by immunohistochemistry.
Results: Human mucosal epithelial cells in culture can be harvested
as an organized cell patch with high efficiency (92.3 ± 4.5%) and
high cell viability (89.5 ± 5.5%). Cells harvested from tissue culture
plates divided and became confluent within 21 days.
Immunohistochemistry demonstrates that the transferred ECM along
contains laminin and type IV collagen.
Conclusions: We were able to harvest mucosal epithelial cells as an
organized monolayer from tissue culture plate along with their
intercellular extracellular matrix. This approach may provide a
practical technique for isolating and transplanting cultivated
autologous epithelial cells to reconstruct the damaged ocular surface
in Steven-Johnson syndrome, chemical and thermal injury, and ocular
cicatricial pemphigoid.
Commercial Relationships: Allen Ho, None; Li-Fang Wang, None
Program Number: 545 Poster Board Number: B0182
Presentation Time: 10:30 AM - 12:15 PM
Medical management of limbal stem cell deficiency with antiinflammatory therapy and tear film optimization
Bryan Kim1, Pejman Bakhtiari1, Kamran Riaz2, Clara C. Chan3,
Jeffrey Welder1, Surendra Basti2, Ali R. Djalilian1. 1Ophthalmology,
University of Illinois Eye and Ear Infirmary, Chicago, IL;
2
Ophthalmology, Northwestern University Feinberg School of
Medicine, Chicago, IL; 3Ophthalmology, University of Toronto,
Toronto, ON, Canada.
Purpose: To characterize the clinical features and medical
management of cases with limbal stem cell deficiency (LSCD) that
were reversible with anti-inflammatory therapy.
Methods: Retrospective case series of 23 patients (35 eyes) at 3
tertiary referral centers who were seen between 2007 and 2011.
These patients initially had clinical findings consistent with LSCD
but then had resolution of these findings with only medical
management. Main outcome measures included comparison of ocular
surface findings during and after medical treatment and changes in
visual acuity.
Results: Mean patient age was 41 years (range, 18-77) with 9 males
and 14 females. Etiologies of LSCD included contact lens wear (27
eyes), contact lens wear in the setting of ocular rosacea (3 eyes),
benzalkonium chloride (BAK) toxicity (2 eyes), and idiopathic (3
eyes). Clinical findings included progressive epitheliopathy with
associated opaque epithelium arising from the limbus, loss of limbal
architecture, and late fluorescein staining in a wavy or whorl pattern.
Extent of limbal disease involvement varied from 30 to 360 degrees
of limbus, with the superior limbus as the most common site of
involvement (31 eyes). Medical management was initiated in all
patients who had persistent disease after 3 months of conservative
measures (e.g. discontinuing contact lens wear). The treatments
included: topical corticosteroids (11 eyes), topical cyclosporine (11
eyes), topical vitamin A (6 eyes), doxycycline (3 eyes), and punctal
occlusion (15 eyes). Following treatment, all 35 eyes achieved a
stable ocular surface and resolution of LSCD over a mean follow-up
of 7.9 months (range, 2-36 months). 32 eyes experienced
improvement in visual acuity from an initial mean log MAR of 0.363
(20/46) to a post-treatment mean log MAR of 0.136 (20/27)
(P<0.001). 3 remaining eyes had unchanged visual acuity but had
good starting vision (20/20 - 20/25).
Conclusions: LSCD can result from dysfunction of limbal stem cells
or progenitor cells presumably due to disturbances to the limbal stem
cell niche. Our results highlight the potentially reversible nature of
this disease and support early intervention with medical therapy
aimed at improving the tear film and suppressing inflammation.
Commercial Relationships: Bryan Kim, None; Pejman Bakhtiari,
None; Kamran Riaz, None; Clara C. Chan, Alcon Labs Inc. (R),
Bausch & Lomb (R), Allergan (R); Jeffrey Welder, None; Surendra
Basti, None; Ali R. Djalilian, None
Support: The work by SB and ARD is supported in part by
unrestricted grants from “Research to Prevent Blindness” to
Departments of Ophthalmology at Northwestern University Feinberg
School of Medicine and University of Illinois Eye and Ear Infirmary,
respectively. ARD is the recipient of a career development award
from the National Eye Institute, NIH and from Research to Prevent
Blindness.
Program Number: 546 Poster Board Number: B0183
Presentation Time: 10:30 AM - 12:15 PM
Neuroprotectin D1 Stimulates the Expression and Secretion of
Nerve Growth Factor in Corneal Epithelial Cells
Azucena H. Kakazu, Nicolas G. Bazan, Haydee E. Bazan.
Ophthalmology/Neuroscience Center, LSU Health Sciences Center,
New Orleans, LA.
Purpose: Nerve growth factor (NGF) is a neurotrophic factor
expressed in the corneal epithelium that promotes cell proliferation
and wound healing. It is responsible for the axonal growth and
survival of sensory neurons. Neuroprotectin D1 (NPD1) is a lipid
mediator derived from docosahexaenoic acid (DHA) with antiinflammatory and neuroprotective actions. Synthesis of NPD1 is
stimulated in corneal epithelial cells treated with pigment epithelial
derived factor (PEDF) in conjunction with DHA. Recent studies in
our laboratory showed that topical treatment of NPD1, applied to
rabbit corneas after experimental surgery, increase corneal nerve
regeneration and enhanced neurite outgrowth of trigeminal ganglion
neurons in culture (ARVO 2012). The exact mechanism by which
NPD1promotes nerve regeneration is not understood. The purpose of
this study was to investigate if NPD1 and/or its precursors PEDF plus
DHA stimulate NGF synthesis.
Methods: First passage rabbit corneal epithelial cells (RCEC) were
used. The cells were grown in serum-free medium (CnT-20). Once
they reached 70-80 % confluence, the cells were starved overnight
and then stimulated with 50nM NPD1 or with the combination of
50ng/ml PEDF and 50nM DHA for different times. Changes in NGF
mRNA expression were assayed by PCR performed using Taq PCR
Master Mix Kit (Qiagen) with specific primers for rabbit. Secretion
of NGF peptide was measured in the tissue culture supernatant by
ELISA.
Results: NGF gene expression increased significantly after 3h and 6h
of NPD1 stimulation; at 16h gene expression started to decrease.
There was an increase of NGF secreted into the medium from the
cells in the presence of NPD1 or PEDF+DHA. After 48h, NGF
stimulation increased between 40 and 50% when NPD1 was added to
the medium; in presence of PEDF plus DHA, the NGF increment was
around 30%.
Conclusions: The results suggest that NPD1 and its precursors PEDF
plus DHA promote regenerative corneal innervation by modulating
NGF gene expression, followed by its synthesis and secretion.
Commercial Relationships: Azucena H. Kakazu, None; Nicolas G.
Bazan, None; Haydee E. Bazan, None
Support: NH Grant EY019465
Program Number: 547 Poster Board Number: B0184
Presentation Time: 10:30 AM - 12:15 PM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Multicentric study validation of a new molecular method for
Limbal Stem Cell Deficiency diagnosis based on MUC5AC
transcript detection in corneal epithelium by reverse dot-blot
strip
Tatiana M. Suarez-Cortes1, Iker Garcia1, Jaime Etxebarria2, Jesus
Merayo-Lloves3, Josep Torras4, Ana Boto-de-los-Bueis5, David DiazValle6, Rosalia Mendez6, Xabier Landaluce1, Arantxa Acera1.
1
Biomed R & D, Bioftalmik, Derio, Spain; 2Department of
Ophthalmology, Cruces Hospital, Plaza Cruces 12, Baracaldo, Spain;
3
Fundación de Investigación Oftalmológica, Oviedo, Spain;
4
Department of Ophthalmology, Hospital Clinic de Barcelona,
Barcelona, Spain; 5Department of Ophthalmology, Hospital La Paz,
idiPaz, Madrid, Spain; 6Department of Ophthalmology, Hospital
Clínico San Carlos, Madrid, Spain.
Purpose: To validate the efficacy, accuracy and sensitivity of a PCRstrip based on reverse dot-blot for detection of MUC5AC transcript
as indicative of the presence of goblet cells in cornea of patients with
Limbal Stem Cell Deficiency (LSCD), and to evaluate the correlation
with clinical diagnosis.
Methods: Eighty-five corneal impression cytology (IC) samples
from 54 subjects were analyzed in the study: 44 clinically diagnosed
LSCD corneas and 41 healthy corneas. Sixteen conjunctival
impression cytology (IC) samples were assayed as positive control. A
total of 101 impression cytology samples were analyzed in the study.
All the corneal impression cytologies were processed by RNA
extraction, retrotranscription, and analyzed by the presence of goblet
cells in the cornea by the PCR-strip based on reverse blotting system
(Limbokit).
Results: The total IC samples analyzed indicated that 43 of 44
samples clinically diagnosed as LSCD were confirmed positive for
MUC5AC, 33 of 41 healthy corneas were confirmed negative for
MUC5AC, 4 healthy corneas were found positive, and 4 were
rendered inconclusive results. All conjunctival impression cytologies
used as positive control were confirmed MUC5A positive. The data
indicate a global correlation of 91.1%. (p=0.0001). Confirmation of
PCR results in TAE-agarose gels indicated that reverse dot-blot strip
was more sensitive for bands detection and enhances visualization of
results. The overall sensitivity, specificity, Positive Predictive Value
(PPV) and Negative Predictive Value (NPV) of Limbokit visualized
on PCR-strips, were 98%, 89%, 91% and 97% respectively.
Conclusions: The PCR-strip test based on reverse blotting was found
to be a sensitive technique for detection of MUC5AC transcript in
corneal epithelium. The test results correlate well with clinical
diagnosis of characterized LSCD cases. The overall sensitivity,
specificity, and positive and negative predictive values were
satisfactory for diagnostic purposes. The PCR-strip based test
(Limbokit) constitutes a robust system for the detection of MUC5AC
in corneal epithelium and may be used for early detection and for
mild cases of Limbal Stem Cell Deficiency, and also as an objective
clinical tool for monitoring of treatments and surgical decisions.
Commercial Relationships: Tatiana M. Suarez-Cortes, Bioftalmik
S.L. (E); Iker Garcia, Bioftalmik Applied Research (P); Jaime
Etxebarria, bioftalmik (C); Jesus Merayo-Lloves, Ferrara & Hijos
SL (I); Josep Torras, None; Ana Boto-de-los-Bueis, None; David
Diaz-Valle, None; Rosalia Mendez, None; Xabier Landaluce,
Bioftalmik (E); Arantxa Acera, BIOFTALMIK SL (E)
Support: NEOTEC Program, Grant IDI-20080118
Program Number: 548 Poster Board Number: B0185
Presentation Time: 10:30 AM - 12:15 PM
Optimized culturing conditions for limbal epithelial cells
cultivated on semi-synthetic collagen matrices
Corinna Petsch1, Ursula Schlotzer-Schrehardt1, Markus Frey2,
Johannes Menzel-Severing1, Friedrich E. Kruse1, Bjoern O.
Bachmann1. 1Department of Ophthalmology, University of ErlangenNuremberg, Erlangen, Germany; 2RESORBA Wunversorgung
GmbH & Co.KG, Nuremberg, Germany.
Purpose: To optimize culturing conditions for limbal epithelial cells
on variants of a semi-synthetic collagen matrices with different in
vivo degradation characteristics.
Methods: Limbal epithelial stem cells were clonally enriched on 3T3
feeder cells and subcultivated on 3 variants (two crosslinked and one
non-crosslinked variant) of a semi-synthetic type I collagen substrate
(RESORBA, Germany). For clonal enrichment and subcultivation on
the collagen matrices 3 different cell culture media were evaluated:
MCDB151, DMEM/F12-1 (with bovine pituitary gland extract) and
DMEM/F12-2 (without bovine pituitary gland extract). After fixation
cell cultures were examined concerning cell adhesion, proliferation
and cellular phenotype by light and electron microscopy as well as
immunohistochemistry.
Results: Immunohistochemistry as well as light and electron
microscopy revealed no differences in adhesion, proliferation and cell
sheet formation between cell cultures on either variant of the collagen
matrix. When cultured with MCDB151 or DMEM/F12-1 monolayer
formation of limbal epithelial cells was seen, while the use of
DMEM/F12-2 resulted in a multilayered cell sheet.
By immunohistochemistry, E-Cadherin (as a marker for adherens
junctions) and K3/12 (as a differentiation marker) were localized in
all cell layers. Integrinα6 (marker for hemidesomsomes) and p63 (a
putative stem cell marker) were expressed in the basal cell layer. P63
was also apparent in upper layers of cells cultured on non-crosslinked
collagen matrices.
Electron microscopically hemidesmosomes were seen on cells of the
basal cell layer of cultures on either collagen substrate when cultured
with DMEM/F12-1 or DMEM/F12-2.
Conclusions: Crosslinked and non-crosslinked variants of a semisynthetic collagen matrix are suitable for the cultivation of limbal
epithelial cells. The use of DMEM/F12-2 with either collagen matrix
resulted in a multilayered cell sheet of cultured limbal epithelial cells.
The ability to serve as a growth substrate for limbal epithelial cells
and its known biocompatibility with the cornea indicates that the used
collagen matrices might be suitable for cultivation and transplantation
of ex vivo expanded limbal epithelial cells.
Commercial Relationships: Corinna Petsch, RESORBA
Wundversorgung GmbH &Co.KG, Nuremberg, Germany (F); Ursula
Schlotzer-Schrehardt, None; Markus Frey, RESORBA Medical
GmbH (E); Johannes Menzel-Severing, None; Friedrich E. Kruse,
None; Bjoern O. Bachmann, None
Support: Medical Valley EMR Erlangen Project A-06, sponsored by
the Federal Ministry of Education and Resarch, Germany (BMBF)
Program Number: 549 Poster Board Number: B0186
Presentation Time: 10:30 AM - 12:15 PM
CD45+F4/80+ immune cells are rapidly recruited to the wound
edge after corneal debridement wounds
Gauri Tadvalkar1, Sonali Ghosh1, Ahdeah Pajoohesh-Ganji1, Janice
L. Walker2, A S. Menko2, Mary Ann Stepp1. 1Anatomy &
Regenerative Biology, George Washington University Medical
Center, Washington, DC; 2Pathology, Anatomy and Cell Biology,
Thomas Jefferson University, Philadelphia, PA.
Purpose: A population of leader cells was found to direct migration
of lens epithelial cells in response to wounding. The current studies
were carried out to determine 1) whether leader cells are involved in
the response of the corneal epithelium to injury, and 2) test the
hypothesis that leader cells are immune cells.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Methods: 1.5 mm debridement wounds were made manually in 8
week old BALB/c using one of two techniques: a rotating burr (RB)
or a dulled blade (DB). Mice were either sacrificed at the time of
wounding (Time 0, T0) or allowed to heal for 1 or 6 hrs. All eyes
were fixed within 5-10 min after injury. Corneas were used for whole
mount immunofluorescence studies to localize CD45 (the common
leukocyte antigen), F4/80 (a monocyte marker), GL3 (an epitope
present on γδT cells), and vimentin, at the leading edges of the
wounded corneas. For each variable studied, no fewer than 3 corneas
from 3 different mice were used.
Results: CD45+ cells appear at the leading edge in the T0 corneas.
T0 DB wounds recruit more F4/80+ cells compared to RB wounds
and this difference persists through the 6 hr time point. Similar levels
of F4/80+ cells were present at T0 and 1 hr after wounding. There
were several detectable vimentin+GL3+ cells present at the leading
edge at T0 and 1 hr post-injury for both RB and DB wounds. By
contrast, after 6 hrs, GL3+ cells were absent after DB, and only rarely
seen after RB wounding.
Conclusions: The rapid migration of leader cells to the wound edge
following mock cataract surgery in the lens is also seen in the cornea
after manual debridement wounding. The numbers of leader cells
recruited at T0 and 6 hrs after wounding varied depending on the type
of wound. All of the leader cells are positive for CD45, with the
majority being positive also for F4/80. A subpopulation of the leader
cells are vimentin+GL3+ but these cells are more transient than the
F4/80+ cells since they are largely absent by 6 hrs after wounding.
Unlike the mouse cornea, the majority of the leader cells in the
chicken lens wound model express vimentin. While the functions of
these rapidly appearing immune cells is not clear, our studies indicate
that wound healing in chicken and mouse and in diverse tissues
including the lens and cornea share a similar property: rapid
recruitment of immune derived cells to the leading edge.
Commercial Relationships: Gauri Tadvalkar, None; Sonali
Ghosh, None; Ahdeah Pajoohesh-Ganji, None; Janice L. Walker,
None; A S. Menko, None; Mary Ann Stepp, None
Support: EYO08512 (MAS), EY021784 (ASM,MAS)
Program Number: 550 Poster Board Number: B0187
Presentation Time: 10:30 AM - 12:15 PM
Basement Membrane Removal Reduces Inflammation in the
Mouse Cornea and Enhances Wound Healing
Sonali Pal-Ghosh1, Ahdeah Pajoohesh-Ganji1, Gauri Tadvalkar1,
Daniel R. Saban2, Mary Ann Stepp1. 1Anatomy & Regenerative
Biology, George Washington University Medical Center,
Washington, DC; 2Ophthalmology and Immunology, Duke
University School of Medicine, Durham, NC.
Purpose: An in vivo mouse model has been developed that
reproducibly induces recurrent epithelial erosions in wild-type mice
spontaneously within two weeks after a single 1.5 mm corneal
debridement wound using a dulled blade. Using a rotating burr rather
than a dulled blade to create a 1.5 mm wound allows mice to heal
without developing erosions. These experiments were conducted to
determine the cause of the difference in healing outcomes after dulled
blade compared to rotating burr wounds.
Methods: A 1.5 mm area of corneal epithelium was removed from
the corneal surface of adult C57BL/6 mice using either a dulled blade
or rotating burr. Corneas were allowed to heal in vivo for 0, 3, 6 hr or
5 days. After sacrifice, tissues were used for immunofluorescence,
QPCR, flow cytometry, and/or chemokine protein array studies. Flow
experiments were repeated three times. QPCR was performed on
control (n=8) and 6 hr dulled blade or rotating burr wounded (n=8)
corneas. For QPCR studies, RNA isolated from epithelial cells only
and whole dissected corneas were compared. All experiments were
repeated at least twice.
Results: Data show that 1) erosions form after dulled blade but not
after rotating burr wounds, 2) the basement membrane (BMZ) is left
behind after dulled blade wounds but is removed by the rotating burr,
3) there are more monocytes (CD45+/ Ly6C hi/ Ly6G+/ CD11b hi/
F480 low/ CD11c+) and γδ T cells (CD45+/GL3 hi) recruited 6 hr
after dulled blade wounds, and 4) chemokine array studies show that,
at the time of wounding and 5 days later, chemokines are elevated
after dulled blade compared to rotating burr wounds.
Conclusions: Despite the fact that rotating burr wounds remove the
BMZ, damage more of the subbasal nerves, and induce more
cytokine mRNAs within corneal epithelial cells, they heal without
developing erosions. Improved healing after trauma is associated
with basement membrane removal and the reduced influx of
monocytes and γδT cells 6 hr after wounding. Data from chemokine
arrays indicate more CCL8 and CCL12 and complement proteins (5a
and Factor D) retained on the corneal stroma after dulled blade
wounds that may account for the early increase in monocyte and γδT
cells seen after dulled blade wounds.
Commercial Relationships: Sonali Pal-Ghosh, None; Ahdeah
Pajoohesh-Ganji, None; Gauri Tadvalkar, None; Daniel R.
Saban, Schepens Eye Res Inst, Mass Eye and Ear, (P), Eleven
Biotherapuetics (R); Mary Ann Stepp, None
Support: EYO08512 (MAS), EY021784 (ASM,MAS)
Program Number: 551 Poster Board Number: B0188
Presentation Time: 10:30 AM - 12:15 PM
Schirmer Testing and Corneal Surface Findings in Chronic
Graft-Versus-Host Disease
Kathleen M. Williamson1, Evan Allan3, Michelle P. Lin2, Michael C.
Wu1. 1Ophthalmology, University of Washington, Seattle, WA;
2
Ophthalmology, Cleveland Clinic, Cleveland, OH; 3Ophthalmology,
University of Oklahoma, Oklahoma City, OK.
Purpose: To report whether low Schirmer testing scores are
predictive of ocular surface findings in patients with chronic graftversus-host disease (cGVHD) diagnosed after hematopoietic cell
transplantation.
Methods: Retrospective study of 429 consecutive patients with
cGVHD referred for ophthalmic examination between 1990 and
2006. Office visit records were reviewed for extraction of data
including visual acuity, Schirmer testing scores, slit-lamp
biomicroscopic findings (including corneal staining with fluorescein),
and fundoscopic abnormalities. In this analysis, we considered the
relationship between Schirmer testing scores and corneal staining.
Two arbitrary cutoff values were used to define ‘low’ Schirmer
scores, 5mm or less and 10mm or less. Analysis was then performed
to determine if there was an association between ‘low’ scores and
corneal surface staining.
Results: In our population of patients with cGVHD, Schirmer testing
scores of 5 mm or less are associated with presence of corneal
fluorescein staining (p<0.0001). Of those patients with Schirmer
scores of 5 mm or less, 44.4% were found to have corneal staining
versus 12.5% of patients with scores greater than 5 mm. Similarly,
Schirmer testing scores of 10 mm or less are also associated with the
presence of corneal fluorescein staining (p<0.0001). Of those patients
with Schirmer scores of 10 mm or less, 32.7% were found to have
corneal staining versus 7.1% of patients with scores greater than 10
mm.
Conclusions: Schirmer testing scores of 5mm or less and 10mm or
less are associated with presence of corneal fluorescein staining in
our population of patients with cGVHD.
Commercial Relationships: Kathleen M. Williamson, None; Evan
Allan, None; Michelle P. Lin, None; Michael C. Wu, None
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Program Number: 552 Poster Board Number: B0189
Presentation Time: 10:30 AM - 12:15 PM
Boric Acid-based Multipurpose Contact Lens Care Solutions
(MPSs) Cytotoxicity Effect on Human Corneal Epithelial Cells
Kissaou T. Tchedre1, Masaki Imayasu2, Yuichi Hori3, H D.
Cavanagh4. 1R&D and Innovation Center, Menicon LTD, Le Mans,
France; 2R&D center, Menicon, Co. ltd, Kasugai, Japan;
3
Ophthalmology, Toho University Sakura Medical Center, Sakura,
Japan; 4Ophthalmology, Univ Texas Southwestern Med Ctr, Dallas,
TX.
Purpose: The purpose of this study is to determine whether
commercially available new multipurpose contact lens care solutions
(MPSs) have any cytotoxicity effect on human corneal epithelial
(HCE-T) cells. MPSs effect on membrane-associated mucins (MUC1
and MUC16) expressions in the Rat cornea was also assessed.
Membrane-associated mucins are one of the major components of the
ocular surface that play a vital role in the maintenance of the ocular
surface integrity
Methods: HCE-T cells were treated with different concentrations of
MPS-F (1ppm PHMB, no boric acid), MPS-G (1.3ppm PHMB, 1ppm
PQ-1, boric acid), MPS-H (1.6 ppm, Alexidine, 3ppm PQ-1, boric
acid), MPS-I (1ppm PHMB, boric acid), and MPS-J (5ppm ALDOX,
10ppm PQ-1, boric acid): 100% treatment for 30 minutes and 10%
treatment for 24 hours. Cell death was measured by using a
viability/cytotoxicity assay kit. Winstar Rats were also subjected to
MPSs (1 drop in the right eye every 10 minutes for 1 hour). The left
Eye was used as control (1 drop of PBS every 10 min for 1 hour).
Cornea lysates were subsequently prepared and used for Western blot
analysis for MUC1 and MUC16.
Results: The viability/cytotoxicity assay result showed that MPSs
containing boric acid induce cell death in HCE-T cells. The western
blot result showed that boric acid-based MPS down-regulate
membrane-associated mucins in the cornea while MPSs without boric
acid had no effect on membrane-associated mucins.
Conclusions: The concentration of boric acid used in commercially
available multipurpose contact lens care solutions should be chosen
carefully to avoid MPS-related ocular surface damage. Ocular surface
damage simultaneously promotes microbial pathogens and potentially
increases clinical rates of infection.
Commercial Relationships: Kissaou T. Tchedre, Menicon, Co. Ltd
(E); Masaki Imayasu, Menicon Co., Ltd. (E); Yuichi Hori, None; H
D. Cavanagh, Menicon Ltd (C)
Program Number: 553 Poster Board Number: B0190
Presentation Time: 10:30 AM - 12:15 PM
The Effect of Amniotic Membrane De-epithelialization Method
on its Biological Properties and Ability to Promote Limbal
Epithelial Cell Culture
Gary Hin-Fai Yam1, Ting Zhang1, Andri K Riau1, Roger W.
Beuerman1, Donald T. Tan2, Jodhbir S. Mehta1, 2. 1Singapore Eye
Research Institute, Singapore, Singapore; 2Singapore National Eye
Center, Singapore, Singapore.
Purpose: To characterize the de-epithelialized human amniotic
membrane (HAM) and compare cell attachment and proliferation
efficiencies.
Methods: HAM was de-epithelialized by 20% ethanol (AHAM), 1.2
U/ml Dispase (DHAM), 0.02% EDTA (EHAM), 0.25% trypsinEDTA (THAM) and 5M urea (UHAM), respectively, followed by
gentle scrapping with a #15 blade. Surface topology, extracellular
matrix (ECM) and growth factor content were characterized and
compared to intact HAM by electron microscopies (EM), atomic
force microscopy (AFM), immunohistochemistry and western
blotting. Primary human limbal epithelial cells (LEC) attachment and
proliferation efficiencies were assayed. Statistical significance was
calculated by SPSS and Fisher’s Least Significant Difference test.
Results: EHAM, THAM and UHAM had intact basal lamina and
smooth basement membrane surface shown under transmission and
scanning EM and AFM. Cell remnants stayed on AHAM. Disrupted
basement membrane and stroma was found in DHAM.
Immunostaining intensity quantification and hierarchical clustering
revealed that ECM composition of EHAM and UHAM resembled to
intact HAM. In contrast, DHAM and THAM had drastic loss of ECM
and growth factor content. LEC attachment efficiency at 24 hours
post-seeding was the highest in EHAM (51% as on conventional
culture surface), followed by UHAM and AHAM. However, cell
proliferation indices at day 10 of culture were similar among different
HAM substrates, suggesting repair of ECM and basement membrane
by growing epithelial cells.
Conclusions: Urea denudation preserved the basement membrane
integrity, ECM and growth factor composition, and had higher cell
attachment and proliferation efficiencies. With its short processing
time, urea treatment offers a novel alternative for HAM deepithelialization.
Commercial Relationships: Gary Hin-Fai Yam, None; Ting
Zhang, None; Andri K Riau, None; Roger W. Beuerman, Allergan
(F), SERI (P), Santen (R); Donald T. Tan, Network Medical
Products (P), Carl Zeiss Meditec (F), Alcon Labs (F), Bausch &
Lomb (F), Allergan (F), Santen (F); Jodhbir S. Mehta, None
Support: Singapore National Medical Research Council grants IBG
and R485/34/2006
Program Number: 554 Poster Board Number: B0191
Presentation Time: 10:30 AM - 12:15 PM
Knockdown of MUC16 Alters Tight Junctions of Corneal
Epithelial Cells Resulting in Decreased Transepithelial
Resistance
Ilene K. Gipson, Sandra J. Spurr-Michaud, Ann S. Tisdale. Harvard
Med Sch/Dept Ophthal, Schepens Eye Research Inst/MEEI, Boston,
MA.
Purpose: MUC16 is a major membrane associated mucin of the
human corneal epithelium. We have shown that MUC16 is a barrier
to rose bengal dye penetrance and pathogen adherence. We also
demonstrated that MUC16’s cytoplasmic tail associates with actin
through the ezrin, radixin, moesin family of actin linking proteins.
Because MUC16 may be associated with actin filaments that insert
into the apical web of actin filaments that terminate in tight junctions,
the purpose of this study was to determine the role of MUC16 in tight
junction formation and function as measured by transepithelial
resistance (TER).
Methods: An immortalized human corneal limbal epithelial cell line
(HCLE) (Gipson et al, 2003) and the HCLE cell line stably
transfected with siRNA to MUC16 in which MUC16 levels were
reduced by 70% and a vector control line were used. Cells were
cultured for optimal mucin expression and stratification. Assays
comparing the cell lines included measurement of apical cell size,
localization and mRNA levels of Z01, a tight junction protein, and
TER using an Evum2 Epithelial Voltohmmeter.
Results: HCLE cells knocked down (KD) for MUC16 showed a
significant 2-3 fold (p<0.001) increase in apical cell size as compared
to vector and non-transfected controls. ZO1 localization in the
MUC16 KD cells showed long discontinuities along apical cell
peripheries as well as regions of punctate localization as compared to
the control cell lines. Corroboratively, message levels of ZO1 were
significantly lower in the MUC16 KO cells (p<0.01 to controls). The
alterations in ZO1 localization and expression in the MUC16 KD
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
cells correlated with a significant decrease in TER (39 +3 ohms)
compared to controls (127 + 5 ohms, p<0.001).
Conclusions: A decrease in MUC16 in corneal apical cells results in
tight junction discontinuities and a decrease in junction function as
measured by TER. These data suggest that MUC16 through its
association to actin within the apical cytoskeletal web that terminates
laterally in tight junctions, influences tight junction formation and
function. We have previously shown that as apical cells increase in
size with time of residence at the ocular surface, the amount of
MUC16 per unit membrane area on the cell decreases. Perhaps
MUC16 diminution leads to disruption of tight junctions that in turn
leads to cell desquamation.
Commercial Relationships: Ilene K. Gipson, None; Sandra J.
Spurr-Michaud, None; Ann S. Tisdale, None
Support: NIH Grant EY03306 to IKG
Program Number: 555 Poster Board Number: B0192
Presentation Time: 10:30 AM - 12:15 PM
Rapamycin helps to maintain colony forming efficiency in
corneal epithelial cells
Farnoud YOUSEFIMILANI, Behrad Y. Milani, Hossein M. Sagha,
Ali R. Djalilian. Ophthalmology and visual siences, Univ of Illinois
at Chicago, chicago, IL.
Purpose: Factors that maintain the limbal epithelial stem/progenitor
cells in vitro have not been completely defined. In this study we
examined the effect of rapamycin, an inhibitor of the mTOR
pathway, on the colony forming efficiency of corneal epithelial cells.
Methods: Primary human corneal epithelial cells were grown in
KSFM (keratinocyte serum free media) and at the same time treated
with a range of concentrations of Rapamycin. Control cultures were
treated with the vehicle (DMSO). After growing in Rapamycin or
DMSO for 7-10 days, the cells were passaged and plated on
mitomycin C treated fibroblasts (no further rapamycin or DMSO
treatment given). Colony forming efficiency was determined by
counting the number of colonies visualized by crystal violet.
Results: Rapamycin appeared to have toxic effects at higher
concentrations but at low concentrations it appeared to reduce the
proliferation of corneal epithelial cells compared to DMSO. Cells that
were treated with Rapamycin subsequently demonstrated higher
colony forming efficiency compared to DMSO.
Conclusions: These results suggest that rapamycin helps to maintain
colony forming efficiency in corneal epithelial cells. It is unclear at
this time whether these effects are simply due to the fact that
rapamycin also reduces proliferation. Further gene expression studies
are needed to determine whether rapamycin helps to maintain cells in
a less differentiated state.
Commercial Relationships: Farnoud YOUSEFIMILANI, None;
Behrad Y. Milani, None; Hossein M. Sagha, None; Ali R.
Djalilian, None
Support: National Eye Institute of the National Institutes of Health
with Career Development Grant K08EY017561-A1 (ARD) and Core
Grant EY01792, the Cless Family Foundation, and a Career
Developmental Award (ARD) and unrestricted departmental grant
from Research to Prevent Blindness.
Program Number: 556 Poster Board Number: B0193
Presentation Time: 10:30 AM - 12:15 PM
Average Corneal Epithelial Thickness Pattern of Normal, PostLASIK, Post-PRK, KCN, Contact Lens Wearer, and Dry Eyes
Using RTVue FD-OCT System
Yao Nie, Qienyuan Zhou, Kelly A. Soules, Ben K. Jang. Optovue Inc,
Fremont, CA.
Purpose: To generate the average corneal epithelial thickness pattern
and compare the epithelial thickness distribution of normal, postLASIK, post-PRK, keratoconus (KCN), contact lens (CL) wearer and
dry eyes, using RTVue Fourier-domain (FD) optical coherent
tomography (OCT) system with Corneal Adaptive Module (CAM)
(Optovue, Fremont, CA).
Methods: A total of 16 normal, 12 post-LASIK, 8 post-PRK, 39
KCN, 28 CL wearer and 30 dry eye subjects from 4 clinical sites
were included, following WIRB-approved study protocols. One eye
per subject was selected, each having 3 repeated
“Pachymetry+Cpwr” scans with central pupil alignment. Scans with
any visible segmentation error were excluded from the following data
processing and analysis. To generate the average epithelial thickness
pattern, the average epithelial thickness map of repeated scans is
calculated for each eye, which is flipped for left eyes to make the
temporal(T)/nasal(N) side on the left/right, and then averaged across
eyes within each group. The average thicknesses of the central (2mm
diameter), superior(S) and inferior(I) (2-5mm) zones, and the
variation (assessed by standard deviation SD) of the central 5mm
diameter zone were calculated from the epithelial thickness map. The
mean and SD for these measurements were generated across eyes
within each group.
Results: The average epithelial thickness pattern for each pathology
group is shown in Fig. 1. The assessments of the zonal thickness and
variation are summarized in Table 1. As shown, the normal, CL
wearer and dry eyes are thicker in the I-N region than in the S-T
region. The post-LASIK and post-PRK eyes are thicker inferiorly,
with the overall thickness noticeably greater than the other groups.
The KCN eyes have the greatest zonal variation, with the S-N region
thicker than the I-T region. The SD of zonal thickness across eyes is
the smallest among normal eyes, and the greatest among post-PRK
and KCN eyes.
Conclusions: Epithelial thickness and pattern are affected by corneal
pathologies and laser refractive surgery (LRS) as measured with
automated epithelial thickness mapping function in RTVue FD-OCT.
The results appear consistent with existing knowledge of epithelial
change in KCN and post-LRS eyes. Note the sample sizes of the
post-LASIK and post-PRK groups are small, which should be
considered when interpreting the above study results.
Commercial Relationships: Yao Nie, Optovue, Inc. (E); Qienyuan
Zhou, Optovue, Inc. (E); Kelly A. Soules, Optovue (E); Ben K.
Jang, Optovue (E)
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Clinical Trial: NCT01712022
Program Number: 557 Poster Board Number: B0194
Presentation Time: 10:30 AM - 12:15 PM
Bone Marrow derived Mesenchymal Stem Cells for
Reconstructing the Limbal Niche
Ali R. Djalilian1, Behrad Y. Milani1, Hossein M. Sagha1, Peiman
Hematti2. 1Ophthalmology, Univ of Illinois at Chicago, Chicago, IL;
2
Medicine, University of Wisconsin, Madison, WI.
Purpose: In patients with limbal stem cell deficiency, in addition to
the loss of the limbal epithelial stem cells, the limbal niche is
invariably damaged/destroyed. Therefore, strategies are needed to
reconstruct the limbal niche. Mesenchymal type cells have been
shown to be an important part of the niche. In this study, we
examined whether mesenchymal stem cells (MSC) from the bone
marrow (BM) can function as niche support cells.
Methods: Human bone marrow MSCs were cultured in serum
containing media. Primary human corneal epithelial cells were grown
in KSFM before being passaged onto plates with BM-MSC. Colony
forming efficiency of epithelial cells co-cultured with BM-MSC was
examined. Conditioned media from BM-MSC and limbal fibroblasts
were compared in terms of their ability to promote epithelial growth.
BM-MSC were also grown in decellularized human corneas to
examine their growth and differentiation in 3D.
Results: As reported by others, BM-MSC supported colony
formation of corneal epithelial cells, which appeared to be dependent
on the passage number of the BM-MSC. Pre-treatment of BM-MSC
with mitomycin C reduced their ability to support corneal epithelial
cell growth as evident by the effect of conditioned media (non-MMC
vs MMC treated) on corneal epithelial cells. BM-MSC proliferated in
3D culture conditions in decellularized human cornea adopting a
keratocyte-like phenotype.
Conclusions: These results indicate that BM-MSC can support the
clonal growth of corneal epithelial cells. More studies are needed to
determine the optimal growth conditions for their use as limbal niche
cells.
Commercial Relationships: Ali R. Djalilian, None; Behrad Y.
Milani, None; Hossein M. Sagha, None; Peiman Hematti, None
Support: National Eye Institute of the National Institutes of Health
with Career Development Grant K08EY017561-A1 (ARD) and Core
Grant EY01792, the Cless Family Foundation, and a Career
Developmental Award (ARD) and unrestricted departmental grant
from Research to Prevent Blindness.
Program Number: 558 Poster Board Number: B0195
Presentation Time: 10:30 AM - 12:15 PM
Decreased Phosphorylation at the Serine-2 Residue of the RNA
Polymerase II in Corneal Epithelial Stem Cells
Satoshi Kawasaki1, Katsuhiko Shinomiya1, Keita Aoi2, 1, Shigeru
Kinoshita1. 1Ophthalmology, Kyoto Prefectural Univ of Med, Kyoto,
Japan; 2Faculty of Life and Medical Sciences, Doshisha University,
Kyotanabe, Japan.
Purpose: Continuous regeneration of stem cells is known to be
essential for maintaining the life-long homeostasis of any types of
cells in the body of animals. In the stem cells of ocular surface
epithelia, several positive and negative markers have been reported.
Recent studies have shown that stem cells are adapted to hypoxic
conditions with slow cell cycling, low transcriptional activity, lower
mitochondrial respiration, and higher glycolysis for the generation of
adenosine-5'-triphosphate (ATP). The purpose of this present study
was to report a new marker for corneal epithelial stem cells based on
low transcriptional activity.
Methods: Corneal tissue samples obtained from humans, rabbits, and
mice were cryo-embedded, sliced into thin sections, fixed, and
immunostained with antibodies against the phosphorylated serine-2
of the RNA polymerase II (RNAP II), a marker of activated
transcription, as well as several positive or negative markers of
corneal epithelial stem cells such as N-cadherin, p63, and keratin 12.
Results: In all of the tissue samples, a decreased level of
phosphorylation at the serine-2 residue of the RNAP II was found at
the basal cells of the limbal epithelium. Immunostaining with
antibodies against the above-described established markers for
corneal epithelial stem cells proved that those cells were, in fact,
corneal epithelial stem cells.
Conclusions: The findings of this study show that phosphorylated
serine-2 of the RNAP II is a new negative marker for corneal
epithelial stem cells. Corneal epithelial stem cells may have a general
feature of stem cells in terms of low transcriptional activity.
Commercial Relationships: Satoshi Kawasaki, None; Katsuhiko
Shinomiya, None; Keita Aoi, None; Shigeru Kinoshita, Senju
Pharmaceutical Co (P), Santen Pharmaceutical Co (P), Otsuka
Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)
Support: 24592675
Program Number: 559 Poster Board Number: B0196
Presentation Time: 10:30 AM - 12:15 PM
Effect of Specular Focal Distance on Endothelial Cell Counting
Accuracy
Jackie Hai, Vivian Xue. HAI Laboratories, Inc., Lexington, MA.
Purpose: To quantify the relationship between specular focal
distance and image distortion, in order to determine a predictive
model of cell counting error based on amount of deviance from true
endothelial cell shape and size.
Methods: High resolution specular image of a standard calibration
lens was captured in focus to establish baseline pachymetry of 0μm.
Subsequent images captured 30μm, 40μm, 50μm, 60μm, 70μm,
80μm, 90μm and 100μm from the baseline were analyzed for changes
in area caused by optical distortion. Analysis was carried out in a
single-blind trial with 10 sample counts performed for each specular
image to determine mean areas.
Results: Given a true area of 10,000μm2 for the baseline distance of
0μm, a negative correlation was found to exist between focal distance
(F) and mean area (A). For F=30μm, A=9666μm2; F=40μm,
A=9444μm2; F=50μm, A=9180μm2; F=60μm, A=9055μm2; F=70μm,
A=8869μm2; F=80μm, A=8767μm2; F=90μm, A=8694μm2;
F=100μm, A=8467μm2. Using simple linear regression, the cell
density error (E) can be extrapolated based on deviance from true
area. For F=10μm, E=1.26%; F=20μm, E=2.90%; F=30μm,
E=4.58%; F=40μm, E=6.53%; F=50μm, E=8.12%; F=60μm,
E=9.99%; F=70μm, E=11.92%; F=80μm, E=13.91%; F=90μm,
E=15.98%; F=100μm, E=18.13%.
Conclusions: Cell counting error can be considered negligible at 029μm, non-negligible at 30-59μm and problematic at 60-100μm of
focal deviance.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Specular images of donor cornea captured 100 micrometers out of
focus, 60 micrometers out of focus, 30 micrometers out of focus, and
0 micrometers out of focus (i.e. completely in focus).
Table: Projected cell density counting error based on deviance from
true focus and true area.
Commercial Relationships: Jackie Hai, HAI Laboratories, Inc. (E);
Vivian Xue, HAI Laboratories, Inc. (E)
Program Number: 560 Poster Board Number: B0197
Presentation Time: 10:30 AM - 12:15 PM
Evaluation of Terrien Marginal Degeneration Using Anterior
Segment Optical Coherence Tomography
Xiaoqiang Liu, Fang Wang. shanghai tenth people's hospital,
Shanghai, China.
Purpose: To investigate the morphologic characteristics of Terrien
marginal degeneration (TMD) using anterior segment optical
coherence tomography (AS-OCT).
Methods: Slitlamp microscopy, AS-OCT and corneal topography
were performed in ten eyes of six patients (four male, two female)
with TMD.
Results: Clinically, all patients demonstrated typical TMD.
Unilateral isolated TMD was presented in two female patients. ASOCT clearly demonstrated the extension of the ectatic part, thickness,
and structure of the cornea in all cases. A characteristic thinned zone
of peripheral cornea was showed in all affected eyes. Hydrop in the
ectatic part of cornea was seen in one patient. The topographic maps
showed irregular astigmatism in four eyes.
Conclusions: The AS-OCT detects the microstructure of cornea
clearly and can be used to monitor the progression of TMD.
Commercial Relationships: Xiaoqiang Liu, None; Fang Wang,
None
Program Number: 561 Poster Board Number: B0198
Presentation Time: 10:30 AM - 12:15 PM
Human graft cornea imaging with full-field optical coherence
tomography
Wajdene Ghouali1, Kate Grieve2, Otman Sandali1, Elena Basli1,
Julien Bullet1, Laurent Laroche1, Vincent Borderie1. 1CHNO des
Quinze-Vingts, Paris, France; 2Institut Langevin, Paris, France.
Purpose: There is currently no efficient method for the study of
epithelium and stroma of human graft corneas. The aim of our study
is to evaluate the performance of a full-field optical coherence
tomograhy system in the evaluation of human graft corneas.
Methods: Our study was carried out using a full-field OCT system
from LLTech ®, developed for non-invasive imaging of tissue
structures in depth. Images were acquired on human donor corneas
(in normal and oedematous conditions) and surgical specimens of
pathological corneas (Fuchs dystrophy, keratoconus, stromal
keratitis)
Results: The Full-field OCT device from LLTech® enables three
dimensional images to be obtained with ultrahigh resolution (1
micron in all directions) comparable to traditional histological
sections. This allows a precise visualisation of the cells and the
different structures (epithelium, Bowman’s membrane, stroma,
Descemet’s membrane and endothelium) in normal corneas (figure
1), but also in pathological corneas (even in the presence of an
oedema), with specific lesions in each condition.
Conclusions: Optical microscopy, with a detailed view of the corneal
endothelium and a cell density determination, remains the « gold
standard » in the study of human cornea grafts. However, full-field
OCT, thanks to a more complete anatomical stydy of the cornea,
could be helpful in the evaluation and the selection of human cornea
grafts whose quality plays a major role in the outlook of the corneal
transplant
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
were seen in the posterior stroma. In LCD, hyperreflective linear and
branching structures with poorly demarcated margins were noted in
the stroma. In PPMD, a variety of vesicular and linear abnormalities
could be identified at the level of the endothelium. In FD, Descemet's
membrane was thickened with round hyporeflective structures
(guttae) at level of the endothelium,with fibrosis and activated
keratocytes seen in the stroma.
Conclusions: AS-OCT and IVCM are non-invasive, high-resolution
imaging modalities useful in the visualization of the corneal
microstructural changes related to corneal dystrophies. These new
imaging technologies are rapidly becoming widely used instruments
for both research and patient care, and may be useful in elucidating
the pathogenesis and natural course of corneal dystrophies.
The figure 1 shows full-field OCT images of the cornea from the 3D
data stack. The upper four images are en face optical slices
descending through the sample depth: EP epithelium; BM Bowman's
membrane; ST stroma; DM+E Descemet's membrane and
endothelium. The lower image is a depth slice through the cornea
showing the different layers as labelled above. Scale bars show
100μm.
Commercial Relationships: Wajdene Ghouali, None; Kate
Grieve, None; Otman Sandali, None; Elena Basli, None; Julien
Bullet, None; Laurent Laroche, None; Vincent Borderie, None
Program Number: 562 Poster Board Number: B0199
Presentation Time: 10:30 AM - 12:15 PM
High-Resolution Anterior Segment Optical Coherence
Tomography and in vivo Confocal Microscopy in the Evaluation
of Corneal Dystrophies
Dennis E. Cortes1, 2, Jennifer Li1, Michael Chen1, Raju Poddar1,
Robert J. Zawadzki1, John S. Werner1, Mark J. Mannis1. 1Department
of Ophthalmology & Vision Science, UC Davis Eye Center,
Sacramento, CA; 2Department of Ophthalmology, Pontificia
Universidad Católica de Chile, Santiago, Chile.
Purpose: To describe the findings observed with high-resolution
anterior segment optical coherence tomography (AS-OCT) and in
vivo confocal microscopy (IVCM) in patients with corneal
dystrophies.
Methods: Five patients with common corneal dystrophies seen in
clinical practice were evaluated with a high-resolution spectraldomain AS-OCT (Heidelberg Engineering, Germany) and IVCM
using the Heidelberg Retina Tomograph 3 Rostock Cornea Module
(Heidelberg Engineering, Germany). A high-speed swept-source
OCT (SS-OCT) prototype was also used to reconstruct the threedimensional structures of the cornea.
Results: Patients included in this study had the following corneal
dystrophies: epithelial basement membrane dystrophy (EBMD),
granular cornea dystrophy (GCD), lattice cornea dystrophy (LCD),
posterior polymorphous corneal dystrophy (PPMD) and Fuchs’
endothelial dystrophy (FD). AS-OCT and IVCM provided precise
information about the pathology in the different layers of the cornea
in all patients. There was a significant correlation between the two
imaging modalities.
In EBMD, we identified an abnormal epithelial basement membrane
protruding into the corneal epithelium and clusters of epithelial cells
in the tear film. In GCD, the majority of opacities were visualized in
the anterior two-thirds of the corneal stroma although rare opacities
Patient with EBMD evaluated with in vivo confocal microscopy
(IVCM) and anterior segment OCT (AS-OCT).
Patient with granular dystrophy evaluated with in vivo confocal
microscopy (IVCM) and anterior segment OCT (AS-OCT)
Commercial Relationships: Dennis E. Cortes, None; Jennifer Li,
None; Michael Chen, None; Raju Poddar, None; Robert J.
Zawadzki, None; John S. Werner, None; Mark J. Mannis, None
Support: NEI 014743, RPB
Program Number: 563 Poster Board Number: B0200
Presentation Time: 10:30 AM - 12:15 PM
Analysis Of Early Postoperative Morphologic Features Of Clear
Corneal Incisions Created With The Femtosecond Cataract
Laser Using Anterior Segment Optical Coherence Tomography:
Comparison Of Intended Versus Achieved Wound Parameters
Surendra Basti, Dilraj S. Grewal. Ophthalmology, Northwestern
University Feinberg School of Medicine, Chicago, IL.
Purpose: To analyze the morphology of clear corneal incisions (CCI)
performed using a Femtosecond Cataract Laser (Catalys, Optimedica,
Santa Clara, CA) in patients undergoing femtosecond laser-assisted
cataract surgery using spectral domain anterior segment optical
coherence tomography (AS-OCT).
Methods: Morphology of clear corneal incisions created with the
Femtosecond cataract laser was studied with AS-OCT. An intended
triplanar incision was programmed into the Catalys femtosecond laser
software. The first plane was at 90 degree to the corneal surface and
extended to 40% corneal depth, the second plane was an angled
intrastromal plane and the third plane was at 45 degrees to the
posterior corneal surface and reached the second plane at 70% depth.
The intended incision width was 2.85 mm and length was 1.8 mm.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
AS-OCT was performed on the first postoperative day using the
Cirrus HD-OCT (Carl Zeiss Meditec, Dublin, CA). The clear corneal
incision length, incision depth, angles of the tri-planar corneal
incision, and wound gaping were measured using software calipers.
The variability in wound length, depth and angle were calculated and
compared to the programmed software settings. Five architectural
features were used to describe the clear corneal incisions: gaping of
the wound at the epithelial side, gaping of the wound at the
endothelial side, within wound gape, misalignment of the roof and
floor of the incision at the endothelial side and local Descemet’s
membrane (DM) detachment. This study is ongoing and will include
30 eyes.
Results: On analysis of first post-operative day AS-OCT images of
the initial 3 eyes undergoing femtosecond cataract extraction, 2/3
eyes had endothelial side wound gape, 1/3 had epithelial wound gape,
2/3 eyes had a within wound gape and 3/3 eyes had a focal DM
detachment. All three eyes had visible 3-plane profile on AS-OCT.
The clear corneal incision length was within 100 microns of the
intended length. The incision depth was within 8 percent of the
intended depth.
Conclusions: Our initial results suggest that clear corneal incisions
using the femtosecond cataract laser were close to the intended size
and depth. A significant proportion of eyes had marginal and stromal
wound gape and focal DM detachment.
Commercial Relationships: Surendra Basti, None; Dilraj S.
Grewal, None
Support: An Unrestricted Grant From Research To Prevent
Blindness, Inc., New York, New York
Program Number: 564 Poster Board Number: B0201
Presentation Time: 10:30 AM - 12:15 PM
Epithelial Basement Membrane Dystrophy: A Study with in vivo
Confocal Microscopy and High-Resolution Anterior Segment
Optical Coherence Tomography
Peter Wu1, Dennis E. Cortes1, 2, Jennifer Li1, Michael Chen1, Mark J.
Mannis1. 1Ophthalmology & Vision Sciences, University of
California Davis Eye Center, Sacramento, CA; 2Ophthalmology,
Universidad Catolica de Chile, Santiago, Chile.
Purpose: To report the pathological changes of patients with
epithelial basement membrane dystrophy (EBMD), the most common
hereditary anterior corneal dystrophy, using in vivo confocal
microscopy (IVCM) and high-resolution anterior segment optical
coherence tomography (AS-OCT).
Methods: Five patients with EBMD seen in clinical practice were
evaluated with a high-resolution spectral-domain AS-OCT
(Heidelberg Engineering, Germany) and IVCM using the Heidelberg
Retina Tomograph 3 Rostock Cornea Module (Heidelberg
Engineering, Germany).
Results: AS-OCT revealed hyperreflective material in the posterior
epithelium and anterior stroma. These findings were correlated with
IVCM findings that showed multiple linear and curvilinear
hyperreflective structures, corresponding to abnormal epithelial
basement membrane extending into the corneal epithelium.
Additionally, IVCM revealed hyperreflective deposits in the anterior
stroma with signs of activation of anterior keratocytes, intraepithelial
microcysts, and clusters of epithelial cells in the tear film.
Conclusions: The acquisition of high-resolution imaging of the
cornea with IVCM and AS-OCT provides new insights into the
microstructural characteristics of EBMD and may be useful
modalities in elucidating the pathogenesis and natural course of this
corneal dystrophy.
A: slit lamp photo of EBMD B: in-vivo confocal microscopy (IVCM)
revealing linear hyperreflective structures corresponding to abnormal
epithelial basement membrane extending into the corneal epithelium
C: anterior segment OCT (AS-OCT) revealing hyperreflective
material in the posterior epithelium and anterior stroma
A: slit lamp photo of EBMD B: oblique section of in-vivo confocal
microscopy revealing an area of fibrosis at the sub-epithelial level C:
anterior segment OCT (AS-OCT) demonstrating hyperreflective
material in the posterior epithelium and anterior stroma
Commercial Relationships: Peter Wu, None; Dennis E. Cortes,
None; Jennifer Li, None; Michael Chen, None; Mark J. Mannis,
None
Program Number: 566 Poster Board Number: B0203
Presentation Time: 10:30 AM - 12:15 PM
Study Of Ocular Surface impairment in children presenting
ongoing dry eye with MGD with Meibomian Gland Analysis
Dominique Bremond-Gignac1, 2, Solange Milazzo1. 1Ophthalmology,
St Victor Center, University Hospital of Amiens, Picardie Jules
Verne University, Amiens, France; 2CNRS IRIS UMR8194, Paris V
University, Paris, France.
Purpose: To evaluate in a retrospective study, the ocular surface
impairment in children presenting ongoing MGD with an analysis of
meibomian glands. Tear film quality is conditionned by meibomian
gland production of the lipid layer. Anomaly of this production in
children can lead to ocular surface impairment and an evaluation of
Meibomian Glands is a key point of the exploration of production
function.
Methods: Our retrospective study included 12 children (22 eyes
evaluated) with a mean age 11yo, range 4 to 17yo, of two groups that
presented at ocular consultation. Group a, 6 with ongoing MGD,
group b 6 control children who had been tested with no ocular surface
impairment. All children underwent a Meibomian Gland Analysis
with meibography images with infrared illumination acquired with
Cobra system. An evaluation with Phoenix software calculated area
of loss of glands. The density of inferior eyelids Meibomian glands
had also been evaluated.
Results: In group a, all children presented clinical signs of MGD due
to ocular blepharitis or dry eye. In group b no ocular signs of
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
conjunctiva or ocular surface anomalies were found. In group a area
loss of meibomian gland was evaluated from 41% to 59%. Density of
glands was low. In group b area loss of meibomian gland was
evaluated from 10% to 42%. In younger children (4 to 7 yo) density
of glands was tight and lenght size shorter.
Conclusions: In children, few data are known about Meibomian
Gland development and function. Our study is suitable in children
with dry eye for the evaluation of Meibomian Glands and can be a
useful tool. A larger study has to be performed for a better
understanding of dry eye ocular surface impairment in children.
Commercial Relationships: Dominique Bremond-Gignac, None;
Solange Milazzo, None
Program Number: 567 Poster Board Number: B0204
Presentation Time: 10:30 AM - 12:15 PM
Gender differences in ocular biometric data
yoona jang1, Hiroshi Uozato1, 2, Takushi Kawamorita1, 2, Yuko
Shibata3. 1Department of Visual Science, Kitasato University
Graduate School, Sagamihara, Japan; 2Ophthoptics and Visual
Sciences, Kitasato University School of Allied Health Science,
Sagamihara, Japan; 3Ophthalmology and Visual Sciences, Kitasato
University Graduate School, Sagamihara, Japan.
Purpose: To compare the differences in ocular biometric data with
regard to gender
Methods: Seventy-two eyes from 38 healthy subjects (mean age =
21.4 ± 2.7 years, range: 18 to 31 years) including 19 males (mean age
= 22.5 ± 2.6 years) and 19 females (mean age = 20.3 ± 2.5 years)
were recruited. Anterior chamber depth (ACD) and axial length (AL)
were measured with IOL Master TM (Carl Zeiss Meditec, Jena,
Germany). ACD was defined as the length between the center of
anterior surface of cornea and the center of anterior lens surface.
Angle lambda (kappa), distance from central pupillary eccentricity to
thinnest corneal thickness, corneal curvature(CR), corneal
power(CP), central corneal thickness(CCT) were measured with
pachymetry with a dual scheimpflug imaging system GalileiTM
(Ziemer Ophthalmic Systems, Port, Switzerland ). The statistical
significance of the gender differences between measurements was
evaluated by Student’s t-test.
Results: The mean ACD was 3.77 ± 0.32 mm for males and 3.65 ±
0.25 mm for females. Males had significantly deeper ACD than
females (p = 0.049). The mean angle lambda, AL and CCT were 3.15
± 1.60 °, 25.31 ± 1.59 mm, 552.01 ± 30.05 μm for males and 3.86 ±
1.58 °, 24.79 ± 1.15 mm, 551.81 ± 20.23 μm for females,
respectively. Males had significantly larger angle lambda than
females (p = 0.028). There was no statistically significant difference
in the AL and distance from central pupillary eccentricity to thinnest
corneal thickness in gender comparison, but there are tendency
associated with differences gender (p = 0.056, p = 0.054). There was
no statistically significant difference in mean CR, CP and CCT
between males and females.
Conclusions: Both ACD and angle lambda had statistically
significant difference with gender. And, AL and distance from central
pupillary eccentricity to thinnest corneal thickness were tendency
associated with gender difference. However, gender did not show any
statistically significant effect on cornea as CR, CP and CCT.
Therefore, consider to gender would be an important factor to ocular
biometric examinations for refractive and cataract surgeries.
Commercial Relationships: yoona jang, None; Hiroshi Uozato,
None; Takushi Kawamorita, None; Yuko Shibata, None
138 Dry Eye and Lacrimal Gland I
Sunday, May 05, 2013 1:00 PM-2:45 PM
Exhibit Hall Poster Session
Program #/Board # Range: 900-954/B0205-B0259
Organizing Section: Cornea
Program Number: 900 Poster Board Number: B0205
Presentation Time: 1:00 PM - 2:45 PM
Does Altering the Volume of an Artificial Tear Applied to the
Eye Influence Vision?
William H. Ridder, Monisha Paripatyadar, Lisa Wahl. Basic &
Visual Science, Southern California College of Optometry, Fullerton,
CA.
Purpose: Artificial tears (AT) are the most common treatment for
dry eye. Several studies have shown that immediately after an AT is
placed on the eye vision is degraded for a short time. The purpose of
this investigation was to determine if there is a correlation between
the volume of AT placed on the eye and the degradation in vision.
Methods: Six normal, adult subjects took part in this project. Snellen
acuity was better than 20/25 in the eye tested. The subjects had 1
hour of training with the contrast sensitivity (CS) measurement
technique before data collection. CS to a 14 cpd sine wave grating
(16 ms stimulus presented at 1 sec after the blink) was continually
tracked (using a 2 AFC technique) before and after (minimum of 25
minutes) an AT (Refresh Optive, Allergan, Inc.) was applied. The
volumes of the ATs applied, randomized across visits, were 25, 45, or
65 µl (Pos D Pipette, Rainin Inst.). The data for different drop
volumes were collected on separate days. The magnitude of the loss
in normalized contrast sensitivity was compared across drop
volumes.
Results: The average loss in normalized CS (mean ± SD) for the
drop volumes were: 0.46 ± 0.182, 0.50 ± 0.171, and 0.60 ± 0.293 for
25, 45, and 65 µl, respectively. A paired t-test found a significant
difference in the CS loss between the 25 and 65 µl drop sizes (p =
0.05, t = 2.46). No other comparisons were significant (p > 0.05).
Conclusions: The CS decreased as the volume of the applied AT
increased. This may be the result of a greater disruption in the tear
layer as the AT drop volume increases.
Commercial Relationships: William H. Ridder, None; Monisha
Paripatyadar, None; Lisa Wahl, None
Program Number: 901 Poster Board Number: B0206
Presentation Time: 1:00 PM - 2:45 PM
Effects of Omega-3 Fatty Acid and Hyaluronic acid Mixture Eye
Drops on the Ocular Surface in Desiccating Stress induced
Murine Dry Eye
Zhengri Li1, Jung-Han Choi2, Hyo Seok Lee3, Ji-Suk Choi4, Han Jin
Oh5, Kyung Chul Yoon6. 1Chonnam National Uinversity and hospital,
Gwangju, Republic of Korea; 2Chonnam National Uinversity and
hospital, Gwangju, Republic of Korea; 3Chonnam National
Uinversity and hospital, Gwangju, Republic of Korea; 4Chonnam
National Uinversity and hospital, Gwangju, Republic of Korea;
5
Chonnam National Uinversity and hospital, Gwangju, Republic of
Korea; 6Chonnam National Uinversity and hospital, Gwangju,
Republic of Korea.
Purpose: To investigate the effects of topical application of omega-3
Fatty Acid (FA) and hyaluronic acid (HA) mixtures on inflammation
and oxidative stress markers in a mouse model of experimental dry
eye (EDE).
Methods: Eye drops consisting of 0.02%, or 0.2% omega-3 FA and
0.1% HA mixtures, or 0.1% HA were applied in desiccating stress
induced murine dry eye. Corneal irregularity and corneal fluorescein
staining scores were measured at 5 and 10 days after treatment.
Levels of interleukin (IL)-1β, IL-17, and interferon gamma-induced
protein (IP)-10 were measured in the conjunctiva using a multiplex
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
immunobead assay at 10 days. The concentrations of HEL and 4HNE were measured with enzyme-linked immunosorbent assays in
conjunctiva tissue.
Results: Although all parameters were lower in the 0.02% FA
mixture and HA-treated groups compared with the EDE group, the
effect was not statistically significant. However, mice treated with
0.2% omega-3 FA mixture showed a significant improvement in
corneal irregularity and corneal fluorescein staining compared with
EDE and HA-treated mice. A significant decrease in the levels of IL1β, IL-17, and IP-10 were observed in the 0.2% FA mixture-treated
group, compared with the other groups. In the 0.2% FA mixturetreated groups, the concentrations of HEL and 4-HNE were also
lower than the other groups.
Conclusions: Topical omega-3 FA mixture eye drops can improve
corneal irregularity and corneal epithelial barrier disruption, and
decrease inflammatory cytokines and oxidative stress markers on the
ocular surface. These results suggest that mixture of 0.2% Omega-3
FA and HA can be useful for treatment of dry eye treatment.
Commercial Relationships: Zhengri Li, None; Jung-Han Choi,
None; Hyo Seok Lee, None; Ji-Suk Choi, None; Han Jin Oh,
None; Kyung Chul Yoon, None
Program Number: 902 Poster Board Number: B0207
Presentation Time: 1:00 PM - 2:45 PM
Evaluation of a Topical Cis-urocanic acid in a Murine CAE™
Model of Dry Eye Disease
Andy Whitlock1, Laura Belen1, Jennifer Brackett1, Burkhard Blank2.
1
Ora, Inc., Andover, MA; 2Laurantis Pharma, Turku, Finland.
Purpose: The goal of this study was to test the potential of Cisurocanic acid (Cis-UCA) in a topical formulation as a treatment for
dry eye.
Methods: The study employed a murine model of dry eye disease
that combines of environmental and pharmacologic induction of
pathologic signs of disease. Cis-UCA was provided by the sponsor as
a clear solution at concentrations of 0.0, 0.5, 1.0, and 2.5%.
Restasis®, dry eye treatment (scopolamine and chamber only), and
naive (room air) controls were included in the study as well. Mice
were dosed topically three times a day in both eyes for a total of 22
days. Animals received 3 days of prophylactic topical treatment (no
scopolamine or chamber) and 14 days of dry eye treatment (with
scopolamine and chamber).Fluorescein staining evaluations were
performed using Ora’s novel Micron III imaging system at various
time points throughout the study. Corneal images were evaluated
using Ora’s proprietary clinical scale as well as a custom image
analysis algorithm.
Results: 1.0%Cis-UCA was most effective in reducing corneal
fluorescein staining as compared to vehicle and the dry eye treatment
controls. Throughout the study duration, the average staining of all
the eyes in the group was maintained at about 8 units, whereas the
vehicle and dry eye treatment groups steadily increased to a
maximum average staining of approximately 12 units by Day 17. The
average corneal staining for mice in the 1.0%Cis-UCA treatment
group was statistically lower than both the vehicle and the dry eye
treatment group (p<0.05 for both). The 0.5 and 2.5% Cis-UCA
groups did not show a statistically significant reduction in staining
values versus either control. Restasis®, which was included in the
experiment as a clinical comparator, showed no statistical separation
throughout the experiment and did not provide a significant reduction
in corneal staining at Day 17.
Conclusions: Topical treatment with 1% Cis-UCA significantly
reduced corneal staining in a pre-clinical model of dry eye disease.
This concentration was superior to both higher or lower
concentrations, suggesting that the dosing range used in this study
provided an optimal compound concentration for topical ocular
therapy. The 1% Cis-UCA formulation was superior to the positive
comparator at all data points, providing further support for its
potential as a therapy for dry eye disease.
Commercial Relationships: Andy Whitlock, Ora, Inc. (E); Laura
Belen, Ora, Inc. (E); Jennifer Brackett, Ora, Inc. (E); Burkhard
Blank, None
Program Number: 903 Poster Board Number: B0208
Presentation Time: 1:00 PM - 2:45 PM
Changes In Corneal Cold Thermoreceptor Activity Induced By
Repetitive Allergen Challenges, In A Guinea-Pig Model Of
Allergic Conjunctivitis
M Carmen Acosta, Carolina Luna, Susana Quirce, Carlos Belmonte,
Juana Gallar. Instituto de Neurociencias, Universidad Miguel
Hernandez-CSIC, San Juan, Spain.
Purpose: To test in a guinea-pig model of allergic conjunctivitis
whether exposure to the allergen evokes changes in blinking, tearing
and corneal cold sensory nerve activity and the contribution of
TRPA1 and TRPV1 channels of sensory nerves to the allergic
response.
Methods: Sensitization to ovalbumin (OVA) was evoked by i.p.
injection of 100µg OVA+20mg Al(OH)3 in 1ml PBS. On days 14th
to 18th, 10% OVA (10µl drop) was applied to both eyes and tearing
and blinking rates were measured immediately afterwards during 5
min. Nerve activity from cold-sensitive corneal nerve terminals
(CNTs) was recorded in the superfused cornea in vitro, in response to
thermal changes (exposure to bath temperature variations from the
basal temperature of 34°C, down to 20°C or up to 50°C) at different
days after the allergic challenge. In some experiments a 10µl drop of
the TRPA1 channel blocker HC-030031, 10µM or of the TRPV1
channel blocker Capsazepine, 5mM was applied topically, 20 to 60
min before ocular instillation of the OVA.
Results: Tearing and blinking rates were significantly increased and
the response to cooling of CNTs was decreased after a single
exposure to the allergen (Table 1). These effects were more
pronounced after repeated exposures to OVA (RE) (see table)
Blockade of TRPA1 and TRPV1 channels attenuated the tearing and
blinking response to repeated exposure to the allergen. Only
capsazepine reversed in part the depression of cold receptors by the
allergic inflammation, possibly through its anti-inflammatory effect.
Conclusions: Inflammatory mediators released during allergic
conjunctivitis, inhibit TRPM8 decreasing cold receptor activity, and
activate TRPV1 and TRPA1 channels enhancing blinking through
polymodal nociceptor excitation.
Commercial Relationships: M Carmen Acosta, None; Carolina
Luna, None; Susana Quirce, None; Carlos Belmonte, None; Juana
Gallar, None
Support: Supported by: SAF2011-22500, GV/2007/030 and in part
by CSD2007-00023, IPT-2011-1110-900000 and BFU2008-04425
(Ministerio de Ciencia e Innovación, Spain, and FEDER, EU).
Program Number: 904 Poster Board Number: B0209
Presentation Time: 1:00 PM - 2:45 PM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Long-Term Corneal Sensitivity after PRK determined by Noncontact Gas Esthesiometry
Juana Gallar1, Jukka A. Moilanen2, M Carmen Acosta1, Juha M.
Holopainen2, Carlos Belmonte1, Timo M. Tervo2, Waldir Neira2, 1.
1
Instituto de Neurociencias, Universidad Miguel Hernandez-CSIC,
San Juan de Alicante, Spain; 2Ophthalmology, University of
Helsinki, Helsinki, Finland.
Purpose: To evaluate the long-term (5-12 years) evolution of corneal
sensitivity to mechanical and chemical stimuli after photorefractive
keratectomy (PRK).
Methods: Nineteen patients (12 male, 7 female; mean age 35.3;
range: 21-55 years at the time of surgery) who underwent PRK for
low to moderate myopia (mean spherical equivalent -3.0 D; range 2.5 to -8.0 D), and 14 control individuals (5 male, 9 female; mean
age: 40.3, range: 28-54 years) were examined at 1 week, and 0.5, 2, 5
and at least 10 years (range 10.5-12 years) after surgery. Corneal
sensitivity to mechanical (air flow between 0 and 260 ml/min) and
chemical (0 to 80% CO2 in the air, at subthreshold flow) stimulation
was tested with non-contact gas esthesiometry (original Belmonte
esthesiometer: 1 week-2 years; modified Belmonte-CRCERT
esthesiometer: > 5years) using 0-10 Visual Analogue Scales (VAS)
and performing intensity-response curves. The sensation threshold
value and the slope of the intensity-response curve were calculated.
Results: Corneal sensitivity to mechanical stimulation was present
but significantly reduced 1 week after surgery when compared with
controls (mechanical thresholds: 203±49 vs. 105±14 ml/min; slopes:
0.0044 vs. 0.0219 VAS unit/ml of flow, respectively). The decrease
of corneal mechanical sensitivity was more pronounced 0.5 years
after PRK and remained below control values 2 and 5 years
postoperatively (thresholds: 267±18; 260±10, and 249±13 ml/min,
respectively). Corneal sensitivity to chemical stimulation was only
slightly modified after PRK (slightly enhanced at 1 week but slightly
reduced at 0.5, 2 and 5 years). Sensitivity to both mechanical and
chemical stimulation presented normal values 10 years after PRK
(mechanical threshold: 91±12 ml/min, slope: 0.0218 VAS unit/ml of
flow).
Conclusions: Immediately hyperesthesia to chemical sensitivity is
attributable to sensitization. The long-lasting reduction of corneal
sensitivity to mechanical stimulation after PRK indicates that the
transduction capacity to mechanical forces of injured nerve fibers is
permanently impaired, contributing to the altered sensations
experienced after PRK. New regenerating nociceptive corneal nerves
slowly invade the denervated area, being responsible of the recovery
of mechanical and chemical sensitivity observed at 10 years after
PRK.
Commercial Relationships: Juana Gallar, None; Jukka A.
Moilanen, Adventus Technology Inc. (C); M Carmen Acosta,
None; Juha M. Holopainen, None; Carlos Belmonte, None; Timo
M. Tervo, None; Waldir Neira, None
Support: SAF2011-22500, and in part by BFU2008-04425,
CSD2007-00023 and IPT-2011-1110-900000 (Ministerio de
Economía y Competitividad, Spain, and FEDER, EU), Evald and
Hilda Nissi Foundation and Finnish Eye Foundation (Finland)
Program Number: 905 Poster Board Number: B0210
Presentation Time: 1:00 PM - 2:45 PM
The Ocular Surface Sensory Response to Tear Film Instability
With and Without a Contact Lens
Carolyn G. Begley1, Jun Zhang1, Ping Situ1, Ziwei Wu1, Trefford L.
Simpson2. 1School of Optometry, Indiana University, Bloomington,
IN; 2Optometry and Vision Science, University of Waterloo,
Waterloo, ON, Canada.
Purpose: Tear film instability (TFI) is a core mechanism of dry eye
(DEWS, 2007), but its sensory impact on the ocular surface remains
poorly understood. In this study, we test the hypothesis that TFI
directly stimulates ocular surface sensory neurons and that wearing a
soft contact lens (CL) partially blocks this stimulation.
Methods: Ten adapted CL wearers participated in 2 study visits.
While not wearing CLs, subjects were seated behind a slit lamp
biomicroscope and were asked to keep one eye open as long as
possible (maximum blink interval=MBI) while fluorescein TFI was
monitored and subjects simultaneously indicated the level of
discomfort using a “discomfort knob” (DK) potentiometer (0-10
scale). The MBI procedure was repeated 10 times. Discomfort and
burning sensations during and after each trial were rated using 0-10
visual analogue scales (VAS). The entire procedure was repeated at a
second study visit while wearing CLs for a fixed 30 sec MBI with
retroillumination used to view TFI over CLs.
Results: The discomfort intensity and slope measured by the DK was
significantly lower (paired t-test, p<0.004) while wearing a CL
(AVG±SD; End DK=4±2; slope=0.15±0.08DK/sec) versus no CL
(AVG±SD; End DK=9±2; slope=0.66±0.23DK/sec). Likewise, the
VAS ratings during and after MBI trials for discomfort (AVG±SD
with CL: 3.17±1.70 and 3.05±1.71; no CL: 6.38±2.11 and 4.64±2.71)
and burning (AVG±SD with CL: 2.56±1.90 and 2.33±1.87; no CL:
5.90±2.63 and 4.56±2.89) were significantly lower with CLs
(ANOVA p=0.013 and 0.011 for discomfort and burning,
respectively).
Conclusions: These results support the hypotheses that TFI provides
nociceptive stimulation to the cornea, perhaps due to increased
osmolarity or surface drying during TFI. This effect is partially
blocked and altered (less burning) by wearing a CL, which suggests
that TFI over the CL surface may stimulate the ocular surface
through different mechanisms.
Example of discomfort intensity by DK during all trials for one
subject
Commercial Relationships: Carolyn G. Begley, Santen, Inc. (C),
ohnson & Johnson Vision Care, Inc. (C), ohnson & Johnson Vision
Care, Inc. (F); Jun Zhang, None; Ping Situ, None; Ziwei Wu,
None; Trefford L. Simpson, None
Support: National Eye Institute R01EY021794
Program Number: 906 Poster Board Number: B0211
Presentation Time: 1:00 PM - 2:45 PM
Lacrimal gland removal increases primary afferent driven
spontaneous blinking and produces ocular hyperalgesia in the rat
Ian D. Meng, Stephen T. Barton, Andrew L. Twaite. Biomedical
Sciences, University of New England, Biddeford, ME.
Purpose: Dry eye syndrome produces ocular pain yet an animal
model for assessing nociceptive responses in the dry eye condition is
lacking. The aim of this study was to characterize spontaneous and
evoked pain in the rat after unilateral removal of the infra- and
exorbital lacrimal glands.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Methods: In male SD rats, spontaneous blinking and eye wipe
behaviors elicited by hypertonic saline (2.5 and 5.0 M) was examined
2 and 8-10 weeks following unilateral removal of the infra- and
exorbital lacrimal glands and in age matched controls. Furthermore,
the effect of topical lidocaine (4%) on spontaneous blinking and the
effect of morphine (5 mg/kg, s.c.) on spontaneous blinking and eye
wipe responses was determined in dry eye and control animals.
Results: Lacrimal gland removal resulted in an increase in
spontaneous blinking in the ipsilateral eye that remained consistently
elevated over an 8-week period. Lidocaine application reduced
spontaneous blinking down to control levels, whereas glycerol and
artificial tear solutions reduced spontaneous blinking. The time spent
eye wiping was also enhanced in response to hypertonic saline (2.5
and 5.0 M) at both the 2 and 8 wk time-points. Morphine attenuated
both spontaneous blinking and the response to hypertonic saline in
dry eye animals.
Conclusions: These results indicate that dry eye produced by
lacrimal gland removal produces hyperalgesia in the rat, as quantified
in the eye wipe assay. In addition, spontaneous blinks in dry eye
animals and their reduction by morphine and topical anesthesia,
indicate the presence of persistent irritation elicited by the activation
of corneal nociceptors.
Commercial Relationships: Ian D. Meng, None; Stephen T.
Barton, None; Andrew L. Twaite, None
Support: NIH Grant R01EY021230
Program Number: 907 Poster Board Number: B0212
Presentation Time: 1:00 PM - 2:45 PM
Alpha Helicity of Lacritin’s C-Terminal Syndecan-1 Binding
Domain is Enhanced by Interaction with the Mutual Binding
Region in Syndecan-1
Jeff Romano1, Jacob Irwin2, Inchan Kwon2, Gordon W. Laurie1. 1Cell
Biology, University of Virginia, Charlottesville, VA; 2Chemical
Engineering, University of Virginia, Charlottesville, VA.
Purpose: Lacritin is a small tear protein (~18 kDa) with mitogenic,
cytoprotective and prosecretory activities. When added topically to
rabbit eyes, lacritin promotes basal tearing. Lacritin targets the
corneal epithelial cell surface protein syndecan-1 (SDC1), a widely
expressed heparan sulfate proteoglycan via a heparanase dependent
mechanism that facilitates interaction between lacritin amino acids
100-109 in its C-terminal alpha-helix and the SDC1 core protein
sequence GAGAL nestled in SDC1’s heparan sulfate rich Nterminus. Since GAGAL is hydrophobic, we wondered whether it
might influence development of lacritin’s C-terminal alpha helix
necessary for binding. Here we performed comparative circular
dichroism analyses of lacritin peptide 1898 (aa 95-119) alone, and in
the presence of SDC1 peptide 1 (aa 20 - 30), scrambled SDC1
peptide 1, or the equivalent region in SDC4 (SDC4 peptide).
Methods: Synthetic peptides were generated by Genescript to a
purity of >95%, dissolved in 10mM dodecylphosphocholine and
subjected to CD analysis in the 190-250 nm range with 0.1 nm pitch,
50 nm/min scan speed, 8 second response time and a 2 nm
bandwidth. A 1 mm pathlength quartz cuvette with cylindrical
geometery (Hellma) was used, stored in 2% Hellmanex prior to
usage, washed vigorously 3 times with ddH2O, subsequently washed
with 100% ethanol, and then dried completely with pure N2 before
and between samples. The spectra displayed represent the average of
at least 5 sample runs. Between sample runs, the dd H2O blank was
monitored to ensure protein buildup in the cuvette was not occurring.
Results: Lacritin amino acids 95-119 (81 µM) assumed an alpha
helical structure in dodecylphosphocholine. This was enhanced by
SDC1 peptide 1 (57 µM), but not by scrambled SDC1 peptide 1.
SDC4 peptide was partially effective, but less so than SDC1 peptide
1 - in keeping with less hydrophobicity and no interaction of lacritin
with SDC4. Alone, SDC1 peptide1, scrambled SDC1 peptide 1 and
SDC4 peptide assumed a random coil structure.
Conclusions: Ligation of lacritin’s C-terminal amphiphathic alpha
helix with GAGAL in SDC1 is enhanced by mutual hydrophobicity
that improves alpha helicity.
Commercial Relationships: Jeff Romano, 7,932,227 (P), 7,648,964
(P), 7,459,440 (P), 7,320,870 (P); Jacob Irwin, None; Inchan
Kwon, None; Gordon W. Laurie, UVa Patent Foundation (F)
Support: NH Grant EY018222
Program Number: 908 Poster Board Number: B0213
Presentation Time: 1:00 PM - 2:45 PM
Lacritin accelerated autophagy promotes clearance of aggregated
proteins and is dependent on stress
Keith B. Zimmerman1, Milton F. Tyler1, Ningning Wang1, Ronald
Raab2, Robert L. McKown2, Gordon W. Laurie1. 1Cell Biology,
University of Virginia, Charlottesville, VA; 2Integrated Science &
Technology, James Madison University, Harrisonburg, VA.
Purpose: Lacritin is a natural tear protein that promotes the survival
of human corneal epithelial (HCE) cells stressed with inflammatory
cytokines INFG and TNF, both of which are elevated in dry eye.
Survival is dependent on lacritin stimulated autophagic flux, which is
likely to remove stress-aggregated and damaged proteins. We have
now addressed this assumption by transduction of mCFP tagged
Huntingtin mutant constructs Htt103Q and Htt25Q in HCE cells
stably transduced with autophagy marker LC3B double tagged with
mCherry and EGFP. Htt103Q forms toxic aggregates whereas
Htt25Q remains soluble, thus only Htt103Q cells are significantly
stressed.
Methods: Retroviral pBabe-puro-mCherry-EGFP-LC3B and pBabeHygro-Htt103Q-mCFP or pBabe-Hygro-Htt25Q-mCFP were
expanded in HEK293T cells, and used to co-transduce human corneal
epithelial (HCE) cells (Riken). Cells grown on glass cover slips were
then treated either with 10 nM lacritin or inactive C-25 lacritin for 10,
30, 60 min. After washing, cells were fixed, rinsed, mounted and
examined in a Zeiss LMS 700 confocal microscope. Colocalization of
mCherry with mCFP was analyzed using Fiji.
Results: Lacritin, but not C-25, stimulated the autophagic capture
and transfer of toxic Htt103Q aggregates into autolysosomes. Capture
and transfer peaked at 30 min. Transfer was monitored over time as
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
EGFP (mCherry-EGFP-LC3B) was quenched by autolysosomal
acidity , unlike pH insensitive mCherry. No lacritin stimulated
autophagy was evident in Htt25Q cells in keeping with the absence of
stress.
Conclusions: Lacritin signaling rids cells of toxic aggregated
proteins that develop during stress by rapidly and transiently
stimulating autophagy. Autolysosomal digestion products then feed
into and restore oxidative phosphorylation.
Commercial Relationships: Keith B. Zimmerman, None; Milton
F. Tyler, None; Ningning Wang, None; Ronald Raab, None;
Robert L. McKown, EyeRx Research, Inc. (I); Gordon W. Laurie,
UVa Patent Foundation (F)
Support: NH Grant EY018222
Program Number: 909 Poster Board Number: B0214
Presentation Time: 1:00 PM - 2:45 PM
Lacritin as the Primary Epithelial Survival Activity in Basal
Human Tears Reveals Novel Features About Autophagic
Survival Signaling
Ningning Wang1, Ronald Raab2, Robert L. McKown2, Cindy M.
Hutnik3, Gordon W. Laurie1. 1Cell Biology, University of Virginia,
Charlottesville, VA; 22Integrated Science and Technology, james
madison university, harrisonburg, VA; 3Depts. of Ophthalmology &
Pathology, Ivey Eye Institute, London, ON, Canada.
Purpose: As the fluid thought to support the survival of the avascular
corneal epithelium, tears are rich in proteins with growth and survival
capabilities - including lacritin, a tear prosecretory mitogen that
supports the survival of stressed corneal epithelial cells in culture,
and promotes basal tearing in rabbits and monkeys. Here we ask
whether tears are indeed prosurvival, if so does tear lacritin
contribute, and what is the FOXO3 survival mechanism?
Methods: Human corneal epithelial (HCE; Riken) cells were
sensitized with INFG, and then treated with TNF in the in the
presence of normal or dry eye tears, normal tears mock or lacritin
immunodepleted, or with dry eye tears supplemented with
recombinant lacritin or negative control C-25. FOXO3 translocation
served as a readout for cell stress (nuclear) or survival (cytoplasmic).
FOXO3 immunoprecipitates were used to screen for FOXO3 binding
partners.
Results: Normal and mock depleted tears, but not lacritin depleted
tears promoted the survival of INFG/TNF stressed HCE cells. Tears
from dry eye patients supplemented lacritin promoted cell survival,
but not dry eye tears alone nor C-25 supplemented dry eye tears. We
discovered that autophagy mediator ATG101 binds FOXO3 in
stressed cells treated with lacritin, and that shRNA depletion of
ATG101 or FOXO3 abrogates lacritin stimulated autophagy and
survival.
Conclusions: Lacritin appears to be the primary survival factor in
basal human tears. Surprisingly it is not compensated by other tear
factors when depleted. Lack of dry eye tear survival activity can be
rescued with recombinant lacritin, suggesting efficacy in treating
human dry eye. Lacritin rapidly stimulates FOXO3/ATG101 ligation.
ATG101 is known to bind and stabilize the ATG13/ULK1 complex
responsible for initiating autophagy - thus explaining how lacritin so
rapidly stimulates autophagy.
Commercial Relationships: Ningning Wang, None; Ronald Raab,
None; Robert L. McKown, EyeRx Research, Inc. (I); Cindy M.
Hutnik, None; Gordon W. Laurie, UVa Patent Foundation (F)
Support: EY018222, EY013143 (to GWL).
Neutralization of Ocular Surface TNF-α Reduces Ocular Surface
and Lacrimal Gland Inflammation Induced by In Vivo Dry Eye
Yongwoo Ji, Yu Jeong Byun, Jin Sun Kim, Eunae Jeong, Hyung Keun
Lee. Institute of Vision Research, Department of Ophthalmology,
Yonsei University College of Medicine, Seoul, Republic of Korea.
Purpose: The purposes of this study are to investigate the
effectiveness of tumor necrosis factor (TNF)-α blocker for treatment
of dry eye (DE)-induced inflammation and determine a more
effective method to suppress lacrimal gland inflammation. Efficacy
of topical vs. systemic administration of TNF-α blockers was
determined using a murine DE model.
Methods: The TNF-α blocker HL036 was developed by modification
of the TNF receptor I. Protein purity, binding affinity, and clearance
of TNF-α was compared with etanercept. Using DE induced
C57BL/6 mice, corneal erosion, tear secretion, and goblet cell counts
were measured after subcutaneous or topical treatment with
etanercept or HL036. Inflammatory cytokines in cornea and lacrimal
glands were determined by quantitative reverse transcription
polymerase chain reaction and enzyme-linked immune absorbent
assay.
Results: HL036 showed TNF-α binding affinity comparable to
etanercept, as measured by surface plasmon resonance. HL036
concentration was significantly higher in cornea and anterior segment
than etanercept, and effectively eliminated TNF-α on ocular surfaces.
Etanercept was preferentially concentrated in the posterior segment.
Corneal erosion, goblet cell counts, and tear secretion were improved
only with topically applied etanercept and HL036. Ocular surface
interferon-γ (IFN-γ), interleukin-6 (IL-6), IL-21, and IL-22 were
significantly decreased by topical HL036. DE induced lacrimal gland
IFN-γ and IL-6 expression was effectively suppressed by topical
etanercept, glucocorticoid, and HL036.
Conclusions: Topical TNF-α blockers effectively suppressed
lacrimal gland and corneal inflammation by suppressing IFN-γ, IL21, and IL-6. Differences in TNF-α affinity, clearance, and local
concentration of blockers may account for the anti-inflammatory
effects in different ocular regions.
Program Number: 910 Poster Board Number: B0215
Presentation Time: 1:00 PM - 2:45 PM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Commercial Relationships: Yongwoo Ji, None; Yu Jeong Byun,
None; Jin Sun Kim, None; Eunae Jeong, None; Hyung Keun Lee,
None
Support: This work was supported by the Industrial Strategic
Technology Development Program, (Project No.: 10040233,
Molecular engineering and drug development of anti-TNF antibody
fragment for local inflammatory disease) funded by the Ministry of
Knowledge Economy (MKE, Korea) and Basic Science Research
Program (Grant no: 2012-0001384) and Advanced Science Research
Program (grant no.: 2012R1A2A2A02009081) through the National
Research Foundation of Korea (NRF), funded by the Ministry of
Education, Science, and Technology, Korea.
Program Number: 911 Poster Board Number: B0216
Presentation Time: 1:00 PM - 2:45 PM
Protection of barrier function and anti-inflammatory effects of
rebamipide in human corneal epithelial cells
Hiroshi Tanaka1, 2, Ken Fukuda3, Waka Ishida3, Yosuke Harada3,
Tamaki Sumi3, Atsuki Fukushima3. 1Machida Hospital, Kochi, Japan;
2
Opthalmology, Kyoto Prefectural Univ of Med, Kyoto, Japan;
3
Opthalmology, Kochi Medical School, Nankoku, Japan.
Purpose: Dry eye causes the tear hyperosmolality which stimulates
an inflammatory cascade and disrupts corneal barrier function.
Rebamipide which has mucin secretagogue activity is used for the
treatment of dry eye in Japan. This study examined the effects of
rebamipide on barrier function and cytokine expression in a human
corneal epithelial (HCE) cell line.
Methods: Barrier function of HCE cells was evaluated by
measurement of transepithelial electrical resistance. The subcellular
localization of the tight-junction protein zona occludens (ZO)-1 was
examined by immunofluorescence analysis. The release of cytokines
was determined with enzyme-linked immunosorbent assays, and the
intracellular abundance of cytokine mRNAs was quantitated by
reverse transcription and real-time polymerase chain reaction
analysis. Degradation of the NF-κB inhibitor IκBα was detected by
immunoblot analysis.
Results: Rebamipide increased barrier function of HCE cells in a
concentration-dependent manner as well as blocked both the loss of
barrier function and the disappearance of ZO-1 from the cell surface
induced by tumor necrosis factor (TNF)-α or IL-1β. Rebamipide also
suppressed cytokine-induced expression of interleukin-6 and
interleukin-8 at the mRNA and protein levels as well as inhibited the
TNF-α-induced degradation of IκBα.
Conclusions: Together with its mucin secretagogue activity,
rebamipide is effective for the treatment of dry eye thorough the
mechanism by up-regulation of barrier function and antiinflammatory effects.
Commercial Relationships: Hiroshi Tanaka, None; Ken Fukuda,
None; Waka Ishida, None; Yosuke Harada, None; Tamaki Sumi,
None; Atsuki Fukushima, Otsuka Pharmatheutical Co., Ltd (F)
Program Number: 912 Poster Board Number: B0217
Presentation Time: 1:00 PM - 2:45 PM
Expression and secreting mechanism of mucins induced by DA6034 in cultured human conjunctival epithelial cells
Tae-im Kim, Hun Lee, Ji Won Jung, Eung Kweon Kim. Vision
research institute, Department of Ophthalmology, Yonsei University
College of Medicine, Seoul, Republic of Korea.
Purpose: The purpose of this study was to investigate the mechanism
of ocular mucin secretion mediated by DA-6034 in cultured human
conjunctival epithelial cells.
Methods: After application of 100 μM DA-6034, in cultured human
conjunctival epithelial cells, expressions of MUC1, MUC4,
MUC5AC, MUC16, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors
were measured by reverse transcription-polymerase chain reaction
(RT-PCR), real-time PCR, and Western blot analysis. The effects of
100 μM DA-6034 on expression of mucin-secreting cells were
measured by the counting of Periodic Acid-Schiff (PAS) positive
cells. The effects of DA-6034 on Cl- currents were measured by
perforated patch clamp. The changes of Intracellular Ca2+
concentrations ([Ca2+]i) mediated by 100 μM DA-6034 were
measured by loading cultured human conjunctival epithelial cells
with fluorescent calcium indicator Fluo-4/AM and 0.05% pluronic F127. The level of intracellular Ca2+ was monitored by fluorescence
monitoring camera
Results: RT-PCR and real-time PCR showed that DA-6034 increased
MUC1, MUC4, MUC5AC, MUC16, P2Y2, P2Y4, and P2Y6
receptor gene expression. Western blot analysis showed that DA6034 increased MUC5AC and MUC16 protein expression. DA-6034
induced the expression of mucin-secreting cells as demonstrated by
PAS staining. DA-6034 stimulated not only active Cl- transport but
also [Ca2+]i increases in cultured human conjunctival epithelial cells.
[Ca2+]i increases were demonstrated by emitted fluorescence.
Conclusions: We concluded that DA-6034 stimulated mucin
production and secretion accompanied with the enhanced expression
of P2Y2, P2Y4 and P2Y6 receptor in cultured human conjunctival
epithelial cells. In addition, DA-6034 stimulated active Cl- transport
and [Ca2+]i increases.
Commercial Relationships: Tae-im Kim, None; Hun Lee, None; Ji
Won Jung, None; Eung Kweon Kim, None
Program Number: 913 Poster Board Number: B0218
Presentation Time: 1:00 PM - 2:45 PM
Regulators of MUC5AC gene expression in conjunctival epithelia
of dry eye patients
Rosa M. Corrales, Stephen C. Pflugfelder. Ophthalmology, Baylor
College of Medicine, Houston, TX.
Purpose: T helper (Th) cytokines and transcription factors have been
found to modulate goblet cells differentiation and morphology in
mice. We investigated expression of T helper 1 (Th1) and Th2
cytokine receptors and transcription factors in the conjunctival
epithelia of a dry eye population and compared them with healthy
subjects.
Methods: Conjunctival impression cytology (CIC) samples were
obtained from 16 dry eye patients (tear deficient with a Schirmer-1
score < 5mm wetting in 5 minutes) and 13 healthy subjects. RNA
was extracted, cDNA synthesized and real-time PCR performed
using Taqman probes to evaluate expression of the following genes:
MUC5AC, keratin 7 (K7), forkhead box protein A2 (FOXA2), SAM
pointed domain-containing Ets transcription factor (SPDEF),
Krueppel-like factor 4 (KLF4), IFN-γR, IL-13-Ra1 and IL13Ra2.
The housekeeping gene hypoxanthine phosphoribosyltransferase
1(HPRT-1) was amplified to normalize levels of expression. No
template controls and RNA were studied for evidence of
contamination. Results were analyzed using the comparative Ct
method using values from healthy subjects as calibrator. The student
t-test for independent samples was used to compare means between
groups.
Results: FOXA2 expression was below the level of detection. K7
and MUC5AC transcripts were decreased in dry eye samples.
Expression of the IL-13 signaling receptor (IL-13Rα1) and SPDEF
were unchanged, while expression of the IL-13 decoy receptor, IL13Rα2, as well as IFN-γR and KLF4 were significantly increased in
the dry eye conjunctival epithelium.
Conclusions: These results indicate that dry eye disrupts signaling
pathways in the conjunctival epithelium that are involved in goblet
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
ells differentiation, resulting in decreased signaling by the Th2
cytokine Il-13 and increased signaling by the Th1 cytokine IFN-γ.
Expression of the goblet cell transcription factor KLK4 increased.
Commercial Relationships: Rosa M. Corrales, None; Stephen C.
Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb
(C), Baylor College of Medicine (P)
Support: NIH Grant EY11915 (SCP), Research to Prevent Blindess,
Oshman Foundation, William Stamps Farish Fund, Hamill
Foundation
Program Number: 914 Poster Board Number: B0219
Presentation Time: 1:00 PM - 2:45 PM
Decellularization of porcine lacrimal gland tissue for
development of a lacrimal gland scaffold
Kristina Spaniol1, Alexander Kunze1, Marco Metzger2, Gerd
Geerling1, Stefan Schrader1. 1ophthalmology, university hospital
Düsseldorf, Düsseldorf, Germany; 2tissue engineering and
regenerative medicine, university hospital Würzburg, Würzburg,
Germany.
Purpose: In cases of severe dry eye due to lacrimal gland
insufficiency engineering of a lacrimal gland tissue construct in vitro
might be a promising future treatment approach. Aim of this study
was to evaluate structure and main basement membrane components
of native porcine lacrimal gland tissue before and after a
decellularization process in order to develop an acellular scaffold for
lacrimal gland regeneration.
Methods: Lacrimal glands were extracted from six domestic pigs
after euthanasia. Six biological replicates were used for this study.
Each gland was cut into four pieces, two of them were left native and
the other two were decellularized using sodium desoxychelate in
ultra-pure water over night. Tissue pieces were embedded in paraffin
as well as OCT and the morphology of native and decellularized
tissue was examined histologically by hematoxylin and eosin (H&E)
staining. Additionally, expression of basement membrane markers,
such as laminin, collagen IV and fibronectin was evaluated by
immunostaining.
Results: Histology showed an intact connective tissue matrix after
the decellularization process. Immunohistochemistry revealed the
expression of major basement membrane components such as
collagen IV and laminin in native lacrimal gland tissue and these
components were still detectable after the decellularization of the
lacrimal gland tissue. Efficacy of the decellularization process was
demonstrated by complete absence of nuclei in the lacrimal gland
tissue, as assessed by DAPI-staining.
Conclusions: Decellularization of lacrimal gland tissue generates an
intact acellular scaffold with preserved acinar structures, containing
major basement membrane components such as collagen IV and
laminin. An intact basement membrane is a prerequisite for essential
cellular processes like adhesion, migration as well as proliferation
and therefore decellularized lacrimal gland tissue might be a
promising scaffold for lacrimal gland regeneration in vitro.
Commercial Relationships: Kristina Spaniol, None; Alexander
Kunze, None; Marco Metzger, None; Gerd Geerling, Alcon (C),
Allergan (C), Thea Pharma (C), Novagali (C), Bausch & Lomb (C),
Tearlab Inc. (C); Stefan Schrader, None
Program Number: 915 Poster Board Number: B0220
Presentation Time: 1:00 PM - 2:45 PM
Functional rabbit acinar cells cultured on decellularized lacrimal
gland scaffold
Hui Lin1, Guoying Sun1, Hong he1, Jennifer Elisseeff1, Samuel C.
Yiu1, 2. 1Wilmer Eye Institute, Johns Hopkins University, Baltimore,
MD; 2Ophthalmology, 2. King Khaled Eye Specialist Hospital,
Riyadh, Saudi Arabia.
Purpose: Aqueous tear-deficient dry eye is a multifactorial chronic
disorder in which the lacrimal glands fail to produce enough tears to
maintain a healthy eye surface. The treatment primarily involves
palliation using ocular surface lubricant. Construction of a
bioengineered lacrimal gland having functional secretory epithelial
cells is a potentially promising option for providing long-term relief
to severe dye eye patients. Previously, we have successfully
decellularized the rabbit lacrimal gland to create a novel scaffold for
3 dimensional culture of acinar cell. Current study investigates the
secretory function of cells seeded and cultured in decellularized
lacrimal gland tissue.
Methods: NZW rabbit lacrimal glands purchased from Pel Freeze
were treated with 1% sodium dodecyl sulfate or 1% Triton X-100 for
36 to 40 hours for decellularization. The rabbit lacrimal gland acinar
cells were isolated by enzyme mixtures of collagenase, hyaluronidase
and DNase. Subsequently, the isolated cells were layered over 5%
Ficoll and centrifuged at 50g for purification. Prior to seeding, the
decellularized lacrimal gland scaffold was treated with Matrigel,
fibronectin or collagen I in different groups, and with Peter’s
complete medium (PCM) in control group. The lacrimal constructs
were cultured in PCM for up to 2 weeks. The live/dead assay for cell
viability was performed on day 2, 7 and 14. The beta-hexosaminidase
secretion assay for assessing acinar cell function was performed on
day 2 and 7. RNA was also extracted to evaluate the gene expression
of beta-hexosaminidase.
Results: Majority of the lacrimal acinar cells in the decellularized
scaffold survived for at least 7 days in all groups, however, cell
viability decreased at day 14. Use of extracellular matrix to treat the
scaffold during the seeding process helped improve cell viability.
Beta-hexosaminidase activity increased in the supernatant medium
following stimulation with 100uM carbachol on day 2 and 7.
Carbachol stimulation was also found to up-regulate mRNA level of
beta-hexosaminidase in the reseeded cells.
Conclusions: Isolated acinar cells successfully cultured in
decellularized lacrimal gland tissue and maintained the cell viability
and secretory function for at least 1 week. Further investigation is
required to optimize the decellularization and seeding process to
improve the duration of cell viability and function.
Commercial Relationships: Hui Lin, None; Guoying Sun, None;
Hong he, None; Jennifer Elisseeff, Collagen Vitrigel (P); Samuel C.
Yiu, None
Support: Partially supported by an unrestricted grant from Research
to Prevent Blindness, New York, NY to the Wilmer Eye Institute
Program Number: 916 Poster Board Number: B0221
Presentation Time: 1:00 PM - 2:45 PM
ENaC in the rabbit lacrimal gland and its changes during
Sjögren’s syndrome and pregnancy
Chuanqing Ding, Michael Lu, Jianyan Huang. Cell &
Neurobiology/Doheny Eye Institute, University of Southern
California, Los Angeles, CA.
Purpose: The epithelial sodium channel (ENaC), which is comprised
of α, β, and γ subunits, plays a critical role in the control of Na+
balance and has been implicated in the development and progression
of exocrine gland pathology. The aim of the present study was to
investigate the expression of ENaC in the rabbit lacrimal gland (LG)
and its potential changes in rabbits with induced autoimmune
dacryoadenitis (IAD), a rabbit model of Sjögren’s syndrome, and in
term-pregnant rabbits, a physiological condition that has been shown
to exhibit altered LG secretion and ocular surface symptoms.
Methods: Total mRNA of α, β, and γ subunits was extracted from
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
whole LG, acinar cells, and epithelial cells from various duct
segments by laser capture microdissection (LCM) for real time RTPCR. LG lysates were processed for Western blot and cryosections of
LG were used for immunofluorescence. Goat polyclonal primary
antibodies of α, β, and γ were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA).
Results: mRNA for both α and γ was expressed in whole LG lysates
while β was undetectable. In rabbits with IAD, the levels of mRNA
for α and γ were 20.9% and 58.9% lower (p<0.05) while no
significant changes were observed in term-pregnant rabbits (p>0.05).
However, we were unable to detect mRNA of any of these three
subunits in LCM samples of epithelial cells due to their extremely
low level. Western blot demonstrated bands for both α (90 kDa) and γ
(85 kDa) but β was undetectable. In rabbits with IAD, densitometry
analysis showed that expression of α decreased 22% while γ
decreased 26% (p<0.05). In pregnant rabbits, however, α expression
was 31% lower while γ was 34% lower (p<0.05). From
immunofluorescence studies, all subunits were present in ductal cells
only with virtually no immunoreactivity detected in acini. No
noticeable changes of their distribution pattern and intensity were
found in rabbits with IAD or pregnant rabbits.
Conclusions: The present data demonstrated the presence of ENaC
in the rabbit LG and its changes in rabbits with IAD and pregnant
rabbits. This suggests that ENaC may contribute to the pathogenesis
of altered LG secretion and ocular surface symptoms in these
animals. The exact mechanisms of how ENaC functions in the LG
under physiological and pathological conditions certainly warrant
further investigations.
Commercial Relationships: Chuanqing Ding, None; Michael Lu,
None; Jianyan Huang, None
Support: NIH Grants EY017731 (CD), EY03040 (Doheny Eye
Institute Core)
Program Number: 917 Poster Board Number: B0222
Presentation Time: 1:00 PM - 2:45 PM
Intermittent fasting prevents lacrimal hypofunction in rat visual
display termin user model: a pivotal role of endogenous D-3hydroxybutyrate
Shigeru Nakamura, Ryuji Hisamura, Toshihiro Imada, Kazuo
Tsubota. Keio university, Tokyo, Japan.
Purpose: Calorie restriction extends life span and retards age-related
chronic diseases. We previously reported that calorie restriction
through intermittent fasting restores lacrimal function in VDT
associated dry eye using rat model. (ARVO 2012). In this condition,
serum ketone bodies concentrations increases approximately 1000
fold from the normal state. D-3-hydroxybutyrate (3-HB), a major
circulating ketone body, has been demonstrated to be effective in
neurological disorders such as Alzheimer’s, Parkinson’s, and found
to attenuate corneal disorder in dry eye condition. In the present
study, we explored the possible role of 3-HB on the attenuation of
lacrimal function by calorie restriction.
Methods: 8-week-old female Sprague-Dawley rats were used for this
study. A series of treatments were performed under continuous
exposure to low-humidity airflow (25 ± 5%, 2 - 4 m/s). Rats were
placed on a swing made of a plastic pipe for 7.5 h/d, and for 16.5
hours, they were placed in individual cages without swing treatment.
This series of treatments was repeated for up to 7 days.
Rats were assigned to three groups: AL, ad libitum-fed animals, and
IF, intermittent fasting rats, which were provided unlimited access to
food every other day, AL+3-HB, 3-HB diluted with saline was
injected into rat dorsal skin at a dose of 2000 mg/kg/day. Change in
tear secretion was measured by the cotton thread test. Serum and
lacrimal glands 3-HB concentrations were measured by enzymatic
method.
Results: A significant decrease in tear secretion was observed in the
AL compared with the initial value. In the IF and AL+3-HB, slight
decreases in the tear secretion were observed, although the
differences were not significant compared with the initial values.
Change in tear secretion was significantly suppressed in the IF (14.3
± 0.81, n=9, p < 0.05) and AL+3-HB (13.9 ± 0.71, n=10, p < 0.05)
compare to the AL day 7(10.1 ± 0.79 n=10) Serum and lacrimal
glands 3-HB concentrations were significantly increased 15 hours
after food deprivation and 0.5 hour to 3 hour after 3-HB injection.
The peak 3-HB concentration of serum and lacrimal gland was
approximately ten-fold compared to initial value.
Conclusions: Our findings suggest that increased 3-HB play an
important role of tear secretion which can be improved by IF in VDT
associated dry eye.
Commercial Relationships: Shigeru Nakamura, Ophtecs Co. Ltd
(E); Ryuji Hisamura, Ophtecs corporation (E); Toshihiro Imada,
Yamada bee farm corporation (F); Kazuo Tsubota, AcuFocus, Inc
(C), Allergan (F), Bausch Lomb Surgical (C), Functional visual
acuity meter (P), JiNS (P), Kissei (F), Kowa (F), Santen, Inc. (F),
Otsuka (F), Pfizer (C), Thea (C), Echo Denki (P), Nidek (F), Ophtecs
(F), Wakasa Seikatsu (F), CEPT Company (P)
Program Number: 918 Poster Board Number: B0223
Presentation Time: 1:00 PM - 2:45 PM
Analysis of chemokines involved in the pathogenesis of
dacryoadenitis in mice deficient for programmed cell death-1
Yutaka Sakurai1, Masataka Ito1, Yoko Karasawa1, Yoshihiko Usui2,
Takaaki Hattori2, Hiroshi Goto2, Masaru Takeuchi1. 1national
defense medecal college, Tokorozawa, Japan; 2tokyo medical
university, tokyo, Japan.
Purpose: Programmed cell death-1 (PD-1, pdcd1) is one of the
costimulatory molecules, negatively regulates the immune responses.
Previously, we reported that PD-1-deficient C57BL/6 mice
spontaneously develop sjogren syndrome-like dacryoadenitis from 3
months after birth.In this study, we investigated chemokines
expression in the lacrimal gland (LG) of pdcd1-/- mice to
characterize T cells involved in the development.
Methods: The LG were obtained from C57BL/6 pdcd1+/+ and
pdcd1-/- mice at 4-,6-,or 12-month-old. The production of CCL1,
CCL2, CCL3, CCL4, CCL5, CCL11, CCL12, CCL17, CXCL1,
CXCL2, CXCL9, CXCL10, CXCL11, CXCL12, and CXCL13 in the
LG of 4-,6-,or 12-month-old pdcd1-/- mice and 4-month-old
pdcd1+/+ mice were measured by mouse cytokine array A kit (R&D
system). In addition, mRNAs were extracted from the LG, and
mRNA expression of CCL3, CCL5, CCL20, CCL22, CXCL9, and
CXCL10 in the LG of 4-month-old pdcd1-/- mice and pdcd1+/+ mice
were analyzed by real-time PCR.
Results: CCL3, CCL5, CXCL9, and CXCL10 associated with Th1
cell migration were produced in the LG of pdcd1-/- mice compared to
pdcd1+/+ mice at 4-month-old. Production of these chemokines
related to Th1 cells in the LG of pdcd1-/- mice increased with aging.
In the mRNA expression, Pdcd1-/- mice had significantly higher
levels of CCL3, CCL5, CXCL9, CXCL10 related to Th1 cells, and
CCL20 related to Th17 cells mRNA transcripts in the LG than
pdcd1+/+ mice at 4-month-old. Especially, mRNA expression of
CCL20 was remarkably higher than others. mRNA expression of
CCL22 related to Th2 cells were also detected, but was apparently
lower than others.
Conclusions: These results indicate that Th1- and Th17-mediated
immune responses are involved in the development of dacryoadenitis
in Pdcd1-/- mice, and that Th17 cells play a pivotal role in the
pathogenesis.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Commercial Relationships: Yutaka Sakurai, None; Masataka Ito,
None; Yoko Karasawa, None; Yoshihiko Usui, None; Takaaki
Hattori, None; Hiroshi Goto, None; Masaru Takeuchi, None
Program Number: 919 Poster Board Number: B0224
Presentation Time: 1:00 PM - 2:45 PM
Potential Regulatory T Cell Mechanisms Of Immune
Suppression In A Mouse Model Of Dry Eye
Katherine S. Held1, Chris S. Schaumburg1, Jianping Gao1, Larry A.
Wheeler1, Margarita Calonge2, Jerry Y. Niederkorn4, Stephen C.
Pflugfelder3, Michael E. Stern1. 1R&D, Allergan, Inc, Irvine, CA;
2
IOBA, University of Valladolid, Valladolid, Spain; 3Baylor College
of Medicine, Houston, TX; 4Ophthalmology, UT SW-Med Center,
Dallas, TX.
Purpose: To evaluate immunosuppressive properties of inducible
and natural T regulatory (Tregs) subsets following induction of
experimental dry eye in mice.
Methods: Flow cytometry was utilized to characterize Tregs within
the periphery during experimental dry eye to determine i) the
frequencies of inducible, iTregs, and natural, nTregs, and ii) the level
of expression markers indicating activation and the capacity for
suppression by metabolic disruption and dendritic cell immune
modulation. C57BL/6 wild-type female mice were exposed to
desiccating environmental stress (DS; subcutaneous scopolamine
injections, humidity <40%, and air flow across wire meshed screened
cages) for 3 or 10 days. Draining cervical lymph nodes were
collected from control or DS mice, and processed for flow cytometric
analysis. nTregs (Foxp3+ Helios+CD25hi) were distinguished from
iTregs (Foxp3+ Helios-CD25hi) using the Helios transcription factor
as a putative marker for thymus-derived Tregs. The expression of
CD44 was used to evaluate effector Tregs, CD73 was used to
evaluate the capacity for metabolic disruption, and CD152 (CTLA4)
and CD223 (LAG3) were used to evaluate the capacity for dendritic
cell targeted immune modulation.
Results: The frequencies of CD4+ or CD8+ iTregs and nTregs were
significantly increased at day 3 DS (p<0.01), while these populations
decreased at day 10 DS, relative to controls. CD44hi expression
increased following DS only in CD4+ iTregs and was significantly
higher than controls by day 10 DS (p=0.006). In addition, both CD4+
iTregs and nTregs showed significantly increased expression of
CD73 at day 10 DS compared to controls (p<0.01). Furthermore,
evaluation of CD4+ nTregs and iTregs at day 3 DS revealed
significant increases in CD152 and CD223 expression compared to
controls (p<0.001).
Conclusions: These results indicate that both nTregs and iTregs
populations within the cervical lymph nodes increase early following
dry eye disease and may elicit their immunosuppressive effects via
CD73 generation of adenosine, CTLA4 down-modulation of
costimulatory molecules on dendritic cells, and LAG3-mediated
inhibition of dendritic cell maturation. Collectively, the data suggests
the potential for multiple pathways of immune suppression by Tregs
in response to dry eye.
Commercial Relationships: Katherine S. Held, Allergan, Inc. (E);
Chris S. Schaumburg, Allergan, Inc (E); Jianping Gao, Allergan,
Inc. (E); Larry A. Wheeler, Allergan Pharm (E); Margarita
Calonge, Allergan (C); Jerry Y. Niederkorn, Allergan (C); Stephen
C. Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and
Lomb (C), Baylor College of Medicine (P); Michael E. Stern,
Allergan, Inc. (E)
Program Number: 920 Poster Board Number: B0225
Presentation Time: 1:00 PM - 2:45 PM
Minocycline controls clinical outcomes and inflammatory
cytokines in moderate and severe meibomian gland dysfunction
HUN LEE1, Ji Won Jung1, Sangyeop Lee1, Eung Kweon Kim1, 2, Taeim Kim1. 1The Institute of Vision Research, Department of
Ophthalmology, Yonsei University College of Medicine, Seoul,
Republic of Korea; 2Cornea Dystrophy Research Institute;
Department of Ophthalmology; Severance Biomedical Science
Institute, and Brain Korea 21 Project for Medical Science, Yonsei
University College of Medicine, Seoul, Republic of Korea.
Purpose: To assess clinical outcomes and tear cytokine levels in
patients with moderate and severe meibomian gland dysfunction
(MGD) after treatment with oral minocycline and artificial tears
versus artificial tears only.
Methods: Sixty eyes of 60 patients with stage 3 or 4 meibomian
gland dysfunction were enrolled. We evaluated the tear film break-up
time, Schirmer test results, corneal and conjunctival fluorescein
staining results, biomicroscopic examination results of lid margins
and meibomian
glands, and tear cytokine levels before and after 1 month and 2
months of oral minocycline and artificial tears (group 1) or artificial
tears only (group 2). Tear samples were collected and analyzed using
a BD Cytometric Bead Array (BD Bioscience, San Jose, California,
USA) for detection of interleukin (IL)-1β, IL-6, IL-7, IL-8, IL-12p70,
IL-17α, interferon-γ, tumor necrosis factor-α, and monocyte
chemotactic protein-1. The Wilcoxon signed-rank test, MannWhitney U test, generalized linear model, and linear mixed model
were performed.
Results: Patients in group 1 showed statistically significant
improvement in all clinical signs and symptoms after 1 month and 2
months of treatment. Patients of group 1 showed more significant
improvement compared with those in group 2. Patients in group 1
also showed statistically significant reductions in IL-6, IL-1β, IL-17α,
tumor necrosis factor-α, and IL-12p70 after 2 months of treatment.
Conclusions: Oral minocycline can provide clinical benefits in
treating moderate and severe meibomian gland dysfunction by
reducing inflammatory cytokine levels.
Commercial Relationships: HUN LEE, None; Ji Won Jung, None;
Sangyeop Lee, None; Eung Kweon Kim, None; Tae-im Kim, None
Support: National Research Foundation of Korea grant (MEST no.
2010-0022006); Converging Research Center Program funded by the
Ministry of Education, Science and Technology (no. 2011K000680)
Clinical Trial: NCT01600625
Program Number: 921 Poster Board Number: B0226
Presentation Time: 1:00 PM - 2:45 PM
Topical administration of L-carnitine on prevention and
treatment of murine dry eye
Qian Garrett1, 2, Xin Zhang3, Yu Wang3, Peter A. Simmons4, Joseph
G. Vehige4, Jinyang Li3, Wei Chen3. 1Brien Holden Vision Institute,
Sydey, NSW, Australia; 2School of Optometry and Vision Science,
University of New South Wales, Sydney, NSW, Australia; 3School of
Ophthalmology & Optometry, Wenzhou Medical College, Wenzhou,
China; 4Allergan Inc, Irvine, CA.
Purpose: L-carnitine has been reported to help maintain human
corneal epithelial cell volume under hyperosmotic stress and
ameliorate aspects of hyperosmotic stress-induced apoptosis. Eye
drops containing L-carnitine have been shown to produce rapid and
consistent improvements in the signs and symptoms of dry eye.
Using a murine dry eye model, the present study evaluated the
efficacy of L-carnitine on prevention and treatment of dry eye.
Methods: Dry eye was induced in mice using an intelligently
controlled environmental system (ICES). L-carnitine (in PBS) or PBS
alone was topically administered to the mouse eyes 4 times daily
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
according to two schedules: Schedule 1 (S1): to model prevention,
dosing started at the beginning of housing in ICES (Day1) and lasted
for 21 days; Schedule 2 (S2): to model treatment, dosing started on
Day22 after the housed mice developed dry eye and lasted until
Day35. The efficacy of the L-carnitine treatment compared to that of
the vehicle (PBS) was tested for ocular surface integrity by
fluorescein staining; corneal epithelial apoptosis by TUNEL or
Caspase-3 assays; goblet cell numbers by PAS staining; and
expression of inflammatory mediators, TNF-α, IL-17, IL-6 or IL-1β
using real-time PCR, on Days 0, 14, 21 and/or 35.
Results: Compared to the vehicle, eyes treated with L-carnitine
showed a significant reduction in corneal fluorescein staining on Day
14, 21 and 35 for S1 (p=0.0001, 0.0001, and 0.017, respectively) and
Day35 for S2 (p=0.0001); the number of TUNEL-positive apoptotic
corneal epithelial cells on Day21 for S1 (p=0.032) or Day35 for S2
(p=0.005); the expression level of TNF-α, IL-17, IL-6, or IL-1β (all
p<0.05); and visible reduction in corneal epithelial expression of
caspase-3; as well as a significant increase in goblet cell density for
both schedules (p=0.003 and 0.0001 on Day21 for S1 and Day35 for
S2, respectively).
Conclusions: Topical application of L-carnitine could limit
progression as well as reduce the severity of dry eye, suggesting Lcarnitine has prophylactic and therapeutic potential in dry eye
management and treatment.
Commercial Relationships: Qian Garrett, Allergan (F); Xin
Zhang, None; Yu Wang, None; Peter A. Simmons, Allergan (E);
Joseph G. Vehige, Allergan, Inc. (E); Jinyang Li, None; Wei Chen,
None
Support: This study was supported by Allergan Inc., Brien Holden
Vision Institute, and the National Natural Science Foundation of
China
Program Number: 922 Poster Board Number: B0227
Presentation Time: 1:00 PM - 2:45 PM
Effect of Evaporative Stress on Meibomian Gland Proliferative
Status and Lipid-Protein Composition
Mikhail Geyfman1, Jeffrey Suhalim2, Tejas N. Shah1, Cintia S. De
Paiva3, Stephen C. Pflugfelder3, Eric Potma2, James V. Jester1.
1
Gavin Herbert Eye Institute, University of California, Irvine, CA;
2
Chemistry, University of California, Irvine, CA; 3College of
Medicine, Baylor, Houston, TX.
Purpose: Evaporative environmental stress incorporating exposure to
high air flow under low humidity conditions combined with
scopolamine-induced lacrimal gland inhibition is a model system that
is widely used to assess ocular surface changes related to evaporative
dry eye. While studies have shown marked inflammatory and
immune effects on the ocular surface, its effect on meibomian gland
function remains largely unknown. We sought to evaluate the effects
of evaporative stress on meibocyte proliferation and lipid quality.
Methods: To induce environmental stress, ten mice were treated with
scopolamine and placed in a blower hood under reduce
environmental humidity. Five and ten days after treatment, eye lids
from the treated (5 mice each) and untreated control mice (5 mice)
were harvested and embedded in OCT. Cryosections were prepared
and stained with antibodies to Ki67 (marker of cycling cells).
Percentage of positive basal acinar cells was determined. Eyelid
sections were also imaged using coherent Raman Scattering (CRS)
microscopy to characterize the lipid compositional changes in the
gland. Specifically, the protein-to-lipid ratio was obtained by tuning
the laser beams to probe the vibrational signatures of protein and
lipid using the methyl and amide-I vibration for protein and the
symmetric stretch and bending modes of methylene to probe lipid.
Statistical significance of all data was determined using all pairwise
multiple comparison ANOVA (Holm-Sidak method).
Results: Evaporative stress caused a marked 3 fold increase in basal
acinar cell proliferation from 18.3 ± 11.1% in untreated mice to 64.4
± 19.9% and 66.6 ± 13.4% after 5 and 10 days exposure, respectively
(P < 0.001). In addition, evaluation of the lipid compartment of the
meibomian gland by CRS showed wider variation in the protein-tolipid ratio throughout the gland suggesting alterations in meibocyte
differentiation and lipid synthesis
Conclusions: This data is consistent with a model that evaporative
stress may have a direct effect on Meibomian gland function leading
to a significant increase in both basal acinar cell proliferation and
possibly altered meibocyte differentiation and lipid production. A
provocative hypothesis that remains to be tested is that prolonged
evaporative stress may causes stem cell exhaustion, and eventual
meibomian gland atrophy leading to evaporative dry eye disease.
Commercial Relationships: Mikhail Geyfman, None; Jeffrey
Suhalim, None; Tejas N. Shah, None; Cintia S. De Paiva, Glaxo
Smith Kline (C), Baylor College of Medicine (P); Stephen C.
Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb
(C), Baylor College of Medicine (P); Eric Potma, None; James V.
Jester, None
Support: NIH/NEI EY021510, Research to Prevent Blindness, Inc.
and the Discovery Eye Foundation
Program Number: 923 Poster Board Number: B0228
Presentation Time: 1:00 PM - 2:45 PM
Does EDAR Gene Affect Meibomian Glands Development in
Human?
Yujing YANG, JianJiang Xu, Jiaxu Hong. Ophthalmology, Shanghai
EENT Hospital of Fudan University, Shanghai, China.
Purpose: EDAR gene plays an important role in the development of
hair,teeth and other ectodermal derivatives. Asian-specific mutation
in derived EDAR allele was found associated with enlargement of
meibomian glands(MGs) in mouse. This study was conducted to test
the hypothesis that EDAR contributes to population differentiation
regarding MG morphology.
Methods: The derived EDAR allele attained high frequency in East
Asian and moderate in Uygur, absent in European. We recruited 37
unrelated individuals and divided them into 3 groups according to
ethnicity (Chinese(Ch):17; Uygur(Uy):13; Europeans(Eu):7). DNA
was extracted from blood samples for genotyping. MG morphology
was observed by laser scanning confocal microscopy(LSCM) in vivo,
we measured glandular acinar unit density(MGAUD) and acinar
longest/shortest diameter(MGALD/MGASD) as main parameters for
comparative analysis to investigate the association of genotype and
phenotype.
Results: We designed primers to genotype EDAR V370A
polymorphism (rs3827760), the allele frequency was consistent with
expectations under Hardy-Weinberg equilibrium,as reported
previously.
LSCM demonstrated that MGASD was significantly smaller in
Ch(31.6±7.5μm)compared with Uy(43.2±21.6μm;p=0.0368, MannWhitney U test) and Eu(48.0±19.2μm;p=0.0252). However, values of
MGAUD(Ch:114.6±28.0/mm2; Uy:111.0±39.5/mm2;
Eu:117.8±35.6/mm2) and MGALD(Ch:89.5±13.0μm;
Uy:82.7±19.5μm; Eu:82.5±14.5μm) had no significant differences
(p>0.05) among populations.
In order to study the relation between genotype and phenotype, we
performed both additive and dominant gene model then did linear
regression analysis, results of which indicated that EDAR V370A
was not correlated with MG density and diameter(p>0.05,Spearman).
Conclusions: Genotyping revealed that difference of derived EDAR
allele frequency did exist among populations. Based on limited
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
phenotype data provided by LSCM, we didn’t demonstrate the impact
of population difference on MG morphological pattern, no
association was found between genotype and phenotype.
Due to small sample size and other reasons, we didn't get outcomes
consistent with those in previous animal experiments. Further
population-based association studies need to be carried out to
unequivocally test our hypothesis and reach a definite conclusion.
FIGURE 1. Meibomian glandular acinar units imaged by LSCM.
(A)Chinese; (B)Uyghur;(C)European.
TABLE 1. Correlation between EDAR V370A and MGs
Commercial Relationships: Yujing YANG, None; JianJiang Xu,
None; Jiaxu Hong, None
Program Number: 924 Poster Board Number: B0229
Presentation Time: 1:00 PM - 2:45 PM
Notch regulation of PPAR-gamma and development of
meibomian gland dysfunction
Mindy K. Call, Katy Fischesser, Matthew O. Lunn, Winston Kao.
Ophthalmology, University of Cincinnati, Cincinnati, OH.
Purpose: Meibomian gland dysfunction (MGD) is a chronic, diffuse
abnormality of the meibomian glands, which is characterized by
terminal duct obstruction and/or qualitative/quantitative changes in
glandular secretion resulting in alterations of the tear film, eye
irritation and inflammation. One particular molecule thought to play
an important role in lipid secretion is peroxisome proliferator
activated receptor gamma (PPARλ). Given the importance of PPARλ
in lipid metabolism, this study aims to elucidate its role in meibomian
gland formation/function and the development of MGD. The
hypothesis is that loss of PPARλ will disrupt meibomian gland
formation/function resulting in a hyposecretory state. In addition this
study will examine the role of Notch signaling in regulating PPARλ
expression.
Methods: Transgenic animals, utilizing both Le-cre and K14-rtTA
driver mice, were used to ablate PPARλ and Jag1 and to express
dnMAML. Animals were examined for signs of MGD/dry eye via
tear solution assessment, HRT II, slit lamp, lissamine green staining,
oil red O staining and lyve 1 staining. In addition, eyes were collected
at various time points to assess expression of Notch signaling
components. All reported research was conducted in compliance with
the ARVO Statement for the Use of Animals in Ophthalmic and
Vision Research and approved by the IACUC of the University of
Cincinnati.
Results: Loss of PPARλ resulted in abnormalities in meibomian
gland morphogenesis leading to dry eye-like symptoms. Specifically
there was an increase in the number of cell layers lining each acinus,
fewer and more condensed lipid droplets, “swollen” eyelids,
increased protein concentration in the tears and increased
lymphangiogenesis. Inhibition of the notch signaling pathway using
dnMAML mice resulted in a significant meibomian gland defect.
With a phenotype similar to what was seen in the absence of PPARλ.
Loss of Jag1 did not have as profound of an effect on meibomian
gland formation or function, which may be explained by
compensation by other Notch ligands.
Conclusions: Together these data, suggest that PPARλ is critical for
the proper formation and function of the meibomian glands and also
demonstrate a mouse model that can be used to study MGD. Notch
signaling also appears to be an important regulator of PPARλ.
Commercial Relationships: Mindy K. Call, None; Katy
Fischesser, None; Matthew O. Lunn, None; Winston Kao, None
Support: from NIH/NEI EY013755, Research to Prevent Blindness,
Ohio Lions Eye Research Foundation
Program Number: 925 Poster Board Number: B0230
Presentation Time: 1:00 PM - 2:45 PM
Tear Meniscus Dimensions and Location of Marx’s Line in
Meibomian Gland Dysfunction
Jason Feuerman, Stephen C. Pflugfelder. Ophthalmology, Baylor
College of Medicine, Houston, TX.
Purpose: In patients with tear film instability and meibomian gland
dysfunction (MGD), Marx’s line (ML) is often displaced anteriorly.
Bron, et al, suggest that ML may be formed as a result of a relatively
increased solute concentration at the peripheral apex of the tear
meniscus, which may thus play a significant role in the
pathophysiology of primary MGD. We hypothesized that tear
meniscus height, area, and length of anterior excursion along the lid
margin positively correlate with the extent of anterior migration of
ML in patients with MGD.
Methods: We performed a retrospective analysis of consecutive
patients with eye irritation symptoms, MGD with tear film instability,
no aqueous tear deficiency, and eyelid photographs and OCTs of
sufficient quality for analysis. 32 eyes of 17 patients were included.
Photographs of lissamine green-stained lower eyelids were analyzed
using the ImageJ analysis software. The furthest anterior migration of
ML was measured in 3 zones: temporal, central, and nasal. For each
eye, the tear meniscus height, area, and length of anterior excursion at
the center of the lower eyelid were measured using Fourier-Domain
RTVue-100 optical coherence tomography (OCT). Spearman
correlation analysis was performed.
Results: The mean eye age was 68 ± 10 years. In the temporal zone,
there was a statistically significant positive correlation between the
furthest anterior migration of ML and the central tear meniscus height
(r = 0.468, p = 0.007), area (r = 0.492, p = 0.004), and anterior
excursion (r = 0.448, 0.010). In the central zone and the nasal zone
there was no significant correlation between the furthest anterior
migration of ML and any of the OCT measurements.
Conclusions: In patients with symptomatic MGD, central tear
meniscus height, area, and anterior excursion positively correlate
with the furthest anterior migration of ML in the temporal zone, but
not in the central or nasal zone. A possible explanation is that aging
changes such as conjunctivochalasis, which tends to be more
pronounced temporally, physically impedes lateral tear migration
leading to increased central tear pooling, while also promoting
anterior excursion of the tear meniscus temporally by acting as a
bridge. By this process, the solute gradient mechanism could
contribute to the initiation of MGD. MGD could also just be initiated
by increased exposure of meibomian gland orifices to tears,
regardless of their osmolarity.
Commercial Relationships: Jason Feuerman, None; Stephen C.
Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb
(C), Baylor College of Medicine (P)
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Support: NIH Grant EY11915 (SCP), Research to Prevent
Blindness, Oshman Foundation, William Stamps Farish Fund, Hamill
Foundation
Program Number: 926 Poster Board Number: B0231
Presentation Time: 1:00 PM - 2:45 PM
Commensal microflora of eyelid margins and meibomian gland
function in normal humans
Hua Zhu1, 2, Judith L. Flanagan1, 2, Nisha S. Yeotikar1, Negar Babaei
Omali1, Daniel Tilia1, Shamil Y. Iskandar1, Brien A. Holden1, 2, Eric
B. Papas1, 2. 1Brien Holden Vision Institute, Sydney, NSW, Australia;
2
School of Optometry and Vision Science, University of New South
Wales, Sydney, NSW, Australia.
Purpose: The etiology of meibomian gland dysfunction remains
unclear. The aim of our study was to assess the association between
the commensal microflora of the eyelid margins and the function of
the meibomian glands in different age groups of normal subjects.
Methods: 103 subjects, aged from 25 to 65 years old, with no
systematic conditions, pre-existing ocular irritation, injury or
infection, were included in the study. An ocular swab was taken from
the lower lid margin of the left eye of each subject. Conventional
cultivation techniques were used for isolation and identification of
microorganisms. Meibum quality (MQ) and meibomian gland
expressibility (MGE) of the lower eyelid were graded following the
guidelines of TFOS workshop on meibomian gland dysfunction.
Meibography was performed on the lower eyelids using an infra-red
camera, and meibomian gland loss factors (MGLF, degrees 0 to 4)
were assessed. The association between microorganism counts and
meibomian gland functional grading/s (including MQ, MGE, or
MGLF) was assessed.
Results: The most commonly identified microorganisms were
commensal skin bacteria including Propionibacterium species (87%),
and coagulase negative staphylococci, mainly Staphylococcus
epidermidis (80%). A significantly higher number of microorganisms
(348 ± 231 cfu per swab) was found on eyelid swabs collected from
subjects with an MQ grade of 4 (no meibum expressed) and an MGE
grade of 3 (no glands expressible) compared to the MQ grades 0-2 (<
157 ± 165 cfu per swab; p < 0.05) and MGE grades 0-2 (< 131 ± 134
cfu per swab; p < 0.01). The average number of microorganisms
recovered from eyelid swabs was significantly lower in the younger
(25 - 44 years old) female group (50 ± 69 cfu per swab) compared to
the older (≥ 45 years age) female group (145 ± 170 cfu per swab, p <
0.05) and male group in general (191 ± 169 cfu per swab, p < 0.05).
Concomitantly, more younger women (63%) than either older women
(35%) or males (33%) presented with MQ grade 0 (p < 0.05).
Conclusions: Higher numbers of commensal bacteria on the eyelids
are associated with clinical measures of decreased meibum quality
and function, as well as increased age in female population. It
remains unknown whether the increased number of bacteria is a
causative agent to the compromise of meibomian gland function, or a
consequence, either of such changes or other systemic factors, e.g.
reduced sex hormones in elderly women.
Commercial Relationships: Hua Zhu, Brien Holden Vision
Institute (E); Judith L. Flanagan, None; Nisha S. Yeotikar, None;
Negar Babaei Omali, None; Daniel Tilia, Brien Holden Vision
Institute (E); Shamil Y. Iskandar, Brien Holden Vision Institute (E);
Brien A. Holden, Allergan (F), AMO (I); Eric B. Papas, None
Clinical Trial: ACTRN12612000703808
Program Number: 927 Poster Board Number: B0232
Presentation Time: 1:00 PM - 2:45 PM
Confirmation of Squalene in Tears and Sebum and It’s Potential
Function
Douglas Borchman1, Georgi A. Georgiev2, Marta C. Yappert4,
Norihiko Yokoi3. 1Department of Ophthalmology and Visual
Sciences, University of Louisville, Louisville, KY; 2Model
Membranes Lab, Department of Biochemistry, Faculty of Biology,
St. Kliment Ohridski University of Sofia, Sofia, Bulgaria;
3
Department of Ophthalmology, Kyoto Prefectural University of
Medicine, Kyoto, Japan; 4Department of Chemistry, University of
Louisville, Louisville, KY.
Purpose: Squalene (SQ) is found on the skin in sebum and in
meibum on the surface of the eye lid and tears. A 1H NMR resonance
at 5.2 ppm has been tentatively used to quantify SQ in human sebum
and meibum. When the relative intensity of the resonance in the
NMR spectra of human meibum is low as in dry eye associated with
meibomian gland dysfunction, the tear film is unstable and patients
have signs and symptoms of dry eye. When the intensity of the
resonance is restored with azithromycin or doxycycline treatment,
tear film stability is restored and patients no longer are afflicted by
symptoms of dry eye. In this study, we confirmed that the 5.2 ppm
resonance associated with human sebum and eye lid meibum lipid
(ELML) is due to SQ.
Methods: Infrared spectroscopy was used to measure SQ-wax
interactions. 1H and 13C NMR resonance assignments of SQ were
confirmed in human meibum, human sebum, and in human eye lid
lipid using heteronuclear single quantum correlation spectroscopy.
Langmuir trough and Brewster angle microscopy were used to study
the surfactant properties of SQ and SQ mixed with human meibum.
Evaporataion rates were measured in vitro by gravimetric analysis.
Results: The content of SQ in human sebum was found to be 28
mole %. We confirmed the presence of SQ in ELML (4 mole %) but
not meibum. SQ was 87 % disordered and did not undergo a phase
transition between 20 and 80o C. SQ at 20% by weight had no
significant effect on the phase transition temperature, minimum or
maximum frequency or cooperativity of wax but a mixture of wax
and SQ decreased the rate of evaporation of buffer with increasing
thickness. SQ did not possess surfactant properties and when mixed
with human meibum did not contribute to the surface pressure of
films at physiological surface pressure.
Conclusions: Due to the lack of SQ-SQ and SQ-wax interactions, SQ
is likely to spread over the surface of skin or the tear film. It is
possible that the thin layer of SQ could reduce the rate of evaporation
on the surface of the skin and offer antioxidant, antibacterial, and
anti-inflammatory protection to the skin and tears. At low surface
pressures, SQ filled thinner regions of meibum films. It is this
property of SQ that could potentially stabilize the tear film during
break up by migrating to the areas without a tear film lipid layer
offering protection to the cornea.
Commercial Relationships: Douglas Borchman, None; Georgi A.
Georgiev, Rohto Pharmaceuticals (F), Santen (F), Menicon (F);
Marta C. Yappert, None; Norihiko Yokoi, Santen Pharmaceutical
(F), Otsuka Pharmaceutical (F), Kowa (P), Kissei Pharmaceutical
(C), Menicon (F), Alcon Japan (F), White medical (F), Nitten (F),
Rohto (C), Nidek (F), Johson & Johnson (F)
Support: Supported the Kentucky Lions Eye Foundation, and an
unrestricted grant from Research to Prevent Blindness Inc.
Program Number: 928 Poster Board Number: B0233
Presentation Time: 1:00 PM - 2:45 PM
Identification of biomarkers for disease progession of
Keratoconus
Arkasubhra Ghosh1, Rohit Shetty1, Lei Zhou2, Sharon D'Souza1,
Roger W. Beuerman2. 1Molecular Signalling and Gene Therapy,
Narayana Nethralaya, Bangalore, India; 2Singapore Eye Research
Institute, Singapore, Singapore.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Purpose: Keratoconus (KC) is a corneal disease, which is
characterized by corneal thinning leading to loss of visual acuity
through ectasia and irregular astigmatism. Clinically, the symptoms
of KC can be highly variable and in part, depend on the stage of the
progression of the disorder as well as associated eye-rubbing, itching,
etc. There is currently no effective management for KC, nor any
adequate biomarkers to predict the outcome of clinical severity of the
disease. Furthermore, there is considerable confusion in the field
regarding the pathophysiology of the disease and key involvement of
inflammation. To that end, this study was designed to address these
questions.
Methods: We correlated the clinical features associated with KC in a
cohort of 54 patients with tear proteomic profiles. This noninterventional study was approved by the Narayana Nethralaya IRB.
Clinical grades and associated symptoms were graded as per the
Amsler-Krumeich classification (Grade I: 32 patients, Grade II: 14
patients, Grade III: 8 patients). Quantitative Proteomic analysis was
carried out using iTRAQ with nanoLC-MS/MS on 25μg total tear
protein from each patient and pooled healthy control subjects (n = 12)
for comparison.
Results: We observed that eye-rubbing correlated with increasing
clinical grades and morbidity of KC. In total, over 1000 tear proteins
(< 1% false discovery rate) were identified from the whole study.
Quantitative proteomic results from a group of up-regulated and
down-regulated proteins (ratio for KC/control > 1.5, or < 0.67)
revealed a positive correlation between several tear proteins (LCN1,
PLA2G2A, SCGB2A1, MSLN and CRYAB) and KC clinical grades
(Grade I, II and III). A negative correlation between a group of tear
proteins (ALB, IGHG2 & G3, A2M, complement factors C3, C4A,
C6 & H, ORM1, KNG1, PRDX1, SERPIN-F1, GSN) and KC clinical
grades was also observed.
Conclusions: These proteins could be used alone or in combination
as biomarkers for predicting the progression or severity of
keratoconus. These proteins also shed light on the underlying
deregulation of important inflammatory and immunomodulatory
pathways that may be key drivers of KC pathophysiology.
Commercial Relationships: Arkasubhra Ghosh, None; Rohit
Shetty, None; Lei Zhou, Singapore Eye Research Institute (P);
Sharon D'Souza, None; Roger W. Beuerman, Allergan (F), SERI
(P), Santen (R)
Clinical Trial: J0002GQQ
Program Number: 929 Poster Board Number: B0234
Presentation Time: 1:00 PM - 2:45 PM
Several Tear Protein Levels Of Breast Cancer Patients Differ
from Healthy Participants: A Microarray Study
Ksenia Keller1, 2, Julia Pieter2, Daniel Boehm2, Norbert Pfeiffer3,
Franz H. Grus1. 1Experimental Ophthalmology, University Medical
Center, Mainz, Germany; 2Gynecology and Obstetrics, University
Medical Center, Mainz, Germany; 3Ophthalmology, University
Medical Center, Mainz, Germany.
Purpose: Currently no molecular biomarkers for early detection of
breast cancer (BC) are available. The explorations of cancer
proteome revealed several new findings according significant level
changes of proteins. Even in the distant body fluids like tears we
reported over 20 de- or increased proteins in pooled BC and healthy
(CTRL) samples in our previous study. Here we report our additional
study regarding individual tear protein profiling using high sensitive
antibody-microarray plattform
Methods: Tear fluid was obtained from 38 women, whereas 19 of
them were diagnosed with primary non-metastasized BC, with the
help of Schirmer test. Tear proteins were eluted overnight with 0.1%
n-dodecyl-beta-D-maltoside. Proteins were precipitated and their
concentration was determined. 5 µg of labeled proteins from each
participant were incubated on microarrays with antibodies against
complement proteins, other immune components and high abundant
tear proteins. After signal visualization a semi-quantitative
comparison of protein levels was performed
Results: We detected several significantly different distributed
proteins in BC and CTRL groups. For example, the interleukins IL2
and IL1beta were decreased in BC samples (p=0.025 and p=0.017,
respectively). Furthermore we used the artificial neuronal networks to
test the discrimination ability of the putative biomarkers. Thus the
best combination of biomarkers revealed the sensitivity of 89% and
specificity of 100%. An area under the curve of 0.91 was achieved
Conclusions: This pilot study provides more findings to our previous
tear protein profiling investigations. Obviously also in other body
fluids besides serum and tumor microenvironment several protein
deregulations occur leading perhaps to changed signal cascades and
aberrant reactions. This study also shows the immediate involvement
of immune system, whereas its component levels are changed in
comparison to CTRL subjects. Further explorations are ongoing for
the verifying the results in a larger study population for establishment
of putative BC biomarkers
Commercial Relationships: Ksenia Keller, None; Julia Pieter,
None; Daniel Boehm, None; Norbert Pfeiffer, Sensimed AG (F),
Sensimed AG (R), MSD (F), MSD (R), Alcon (F), Allergan (F),
Novartis (F), Novartis (R), Bayer (F), Heidelberg Engineering (F),
Bausch&Lomb (F), Boehringer-Ingelheim (F), Carl Zeiss Meditech
(F), Chibret (F), Nidek (F), Pfizer (F), Santen (F), Santen (R),
Topcon (F), Ivantis Inc (F), Ivantis Inc (R); Franz H. Grus, None
Support: partly founded by MAIFOR
Program Number: 930 Poster Board Number: B0235
Presentation Time: 1:00 PM - 2:45 PM
Method development for analysis of tear proteins using selected
reaction monitoring
Simin Masoudi1, 2, Ling Zhong3, Mark Raftery3, Mark D. Willcox2.
1
Brien Holden Vision Institute, Sydney, NSW, Australia; 2School of
Optometry and Vision Science, University of New South Wales,
Sydney, NSW, Australia; 3Bioanalytical Mass Spectrometry Facility,
University of New South Wales, Sydney, NSW, Australia.
Purpose: Previous studies of dry eye and contact lens (CL) related
dry eye have shown qualitative differences in lactoferrin, lysozyme,
lipocalin 1, prolactin-induced protein and proline rich protein 4. The
aim of this study was to develop a method for simultaneous detection
and accurate quantification of these five proteins in human tears
using selected reaction monitoring (SRM) mass spectrometry.
Methods: Basal tears were collected from normal subjects (male=1,
female=6, CL=3, No CL=4).Tears were processed as described
before with modifications. SRM transitions from two peptides
representing each protein were selected using Skyline software then
analyzed using nano-flow liquid chromatography mass spectrometry.
Calibration standards were generated using unlabelled synthetized
peptides diluted to a range of 1 to 1000fmol/µL in the presence of a
fixed amount of stable-isotopically labelled peptides (50fmol/µL).
The ratios of the peak areas of the unlabelled/labelled peptides were
plotted against the concentrations of the corresponding unlabelled
peptides. The concentration of endogenous proteins (µg/µL) in tear
samples was deduced using the peak area ratio of the endogenous
peptides to labelled peptides.
Results: The limits of quantification for the selected peptides were
below 50pg/μl. The recovery of peptides from spiked digested tears
was ≥56% and the coefficient of variation values were ≤16% which
show good precision of the method. For lactoferrin
(1.20±0.77μg/μL), lysozyme (2.11±1.50μg/μL) and lipocalin-1
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
(1.751±0.99μg/μL) the findings were consistent with findings from
previous ELISA studies. Tear levels of prolactin-induced protein
(0.09± 0.06μg/μL) and proline rich 4 (0.80±0.50μg/μL) are reported
here for the first time.
Conclusions: SRM method can be used for simultaneous detection
and quantification of the five selected proteins in low volume human
tear samples (2.5µl per sample) without prior purification of each
protein component or need for antibodies.
Commercial Relationships: Simin Masoudi, None; Ling Zhong,
None; Mark Raftery, None; Mark D. Willcox, Allergan Inc (C),
Allergan Inc (R), Brien Holden Vision Institute (P), Bausch + Lomb
(C), Basuch + Lomb (R)
Program Number: 931 Poster Board Number: B0236
Presentation Time: 1:00 PM - 2:45 PM
Dry Eye and Systemic Diseases
Claudia Henrich, Esen K. Akpek. Cornea, The Wilmer Eye Institute
at Johns Hopkins, Baltimore, MD.
Purpose: To evaluate the prevalence of associated inflammatory
systemic diseases in a cohort of patients with dry eye syndrome.
Methods: Medical records of patients who presented to the Wilmer
Eye Institute Ocular Surface Diseases and Dry Eye Clinic with dry
eye symptomatology during a period of two years (January 2010 to
December 2011) were reviewed retrospectively. Patients were
divided into two groups: patients with clinically significant dry eye
disease (defined as Schirmer test result without topical anesthesia
≤10 mm at 5 minutes in either eye or bulbar conjunctival staining
with lissamine green scored based on Oxford scale ≥1 in either eye)
and patients with dry eye symptomatology but without the clinical
findings.
Results: A patient list of 326 patients was generated electronically
using a diagnostic code of ICD 375.15. Sixty two patients were
excluded as they presented primarily with other ocular surface
problems and dry eye was a secondary diagnosis. Two hundred and
sixty four patients with a primary diagnosis of dry eye were included
in the analysis. The majority of the patients (215/264, 81.4%) were
female. Two hundred and seventeen (217/264; 82.2%) had clinically
significant dry eye. About half of the patients (121/264, 45.8%) had
an underlying inflammatory systemic disease on presentation. One
hundred and nine of them (109/121, 90.1%) had a clinically
significant dry eye. Thirty one patients (31/264, 11.7%) had primary
Sjögren Syndrome, 38 (38/264; 14.4%) had thyroid disease (either
hypothyroidism, Graves disease or Hashimotos thyroiditis), 13
(13/264, 4.9%) had rheumatoid arthritis, 42 (42/264, 15.9%) had
other rheumatic diseases.
In 50 patients (50/143, 35.0%) without a previously known systemic
disease (regardless of the severity of the dry eye) a further work up
was performed based on review of systems. In 12 patients (12/50,
24.0%) a diagnosis based on the work up was established: 10 patients
(10/50, 20.0%) were diagnosed with thyroid eye disease, 2 patients
(2/50, 4.0%) were diagnosed with Sjögren syndrome or presumed
Sjögren syndrome, one (1/50, 2.0%) was diagnosed with a mixed
connective tissue disease.
Conclusions: Dry eye is a very common condition. Although usually
a local disease with a benign course, prevalence of systemic disease
association seems to be significant. Based on clinical suspicion and
review of systems, further diagnostic testing might uncover some of
these previously undiagnosed conditions.
Commercial Relationships: Claudia Henrich, None; Esen K.
Akpek, Alcon (F), Allergan (F), Baush & Lomb (C)
FODE Highlights Impression Based Mucin Defects on the Ocular
Surface in Dry Eye Patients: Kinetics and Retrieval by Tear
Lipocalin
Po-Ting Yeh1, 2, Ben J. Glasgow2, 3, Richard Casey2. 1Ophthalmology,
National Taiwan University Hospital, Taipei, Taiwan;
2
Ophthalmology, Jules Stein Eye Institute, David Geffen School of
Medicine at UCLA, Los Angeles, CA; 3Pathology and Laboratory
Medicine, David Geffen School of Medicine at UCLA, Los Angeles,
CA.
Purpose: A fluorescent probe was used to identify mucin depleted
areas on the ocular surface and test the hypothesis that tear lipocalin,
retrieves lipids from the eyes of normal and dry eye subjects.
Methods: Fluorescein labeled octadecyl ester, FODE, was
characterized by mass spectrometry and absorbance
spectrophotometry. The use of FODE to define mucin defects was
studied with impression membranes under conditions that selectively
deplete mucin. The kinetics of FODE removal from the ocular
surface was analyzed by sampling tears from control and dry eye
patients at various times. The tear protein-FODE complexes were
isolated by gel filtration and ion exchange chromatographies,
monitored with absorption and fluorescent spectroscopies and
analysed by gel electrophoresis. Immunoprecipitation verified FODE
complexed to tear lipocalin.
Results: FODE exhibits an isosbestic point at 473nm, pKa of 7.5,
and red shift relative to fluorescein. The low solubility of FODE in
buffer is enhanced with 1% Tween 80 and ethanol. FODE adheres to
the ocular surface of dry eye patients. FODE produced visible
staining at the contact sites of membranes, which correlated with
removal of mucin. Despite the fact that tear lipocalin was reduced in
dry eye patients, FODE removal followed similar rapid exponential
decay functions for all subjects. The majority of FODE was eluted
bound to tear lipocalin.
Conclusions: Tear lipocalin retrieves lipid rapidly from the human
ocular surface in mild to moderate dry eye disease and controls. With
improvements in solubility, FODE may have potential as a
fluorescent probe to identify mucin depleted areas.
FPLC of tear samples from dry eye subjects. (●) Absorbance at 280
nm (○) Fluorescence intensity (λem=511 nm). Inset left, coomasssiestained tricine-SDS-PAGE. Lane 1, fx 4-6. Lane 2, fx 8-10. Lane 3,
fx 14-16. Lane 4, fx 21-23. Lane 5, fx 26-28. Lane M, molecular
weight markers. Lactoferrin (Lf), lysozyme (Ly) and tear lipocalin
(TL) are indicated at left. Inset right, fluorescence of anion exchange
chromatography performed on fractions 21-23 of control subjects.
Fluorescence was seen only in the elution buffer (high salt) fractions.
Gel filtration chromatography of control sample was nearly identical
to that of dry eye.
Program Number: 932 Poster Board Number: B0237
Presentation Time: 1:00 PM - 2:45 PM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
FODE staining of sites where Schirmer test II was performed
(arrows). Photos taken after: (A) 3 minutes (B) 5 minutes.
Commercial Relationships: Po-Ting Yeh, None; Ben J. Glasgow,
None; Richard Casey, None
Support: NIH EY11224 (BG), EY000331 (Core), NIH S10RR023718 A (Shared Instrumentation Grant) and the Edith and Lew
Wasserman Professorship (BG)
Program Number: 933 Poster Board Number: B0238
Presentation Time: 1:00 PM - 2:45 PM
Factors associated with dry eye: The Korea National Health and
Nutrition Examination Survey 2010
Tyler Hyung Taek Rim1, Ji Min Ahn2, Sangchul Yoon1, Tae-im Kim1,
Eung Kweon Kim1, Kyoung Yul Seo1. 1Department of
Ophthalmology, Yonsei University College of Medicine, Seoul,
Republic of Korea; 2Ophthalmology, Siloam Eye Hospital, Seoul,
Korea, Seoul, Republic of Korea.
Purpose: To assess socio-demographic and health-related risk factors
associated with dry eye.
Methods: In 2010, a total of 5,726 randomly selected national
representative participants of the Korea National Health and Nutrition
Examination Survey underwent additional ophthalmologic
examinations by the Korean Ophthalmologic Society. Subjects were
asked to respond to a question of “Until now have you ever been
diagnosed before of having dry eye, either eye, by a doctor?” with
possible choices of “no” or “yes.” Independent variables were
divided into four categories: 1) socio-demographic factors, 2) health
examination variables, 3) health behavioral risk factors and 4)
variables concerning the eyes. The risk factors for dry eye were
identified by using a multivariate logistic regression analysis.
Results: The prevalence of dry eye was 8.2% (95% confidence
interval (CI) 7.3-9.2) in subjects aged 19 or older. History of any eye
surgeries (adjusted Odds Ratio (aOR)=2.2, 95% CI, 1.7-2.8), female
sex (aOR=2.2, 95% CI, 1.5-3.3), extremely stressful status(aOR=1.8,
95% CI, 1.0-3.1), and hypercholesterolemia of ≥240 mg/dL
(aOR=1.3, 95% CI, 1.0-1.7) were independent risk factors for dry
eye. Binge alcohol users (aOR=0.6, 95% CI, 0.5-0.9) and subjects
with occupations of farming, fishing, and forestry (aOR=0.2, 95% CI,
0.1-0.4) were less likely to have dry eye compared to their reference
groups of non-drinkers and subjects with occupations in
administration, management or white-collar professionals,
respectively. Among subjects who received an eye surgery, ptosis
surgery (aOR = 6.2, 95% CI, 1.4-27.1), cataract surgery(aOR = 1.8,
95% CI, 1.2-2.6), and refractive surgery(aOR=2.9, 95% CI, 1.8-4.5)
were important risk factors.
Conclusions: Ophthalmologists should be aware that past
experiences of eye surgeries could be one of the most important risk
factors of dry eye, and should focus on treating dry eye problems in
subjects who will receive or have already received an eye surgery.
FIGURE 1. Flowchart of the step approach for identifying risk
factors of dry eye using univariate and multivariate analysis.
FIGURE 2. An association between type of eye surgery and dry eye.
Commercial Relationships: Tyler Hyung Taek Rim, None; Ji Min
Ahn, None; Sangchul Yoon, None; Tae-im Kim, None; Eung
Kweon Kim, None; Kyoung Yul Seo, None
Support: The authors state that they have no financial or conflicts of
interest to disclose.
Program Number: 934 Poster Board Number: B0239
Presentation Time: 1:00 PM - 2:45 PM
Prevalence of asymptomatic dry eye in patients with Collagen
Vascular Diseases in South India
RAMYA RAVINDRAN, Kalpana Suresh, Varshini Varadaraj.
Ophthalmology, SRMC and RI, Chennai, India.
Purpose: Dry Eye Syndrome (DES) is common in patients with
collagen vascular disorders. The purpose of this study was to identify
asymptomatic DES in patients with collagen vascular disorders in
South India. In our part of the world, awareness regarding the
implications of late diagnosis is still lacking. The objectives were to
identify DES, classify it's severity based on the ocular surface
integrity and to delineate the sub set that had not undergone a prior
ophthalmic evaluation.
Methods: A prospective, cross sectional, non interventional,
observational study was conducted in a university hospital. 75
patients diagnosed with various collagen vascular disorders with no
ocular complaints from the departments of General medicine,
Pediatrics and Rheumatology formed the study group. 75 age and sex
matched controls were used for comparison. Patients with local and
systemic conditions that contribute to DES were excluded. Schirmer's
test, TBUT and rose bengal (RB) staining were performed and scored
according to the Van Bijsterveld scoring system. History of their last
ophthalmic visit was noted. The Mann Whitney U test was used for
statistical analysis.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Results: In our study we noticed a female preponderance (64%). Post
diagnosis with collagen vascular disorders, 76% of the patients had
not undergone an ophthalmic examination. 30.66% of the patients in
the study group had an abnormal Schirmers test compared to 12% of
the controls (p<0.001). 81.33% of the study group had an abnormal
TBUT which was the commonest deranged test in our study versus
8% of the controls(p<0.0001). A RB staining score of > 3 was seen in
52.67% of the study group and 6% of the controls (p<0.0001) .Severe
DES as identified by a RB staining score of 7-9 was seen only in the
study group (11.33%). Overall dry eye was present in 84% of the
study group versus 20% of the controls (p<0.0001).
Conclusions: We noticed a statistically significant difference
between the study and the control group, proving that the presence of
DES was considerably higher than presumed in these patients. We
also noticed that a visit to an Ophthalmologist was a rarity in these
patients as they had no ocular symptoms. This large referral gap
between the primary care physician and the Ophthalmologist is yet to
be bridged. It is of utmost significance for the primary care physician
to be aware of asymptomatic DES for timely referral to prevent
possible complications.
Commercial Relationships: RAMYA RAVINDRAN, None;
Kalpana Suresh, None; Varshini Varadaraj, None
Program Number: 935 Poster Board Number: B0240
Presentation Time: 1:00 PM - 2:45 PM
Prevalence and Risk Factors of Dry Eye Disease Among a
Hospital-based Population
Wei Chen, Jinyang Li, Qinxiang Zheng. Sch of Ophthal &
Optometry, Wenzhou Medical College, Wenzhou, China.
Purpose: To investigate the prevalence of dry eye disease (DED) and
distribution of associated risk factors among a hospital-based
population.
Methods: In this clinic-based, cross-sectional study, consecutive
outpatients aged above 20 years from May 1st 2010 to May 16th
2010 were screened. Symptomatic dry eye patients screened by
questionnaire were then involved with objective tests, including tear
film breakup time test, corneal and conjunctival fluorescein staining,
Schirmer test and slit-lamp examination for assessment of the
meibomian gland. Related risk factors were collected by designed
questionnaires.
Results: We collected the detailed information of clinically defined
moderate to severe dry eye patients among a consecutive hospitalbased population, including age trend, gender structure, frequency of
symptoms and distribution of associated environmental/occupational
risk factors. Of 6657 consecutive outpatients aged above 20 years,
symptomatic dry eye present in 635 (9.54%) subjects. 532 (7.99%)
out of those 635 subjects were clinically diagnosed as defined DED
who combined with positive signs.
The prevalence was significantly higher in patients aged 31-50 years
(p<0.005) and significantly lower in age group of over 70 years
(p<0.001), which demonstrated an inverted U-shaped relationship,
which females (10.41%) was significantly higher as compared to
males (5.21%) (P<0.001). Overexposure to visual display terminal
(VDT) was major risk factor for DED among young men and women
(56.2%). High and low risk of occupational exposure to adverse
environment accounted for a large proportion, 31.2% and 20.9%
respectively. Contact lenses use was closely associated with DED in
young women, and history of ocular surgeries might be another factor
associated with DED in old people. 163 (43.9%) of 371 female dry
eye patients were associated with hormonal changes. The incidence
of meibomian gland dysfunction related DED increased gradually
with age. There were only 10 (1.9%) dry eye patients were associated
with Sjögren's syndrome and all of them were females.
Conclusions: Environmental and occupational factors were strongly
associated with DED and constituted the major proportion in a
hospital-based population. A classification of DED based on
distribution of risk factors was recommended for clinical use.
Commercial Relationships: Wei Chen, None; Jinyang Li, None;
Qinxiang Zheng, None
Support: Supported by National Natural Science Foundation of
China (Grant Number: 81170820)
Program Number: 936 Poster Board Number: B0241
Presentation Time: 1:00 PM - 2:45 PM
Simulation of Acute Symptomatic Exacerbation in Dry Eye (DE)
Patients After a Two Hour Exposure to Adverse Conditions in an
Environmental Chamber
Margarita Calonge1, 2, Marisa Tesón1, Alberto López-Miguel3,
Amalia Enriquez-De-Salamanca1, 2, Vicente Martín-Montañez1,
Maria-Jesus Benito1, María E. Mateo1, Jose M. Herreras1, 2, Michael
E. Stern4, Maria J. Gonzalez-Garcia1, 2. 1Ocular Surface Group,
IOBA-University of Valladolid, Valladolid, Spain; 2Biomedical
Research Networking center in Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Valladolid, Spain; 3VISIÓN I+D, SL,
Valladolid, Spain; 4Allergan, Inc, Irvine, CA.
Purpose: To simulate, in an environmental chamber, the acute
exacerbation in symptoms and signs that Dry Eye (DE) patients often
suffer when they are exposed to adverse conditions, usually
consisting of low humidity and/or air flow.
Methods: Twenty mild-moderate DED patients (6 males, 14 females;
64.7 ± 1.8 years old) were subjected to a controlled adverse condition
(5% relative humidity, 23°C temperature and localized air flow) for
two hours in an environmental chamber (CERLab, IOBA, University
of Valladolid, Spain). The following diagnostic tests were performed
immediately before and after exposure: modified SIDEQ
questionnaire, tear osmolarity, phenol red thread test, conjunctival
hyperemia, fluorescein tear-breakup time (T-BUT), Schirmer’s test,
corneal staining (Oxford and Baylor schemes), and conjunctival
staining (Oxford scheme). Additionally, 16 molecules were analyzed
in unstimulated tears (4 μl) by multiplex bead analysis in a Luminex
IS-100: EGF, CX3CL1/fractalkine, IFN-γ, IL-10, IL-12p70, IL-17A,
IL-1β, IL-1RA, IL-2, IL-6, CXCL8/IL-8, CXCL10/IP-10,
CCL5/RANTES, TNF-α, VEGF, and MMP-9.
Results: After 2 hours in the controlled adverse condition, DE
patients showed a significant (p<0.01) increase in corneal global
staining (from 0.65 ± 0.15 to 1.10 ± 0.14) as well as in the nasal (p<
0.05), temporal (p<0.01) and inferior (p< 0.001) areas. Fluorescein TBUT values significantly (p<0.001) decreased from 2.75 ± 0.53 to
1.90 ± 0.24. Interestingly, all patients complained about their eyes
feeling remarkably worse after exiting the chamber but SIDEQ
questionnaire failed to show that. Tear IL-6 levels significantly
(p<0.05) increased from 38.6 ± 8.5 to 55.7 ± 9.7 pg/ml after
exposure. Tear MMP-9 levels greatly increased, also significantly
(p<0.01), from 894.8 ± 260.1 to 3829.0 ± 1202.3 pg/ml after the
exposure.
Conclusions: Mild to moderate DE patients can be “acutely”
inflamed in an environmental chamber resembling the sudden
episodes of worsening that DE patients frequently suffer in their daily
life. These exacerbations should be taken into account not only for
the easiness of clinical trial recruitments and the reliability of the end
points, but also for the design of therapeutics that can ameliorate
these inflammatory episodes.
Commercial Relationships: Margarita Calonge, Allergan (C);
Marisa Tesón, None; Alberto López-Miguel, VISIÓN I+D, SL (E);
Amalia Enriquez-De-Salamanca, Allergan Inc. (F); Vicente
Martín-Montañez, None; Maria-Jesus Benito, None; María E.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Mateo, None; Jose M. Herreras, None; Michael E. Stern, Allergan,
Inc. (E); Maria J. Gonzalez-Garcia, None
Support: The present study was partially supported by (1) Junta de
Castilla y León: VA145A11-2 and 04/09/VA/0158; (2) University of
Valladolid FPI-UVa-2008; (3) Ministry of Economy and
Competence: PTQ-09-02-01913, CDTI: IDI-20060676, CICYT:
SAF2010-15361.
Program Number: 937 Poster Board Number: B0242
Presentation Time: 1:00 PM - 2:45 PM
Reproducibility of tearfilm osmolarity measurement assessed
with electrical impedance in patients with dry eye syndrome and
healthy subjects
Gerhard Garhofer1, Semira Kaya1, Johannes Nepp2, Doreen
Schmidl1, Leopold Schmetterer1, 3. 1Department of Clinical
Pharmacology, Medical University of Vienna, Vienna, Austria;
2
Department of Ophthalmology, Medical University of Vienna,
Austria, Austria; 3Department of Biomedical Engineering and
Physics, Medical University of Vienna, Austria, Austria.
Purpose: Dry Eye Syndrome (DES) is a common medial condition
that affects up to 20% of adults aged 45 years or older. Standard
clinical tests are limited by their poor reproducibility and show only a
weak correlation to patients complains. It has been hypothesized that
measurement of tear film osmolarity may be a new and promising
non-invasive approach for the diagnosis and monitoring of treatment
success. The current study investigated the short time and day-to-day
reproducibility of the Tear Lab Osmolarity system.
Methods: 20 patients with DES and 20 age and sex matched
volunteers were included in this study. Tear film osmolarity was
measured in both groups on three consecutive study days to calculate
day-to-day reproducibility. To assess short time reproducibility of
osmolarity measurements were taken 3 times per study day. Tear film
break up time (BUT) and Schirmer 1 test were assessed by standard
clinical tests. Coefficient of variation was calculated as a measure of
reproducibility.
Results: Schirmer test and BUT was lower in patients with DES
compared to healthy subjects on all three study days (p<0.05).Tear
film osmolarity was significantly higher in patients with dry eye
compared to healthy controls on day 1.(DES:311±11 mOsml/l;
healthy: 301±12 mOsml/l, p=0.02 between groups) This was also true
for day 2 (DES:309±11 mOsm/l; healthy:299±11mOsml/l, p=0.01
between groups) and day 3 (DES:308±12 mOsml/l; healthy:
298±11mOsml/l, p=0.01 between groups). The overlap between the
groups was, however, high. Calculated test-retest coefficient of
variation (cv) in tearfilm osmolarity on each day was 3.18%±2.89%,
2.81%±2.08% and 2.23%±1.85%, respectively. Day-to-day
variability as calculated by the cv between the three study days was
3.47%±1.69%.
Conclusions: Our results indicate good short-time and day-to-day
reproducibility of the osmolarity measurements. Large scale
longitudinal clinical studies are needed to investigate whether tear
film osmolarity is a suitable surrogate parameter for diagnosis of
DES and monitoring disease progress.
Commercial Relationships: Gerhard Garhofer, None; Semira
Kaya, None; Johannes Nepp, None; Doreen Schmidl, None;
Leopold Schmetterer, None
Clinical Trial: NCT01744457
Program Number: 938 Poster Board Number: B0243
Presentation Time: 1:00 PM - 2:45 PM
Comparison of diagnostic tests in distinct well-defined diseases
related to dry eye syndrome
Monica Alves1, 2, Danilo Ribeiro1, Jacqueline F. Faustino1, Leticia
Bachette1, Francisco Aranha3, Afonso C. Vigorito3, Carmino A. De
Souza3, Jayter S. Paula1, Antonio Augusto V. Cruz1, Eduardo M.
Rocha1. 1Ophthalmology, University of Sao Paulo, Ribeirao Preto,
Brazil; 2Ophthalmology, Pontific Catholic University of Campinas,
Campinas, Brazil; 3Hematology, University of campinas, Campinas,
Brazil.
Purpose: The aim of this study was to describe and compare signs
and symptoms of dry eye syndrome (DES) and ocular surface
disorder in six well-defined and non-overlapping diseases.
Methods: A spontaneous and continuous sample of patients with
Sjogren’s syndrome (SS) (n=27) , GVHD (n=30), Graves orbitopathy
(n=28), facial palsy (n=8), diabetes (n=14) and under chronic
glaucoma treatment (CGT) (n=20) were enrolled. Evaluation
consisted of a comprehensive protocol encompassing: (1) structured
questionnaire - Ocular Surface Disease Index (OSDI); (2) tear
osmolarity (TearLab Osmolarity System-Ocusense); (3) tear film
break-up time (TBUT); (4) fluoresceine and lissamine staining; (5)
Schirmer test; (6) severity grading.
Results: One hundred twenty-seven patients (aged 48.8 yearsold±14.1, male:female ratio=0.46) were enrolled in the study, along
with 24 age and gender matched controls. In all six groups,
significant diferences in the studied parameters were observed
comparing individual patients and controls. OSDI was higher in SS,
facial palsy and GVHD patients (all P < 0.05). Fluoresceine and
lissamine staining was higher in SS and GVHD patients (P<0.05),
who also presented the lowest TBUT and Schirmer test. Graves
orbitopathy and patients under chronic glaucoma treatment presented
tear instability (low TBUT) but staining and Schirmer test were close
to normal. Tear osmolarity in mOsmos/L was consistently higher in
diabetes (318.6±15.9), SS (316.0±23.1) and GHVD patients
(310.7±18.3) compared to controls (295.3±7.8) (p<0.0001).
Conclusions: DES is a complex condition, in which no single test is
a sole indicator. Herein, we have described a comprehensive set of
diagnostic tools that could allow a more detailed evaluation of DES
in several distinct conditions.
Commercial Relationships: Monica Alves, None; Danilo Ribeiro,
None; Jacqueline F. Faustino, None; Leticia Bachette, None;
Francisco Aranha, None; Afonso C. Vigorito, None; Carmino A.
De Souza, None; Jayter S. Paula, None; Antonio Augusto V. Cruz,
None; Eduardo M. Rocha, None
Support: Fapesp, FAEPA
Program Number: 939 Poster Board Number: B0244
Presentation Time: 1:00 PM - 2:45 PM
The Relationship between Ocular Surface Epithelial Damage and
Tear Abnormalities: Osaka Study
Hiroaki Kato1, Norihiko Yokoi1, Aoi Komuro1, Yukiko Sonomura1,
Yuichi Uchino2, Miki Uchino2, Dogru Murat2, Motoko Kawashima2,
Kazuo Tsubota2, Shigeru Kinoshita1. 1Department of Ophthalmology,
Kyoto Prefectural University of Medcine, Kyoto, Japan; 2Department
of Ophthalmology, Keio University School of Medcine, Tokyo,
Japan.
Purpose: In the pathophysiology of dry eye, tear abnormalities result
in epithelial damage of the cornea and conjunctiva. The purpose of
this present study was to investigate the relationship between ocular
surface epithelial damage and tear abnormalities.
Methods: This study involved 279 eyes of 173 subjects (132 eyes of
72 males, 147 eyes of 101 females, mean age: 49 years) from normal
subjects and dry eye patients. In all eyes, tear meniscus radius (TMR,
mm), spread grade of the tear film lipid layer (SG, 1-4: 1 being the
best), non-invasive breakup time (NIBUT, seconds), fluorescein
breakup time (FBUT, seconds) and ocular surface epithelial damage
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
(OSED) score (cornea: 15 points maximum, conjunctiva: 6 points
maximum), Schirmer 1 test (ST1, mm) were measured, and the
factors determining OSED score were investigated by multiple
regression analysis in which the parameters were chosen using the
stepwise procedure.
Results: The corneal OSED score was found to be determined by
TMR, SG, and FBUT, and was described as: -0.363 - (3.997 x TMR)
- (3.284 x SG) - (0.189 x FBUT) (R2=0.54, p<0.0001). The
conjunctival OSED score was found to be determined by TMR, SG,
and ST1, and was described as: 0.834 - (2.933 x TMR) + (1.320 x
SG) - (0.025 x ST1) (R2=0.41, p<0.0001).
Conclusions: The findings of this study show that TMR and SG,
factors related to aqueous tear volume and aqueous tear film
thickness, respectively, were significantly related to both corneal and
conjunctival epithelial damage, while FBUT, a factor related to tear
film stability, was significantly related to the corneal OSED score
and ST1, a factor related to lacrimal gland function, was related to the
conjunctival OSED score.
Commercial Relationships: Hiroaki Kato, Santen Pharmaceutical
(F); Norihiko Yokoi, Santen Pharmaceutical (F), Otsuka
Pharmaceutical (F), Kowa (P), Kissei Pharmaceutical (C), Menicon
(F), Alcon Japan (F), White medical (F), Nitten (F), Rohto (C), Nidek
(F), Johson & Johnson (F); Aoi Komuro, Santen pharmaceutical (F);
Yukiko Sonomura, Santen pharmaceutical (F); Yuichi Uchino,
Santen Pharmaceutical (F); Miki Uchino, Santen Pharmaceutical Co
(F); Dogru Murat, Otsuka Pharmaceuticals (F); Motoko
Kawashima, Santen Pharmaceutical Co., Ltd (F); Kazuo Tsubota,
AcuFocus, Inc (C), Allergan (F), Bausch Lomb Surgical (C),
Functional visual acuity meter (P), JiNS (P), Kissei (F), Kowa (F),
Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), Echo Denki (P),
Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPT Company (P);
Shigeru Kinoshita, Senju Pharmaceutical Co (P), Santen
Pharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon (R),
AMO (R), HOYA (R)
Support: None in the Support
Program Number: 940 Poster Board Number: B0245
Presentation Time: 1:00 PM - 2:45 PM
Association of Dry Eye Disease with Physical Activity and Sleep
Quality: Osaka study
Motoko Kawashima1, Miki Uchino1, Norihiko Yokoi2, Yuichi Uchino1,
Murat Dogru1, Aoi Komuro2, Yukiko Sonomura2, Hiroaki Kato2,
Shigeru Kinoshita2, Kazuo Tsubota1. 1Department of Ophthalmology,
Keio University School of Medicine, Shinjuku ku, Japan;
2
Department of Opthalmology, Kyoto Prefectural University of
Medicine, Kyoto, Japan.
Purpose: To investigate the association between physical activity
and sleep quality, and dry eye disease.
Methods: Data from the cross-sectional study conducted in a certain
company in 2011(the Osaka study) were analyzed. The participants
were classified into definite dry eye disease(DED), probable-DED,
and non-DED by dry eye examination results including Schirmer test,
fluorescein and lissamine green staining, tear film break-up time, and
symptom questionnaire, according to the Japanese Dry Eye diagnosis
criteria. The short form of the International Physical Activity
Questionnaire (IPAQ-J) and the Pittsburg Sleep Quality Index (PSQIJ) were implemented to determine physical activity and sleep quality,
respectively. The participants’ physical activity level in metabolic
equivalent units per week (MET, min/week) was calculated and
classified into high, moderate, and low level on the basis of the
IPAQ. The global PSQI score ranging from 0 to 21 was calculated by
summing the scores of seven sleep variables on a scale of 0 to 3; the
score ≥5.5 indicates poor sleep.
Results: According to the IPAQ, the breakdown of the participants
by physical activity levels was 10.1% for high, 48.7% for moderate,
and 41.2% for low, respectively. (completed N=425) . More
participants tended to score high in the physical activity in non-DED
group (14.4%) than in DED, with a significant difference between
mean MET scores for DED and non-DED (P=0.03). Total mean
PSQI global score was 5.1±2.3 (completed N=383). A total of 45% of
the DED and probable-DED groups reported poor sleep quality while
34% of non-DED group did, with a significant difference in the
global score between probable-DED and non-DED (p=0.006).
Furthermore, statistically significant correlation was observed
between the PSQI score and the dry eye symptoms score
(r=0.27,p<0.001).
Conclusions: These results suggest that both physical activity and
sleep quality were associated with DED. The high level of physical
activity seems to lower the risk of DED. And sleep disturbances seem
to be an influencing factor of DED, especially dry eye symptoms.
Further study with larger longitudinal or intervention trials will allow
us to draw more definitive conclusions.
Commercial Relationships: Motoko Kawashima, Santen
Pharmaceutical Co., Ltd (F); Miki Uchino, Santen Pharmaceutical
Co (F); Norihiko Yokoi, Santen Pharmaceutical (F), Otsuka
Pharmaceutical (F), Kowa (P), Kissei Pharmaceutical (C), Menicon
(F), Alcon Japan (F), White medical (F), Nitten (F), Rohto (C), Nidek
(F), Johson & Johnson (F); Yuichi Uchino, Santen Pharmaceutical
(F); Murat Dogru, None; Aoi Komuro, Santen pharmaceutical (F);
Yukiko Sonomura, Santen pharmaceutical (F); Hiroaki Kato,
Santen Pharmaceutical (F); Shigeru Kinoshita, Senju
Pharmaceutical Co (P), Santen Pharmaceutical Co (P), Otsuka
Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R); Kazuo
Tsubota, AcuFocus, Inc (C), Allergan (F), Bausch Lomb Surgical
(C), Functional visual acuity meter (P), JiNS (P), Kissei (F), Kowa
(F), Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), Echo Denki
(P), Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPT Company
(P)
Support: Santen Pharmaceutical Co., Ltd
Program Number: 941 Poster Board Number: B0246
Presentation Time: 1:00 PM - 2:45 PM
Prospective, Randomized, Multicenter, Double-Blind PlaceboControlled Study of the Safety and Efficacy of 1% D-3Hydroxybutyrate eye drop for the Treatment of Dry Eye
Tetsuya Kawakita1, Etsuko Takamura2, Jun Shimazaki3, Junko
Ogawa4, Miki Uchino5, Kazumi Fukagawa6, Kenichi Yoshino7, Mari
Tanaka8, Machiko Shimmura-Tomita9, Kazuo Tsubota1.
1
Ophthalmology, Keio Univ School of Medicine, Shinjuku-ku, Japan;
2
Tokyo Women's Medical University, Tokyo, Japan; 3Tokyo Dental
College,Ichikawa General Hospital, Chiba, Japan; 4Kitasato Institute
Hospital, Tokyo, Japan; 5Ryogoku Eye Clinic, Tokyo, Japan;
6
Iidabashi Eye Clinic, Tokyo, Japan; 7Yoshino Eye Clinic, Tokyo,
Japan; 8Yatsuekimae azisai Eye Clinic, Tokyo, Japan; 9Ichikawa
shapo Eye Clinic, Yatsuekimae azisai Eye Clinic, Chiba, Japan.
Purpose: To D-3- hydroxybutyrate( BHB) is the most abundantly
produced physiological ketone, is a fatty acid-derived substrate that
plays key role in mammalian energy metabolism. BHB has been
demonstrated to be effective in neurological disorders such as
Alzheimer’s, Parkinson’s, and Huntington’s disease in animal models
and humans We showed that topically applied BHB ameliorates
corneal epithelial erosion and superficial punctate keratopathy, a
hallmark of corneal surface symptoms of dry eye, in a rat dry eye
model (IOVS 2003, 2005). The purpose of this study is to investigate
the safety and efficacy of 1% BHB eye drop in the treatment of dry
eye patient.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Methods: Prospective, randomized, multicenter, double-blind
placebo-controlled study were performed. A total of 65 dry eye
patients were randomly assigned to placebo (n=33) and 1% BHB eye
drop (n=32) and patients were received 6 times a day for 4 weeks.
Corneal fluoresecin staining, cornea and conjuctival rose bengal
staining, tear film BUT, Schirmer test and subjective symptoms were
evaluated.
Results: In the corneal rose bengal score, statistically significant
improvement was observed between the placebo and 1% BHB drop
at 2 and 4 week (P < 0.05).
In the corneal fluorescein staining score, significant improvement
was observed between placebo and 1% BHB eye drop at 2 week in
the patient with Schirmer ≤ 5 mm (n=38, P < 0.05). Trend toward a
significant improvement between placebo group and 1% BHB eye
drop at week 4 in the patient with Schirmer ≤ 5 mm with apparent
foreign sensation (n=23, P < 0.1). There was no significant difference
in tear film status and subjective symptoms between placebo and 1%
BHB eye drop. All of adverse effects were mild ocular symptoms and
spontaneously recovered in both group.
Conclusions: These results indicate that 1% BHB is safe and
effectiveness in the treatment of ocular surface disorder in teardeficient dry eye patient.
Commercial Relationships: Tetsuya Kawakita, None; Etsuko
Takamura, None; Jun Shimazaki, Santen Pharmaceutical Co. (F),
Otsuka Pharmaceutical Co. (F), Abott Medical Optics (F); Junko
Ogawa, None; Miki Uchino, Santen Pharmaceutical Co (F); Kazumi
Fukagawa, None; Kenichi Yoshino, None; Mari Tanaka, None;
Machiko Shimmura-Tomita, None; Kazuo Tsubota, AcuFocus,
Inc (C), Allergan (F), Bausch Lomb Surgical (C), Functional visual
acuity meter (P), JiNS (P), Kissei (F), Kowa (F), Santen, Inc. (F),
Otsuka (F), Pfizer (C), Thea (C), Echo Denki (P), Nidek (F), Ophtecs
(F), Wakasa Seikatsu (F), CEPT Company (P)
Support: Ophtecs
Program Number: 942 Poster Board Number: B0247
Presentation Time: 1:00 PM - 2:45 PM
The Korb-Blackie Lid Light Test
Donald R. Korb1, 2, Caroline A. Blackie1, 2. 1Korb Associates, Boston,
MA; 2TearScience, Morrisville, NC.
Purpose: The Korb-Blackie (KB) lid light test was developed to
investigate the possibility that apparently normal, closed, lids fail to
create the necessary protective seal to prevent ocular surface
dessication during sleeping. The test results are correlated with
symptoms of eye discomfort upon awakening.
Methods: Patients (n=148) reporting for routine eye examinations
that met the inclusion criteria for the study were enrolled. Inclusion
criteria: willingness to participate in the study, over the age of 18, no
lid or closure (e.g. lagophthalmos) abnormalities, no current ocular
inflammation/disease, no ocular surgery within the last 6 months, no
history of lid surgery. After answering a simple questionnaire
regarding their eye comfort upon awakening, patients were asked to
rest their heads back against the head rest of a semi-reclined exam
chair and to close their eyes as if they were falling sleep. A
transilluminator was lightly placed against the closed outer upper
eyelid of each eye. The apparently closed lids were examined for the
presence of any light emanating from the lid area between the lashes.
The examiner positioned their eye level inferiorly such that they were
looking up, to optimize viewing of the designated region. The lids
were divided into three sections: temporal, central and nasal. The
amount of visible light in each section was quantified on a scale of 0 3 where 0 = no light, 1 = minimal, 2 = moderate and 3 = severe. Eye
discomfort upon awakening was quantified on a scale of 0 - 2 where
as 0 = no discomfort, 1 = mild and 2 = significant discomfort.
Results: The mean age of the patients = 53.9±16.2 years (50 males;
98 females). Data for right eyes only are presented. The mean light
score for each lid region was: temporal = 0.3±0.5; central = 1.0±1.0;
nasal = 0.5±0.7, indicating the central region is the least likely to
close completely. The level of eye discomfort upon awakening was
significantly correlated with the number of lid sections (0-3)
emanating light during the KB lid-light test (p<0.0001).
Conclusions: The KB lid light test reveals that light emanating from
between closed lids, which is correlated to symptoms of ocular
discomfort upon awakening, may be linked to the ability of the lids to
prevent ocular surface desiccation during sleeping.
Commercial Relationships: Donald R. Korb, TearScience (F);
Caroline A. Blackie, TearScience (E)
Program Number: 943 Poster Board Number: B0248
Presentation Time: 1:00 PM - 2:45 PM
Black-Spot Formation and Fluorescence Tear Break-up Time
CHENG-CHUN PENG1, Bo Tan2, Meng C. Lin2, 3, Clayton J. Radke1,
3 1
. Chemical and Biomolecular Engineering, University of California,
Berkeley, Berkeley, CA; 2Clinical Research Center, University of
California, School of Optometry, Berkeley, CA; 3Vision Science
Group, University of California, Berkeley, CA.
Purpose: Fluorescence tear break-up time (FBUT) is the most
common diagnostic test for tear-film stability and ocular-surface
health. Appearance of black spots and/or streaks is normally
interpreted as tear-film rupture (i.e., breakup of the tear film down to
the glycocalix). We investigate whether black spots are either deep
depressions in the tear film or actual ruptures.
Methods: Fluorescence intensity is determined by image analysis of
aqueous sodium fluorescein (NaF) solutions (Tan et al, AAO annual
meeting, 2011) in a micron-deep PDMS-microfabricated channels.
Based on the Beer-Lambert law and self-quenching theory (Arbeloa,
J. Chem. Soc., Faraday Trans., 1981), we relate measured emitted
fluorescence intensity to film thickness and NaF concentration. The
theoretical correlation is combined with an evaporation-driven tearfilm-instability model (Peng, ACS Colloid and Surface Science
Symposium, 2012) to predict the dynamic color change of tear film
during an FBUT test.
Results: We generate a color map for various film thickness and NaF
concentrations that ascertains the thickness at which a NaF film turns
black. Fig. 1 demonstrates that a tear film containing 5 x 10-3 g/mL
of NaF that turns black at a thickness of 5 μm. Accordingly, our color
map permits conversion of black-spot images obtained from a FBUT
test into local film tear-film thickness. Instability theory predicts
appearance of a black spot 25 s after the instillation of aqueous NaF
where the tear film thickness reduces from 3.5 to 0.89 μm. Complete
tear rupture does not occur until after 35 s. (See Fig. 2.)
Conclusions: Black spots in FBUT tests indicate local regions of the
tear film with small but finite thicknesses, and not necessarily
complete tear-film rupture. We provide a color map that, for the first
time, allows measured fluorescence intensity in FBUT examinations
to be converted into local tear-film thickness. This result is also
useful to clarify the current discrepancy between FBUT and noninvasive tear break-up time (NIBUT) measurements and provides a
new standardized procedure for FBUT examination with higher
reproducibility.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Fig. 1. Comparison between fluorescence intensity and NaF
concentration from a 5-ìm thick film (filled diamonds) and theory
(line). Colors corresponding to intensity values are also shown.
Fig 2. Predicted dynamic of color change for black-spot formation.
Commercial Relationships: CHENG-CHUN PENG, None; Bo
Tan, None; Meng C. Lin, TearLab Corporation (F), Allergan, Inc.
(F); Clayton J. Radke, novartis corporation (F)
Program Number: 944 Poster Board Number: B0249
Presentation Time: 1:00 PM - 2:45 PM
Surface Chemistry Study Of The Interactions Of Benzalkonium
Chloride and Hyaluronic Acid With Meibomian And Corneal
Lipids
Georgi A. Georgiev1, Norihiko Yokoi2, Slavyana Ivanova1, Rumen
Krastev3, Zdravko Lalchev1. 1Biochemistry, Sofia University "St
Kliment Ohridski", Sofia, Bulgaria; 2Ophthalmology, Kyoto
Prefectural University of Medicine, Kyoto, Japan; 3Biomaterials,
NMI Naturwissenschaftliches und Medizinisches Institut an der
Universität Tübingen, Reutlingen, Germany.
Purpose: Dodecyl dimethyl benzyl ammonium chloride (BAK) is
commonly used preservative in eyedrop formulations, known to
impair the integrity of the tear film lipid layer and of the corneal
epithelium membranes. We studied the capability of high molecular
weight (Mw 1x10+6) hyaluronic acid (HA; Santen Pharmaceutical,
Osaka, Japan) to protect meibomian and corneal lipid films at the
air/water interface from the adverse action of BAK.
Methods: Human meibum was collected from healthy volunteers;
corneal lipids were extracted from SIRC cell culture. The interactions
of BAK with meibomian and corneal lipids at the air/water interface
in presence and absence of HA in the film subphase were examined
in vitro at blink-like compression/expansion of film area by
Langmuir surface balance. The sample’s lateral elasticity and
capability to compress and spread during dynamic area changes were
evaluated through the surface pressure-area isotherms and isocycles.
The lipid films morphology was monitored by Brewster Angle
Microscopy. BAK concentration was kept within the clinical range of
0.005-0.02%; HA was used in the range of 0.01-0.3%. The viability
of SIRC cell cultures treated with BAK, pure and mixed with HA,
was examined.
Results: Pure BAK instantaneously inserted in the lipid films
(corneal or meibomian). The interaction resulted in impaired
spreading and discontinuous patchy structure of the lipid films,
increased surface pressure-area hysteresis and partial displacement of
the lipids by BAK from the surface. The inclusion of HA in the films
subphase opposed to these adverse effects and at ≥ 0.1% HA the
properties of the lipid films were maintained for the entire BAK
concentration range. Identical results were obtained with cell culture
experiments which showed that SIRC cells maintained high viability
at ≥ 0.1% HA.
Conclusions: HA at concentration ≥ 0.1% HA was able to efficiently
suppress the adverse effects of BAK on the properties of meibomian
and corneal lipid films and on the viability of SIRC cells. Thus
mixtures of BAK and HA represent prospective compositions for
eyedrop formulations. Surface chemistry based approach is proposed
for in vitro molecular scale characterization of pharmaceutically
applicable polymers and their interactions with tear film lipids.
Commercial Relationships: Georgi A. Georgiev, Rohto
Pharmaceuticals (F), Santen (F), Menicon (F); Norihiko Yokoi,
Santen Pharmaceutical (F), Otsuka Pharmaceutical (F), Kowa (P),
Kissei Pharmaceutical (C), Menicon (F), Alcon Japan (F), White
medical (F), Nitten (F), Rohto (C), Nidek (F), Johson & Johnson (F);
Slavyana Ivanova, None; Rumen Krastev, None; Zdravko
Lalchev, None
Support: The research is funded by Santen Pharmaceutical.
Program Number: 945 Poster Board Number: B0250
Presentation Time: 1:00 PM - 2:45 PM
Efficacy of rebamipide for laser in situ keratomileusis-associated
dry eye
Yosai Mori, Ryohei Nejima, Ayami Masuda, Yoko Maruyama,
Keiichiro Minami, Kazunori Miyata. Miyata Eye Hospital,
Miyakonojo, Japan.
Purpose: Dry eye syndrome is a major complication after laser in
situ keratomileusis (LASIK), and is occasionally hard to improve
with an artificial tear or hyaluronic acid treatment. Rebamipide is a
quinolinone derivative stimulating mucin secretion as well as
increasing goblet cells on the conjunctiva, resulting in improvement
of ocular surface disorders. Aim of the study is to evaluate the
efficacy of rebamipide for dry eye after LASIK.
Methods: This prospective study comprised 32 eyes of 16 patients
who had LASIK-associated dry eye and had been treated with the
artificial tear or hyaluronic acid eye drop. Rebamipide 2% eyedrop
(Mucosta, Ohtsuka Pharmaceutical) was additionally instilled 4 times
a day for 4 weeks. Tear secretin was examined with the Schirmer test
with anesthesia before and at 4 weeks after the rebamipide treatment.
Tear breakup time (BUT), fluorescein stain, and lissamine green stain
were examined before and at 1 and 4 weeks. Fluorescein staining on
the cornea was evaluated by the summation of area and density
scores (none:0 to severe:3). Lissamine green staining in the
conjunctiva was scored in none:0 to full:18. Questionnaire of 14
symptoms according to Ocular Surface Disease Index was performed
before and at 4 weeks. Change in the examinations after the
additional treatment was evaluated with the Friedman's test following
Scheffe ad-hoc comparison. Symptoms were compared with the
Wilcoxon signed-ranks test.
Results: Mean tear secretin did not change beween before
rebamipide treatment (8.8 mm) and after 4 weeks (8.2 mm). BUT
significantly increased from before (3.4 sec.) to 1 week (4.8 sec.,
P<0.001), and continued up to 4 weeks (5.0 sec.). Fluorescein and
lissamine green staining scores were also improved from 1 week after
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
the rebamipide treatment (P<0.001). There were significant
improvement in 9 symptoms (fatigue, dryness, grittiness, heavy
eyelids, pain, discomfort, blurred vision, light sensitivity, dry place).
Conclusions: Addition of rebamipide 2% improved ocular surface
disorder and dry eye symptoms. It was demonstrated that mucin
secretion in the tear film due to the rebamipide effectively improved
LASIK-associated dry eye.
Commercial Relationships: Yosai Mori, None; Ryohei Nejima,
None; Ayami Masuda, None; Yoko Maruyama, None; Keiichiro
Minami, None; Kazunori Miyata, None
Program Number: 946 Poster Board Number: B0251
Presentation Time: 1:00 PM - 2:45 PM
Models for Interaction of the Tear Film with the Corneal and
Conjunctival Epithelia
Jennifer L. Bruhns1, Richard J. Braun1, Douglas B. Freeman1, Peter
E. King-Smith2, Padmapriya Ramamoorthy2, Jason J. Nichols3.
1
Mathematical Sciences, University of Delaware, Newark, DE;
2
College of Optometry, The Ohio State University, Columbus, OH;
3
College of Optometry, University of Houston, Houston, TX.
Purpose: Evaporation has been shown to be an important factor in
the dynamics of the tear film (TF). The increased osmolarity from
evaporation affects the underlying epithelium. We construct a
mathematical model for the TF thickness (h), osmolarity (c) and the
thicknesses of the underlying epithelia (hi) for both the cornea and
the conjunctiva. The osmolarity in the TF and the epithelial cells is
computed over many blink cycles for different interblink times.
Methods: Equations similar to those of Levin and Verkman for the
epithelial layer thicknesses are coupled to equations for the TF
thickness and osmolarity. Water transport is by osmosis through the
cells to the TF; this is assumed to be the only interaction between
each of the cells and with the TF. Seven epithelial cells of varying
initial thickness represent the corneal epithelium (CoE; see Figure 1);
four cells of different sizes represent the conjunctival epithelium
(CjE). The TF thinning rate is specified by the parameters to match
observed rates; permeability of cell boundaries is chosen to fit
permeability values deduced from thinning rate measurements
(previously described). The TF thickness was deduced from
fluorescence imaging in the quenching regime from 15 subjects who
kept the eye open for 60s. The system of ordinary differential
equations is solved by custom MATLAB programs.
Results: For single interblinks of 6s or 30s with an initial 2.5
micron/min thinning rate, the change in osmolarity in the tear film is
relatively small because osmosis is slow through the cells. For many
blink cycles, with 6s interblinks, the osmolarity remains low in both
the CoE and CjE. For many 30s interblinks in sequence and 2.5
micron/min, the TF osmolarity rises to 394 mOsm, and to 356 mOsm
in the first cell layer of the CoE. In the CjE with an initial thinning
rate of 1.4 micron/min (from measured rate), the TF increases to 334
mOsm, and the first cell rises to 323 mOsm. Cells beneath the first
layer rose to smaller values. These osmolarity values do not
correspond to tear breakup: they are spot values after a fixed repeated
interblink interval.
Conclusions: The model can predict the osmolarities in the TF, CoE
and CjE , for different thinning rates. The cornea is exposed to higher
osmolarities than the conjunctiva for the same initial thinning rate.
Figure 1. A schematic of the model. Evaporation from the top of tear
film drives osmolarity changes.
Commercial Relationships: Jennifer L. Bruhns, None; Richard J.
Braun, None; Douglas B. Freeman, None; Peter E. King-Smith,
None; Padmapriya Ramamoorthy, None; Jason J. Nichols,
Vistakon (R), Vistakon (F), Alcon (R), Alcon (F), Bausch and Lomb
(R)
Support: NSF 1022706, NIH EY021794 and Howard Hughes
Medical Institute
Program Number: 947 Poster Board Number: B0252
Presentation Time: 1:00 PM - 2:45 PM
A New Paradigm for the Structure of the Tear-Film Lipid Layer
Clayton J. Radke1, 4, Liat Rosenfeld2, Colin Cerrretani1, Danielle L.
Lieske2, Michael Toney3. 1chemical and biomolecular engineering,
university of california, berkeley, CA; 2chemical engineering,
stanford university, stanford, CA; 3stanford synchrotron radiation
light source, SLAC national accelerator laboratory, menlo park, CA;
4
vision science group, university of california, berkeley, CA.
Purpose: We explore the unique rheological and structural properties
of human and bovine meibomian lipids (ML) to provide insight into
the on-eye physical behavior the human tear-film lipid layer (TFLL).
Methods: Human ML was collected from healthy individuals.
Bovine ML was exuded from freshly excised eyelids. Bulk
rheological properties of pooled meibomian lipids were measured by
a commercial stress-controlled rheometer; a home-built Interfacial
Stress Rheometer (ISR) probed the interfacial viscoelasticity of
spread layers of meibomian lipids. Small- and wide-angle X-ray
scattering detected the presence and melting of dispersed crystal
structures. A differential scanning calorimeter (DSC) analyzed phase
transitions in bulk samples of bovine meibum.
Results: Bulk and interfacial rheology measurements show that
meibum is extremely viscous and highly elastic. It is also a nonNewtonian, shear-thinning fluid. Small- and wide-angle x-ray
diffraction (SAXS and WAXS), as well as DSC, confirm the
presence of suspended lamellar-crystal structures at physiologic
temperature. A melt transition is detected in the bulk and interfacial
rheology between 29-36 °C. Disappearance of crystalline particles
was detected by SAXS, WAXS, and DSC over the same temperature
range as that of the melt transition in the rheological measurements.
Conclusions: The TFLL is classically thought to be a parallel stack
of molecular layers. Based on our new findings, the proposed
structure for the TFLL shown at physiologic temperature in Fig. 1 is
a highly viscoelastic, shear-thinning liquid suspension consisting of
lipid lamellar crystallites immersed in a continuous liquid phase with
no long-range order. The duplex lipid film exhibits two separate
interfaces, air/lipid and water/lipid, with aqueous protein and
surfactant-like lipids adsorbed at the water/lipid surface. Minor
amounts of water and protein are present in the film. This new picture
of the TFLL overturns the current accepted viewpoint.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Figure 1. Schematic of the TFLL. At eye temperature, asymmetric
crystallites (mostly phase A) are dispersed within an isotropic,
viscoelastic continuous liquid. Polar lipids orient at the aqueous/lipid
interface, but orientation does not persist deep into the bulk lipid
layer. Drawing is not to scale.
Commercial Relationships: Clayton J. Radke, novartis corporation
(F); Liat Rosenfeld, None; Colin Cerrretani, Alcon Corporation
(F); Danielle L. Lieske, None; Michael Toney, None
Support: novartis corporation
Program Number: 948 Poster Board Number: B0253
Presentation Time: 1:00 PM - 2:45 PM
Blood Flow in eye lids and Surface Temperature of
TarsalConjunctiva Decreases in Patients with Obstructive
MeibomianGland Dysfunction
Reiko Arita1, 2, Rika Shirakawa2, Motoko Kawashima3, Kouzo Itoh1,
Sachiko Inoue3, Shiro Amano2, Kazuo Tsubota3. 1Ophthalmology,
Itoh Clinic, Saitama, Japan; 2Ophthalmology, Tokyo University,
Tokyo, Japan; 3Ophthalmology, Keio University, Tokyo, Japan.
Purpose: Meibomian glands secrete lipid into the tear film, thereby
preventing excessive evaporation of the tear film by forming a thin
oily layer on the tear film.The surface temperature in the tarsal
conjunctiva was significantly lower in patients with MGD than those
in normal controls(Arita R, et al. Arch Ophthalmol. in press). The
purpose of this study was to investigate the blood flow in the eye lids
and the surface temperature in the tarsal conjunctiva and to examine
the correlation between the blood flow and the surface temperature in
the tarsal conjunctiva and ocular surface parameters in patients with
MGD.
Methods: Twenty five eyes of 25 patients (11men, 14women; mean
± standard deviation of age, 74.2±10.3 years) who were diagnosed as
obstructive MGD and30 eyes of30 healthy volunteers (18 men, 12
women; 64.6±14.4 years) as a control group.
The blood flow in the eyelid was measured with a newly developed
blood flow meter using a red light. The surface temperature of the
central cornea, andupper and lower tarsal conjunctiva was measured
using ocular surface thermography. Changes in meibomian glands
were scored using non-contact meibography (meibo-score, 0-6). Lid
margin abnormality (0-4), superficial punctuate keratopathy (SPK)
(0-9), and meibum (0-3) was scored. Tear film breakup time (BUT)
was measured. This study was approved by the institutional review
board of Itoh clinic and adhered to the tenets of the Declaration of
Helsinki.
Results: The blood flow in the eye lid was significantly lower in
patients with MGD than in normal controls (P<0.0001)The surface
temperature of the upper /lower palpebral conjunctiva (32.2±0.4,32.0
±0.5°C) was significantly lower in patients with MGD than in normal
controls (33.8±0.3, 33.9 ±0.4°C) (P<0.0001,P<0.0001, respectively ).
Blood flow in the eyelids had a significant negative correlation with
the meiboscore(r=-0.7765, P<0.0001), the surface temperature in the
upper/lower tarsal conjunctiva( r=-0.7725, P<0.0001, r=-0.7605,
P<0.0001), ocular symptom score(r=-0.7072, p<0.0001), lid margin
abnormality score(r=-0.6824, P<0.0001), SPK(r=-0.6505, P<0.0001),
BUT(r=0.2975, P=0.0289), and meibum(r=-0.7864, P<0.0001).
Conclusions: Blood flow in the eye lids was decreased in patients
with obstructive MGD. Decreased blood flow is a likely cause of
obstructive MGD.
Commercial Relationships: Reiko Arita, TOPCON (P), JFC (P);
Rika Shirakawa, None; Motoko Kawashima, Santen
Pharmaceutical Co., Ltd (F); Kouzo Itoh, None; Sachiko Inoue,
None; Shiro Amano, Topcon (P); Kazuo Tsubota, AcuFocus, Inc
(C), Allergan (F), Bausch Lomb Surgical (C), Functional visual
acuity meter (P), JiNS (P), Kissei (F), Kowa (F), Santen, Inc. (F),
Otsuka (F), Pfizer (C), Thea (C), Echo Denki (P), Nidek (F), Ophtecs
(F), Wakasa Seikatsu (F), CEPT Company (P)
Program Number: 949 Poster Board Number: B0254
Presentation Time: 1:00 PM - 2:45 PM
Mathematical Modeling of Tear Break-up and Fluorescent
Intensity
Richard J. Braun1, Carolyn G. Begley2, Adam J. Winkeler2, Peter E.
King-Smith3, Javed Siddique4. 1Dept of Mathematical Sciences,
University of Delaware, Newark, DE; 2School of Optometry, Indiana
University, Bloomington, IN; 3College of Optometry, The Ohio State
University, Columbus, OH; 4Dept of Mathematics, Pennsylvania
State University, York, PA.
Purpose: The local mechanisms involved in the formation and
development of areas of tear breakup (TBU) remain poorly
understood. The purpose of this project is to develop mathematical
theory for variables of interest to compare with experimental images
of TBU to better predict local fluctuations in tear film osmolarity and
fluorescence (FL) during and following TBU.
Methods: Tear films of 10 subjects with a range of tear break-up
times (3-45 sec) were simultaneously recorded at high resolution
following the instillation of 2 microliters of 2% fluorescein. Using
these images, which were aligned by the center of the pupil, as input,
math models were solved for local changes tear film thickness (h),
insoluble surfactant concentration (representing the lipid layer and
affecting evaporation), as well as osmolarity (c) and FL (f)
concentrations inside the tear film. FL concentration was converted to
fluorescent intensity I using the expression involving h and the full
range of f as described by Nichols et al (IOVS 2012;53:5426--32).
Results: Theoretical results show that elevated surfactant
concentration or evaporation rate led to thinner regions where TBU
first occurs. Figure 1 shows results for h, c & I (in the plots, x is
location in space and t is time) with evaporation at 2.5 microns/min
from the initially 2 micron film surface and osmosis from the cornea;
a single breakup regions (blue) is shown. The model predicts locally
elevated concentrations of osmolarity within areas of TBU (red). The
model predicts the FL intensity patterns very similar to the computed
thickness and the observed experimental results (Figure 2: color
contour plot). For the rate of osmosis from the cornea in the
displayed results, the osmolarity increases to 2.4x the isosmolar
value, or 720 mOsm. The sensitive dependence of this result on the
corneal permeability will be studied. Quenching of the FL is captured
as well.
Conclusions: This model, which was developed using experimental
data from subjects with a range of tear film instability, explains
dynamics of areas of TBU and predicts local increases in osmolarity
and the dynamics of fluorescent intensity in areas of TBU.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Figure 1. Thickness h, osmolarity c and intensity I at different times
Figure 2. Color contour plots of h, c, f and I. Blue is smaller, red is
larger. The thickness and intensity plots (left) are quite similar, as are
the plots for osmolarity and fluorescein concentration (right).
Commercial Relationships: Richard J. Braun, None; Carolyn G.
Begley, Santen, Inc. (C), ohnson & Johnson Vision Care, Inc. (C),
ohnson & Johnson Vision Care, Inc. (F); Adam J. Winkeler, None;
Peter E. King-Smith, None; Javed Siddique, None
Support: NIH Grants EY021794, EY017951, and NSF Grant
1022706
Program Number: 950 Poster Board Number: B0255
Presentation Time: 1:00 PM - 2:45 PM
Effects of Stressed Environments on the Ocular Surface
Temperature, Lipid Layer Thickness and Heterogeneity
Ranjini Kottaiyan1, Gheorghe Salahura1, 2, James M. Zavislan2, 1,
Geunyoung Yoon1, 2, James V. Aquavella1. 1Flaum Eye Institute,
University of Rochester, Rochester, NY; 2Center for Vision Science,
The Institute of Optics, Rochester, NY.
Purpose: To investigate the effects of low relative humidity (RH)
and high temperature conditions on the ocular surface temperature
(OST), tear lipid thickness (LT) and uniformity in normal and dry eye
subjects using thermal imaging and quantitative tearscope for
imaging the lipid layer.
Methods: A total of 18 eyes comprising 5 normal, 9 aqueous
deficiency (ADDE) and 4 with meibomian gland dysfunction (MGD)
were included in the study. ADDE and MGD were diagnosed based
on clinical criteria. Measurements were taken with a thermal camera
and a quantitative tearscope before and after 30 minutes acclimation
to three different environmental conditions in our controlled
environmental chamber. The three conditions are 40% RH and 75 °F
(condition 1), 20% RH and 75 °F (condition 2), and 40% RH and 85
°F (condition 3). Subjects were asked to blink once every 5 seconds
for about 3-4 times during the measurements. Thermal data was
analyzed to calculate the average OST in the central 9 mm of the
cornea, while the average LT and uniformity were calculated from
approximately the central one-third of the cornea, excluding the
pupillary region. Changes in OST and LT were compared amongst
the different groups.
Results: The average OST is 34.9 ± 0.2°C, 33.8 ± 0.3°C and 34.6 ±
0.2°C in normal, ADDE and MGD subjects respectively. The average
LT is 40.4 ±8.3 nm, 57.9 ± 30 nm, and 39.6±7.8 nm in normal,
ADDE and MGD subjects respectively at baseline. After 30 min of
acclimation, normal subjects did not show a significant change in
OST under any of the conditions. ADDE, however, showed a
significant increase in OST by 0.6°C, 0.4°C and 0.6°C in conditions
1 (p=0.01), 2 (p=0.02) and 3 (p=0.005) respectively. MGD showed
an increasing trend in OST in conditions 1 and 2, but an interesting
decreasing trend in condition 3 although none of these trends showed
a statistical significance. On the lipid thickness, ADDE showed an
increase in LT by 8.77 ± 10.95 nm and a decrease of 14.47 ± 29.04
nm, in conditions 1 and 2 respectively. Also, MGD showed an
increase of heterogeneity by 0.18 ± 0.19 and a less relative
heterogeneity of 0.14 ± 0.23 in conditions 2 and 3 respectively.
Conclusions: The dry eye groups differ in responding to different
stressed environmental conditions.This finding suggests that the
effects of the ocular stressors on the tear parameters can enhance our
ability to differentiate the various dry eye symptoms.
Commercial Relationships: Ranjini Kottaiyan, None; Gheorghe
Salahura, None; James M. Zavislan, None; Geunyoung Yoon,
Bausch & Lomb (F), Johnson & Johnson (F), Allergan (C), Staar
Surgical (C), CIBA Vision (F), Acufocus (C); James V. Aquavella,
None
Support: Research to Prevent Blindness unrestircted/challenge grant
Program Number: 951 Poster Board Number: B0256
Presentation Time: 1:00 PM - 2:45 PM
Factors Predicting the Ocular Surface Response to Desiccating
Environmental Stress
Anastasia Alex1, 2, Austin Edwards3, 2, J. Daniel Hays3, 2, Michelle
Kerkstra3, 2, Amanda Shih3, 2, Cintia S. De Paiva2, Stephen C.
Pflugfelder2. 1University of Toledo College of Medicine, Holland,
OH; 2Baylor College of Medicine, Houston, TX; 3Rice University,
Houston, TX.
Purpose: To identify factors predicting the ocular surface response to
experimental desiccating stress.
Methods: The ocular surfaces of both eyes of 15 normal and 10 dry
eye subjects were exposed to a controlled desiccating environment
(15-25% RH and 3 L/min airflow) in goggles for 90 minutes. Eye
irritation symptoms, blink rate, tear meniscus dimensions, noninvasive (RBUT) and invasive tear break-up time, and corneal
fluorescein and conjunctival lissamine green dye staining were
recorded before and after desiccating stress. Pre- and post-exposure
measurements were compared and Spearman correlations between
clinical parameters before and after desiccating stress were
calculated.
Results: Conjunctival and corneal dye staining significantly
increased in all subjects following 90-minute exposure to desiccating
environment and the magnitude of change was similar in normal and
dry eye subjects, except superior cornea staining was greater in dry
eye. Irritation severity in the desiccating environment was associated
with baseline dye staining, baseline tear meniscus height and blink
rate after 45 minutes. Desiccation-induced change in corneal
fluorescein staining was inversely correlated to baseline tear
meniscus width, whereas change in total ocular surface dye staining
was inversely correlated to baseline dye staining, RBUT and tear
meniscus height and width. Blink rate from 30-90 minutes of
desiccating environment was higher in the dry eye than normal
group. Blink rate significantly correlated to baseline corneal
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
fluorescein staining and environmental induced change in corneal FL
staining.
Conclusions: Ocular surface dye staining increases in response to
desiccating stress. Baseline ocular surface dye staining, tear meniscus
height and blink rate predict severity of ocular surface dye staining
following exposure to a desiccating environment.
Commercial Relationships: Anastasia Alex, Biocentric
Developments, LLC (P); Austin Edwards, Biocentric Developments
LLC (P); J. Daniel Hays, Biocentric Developments, LLC (P);
Michelle Kerkstra, Biocentric Developments, LLC (P); Amanda
Shih, Biocentric Developments, LLC (P); Cintia S. De Paiva, Glaxo
Smith Kline (C), Baylor College of Medicine (P); Stephen C.
Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb
(C), Baylor College of Medicine (P)
Support: NIH grants R01EY11915 (SCP), Research to Prevent
Blindness, The Oshman Foundation, The William Stamps Farish
Fund, The Hamill Foundation and Allergan, Inc.
Program Number: 952 Poster Board Number: B0257
Presentation Time: 1:00 PM - 2:45 PM
The Effects of Cosmetic Eye Pencil Application on the Tear Film
and Ocular Surface
Alison Ng1, Katharine Evans1, Rachel V. North1, Christine Purslow2,
1 1
. School of Optometry & Vision Sciences, Cardiff University,
Cardiff, United Kingdom; 2School of Health Professions, Plymouth
University, Plymouth, United Kingdom.
Purpose: To investigate and compare the effects of cosmetic eye
pencil on the ocular surface and tear film when applied to periorbital
skin (ELO) or the mucocutaneous junction (ELI).
Methods: 24 healthy female subjects (mean±SD age: 24±5 years)
underwent a 5 day washout period, refraining from all eye cosmetic
use and contact lens wear, prior to study commencement. Subjects
were supplied with pencil eyeliner (MaxFactor Kohl pencil 020
Black, Procter & Gamble, UK) and randomised to either applying
ELO or ELI daily for 7 consecutive days. Subjects crossed over
following a 1 week washout period and applied eyeliner with the
alternative method.
On day 1 and 7 of each method of eyeliner application, the following
clinical parameters were assessed: non-invasive tear break-up time
(NITBUT) and lipid layer thickness (LLT) using a Tearscope
(Keeler, Windsor, UK); bulbar redness, conjunctival and corneal
staining were graded to 0.1 increments on the Efron Grading Scale
and ocular comfort on a scale from 0 to 10 (where 10 indicated
maximal comfort). Additionally, Ocular Surface Disease Index
(OSDI) scores were compared after 7 days of each intervention.
Results: There were no significant differences between the clinical
parameters after 1 day of ELO and ELI application. However, after 7
days of eyeliner use, there was a clinical and statistically significant
improvement in LLT with ELI compared with ELO
(wave→amorphous, p=0.032). Other values for bulbar redness,
NITBUT and conjunctival staining were similar between eyeliner
applications. Subjects reported a slight decrease in comfort scores
after 7 days of ELI compared with ELO (7.8±1.7 vs. 8.3±1.7
respectively), but it was not significant (p=0.162). OSDI scores were
significantly worse after 7 days of ELI compared with ELO
(8.27±8.48 vs. 6.07±7.59 respectively, p=0.046).
Conclusions: The short-term application of eye cosmetic pencils
close to the ocular surface does not appear to be detectable clinically,
but 7 consecutive days of ELI application appears to increase LLT
and dry eye symptomology compared to ELO application. ELI
application is likely to increase the contact of waxes, lipids and other
ingredients from the pencil to the ocular surface compared to ELO,
which may increase the lipid content of the tear film but also disrupt
ocular surface homeostasis, accounting for the changes in symptoms.
Commercial Relationships: Alison Ng, None; Katharine Evans,
None; Rachel V. North, None; Christine Purslow, None
Support: Funding provided by School of Optometry & Vision
Sciences, Cardiff University, Wales, UK
Program Number: 953 Poster Board Number: B0258
Presentation Time: 1:00 PM - 2:45 PM
Effects of Computer Usage on Tear Film Osmolarity and
Precorneal Tear Film Thickness
Stephanie Chu, Kelley Bohm, Kristin O. Chapman, Christopher E.
Starr. Weill Cornell Medical College, New York City, NY.
Purpose: Prolonged computer usage is a common cause of a
constellation of ocular symptoms such as irritation, dryness and
fatigue collectively known as computer vision syndrome (CVS).
Using novel diagnostic tools we aim to objectively quantify the
effects of computer usage on tear film dynamics.
Methods: A prospective cohort study of 20 healthy volunteer
subjects (40 eyes) were evaluated in the morning and again in the
evening after prolonged computer usage. Outcome measures were
tear osmolarity (TearLab Osmolarity System) and precorneal tear
film thickness as measured by a modified Heidelberg ocular
computed tomography (OCT). The average age of patients was 28.5
years (range 24-35), with 60% females. Computer usage times were
measured by an evening survey. Data was analyzed using paired two
tailed t-test, Pearson co-efficient and Chi-square analysis with p<0.05
for significance.
Results: Patients with significant interval computer use (average of
6.55 hours, SD 2.59, range 2-11 hours) had statistically significant
increases in tear film osmolarity between morning and evening
(291.1 +/- 10.2 vs 297.8, +/- 10.9, p=0.0064). The precorneal tear
film thickness did not show a significant difference between morning
and evening measurements (0.039 +/- 0.008, 0.043 +/- 0.016,
p=0.27). 6 patients (30%) identified themselves as having dry eyes,
but tear film osmolarity was not significantly different compared to
those who did not.
Conclusions: Prolonged daily computer usage can cause an increase
in tear osmolarity which may contribute to the symptoms of CVS. In
this small study in healthy volunteers the precorneal tear film
thickness did not change with computer use.
Commercial Relationships: Stephanie Chu, None; Kelley Bohm,
None; Kristin O. Chapman, None; Christopher E. Starr, None
Program Number: 954 Poster Board Number: B0259
Presentation Time: 1:00 PM - 2:45 PM
Migration of Substances Applied Around the Eyelid Margin
Christine Purslow1, Clare Conaty2, Alison Ng2. 1School of Health
Professions, Plymouth University, Plymouth, United Kingdom;
2
School of Optometry & Vision Sciences, Cardiff University, Cardiff,
United Kingdom.
Purpose: Cosmetic products are commonly applied to and close to
the eyelid margins. The primary aim of this study was to demonstrate
the migration of products across the ocular muco-cutaneous junction
and investigate the influence of the position of application on such
migration.
Methods: The study composed of two parts. In Part 1, each eye of
ten male subjects (23.1±3.5yrs) was randomly assigned to receive the
application of petroleum jelly to either the inner (IEL) or the outer
(OEL) eyelid margin prior to measuring tear film stability and lipid
layer patterns using the Keeler Tearscope™, at timed intervals up to
30 minutes, which were then compared to baseline data. In Part 2, six
male subjects (age 22.8±3.1 yrs) attended the laboratory for three
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
sessions during which hydrogel contact lenses were fitted to both
eyes and a fluorescein solution was applied in one of three ways to
one eye only; directly onto the IEL, OEL, or via a liposomal spray to
the closed lid. The lenses were harvested after 20 minutes and
examined using UV spectrophotometry. Differences in UV
absorbance over 400-600nm indicated fluorescein uptake compared
to control lenses.
Results: Part 1: A significant change in the lipid layer pattern of the
tear film was observed after five minutes for IEL (p=0.007), but not
until 20 minutes for OEL (p=0.037). Tear film stability decreased
significantly within five minutes for IEL application (p=0.010), but
not until 20 min for OEL (p=0.045).
Part 2: Contact lenses harvested after IEL application demonstrated
significantly greater absorbance compared to control lenses
(p=0.005). Where fluorescein was applied via spray or OEL, no
significant difference in absorbance compared to controls was
observed (p=0.098 and p=0.124, respectively). Comparing the
application methods, absorbance following IEL was significantly
increased compared to OEL (p=0.014) and spray (p=0.044).
Conclusions: Lipid-based, water-based and liposomal solutions can
migrate across the muco-cutaneous junction when applied in close
proximity to the eye. All three applications studied showed some
migration; however application to the IEL was found to be most
effective in allowing for migration into the tear film. Application of a
lipid based solution to the IEL exhibited migration that was 4 times
faster than OEL application. This has implications for drug delivery
and cosmetic use around the eyelid margins.
Commercial Relationships: Christine Purslow, None; Clare
Conaty, None; Alison Ng, None
139 Ocular Surface and Tear Film
Sunday, May 05, 2013 1:00 PM-2:45 PM
Exhibit Hall Poster Session
Program #/Board # Range: 955-978/B0260-B0283
Organizing Section: Cornea
Program Number: 955 Poster Board Number: B0260
Presentation Time: 1:00 PM - 2:45 PM
Tear Film Biomarker Profiling of Subjects with Dry Eye Disease
by Multiplex Analysis
Suzanne Hagan1, 2, Alan Tomlinson1, Louise Madden1, Anne Marie
Clark2, Katherine M. Oliver1. 1Vision Sciences, Glasgow Caledonian
University, Glasgow, United Kingdom; 2Biological and Biomedical
Sciences, Glasgow Caledonian University, Glasgow, United
Kingdom.
Purpose: Dry eye disease (DED) is a distressing disorder, commonly
associated with ageing, contact lens wear and autoimmune
syndromes. It affects 15%-30% of the over-50s in Western and Asian
populations and is one of the fastest-growing eye problems in this
demographic. DED is significantly underdiagnosed and no “gold
standard” currently exists for its clinical diagnosis. Recent Multiplex
studies of tear fluids from DED subjects have implicated
inflammatory cytokines in this disorder. In this study, we investigated
tear fluids from DED and normal subjects for a panel of cytokines
using the multiplex bead assay.
Methods: This study comprised 15 DED subjects (3 males, 12
females) and 20 healthy controls (2 males, 18 females). All subjects
provided informed consent and the study adhered to the Declaration
of Helsinki tenets. Tear samples were collected from the external
canthus of open eyes, avoiding additional tear reflex. Glass
microcaps were used to collect 1μl tears. Samples were diluted 1:50
and stored at -80C until use. Tear cytokine levels were determined
with a multiplex bead assay (R and D Systems) and quantified using
a Luminex IS200. Briefly, tear samples were incubated with specific
antibody-coated beads for 3h. Washed beads were then incubated
with biotin-labelled secondary antibodies, followed by a streptavidinPE incubation. Standard curves of known cytokine concentrations
were used to calculate protein concentrations and data underwent
analysis by an in-house statistician.
Results: Detectable levels of IL-8 (> 4.05pg/ml) were observed in
12/15 DED subjects (mean 23.1pg/ml) and 16/19 normals (mean
20.6pg/ml). Some variation in IL-8 levels was noted in DED subjects
(4.9-83.8pg/ml), but a trend for increased IL8 in DED subjects was
observed, compared to normals. Moreover, detectable levels of all 7
inflammatory proteins (IL1β, IL2, IL6, IL8, IL17, TNF-α and IFN-γ)
were observed in 2 DED subjects, a result not observed in normals.
Conclusions: Increased IL8 levels in DED suggests a function in
ocular surface inflammation. This data confirms previous Multiplex
bead studies, indicating a role for IL8 in DED. Further studies may
also shed light on roles for the other 6 cytokines detected in 2 DED
subjects. Moreover, this technology appears to be sensitive enough to
detect low abundance proteins in minute sample sizes and therefore
may be useful in screening tear fluids for potential biomarkers of
DED.
Commercial Relationships: Suzanne Hagan, Allergan (F); Alan
Tomlinson, Allergan (E), Allergan (R), Bausch and Lomb (C),
TearLab (C), TearLab (I), Alcon, CibaVision (C), Pfizer (R), Pfizer
(C); Louise Madden, None; Anne Marie Clark, None; Katherine
M. Oliver, Allergan (F)
Program Number: 956 Poster Board Number: B0261
Presentation Time: 1:00 PM - 2:45 PM
Double rows of meibomian gland orifices observed by noninvasive infrared meibography
Rika Shirakawa1, Reiko Arita1, 2, Shima Fukuoka1, 3, Satoshi
Yamamoto4, Kaori Yonehara4, Tsuyoshi Haraguchi4, Shiro Amano1.
1
Ophthalmology, University of Tokyo, Tokyo, Japan;
2
Ophthalmology, Itoh Clinic, Tokyo, Japan; 3Ophthalmology, Toyo
Kyosai Hospital, Tokyo, Japan; 4Topcon Corporation, Tokyo, Japan.
Purpose: Multiple rows of meibomian gland orifices (MGOs), which
were first reported in 1992 by Hykin and Bron, exist mainly in the
upper eyelids of young people. Little is known about the morphology
of the meibomian glands and their influences on the ocular surface.
We observed lid margins and meibomian glands in healthy young
adults to explore the incidence, morphology and function of double
rows of MGOs.
Methods: Subjects were consecutive cases of healthy male
volunteers under 36 years of age. After obtaining written consent, we
measured the width of the eyelid, counted the number of MGOs in
the upper and lower eyelids, obtained a fluorescein staining score,
tear break-up time (BUT), meibum expressibility grade, tear
meniscus height under the slit-lamp microscopy, and performed a
Schirmer tear production test. We also recorded images of
meibomian glands by non-invasive infrared meibography, which
were later reviewed to count the number of meibomian glands in each
lid. We scored a meiboscore (grade 0-3) according to the ratio of the
area of missing glands. “Double rows” were defined where more than
4 orifices were aligned in a second row distinctly separate from the
primary row. This study was approved by the institutional review
board of University of Tokyo School of Medicine, and adhered to the
tenets of the Declaration of Helsinki.
Results: We examined 35 eyes of 35 people (all male, average age
28.9±2.8). We observed double rows of MGOs in the upper eyelids
of 10 eyes (28.5%), whereas none (0%) were observed in the lower
eyelids. Comparing between the double rows group (n=10) and the
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
single row group (n=25), the number of the orifices and number of
the meibomian glands in the upper eyelids are significantly higher in
the double rows group (48.0±5.7 vs 36.3±4.9, p<0.0001 and 29.4±3.4
vs 34.3±7.5, p=0.03, respectively). BUT were significantly longer in
the double rows group (7.4±3.3 sec vs.5.1±2.4 sec, p=0.03). Other
measurements were not significantly different. In 8 out of 10 eyes
with double rows, meibomian glands branched into two narrower
glands just before the lid margin. The other 2 eyes had narrow and
compact meibomian glands.
Conclusions: Double rows of MGOs exist in 28.5% of upper eyelids
of healthy young adult males. BUT is higher in the double rows
group compared with the single row group. There are two types of
morphology for double row MGOs; the dichotomous branching type
and the dense type.
Commercial Relationships: Rika Shirakawa, None; Reiko Arita,
TOPCON (P), JFC (P); Shima Fukuoka, None; Satoshi Yamamoto,
Topcon Corporation (E); Kaori Yonehara, Topcon Corporation (E);
Tsuyoshi Haraguchi, Topcon Corporation (E); Shiro Amano,
Topcon (P)
Program Number: 957 Poster Board Number: B0262
Presentation Time: 1:00 PM - 2:45 PM
P-321, a Novel Long-Acting Epithelial Sodium Channel (ENaC)
Blocker for the Treatment of Dry Eye Disease
Jose L. Boyer1, M. Ross Johnson1, John Ansede1, Karl Donn1,
Richard C. Boucher2, William Thelin1. 1Parion Sciences, Durham,
NC; 2University of North Carolina, Chapel Hill, NC.
Purpose: Dry eye disease (DED) is a multi-factorial disease,
resulting from insufficient tear volume that causes damage to the
ocular surface and symptoms of ocular discomfort. Persistent DED
results in vision impairment and reduced quality of life. One FDA
approved drug is available for the treatment of DED, however, there
remains a large unmet medical need for novel, safe and efficacious
therapies. Parion is developing P-321, a long acting ocular hydrating
agent with a rapid onset of action. This work describes the preclinical
properties of this drug candidate.
Methods: P-321 was studied in a battery of in-vitro and in vivo
pharmacology, toxicology, and tolerability studies in mice, rats, dogs,
and rabbits.
Results: P-321 was designed to be retained on the ocular surface
while producing limited systemic exposure. P-321 is a small
molecule inhibitor of ENaC with an IC50 of 1.9±0.75 nM in human
epithelial cells. P-321 is metabolically stable in plasma, blood, and
hepatocytes. In normal mice, a single ocular administration of P-321
produced four-fold increase in tear volume. In a dry eye animal
model of rats (lacrimal glands excised) exhibiting 60% reduction of
tear volume, a single administration of P-321 restored the tear
volume to levels observed in control animals. The maximal effect of
P-321 was observed within 30 minutes following administration and
was maintained for at least 6 hours. The effect of P-321 on tear
volume was dose-dependent in both normal and dry eye animals, and
the maximal effect was achieved with as low as 0.01%. P-321 is not
bioavailable by the oral route of administration and is rapidly cleared
from the plasma. P-321 is formulated as a non-preserved, aqueous
formulation; preliminary stability studies support storage at room
temperature. The formulation is well tolerated in rabbits and dogs in
ocular repeat dose toxicology studies, indicating an outstanding
safety profile with only minor ocular irritation in one species at
concentrations which are 100-fold greater than maximal
pharmacologically active doses.
Conclusions: P-321 is a novel ocular hydrating agent capable of
restoring normal tear volume with excellent ocular tolerability and
limited systemic exposure. P-321 should prove to be an attractive
candidate for the treatment of DED.
Commercial Relationships: Jose L. Boyer, Parion Sciences (E); M.
Ross Johnson, Parion Sciences (I), Parion Sciences (E), Parion
Sciences (S); John Ansede, Parion Sciences (E); Karl Donn, Parion
Sciences (E); Richard C. Boucher, Parion Sciences (C); William
Thelin, Parion Sciences (E)
Support: NIH Grant EY020705, NIH BrIDGs, NC Biotechnology
Center Grant 2009-CFG-8005
Program Number: 958 Poster Board Number: B0263
Presentation Time: 1:00 PM - 2:45 PM
Novel Topical Inhibitors of the Epithelial Sodium Channel
(ENaC) Promote Sustained Increases in Tear Volume
William Thelin1, M. Ross Johnson1, John Ansede1, Karl Donn1,
Dongfang Yu2, Barbara Grubb2, Driss Zoukhri3, Richard C.
Boucher2, Jose L. Boyer1. 1Research, Parion Sciences, Durham, NC;
2
Cystic Fibrosis Research and Treatment Center, University of North
Carolina at Chapel Hill, Chapel Hill, NC; 3Diagnosis and Health
Promotion, Tufts University, Boston, MA.
Purpose: Through the regulation of electrolyte and water transport,
the ocular surface epithelium plays a critical role in the maintenance
of tear volume and composition. As ENaC serves as the rate-limiting
step for apical sodium transport and osmotic water absorption, we
hypothesized that the inhibition of ENaC on ocular tissues would
increase tear volume, thereby providing a novel therapeutic approach
for treating the underlying cause of dry eye. Therefore we sought to
(1) confirm the expression and activity of ENaC on the ocular
surface; (2) explore the relationship between duration of ENaC
inhibition and changes in tear volume; and (3) evaluate the impact of
ENaC inhibition on ocular health in animal models of dry eye
disease.
Methods: The expression of ENaC in ocular epithelia was measured
in primary culture models, ex vivo tissue explants, and in vivo by
molecular and functional bioelectric assays. The effect of a
chemically diverse series of Parion ENaC blockers on tear volume
was assessed in normal mice and a dry eye rat model (lacrimal
excised + scopolamine rat or “ExLac”) by phenol red thread wicking.
Finally, the effects of ENaC inhibition on improvements in dry eye
signs and ocular health were tested in inflammatory-mediated (IL-1
mouse) and aqueous deficient (ExLac rats) animal models of dry eye.
Results: We confirmed that ENaC subunits are broadly expressed
and functional in the ocular surface epithelia of rodents and man.
When applied topically to normal mice or ExLac rats, Parion ENaC
blockers produce dose-dependent increases in tear volume, with the
duration of effect being a function of the rate of drug clearance from
the tear film. Significantly, we identified a series of ENaC blockers
that are preferentially retained on the ocular surface, which increased
tear volume in ExLac rats to normal levels for >6 hours after a single
dose. In the IL-1 mouse and ExLac rat models of dry eye disease,
treatment with ENaC blockers significantly improved tear volume
and corneal fluorescein staining.
Conclusions: Our studies demonstrate that ENaC inhibitors provide
long-acting increases in tear volume and that restoring volume alone
is sufficient to provide significant improvements in ocular surface
health in models of dry eye disease. Our data provide a clear rationale
for the development of ENaC blockers to treat the underlying ocular
surface desiccation in dry eye disease.
Commercial Relationships: William Thelin, Parion Sciences (E);
M. Ross Johnson, Parion Sciences (I), Parion Sciences (E), Parion
Sciences (S); John Ansede, Parion Sciences (E); Karl Donn, Parion
Sciences (E); Dongfang Yu, Parion Sciences (F); Barbara Grubb,
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to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Parion (F), Parion (I); Driss Zoukhri, Parion Sciences (F); Richard
C. Boucher, Parion Sciences (C); Jose L. Boyer, Parion Sciences (E)
Support: NIH Grant EY020705; NIH BrIDGs; NC Biotechnology
Center Grant 2009-CFG-8005
Program Number: 959 Poster Board Number: B0264
Presentation Time: 1:00 PM - 2:45 PM
Variability of tear osmolarity in dry eye patients and controls
Nicole M. Fuerst1, Mina Massaro-Giordano1, Bridgette E. McCabe1,
Ilaria Macchi2, Maxwell Pistilli1, Gui-Shuang Ying1, Vatinee Y.
Bunya1. 1Ophthalmology, Scheie Eye Institute, Philadelphia, PA;
2
Campus Biomedico University, Rome, Italy.
Purpose: To examine the reproducibility and quantify the variability
of tear osmolarity in different populations of dry eye patients and
controls.
Methods: Seventy-four eyes of thirty-seven subjects (18 Sjogren's
syndrome, 10 blepharitis, and 9 controls) were evaluated for the
variability of measurements using the TearLab system. Controls were
defined as those with no history of symptoms or signs of dry eye
disease. 94% of Sjogren’s syndrome patients and 80% of blepharitis
patients were on systemic or topical dry eye medications at the time
of enrollment. For all subjects, three consecutive osmolarity
measurements were taken at one minute intervals in each eye to
assess the within-session variability. For fifteen subjects, three
measurements were taken at each of 3 time points throughout the day
(9-10am, 12-1pm, 3-4pm) to examine the inter-session variability
over the course of the day. The within-session and inter-session
variability were assessed based on the standard error of measurement
(SEM), calculated from the analysis of variance.
Results: Among all subjects, the within session variation for a single
osmolarity measurement was 14.4 mOsm/l (13.8 for SS, 8.8 for
blepharitis, and 16.2 for controls). When the average of the three
consecutive measurements in a single session were used, the
variability of osmolarity measurement was 8.3 mOsm/l. Betweensession variability was 17.2 mOsm/l for a single osmolarity measure,
and 10.4 mOsm/l for an averaged measurement. The variability of
osmolarity measurements between two eyes of the same subject was
not correlated (pearson rho=-0.05, p=0.76).
Conclusions: A single measurement of tear osmolarity with the
TearLab system had a standard error measurement of 14.4 mOsm/L.
We did not find any correlation between eyes as far as variability of
measurements. Further larger studies would be helpful in studying the
utility of tear osmolarity measurements in different dry eye
populations.
Commercial Relationships: Nicole M. Fuerst, None; Mina
Massaro-Giordano, Tear Lab (R); Bridgette E. McCabe, None;
Ilaria Macchi, None; Maxwell Pistilli, None; Gui-Shuang Ying,
None; Vatinee Y. Bunya, TearLab (F)
Support: Vatinee Bunya: National Eye Institute (K12-EY-015398),
Research to Prevent Blindness, Mina Massaro-Giordano: Research to
Prevent Blindness, Maxwell Pistilli and Gui-Shuang Ying: Vision
Core Grant (P30 EY001583).
Program Number: 960 Poster Board Number: B0265
Presentation Time: 1:00 PM - 2:45 PM
Symptom Burden of Patients with Dry Eye Disease: A Four
Domain Analysis
Joelle Hallak, Sarmad H. Jassim, Sandeep Jain. Ophthalmology and
Visual Sciences, University of Illinois at Chicago, Chicago, IL.
Purpose: Intensity and affective interference are not measured by
current standardized dry eye symptom questionnaires. We
hypothesize that symptom burden of Dry Eye Disease (DED) is
determined with greater accuracy when a 4 domain questionnaire,
that measures symptom persistence, intensity, activity, as well as
affective interference, is utilized.
Methods: The 4 domain DED symptom burden questionnaire was
developed from well-established and validated symptom burden tools
used in other chronic diseases, such as the MD Anderson tool that
assesses pain in cancer patients. A pilot study was performed where
the DED symptom burden questionnaire and the Ocular Surface
Disease Index (OSDI) questionnaires were administered to 15
patients. A weighted item response analysis was performed for the
symptom burden questionnaire (maximum persistence scores were
multiplied with the intensity and average activity and affective scores
were then added to compute a total symptom burden score). The
OSDI (index) score was calculated from OSDI item responses. Tear
production was measured by the schirmer test and correlated with the
symptom burden score and the OSDI index score.
Results: Results from the OSDI questionnaire showed more equal
and comparable scores between patients with significantly different
schirmer measurements than results from the symptom burden
questionnaire. Two patients had OSDI scores of 27 with schirmer
scores of 1 and 14, respectively. The symptom burden scores of these
patients, however, differed 13 versus 34.43. A negative correlation
was shown between symptom burden scores and schirmer scores
whereas a positive correlation was shown between OSDI scores and
schirmer test scores.
Conclusions: Intensity may be an essential domain to include in
symptom questionnaires for DED, specifically when correlating
reported symptoms with clinical signs. A complete analysis of dry
eye symptom burden includes 4 domains (persistence and intensity of
symptoms, and intereference with activity and affect).
Commercial Relationships: Joelle Hallak, None; Sarmad H.
Jassim, None; Sandeep Jain, PCT/US20/51562 (P)
Program Number: 961 Poster Board Number: B0266
Presentation Time: 1:00 PM - 2:45 PM
Frequency and Risk Factors Associated with Ocular Surface
Disease in Patients Attending a Tertiary Care Ophthalmology
Center in Mexico City
Jaime D. Martinez1, Nallely Ramos-Betancourt1, Anat Galor2,
Francisco Beltran1, Jorge Ozorno-Zarate1, Valeria Sánchez Huerta1,
Marco Antonio Torres Vera1, Everardo Hernandez-Quintela1.
1
Asociación para Evitar la Ceguera en México I.A.P, Mexico,
Mexico; 2Bascom Palmer Eye Institute, Miami, FL.
Purpose: The purpose of this study was to ascertain the frequency
and risk factors for ocular surface disease (OSD) among patients in
Mexico attending a tertiary care ophthalmology center.
Methods: 200 consecutive patients seen in an ophthalmologic center
in Mexico City from October 2012 to November 2012 underwent a
comprehensive examination, including measurement of tear film
break-up time (TBUT), fluorescein staining classified by Oxford
scheme, Schirmer test type 1 and evaluation of Meibomian Gland
Dysfunction (MGD). Symptoms of OSD were evaluated by the
Ocular Surface Disease Index (OSDI) and Dry eye Questionnaire
(DEQ-5). Information on demographics, exposures, past medical and
ocular history, and medications was also collected. Symptomatic
OSD was defined as having an OSDI score ≥23 or DEQ-5 score ≥12,
clinical OSD was defined as having a Schirmer test <5, TBUT <5, or
staining >1.
Results: Mean patient age was 46.8 (±15.8) years (range, 16-85); 87
(47.3%) patients were male. The frequency of symptomatic OSD
based on the OSDI score was 57%, with those aged 46-55 years most
likely to have a positive OSDI (64%). The frequency of symptomatic
OSD based on DEQ-5 was 28%, with those aged 36-45 years most
likely to have a positive score (34% compared to 15% for those >66
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
years). Female gender imparted a 1.7 fold increased risk of disease as
defined by the DEQ5 (95% CI 0.92-3.31, p-value 0.09). OSD based
on clinical signs was seen at a frequency of 91%; 69% of patients had
an MGD score >1. The use of a duodenal ulcer medication was found
to be a risk factor for OSD using both the DEQ-5 and OSDI
definitions (OR 2.8 and 13.1, p values < 0.05). Patients with diabetes
mellitus had less OSD by the DEQ5 definition compared to their
counterparts without diabetes (OR 0.12, 95% CI 0.02-0.98, p-value
0.047).
Conclusions: This is the first study to demonstrate the frequency of
symptomatic and clinical OSD in a tertiary care ophthalmology
center in Mexico. The frequency of OSD in our population ranged
from 28% using a symptomatic definition to 91% using objective
measures.
Commercial Relationships: Jaime D. Martinez, None; Nallely
Ramos-Betancourt, None; Anat Galor, None; Francisco Beltran,
None; Jorge Ozorno-Zarate, None; Valeria Sánchez Huerta,
None; Marco Antonio Torres Vera, None; Everardo HernandezQuintela, None
Program Number: 962 Poster Board Number: B0267
Presentation Time: 1:00 PM - 2:45 PM
Blink and Extended Blinks in a Dry Eye Population
Ashley M. LaFond, Patrick Johnston, John D. Rodriguez, Keith J.
Lane, Endri Angjeli. Ora, Inc., Andover, MA.
Purpose: The tracking of spontaneous blink activity has been shown
to permit an investigator to gather important information on the
clinical state of a dry eye subject. Previous research has shown that
the majority of blinks are incomplete with zero lid contact duration
time. During a complete blink, lid duration contact time may vary
from less than 10ms to 80ms. Eyelid closures of up to multiple
seconds, which we refer to as "extended blinks", may also be
observed. Here we investigate the relative incidence of incomplete
and extended blinks in a dry eye population.
Methods: We consider a sample population of 11 non-MGD dry eye
subjects and a control group of 10 normal subjects. Blink information
was obtained using video analysis of each subject viewing a 10
minute nature documentary. Incomplete blinks were tracked by
percent of palpebral fissure closure as ¼, ½ and 3/4. Extended blinks
of multiple seconds were classified per the cutpoints(sec) = (0, 0.1,
1).
Results: Total number of blinks observed for all subjects was 4990
(1414 normal, 3756 dry eye). Of total blinks, 50.6% were incomplete
(dry eye) versus 52% (normal). Dry eye subjects were over 10 times
more likely than normals to exhibit blinks of one second duration or
longer (2.3% vs 0.2% of total respective blinks, p=0.023). Mean lid
closure duration for dry eye subjects was 7.074 (p<.001), 4.274
(p=0.003) and 4.490 (p<.001) times greater than for normals for all
blinks of duration greater than 0 sec, 0.1 sec and 1 sec respectively.
Conclusions: The results suggest that blink duration may play an
important role as a biomarker for dry eye. The relative ease of
tracking blink behavior promises improved tracking of the effects of
treatment and success in clinical trial outcomes.
Commercial Relationships: Ashley M. LaFond, Ora, Inc. (E);
Patrick Johnston, Ora, Inc (E); John D. Rodriguez, Ora, Inc. (E);
Keith J. Lane, Ora, Inc. (E); Endri Angjeli, Ora, Inc. (E)
Program Number: 963 Poster Board Number: B0268
Presentation Time: 1:00 PM - 2:45 PM
Positive- and Negative Regulation of Transcripts Associated with
Formation of Sjögren’s Lesions
Austin K. Mircheff, Yanru Wang. Dept of Physiology & Biophysics,
Univ of Southern California, Los Angeles, CA.
Purpose: Sjögren’s immunopathology involves ectopic lymphoid
tissues that generate autoantigen-specific IgG+ B cells. CCL21
recruits T cells to the lesions; CXCL13 recruits B cells; IL−4 and
IL−10 induce B cell activation and differentiation; and BAFF and
IL−6 support T cell and B cell proliferation. Once formed, the lesions
are self-sustaining.
Methods: Transcript abundances were measured in individual
lacrimal glands from groups of rabbits that had experienced five
different environmental conditions: cool, temperate, hot, dry, and hot
and dry. Transcripts were sorted into clusters on the basis of
deviations from heuristics for quantitative relationships to the
environmental variables and on the basis of strong and significant
correlations.
Results: The abundances of mRNAs for IL−4, IL−6, BAFF, and
IL−10 increased exponentially with increasing dryness. IL−6 and
IL−10 were consistently associated with CTLA-4 and CD8, but IL−4
and BAFF were associated with the T cell surface proteins only
above a threshold of dryness. Each of the four cytokine transcripts
sorted to a different cluster, suggesting that each was expressed by a
different T cell type. Heat augmented BAFF above the exponential
growth heuristic, but in the hot setting BAFF associated with IL−4
inversely (ρ = −0.961, P = 0.038). The combination of heat and
dryness augmented IL−4 above the exponential growth heuristic, and
in that setting BAFF associated with IL−4 positively (ρ = 0.789, P =
0.020). mRNAs for CCL21 and CXCL13 were associated with
mRNAs for CD4, CD3ε, and CD3ζ, but they sorted to different
clusters. CCL21 mRNA increased exponentially with increasing heat.
Dryness suppressed CCL21 below the exponential growth heuristic.
The relationship between CXCL13 and heat was biphasic; it was
suppressed below the exponential growth heuristic in the hot and hot
and dry settings.
Conclusions: Non-diseased lacrimal glands host populations of T
cells expressing transcripts for chemokines and cytokines that may
come to be involved in ectopic lymphoid tissue formation. The
cytokine- and chemokine-expressing cells are regulated differently,
and environmental stresses promoting exponential expansion of one
type or the other would produce quite different immunopathologies.
Negative feedback interactions prevent formation of B cell inductive
tissue. It may be that Sjögren’s pathogenesis depends on abrogation
of the negative regulatory signals.
Commercial Relationships: Austin K. Mircheff, Allergan (C),
Allergan (F); Yanru Wang, Allergan (C)
Support: Unrestricted Grants from Allergan and RPB
Program Number: 964 Poster Board Number: B0269
Presentation Time: 1:00 PM - 2:45 PM
Change of tear meniscus by CL wearing and therapeutic effect of
eye drop for the change
Hitoshi Watanabe, Yukiko Nagahara. Ophtalmology, Kansai Rosai
Hospital, Amagasaki, Japan.
Purpose: Seventy to eighty percent of tear fluid over the ocular
surface is present in upper and lower tear meniscus(TM). We
investigated non-invasively the change of TM height(TMH) before
and after CL wearing by anterior segment OCT and evaluated the
effect of topical application of dry eye drug to improve TMH in CL
wearer.
Methods: The objects are ten healthy normal volunteers; 8 males and
2 females aged from 33 to 50. TMH at the center of lower lid is
determined by anterior segment OCT (CASIA®, Tomey) before and
after wearing one day Acuvue® CL (J&J) . TMH is also investigated
in 5, 15, 30, and 60 minutes after one drop of anti-dry eye drug. As
eye drop, Diquafosol (DQS) is applied to one eye and artificial
tear(AT) to the contralateral eye
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Results: TMH is 2.2±0.4(×100μm) before and is significantly
reduced to 1.3±0.3(×100μm) (59%) after CL wearing(p<0.0001). In
AT treated eyes, TMH is significantly increased up to 15minutes but
up to 60 minutes in DQS treated eyes. Moreover, value of TMH at
15, 30, and 60 minutes after topical application was 67±41, 52±36,
and 34±37μm in DQS treated eyes and 33±21, 8±22, and -1±23μm in
AT treated eyes. The value at each point of DQS treated eyes was
significantly increased compared with that of AT treated eyes (P<
0.04, 0.005 and 0.03, respectively).
Conclusions: Dry eye in CL wearer has some aspect of tear
deficiency by wearing CL and DQS is more effective to treat CL
induced dry eye than artificial tear.
Commercial Relationships: Hitoshi Watanabe, None; Yukiko
Nagahara, None
Program Number: 965 Poster Board Number: B0270
Presentation Time: 1:00 PM - 2:45 PM
Normative Values for the Tear Film of the Rabbit, Dog and
Human
Monica J. Motta1, Peter C. Strom1, Katarzyna Paschalis Trela1,
Andrea Rodrigues2, Christopher J. Murphy1, 3. 1Surgical and
Radiological Sciences, School of Veterinary Medicine, University of
California, Davis, Davis, CA; 2Toxicology, Allergan Inc., Irvine, CA;
3
Ophthalmology & Vision Science, School of Medicine, University
of California, Davis, Davis, CA.
Purpose: The formation and the dynamics of tear films (TFs) are
essential for maintaining the health of the ocular surface.
Abnormalities in the quantity and/or quality of the TF may
compromise these essential functions. The development and
utilization of newer imaging techniques enable more accurate
assessment of the TF than was previously possible. The literature
describing the TF focuses predominantly on the TF of the human
with comparative studies being widely disseminated across diverse
journals and books. TF attributes such as thickness and composition
are poorly documented in the great majority of vertebrate species,
including domestic and lab animal species. The objective of this
review was to create an accessible compilation and comparison of
normative quantitative and qualitative ocular tear film data in 3
different species. The human and two common models in eye
research, the dog and the rabbit.
Methods: Normative data on TF dimensional values, dynamics,
components and properties on all three species were collected
through searching online publication databases, as well as books and
journals available to the authors. The data were then compiled,
organized and evaluated.
Results: Significant differences in TF thickness were identified
between human and rabbit (35-45μm vs. 9.6-10.5μm). There is wide
variation published for human TF thickness (5.1 ± 0.5μm through 3545μm). The values reported varied with measurement technique. We
were unable to find any studies on the entire canine TF thickness but
identified reports on the thickness of individual layers. The thickness
reported for the lipid layer varied widely for both the canine and
human and was technique dependent (0.013-0.586μm; 0.04-0.814μm
reported for the dog and human, respectively). The reported value for
the rabbit was approximately 0.180 μm. Similarly, a range for tear
protein values were reported with values obtained influenced by the
methods used (dog, 2.9-13 g/l; human, 4.21-9.4 g/l; rabbit, 5.4 - 8.2
g/l).
Conclusions: Normative data are essential for providing a basis for
comparison in animal models of tear dysregulation as well as
assessment of animals with spontaneous TF disorders. This review
summarizes the data available in the distributed literature and
highlights the need for complete characterization of the normal TF,
using identical methods in species used for developing and assessing
models of TF disorders.
Commercial Relationships: Monica J. Motta, None; Peter C.
Strom, None; Katarzyna Paschalis Trela, None; Andrea
Rodrigues, None; Christopher J. Murphy, Ocular Services On
Demand (I), Ocular Services On Demand (C), Platypus Technologies
LLC (I), Imbed LLC (I), EyeKor LLC (I), Allergan (C), Genentech
(C), Sarcode (C), Covance (C)
Program Number: 966 Poster Board Number: B0271
Presentation Time: 1:00 PM - 2:45 PM
Intense Pulsed Light as a Treatment for Dry-eye Disease due to
Meibomian Gland Dysfunction
Rolando Toyos. Toyos Clinic, Memphis, TN.
Purpose: To determine the clinical benefits of intense-pulsed-light
therapy for the treatment of
dry-eye disease due to meibomian gland dysfunction.
Methods: A retrospective non-comparative interventional case series
was conducted of 91
patients presenting with severe dry eye syndrome (eligibility tear
breakup time less
than 10 seconds or patient referral). Treatment included intensepulsed-light therapy
and gland expression at a single outpatient clinic over a 30-month
study period
beginning May 2009. Pre/post tear breakup time data were available
for a subset of 78
patients. For all patients, a specially-developed technique for the
treatment of dry eye syndrome was applied as a series of monthly
treatments until adequate improvement in dry eye syndrome
symptoms by physician-judgment or patient-discontinuation.
Results: Primary outcomes included change in tear breakup time by
Oculus Non-Invasive or by
Standard Invasive using Flourescein methods, self-reported patient
satisfaction, and
adverse events.
Physician-judged improvement in dry-eye tear breakup time were
found for 68 of 78
(87%) of patients with 7 treatment visits and 4 maintenance visits on
average
(medians). 93% of patients reported post-treatment satisfaction with
dry eye syndrome
symptoms. Adverse events, most typically redness or swelling, were
found for 13% of
patients. No serious adverse events were found.
Conclusions: While preliminary, study results of intense-pulsed-light
therapy treatment for dry eye
syndrome due to meibomian gland dysfunction are promising. A
multi-site clinical trial
with a larger sample, treatment comparison groups and randomized
controlled trials is
currently underway.
Commercial Relationships: Rolando Toyos, Dermamed (C),
Bausch & Lomb (C), Alcon (C)
Program Number: 967 Poster Board Number: B0272
Presentation Time: 1:00 PM - 2:45 PM
Modeling Blink: Blink Rate or Interblink Interval?
Patrick Johnston1, Lisa Smith1, John D. Rodriguez1, Keith J. Lane1,
George W. Ousler1, Richard Abelson2. 1Ora, Inc., Andover, MA;
2
Statistics & Data Corporation, Tempe, MA.
Purpose: Blink activity is commonly assessed in subjects with dry
eye via blink rate (BR) or interblink interval (IBI). As data the two
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
measurements are equivalent in the sense that one is the reciprocal of
the other (a subject with N blinks over time period T has a BR of N/T
and an average IBI of T/N). As means, however, they are not
equivalent since the reciprocal of mean BR does not equal the mean
of IBI, and this raises the question of preference.
We consider two aspects. First, if a particular blink measurement is
desired (BR or IBI), we assess the merits of normal versus gamma
models. Second, in cases where an investigator is interested in blink
frequency but is not committed to a particular measurement, we
propose a novel method to compare the appropriateness of the two
measurements.
Methods: For a particular measurement (BR or IBI), two-mean
models were fit assuming both normal and gamma distributions and
these were compared using the Akaike information criterion (AIC).
Differences in AIC compare the relative goodness of fit of two
statistical models, the model with the lower AIC being preferred.
Importantly (because normal and gamma models are not special cases
of each other), models compared by AIC need not be nested.
BR and IBI cannot be compared directly because comparisons of AIC
require models with identical outcomes. However, the functional
relationship between BR and IBI permits comparison based on a
generalization of the Box-Cox transformation method. For example,
estimates with outcome IBI = 1/BR using a gamma model are
identical to estimates based on BR using an inverse gamma model,
and it is the latter model that provides the appropriate AIC for
comparison.
Results: The gamma model was preferred to the normal model for
BR by 10 units of AIC, and for IBI by 7 units of AIC. In cases where
either measurement is allowed to represent blink frequency, BR was
preferred to IBI by 4 units of AIC (in both cases using a gamma
model).
Conclusions: It is not uncommon for t-tests to be used in the analysis
of BR and IBI, and while these tests are optimal under normality,
they are not optimal under the superior gamma approximation
indicated by this study. In addition, we have proposed a novel
method to compare BR and IBI, in effect selecting the preferred scale
on which to measure blink frequency. In the present study BR was
preferred to IBI.
Commercial Relationships: Patrick Johnston, Ora, Inc (E); Lisa
Smith, Ora, Inc. (E); John D. Rodriguez, Ora, Inc. (E); Keith J.
Lane, Ora, Inc. (E); George W. Ousler, Ora, Inc. (E); Richard
Abelson, Statistics & Data Corporation (E)
Program Number: 968 Poster Board Number: B0273
Presentation Time: 1:00 PM - 2:45 PM
Zone A (ZA), Posterior Lid Margin Vascularization: An Early
Sign of Ocular Surface Disease (OSD)
Alexander Gan1, Claudia M. Prospero Ponce2, 1, Andrew M. Quinn1,
Patricia Chevez-Barrios2, Alice Z. Chuang1, Richard W. Yee1. 1The
Robert Cizik Eye Clinic.The Richard S. Ruiz Department of
Ophthalmology and Visual Science. University of Texas Health
Science Center at Houston., Houston, TX; 2Ocular Pathology,
Department of Pathology and Genomic Medicine., The Methodist
Hospital, Houston, TX.
Purpose: To analyze the histologic, clinical, and morphological
characteristics of the avascular zone of the inferior posterior lid
margin and its possible clinical significance as a sign of early and late
chronic inflammation in OSD.
Methods: Retrospective chart review of the OSD findings and the
Ocular Surface Disease Index (OSDI) questionnaire were performed
in 49 patients(pts), >20 yrs old seen in a tertiary care center. Inclusion
criteria: pts with a complete ocular surface evaluation including
anterior blepharitis(AB), vascularization(V) of the inferior lid
margin, meibomian gland obstruction(O) and turbidity(T). Basal tear
test(BTT) and lissamine green staining(LGS) and quantification of
the ZA was graded based on the degree of V noted on the everted
posterior inferior lid margin. Exclusion criteria: previous surgery or
on topical anti-inflammatory treatment. OSDI scores were divided
into 2 groups (normal:≤12vs dry eye:>12), ZA was divided into 2
groups normal and severe and were compared to OSD signs using
chi2test. Lower lid biopsy was obtained for histology.
Results: Forty-nine charts were reviewed and analyzed, 14 patients
had normal OSDI and 35 had dry eye OSDI. There was no significant
statistical difference between OSDI groups and all OSD findings
[AB,p=0.08;V,p=0.8;O,p=0.05;T,p=0.7;ZA,p=0.7(77.1%dry eye
OSDI vs 85.7% normal OSDI). LGs and BTT were not statistically
different between the two OSDI groups (p>0.5, p>0.1). Comparing
the ZA groups, 10 pts had normal grading, 39 pts had severe grading.
No significant statistical differences were found between ZA groups
and OSD findings (AB,p=0.6;V,p=0.2;O,p=0.9;T, p=1.0). LGS and
BTT were not statistically different between the ZA group. Patients
with severe ZA grading were found to have normal-mild OSD
findings (AB=84.2%, V=82.1%, O=56.4%, T= 32.4%), in contrast to
patients with severe ZA grading that had severe OSD
(AB=15.8%,V=18.0%,O=43.6%,T=67.6%). Histology showed
inflammatory response and increased number of dilated vessels in the
posterior lid margin.
Conclusions: The OSDI questionnaire did not correlate with any
OSD clinical signs. ZA grading was noted to be severe even in our
cohort with mild disease. ZA vascularization may be a helpful
clinical sign of early OSD.
Commercial Relationships: Alexander Gan, None; Claudia M.
Prospero Ponce, None; Andrew M. Quinn, None; Patricia
Chevez-Barrios, None; Alice Z. Chuang, None; Richard W. Yee,
MTTI (P), Allergan (R)
Program Number: 969 Poster Board Number: B0274
Presentation Time: 1:00 PM - 2:45 PM
Is there a relationship between ocular discomfort and circulating
plasma levels of sex hormones? Preliminary findings
Blanka Golebiowski1, Ulrike Hampel1, 2, Noor Badarudin1, Isabelle
Jalbert1, Michele C. Madigan1, Fiona Stapleton1. 1School of
Optometry and Vision Science, University of New South Wales,
Sydney, NSW, Australia; 2Department of Anatomy 2, Friedrich
Alexander University Erlangen Nürnberg, Erlangen, Germany.
Purpose: Dry eye is a common problem especially for women postmenopause. This study explored the relationship between circulating
levels of plasma sex hormones and ocular dry eye symptoms.
Methods: A cross-sectional, single visit study was conducted. The
study involved a convenience sample of 74 subjects without ocular
surface disease, including 52 females (mean age 35.3±13.4years,
range 18.8-70.3) and 22 males (mean age 34.2±13.8years, range
20.2-75.3). Subjects completed the Dry Eye Questionnaire (DEQ5)
and numerical ratings of discomfort, dryness, foreign body (FB)
sensation, burning and watering. Tear osmolarity (TearLab) and
volume (Phenol Red Thread) were assessed. Venous blood was
collected and plasma concentrations of oestradiol (E2) and total
testosterone (TT) were determined using specific Enzyme-linked
immunosorbent assay. Associations were examined using Pearson’s
or Spearman’s correlations, and differences between groups were
assessed using Independent samples t-test or Mann-Whitney U test,
as appropriate.
Results: Mean group E2 concentration was 65.2±50.9pg/ml in
females and 40.7±23.8pg/ml in males; TT concentration was
0.49±0.29 and 4.3±1.6ng/ml respectively. All symptoms measures
were higher in females (p<0.05). Tear volume was reduced in
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
females (p=0.02); there was no difference in tear osmolarity. In
females, increased ocular symptoms correlated with higher levels of
E2 (DEQ5 Rho=0.36, p=0.01; dryness Rho=0.36, p=0.01; FB
Rho=0.37, p=0.01). Higher TT in females correlated with more FB
sensation (Rho=0.30, p=0.03) and lower tear volume (Rho=-0.30,
p=0.04). No association was found between tear osmolarity and
hormone levels in females. In males, no evidence of a relationship
between hormone levels and ocular symptoms or tear parameters was
apparent. Although concentrations of E2 and TT were reduced with
age in females (E2 Rho=-0.36, p=0.01; TT Rho=-0.37, p=0.01), there
was no association between age and ocular symptoms in either males
or females.
Conclusions: Higher circulating levels of sex steroid hormones
appear to play a role in increased symptoms of dry eye in females
without ocular surface disease, but not in males. This effect does not
appear to be influenced by age. More detailed analysis and
exploration of co-related factors such as levels of free testosterone is
warranted to further explore these relationships in the
pathophysiology of dry eye.
Commercial Relationships: Blanka Golebiowski, TearLab (F);
Ulrike Hampel, None; Noor Badarudin, None; Isabelle Jalbert,
Blackmores Limited (F), TearLab Corporation (F); Michele C.
Madigan, None; Fiona Stapleton, None
Program Number: 970 Poster Board Number: B0275
Presentation Time: 1:00 PM - 2:45 PM
Correlation of Tear Meniscus Dimensions with Clinical
Parameters of Ocular Surface Disease in Subgroups of Dry Eye
Cynthia I. Tung1, 2, Andrew F. Perin1, Koray Gumus1, Stephen C.
Pflugfelder1. 1Ophthalmology, Baylor College of Medicine, Houston,
TX; 2Ophthalmology, University of Texas Medical Branch,
Galveston, TX.
Purpose: Evaluate the relationship between tear meniscus
dimensions and parameters of ocular surface disease in subcategories
of dry eye.
Methods: Prospective analysis comparing tear meniscus dimensions
to clinical ocular surface parameters was performed for 128 eyes
from 64 subjects. Tear meniscus height (TMH) and tear meniscus
area (TMA) were measured using optical coherence tomography
(Optovue). Ocular surface parameters included tear break-up time
(TBUT), corneal staining, conjunctival staining, and Ocular Surface
Disease Index (OSDI). Study groups included meibomian gland
dysfunction (MGD) (n=23; OSDI>20; TBUT<=7); aqueous tear
deficient dry eye (ATDDE) (n=34; OSDI>20; TBUT<=7; Schirmer
I<10); autoimmune disease (AD) including Sjogren's syndrome
(n=16; OSDI>20; TBUT<=7; Schirmer I<10); and normal agematched controls (n=34; OSDI<=20; TBUT>7; Schirmer I>=10).
Statistical analysis was performed using Pearson's correlation and
Student's t-test.
Results: In comparing TMH with corneal staining, correlation was
moderately negative for all comers (R=-0.34; p=0.013) and all dry
eyes (R=-0.31; p=0.015), strongly positive for MGD eyes (R=+0.40;
p=0.059), moderately negative for ATDDE eyes (R=-0.36; p=0.04),
and weakly negative for AD eyes (R=-0.16; p>0.05). Comparison of
TMA with corneal staining showed similar trends with strong
correlation in MGD eyes (R=+0.55; p=0.006), in ATDDE eyes (R=0.40; p=0.018), and in AD eyes (R=-0.43; p>0.05). In comparing
TMH with TBUT, correlation was strongly positive for all comers
(R=+0.41; p<0.0001) and all dry eyes (R=+0.42; p<0.0001), weakly
negative for MGD eyes (R=-0.17; p>0.05), moderately positive for
ATDDE eyes (R=+0.37; p=0.018), and very weakly positive for AD
eyes (R=+0.044, p>0.05). Correlations of tear meniscus dimensions
(TMH, TMA) to conjunctival staining and OSDI in any group were
non-significant. Mean TMH was higher in age-matched controls (344
µm) compared to all dry eyes (234 µm; p=0.012), MGD eyes (302
µm; p>0.05), ATDDE eyes (210 µm; p=0.003), and AD eyes (172
µm; p<0.001).
Conclusions: In ATDDE and AD subjects, lower tear volume is
associated with worse corneal epithelial disease. In MGD subjects,
higher tear volume is associated with corneal epithelial disease,
perhaps due to altered tear composition.
Commercial Relationships: Cynthia I. Tung, None; Andrew F.
Perin, None; Koray Gumus, None; Stephen C. Pflugfelder,
Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb (C), Baylor
College of Medicine (P)
Support: NIH Grant EY11915 (SCP), Research to Prevent
Blindness, NIH Core Grant P30-EY002520, Oshman Foundation;
William Stamps Farish Fund; Hamill Foundation
Program Number: 971 Poster Board Number: B0276
Presentation Time: 1:00 PM - 2:45 PM
Upregulation of chemokine expression in experimental dry eye
requires IL-17A and IFN-γ but not T cells
Terry G. Coursey, Stephen C. Pflugfelder, Cintia S. De Paiva.
Ophthalmology, Ocular Surface Center, Baylor College of Medicine,
Houston, TX.
Purpose: The chemokines CCL20 (CCR6 ligand), and CXCL-9, -10,
-11 (CXCR3 ligands) coordinates migration of CCR6+Th17 cells and
CXCR3+Th1 cells, respectively. Our previous studies have
demonstrated the requirement of Th17 and Th1 cells in the
pathogenesis of dry eye disease. The objective of this study was to
evaluate the role of T cells (specifically Th1 and Th17 cells) in the
upregulation of chemokine expression in an experimental model of
dry eye.
Methods: Desiccating stress (DS) was induced by subcutaneous
injection of scopolamine and exposure to a drafty low humidity
environment in RAG1KO, IL-17KO, IFN-γKO and wild-type (WT)
mice, aged 6-8 weeks for 5 or 10 days (DS5, DS10) or were not
treated. CCL20, CXCL-9, CXCL-10, and CXCL-11 expression in the
cornea and conjunctiva were evaluated by real time quantitative PCR.
Results: Significant upregulation of CCL20 was observed in WT (>
30 fold) and RAG1KO (>7 fold) mice, however it was not
upregulated in IFN-γKO mice and was significantly reduced in IL17AKO mice (~2 fold). Similarly, CXCR3 ligands CXCL10 and -11
expression increased in WT (CXCL10- ~2 fold; CXCL11- ~2 fold)
and RAG1KO (CXCL10- ~ 2 fold; CXCL11 ~ 2 fold) mice but not
IFN-γKO or IL-17KO mice. CXCL9 was upregulated in WT mice
(~5 fold) but not T cell deficient, IFN-γKO, or IL-17KO mice.
Conclusions: Upregulation of chemokine expression in experimental
dry eye requires IL-17A and IFN-γ , but not T cells. Thus, exposure
to DS induces the production of IL-17 and IFN-γ independent of the
adaptive immune response.
Commercial Relationships: Terry G. Coursey, None; Stephen C.
Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb
(C), Baylor College of Medicine (P); Cintia S. De Paiva, Glaxo
Smith Kline (C), Baylor College of Medicine (P)
Support: EY11915 (SCP), Research to Prevent Blindness, the
Oshman Foundation, William Stamps Farish Fund and the Hamill
Foundation
Program Number: 972 Poster Board Number: B0277
Presentation Time: 1:00 PM - 2:45 PM
Differential effect of Th1- and Th2-type cytokines on rat
conjunctival goblet cell function
Laura Garcia-Posadas1, 2, Dayu Li3, 4, Robin R. Hodges3, 4, Marie A.
Shatos3, 4, Yolanda Diebold1, 2, Darlene A. Dartt3, 4. 1Ocular Surface
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Group, IOBA-University of Valladolid, Valladolid, Spain;
2
Networking Research Center on Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Valladolid, Spain; 3Schepens Eye
Research Institute/Massachusetts Eye and Ear, Boston, MA;
4
Department of Ophthalmology, Harvard Medical School, Boston,
MA.
Purpose: To determine if Th1 and Th2 pro-inflammatory cytokines
affect cultured rat conjunctival goblet cells and to measure their
effects on proliferation, intracellular calcium levels ([Ca2+]i) and high
molecular weight glycoconjugate secretion that includes MUC5AC.
Methods: Rat conjunctival goblet cells were grown from tissue
explants. Passage 1 cells were cultured in supplemented RPMI
medium. The expression of goblet (CK-7) and stratified squamous
(CK-4) specific cell markers was analyzed by immunofluorescence
and the lectin UEA-1 was used to identify goblet cell secretory
products. To evaluate proliferation cells were serum starved for 24 h,
treated with the cytokines IFN-γ (3ng/ml), IL-4 (10ng/ml), IL-5
(10ng/ml) or IL-13 (10ng/ml) for 24 h and then stimulated with the
cholinergic agonist carbachol (Cch) or the allergic mediator
histamine for 2 h. Proliferation was measured by WST-8 assay (n=3).
To measure [Ca2+]i, cells were loaded with fura2 and analyzed using
InCyte Im2TM Ratio Imaging System. The effect of Cch and
histamine on [Ca2+]i was studied in cells treated for 15 min or 24 h
with cytokines or buffer (n=5). High molecular weight
glycoconjugate secretion was measured in supernatants from
untreated and 24 h cytokine-treated cells using an enzyme-linked
lectin assay (n=3).
Results: Rat cultured cells expressed goblet cells markers (CK-7 and
secretory products identified by UEA-1), but not stratified epithelial
marker (CK-4). The Th1 cytokine IFN-γ decreased goblet cell
proliferation by 0.79 fold, whereas Th2 cytokines IL-4, IL-5 and IL13, and histamine increased it by 1.94, 2.65, 2.89 and 2.46 fold
respectively. IFN-γ used for 15 min significantly blocked Cchmediated increase in [Ca2+]i (p = 0.009), and IL-4 and IL-13 had the
same effect on histamine-mediated [Ca2+]i increase after 15 min preincubation (p=0.006 and p=0.003, respectively). When cells were
incubated with cytokines for 24 h, only IL-13 maintained the
blockade (p=0.037). IFN-γ significantly blocked Cch-induced
secretion (p=0.002), but Th2 cytokines did not have significant
effects on histamine-induced secretion.
Conclusions: Th1- (IFN-γ) and Th2- (IL-4, IL-5 and IL-13) derived
cytokines have opposite effects on proliferation and secretion from
cultured rat goblet cells. These findings could explain the differences
in goblet cell function found in inflammatory ocular surface diseases,
such as dry eye and allergic conjunctivitis.
Commercial Relationships: Laura Garcia-Posadas, None; Dayu
Li, None; Robin R. Hodges, None; Marie A. Shatos, None;
Yolanda Diebold, None; Darlene A. Dartt, None
Support: FEDER-CICYT Grant MAT2010-20452-CO3-01 and FPI
Scholarship Program BES-2011-046381 and EEBB-I-12-05371
(Ministry of Science and Innovation, Spain), and Regional JCyL
Grant VA132A11-2. NIH EY019470.
Purpose: Benzalkonium chloride (BAK) is the most commonly
found preservative in eye drops, and has been shown to cause ocular
surface inflammation and toxicity. Lacritin is a human tear
glycoprotein secreted from the lacrimal glands that has been found to
be cytoprotective. This study was designed to determine if the
prosecretory and mitogenic properties of lacritin confer protection to
a cultured human corneal epithelial (HCE) cell line, CRL-11515, and
primary HCE cells after exposure to the ocular preservative agent
BAK.
Methods: Recombinant human lacritin and negative control fragment
C-25 were cloned into intein fusion vectors, expressed in E. coli, and
purified on chitin beads and DEAE Sepharose. Metabolic curves
were established after exposure of subconfluent CRL-11515 cells to
BAK or lacritin. Western blot analysis of lipidated LC3 (LC3-II)
provided a measure of autophagy in CRL-11515 cells exposed to
lacritin and/or BAK.
Results: BAK reduced CRL-11515 cellular metabolic activity in a
time and dose dependent manner. BAK-induced cellular stress was
evident by elevated autophagy that increased with rising
concentrations of BAK compared to control (P<0.05). Lacritin
increased HCE cell proliferation at an optimal dose of 1 nM.
Preconditioning HCE cells with 1 nM lacritin for 24 hours prior to
BAK exposure significantly dampened levels of LC3-II (P<0.05) and
promoted a 12% increase in cellular metabolic activity (P<0.01)
when compared to BAK alone.
Conclusions: These results suggest lacritin protects cultured HCE
cells stressed with BAK and it may have the potential to be used as a
topical adjunctive therapy in eyes chronically exposed to BAK.
BAK treatment of CRL-11515 cells increases autophagy. (A) CRL11515 cells treated with BAK (0.001%, 0.004%) for 1 minute
increased cellular lipidated LC3, known as ‘LC3-II‘ with increasing
concentration of BAK (**P<0.01, *P<0.05; n=6). (B) Western blot of
LC3-II with GAPDH as loading control.
Program Number: 973 Poster Board Number: B0278
Presentation Time: 1:00 PM - 2:45 PM
Cytoprotective effect of lacritin on human corneal epithelial cells
exposed to benzalkonium chloride
Mary M. Feng1, Julia Baryla1, Hong Liu1, Gordon W. Laurie2, Robert
L. McKown3, Negin Ashki1, Dinesh Bhayana1, Cindy M. Hutnik1.
1
Department of Ophthalmology, University of Western Ontario,
London, ON, Canada; 2Department of Cell Biology, University of
Virginia, Charlottesville, VA; 3Department of Integrated Science and
Technology, James Madison University, Harrisonburg, VA.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Lacritin treatment of CRL-11515 cells rescues BAK induced
autophagy. (A) LC3-II increased in CRL-11515 cells treated with
BAK (0.004%) compared to control (**P<0.01 vs. control; n=5).
L24h/BAK significantly reduced LC3-II compared to BAK treated
cells (†P<0.05 vs. BAK; n=5). (B) Western Blot of LC3-II with
GAPDH as loading control. BAK = BAK, 1 min. BAK+L = BAK
and lacritin, 1 min. L24h/BAK = Pre-incubation with lacritin, 24
hours, followed by BAK treatment for 1 min.
Commercial Relationships: Mary M. Feng, None; Julia Baryla,
None; Hong Liu, None; Gordon W. Laurie, UVa Patent Foundation
(F); Robert L. McKown, EyeRx Research, Inc. (I); Negin Ashki,
None; Dinesh Bhayana, None; Cindy M. Hutnik, None
Support: Glaucoma Research Society Grant
Program Number: 974 Poster Board Number: B0279
Presentation Time: 1:00 PM - 2:45 PM
Functional characterization of the glucocorticoid receptor in the
cornea
Mahita Kadmiel, John A. Cidlowski. Laboratory of Signal
Transduction, National Institute of Environmental Health Sciences,
Durham, NC.
Purpose: Glucocorticoids have long been in use for the treatment of
ophthalmic diseases for their anti-inflammatory and anti-angiogenic
effects. However, chronic use of glucocorticoids can cause serious
side effects. For example, glucocorticoids are associated with poor
prognosis in wound healing. Nevertheless, the underlying
mechanisms for this complication are poorly understood. The
purpose of this investigation is to determine the precise function of
glucocorticoid receptor (GR) signaling in corneal epithelial wound
healing.
Methods: For nuclear trafficking experiments, Human corneal
epithelial cells (HCE-T) cells were stimulated with 100nM
dexamethasone (Dex; synthetic glucocorticoid) or vehicle (Veh) for 1
hour, fixed, incubated overnight with hGRα antibody and DAPI, and
imaged. For gene expression profiling, HCE-T were treated with
100nM Dex or Veh for 6 hours. Total RNA was extracted, DNasetreated and subjected to whole-genome expression profiling using the
Agilent 4x44 arrays. For in vitro wound healing assays, HCE-T cells
were pre-treated with 100nM Dex or Veh for 24hours. A scratchwound was made and imaged at time zero and after 24hours. Images
were analyzed using Image J software.
Results: Human corneal epithelial cells expressed the glucocorticoid
receptor, which trafficked to the nucleus upon stimulation by Dex.
Microarray analysis in HCE-T cells revealed that glucocorticoids
significantly regulated 2905 probes. Ingenuity Pathway Analysis of
the data identified cell development, movement, morphology and
cell-to-cell signaling as the top significant glucocorticoid-regulated
biological functions. Moreover, in vitro wound healing assays with
HCE-T cells exhibited a remarkable delay in wound healing in cells
treated with Dex. This effect of Dex was inhibited by the GR
antagonist RU486, indicating that the Dex-induced delay in wound
healing is mediated through GR.
Conclusions: Glucocorticoids significantly regulate gene expression
in human corneal epithelial cells. One of the major functions of
glucocorticoids in the corneal epithelium is to regulate wound
healing. The goal of this project is to dissect the molecular
mechanism involved in glucocorticoid/GR-mediated delay in wound
healing. Furthermore, we are generating a mouse model with corneaspecific deletion of GR to elucidate the in vivo role of GR in the
cornea during embryonic and postnatal development.
Commercial Relationships: Mahita Kadmiel, None; John A.
Cidlowski, None
Support: NIH/NIEHS Intramural Research Funding
Program Number: 975 Poster Board Number: B0280
Presentation Time: 1:00 PM - 2:45 PM
Reliability of Oculus Keratograph Meibomian Gland
Assessments
Stephanie Cox1, Catherine Vuong1, Lisa Jones-Jordan2, Kelly K.
Nichols1, Jason J. Nichols1. 1College of Optometry, University of
Houston, Houston, TX; 2College of Optometry, The Ohio State
University, Columbus, OH.
Purpose: The aims of this study were to evaluate the intra-examiner
and inter-examiner reliability of the Gestalt, meiboscore, and
meibomian gland counting grading scales associated with
meibography.
Methods: Sixty subjects were recruited for two study visits separated
by 7 ± 2 days. At each visit, the Oculus Keratograph 4 was used to
obtain a non-invasive break up time (NIK-BUT) and infrared
meibography pictures of each inferior lid’s meibomian glands,
amongst other tests. The meibography images were duplicated, and
each set was randomized and graded by two masked graders using
the gestalt, meiboscore, and meibomian gland count grading scales.
The results were analyzed using kappa and weighted kappa values for
the categorical scales of gestalt and meiboscore. Intraclass correlation
coefficient (ICC) was used to analyze the continuous scale of
meibomian gland counting.
Results: Kappa values for intra-examiner reliability of the same
picture suggest fair to moderate agreement (K = 0.25 - 0.60, Kw =
0.40 - 0.68) when using gestalt grading and fair to substantial
agreement (K = 0.32 - 0.63, Kw = 0.52 - 0.69) when using
meiboscore grading. The ICC to assess intra-examiner reliability
using meibomian gland counting suggests moderate to almost perfect
agreement (ICC = 0.58-0.85). The kappa values of the intra-examiner
reliability of two pictures taken of the same lid at different visits
suggest slight to moderate agreement (K = 0.11 - 0.47, Kw = 0.21 0.57) when using gestalt grading and slight to substantial agreement
(K = 0.15 - 0.56, Kw = 0.21 - 0.63) when using meiboscore grading.
The ICCs to assess intra-examiner reliability across two pictures of
the same lid using meibomian gland counting suggest fair to strong
agreement (ICC = 0.31 - 0.65). The kappa values used to assess interexaminer reliability suggest no better than chance to light agreement
(K = -0.03 - 0.03, Kw = 0.07 - 0.09) when using gestalt grading. The
ICC to assess inter-examiner reliability using meibomian gland
counting suggests moderate agreement (ICC = 0.46).
Conclusions: The Oculus Keratograph provides qualitative, gradable
meibomian gland images. Meibomian gland counting may be the
most reliable way to grade meibography when considering intra- and
inter-examiner grading followed by meiboscore and gestalt. The
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
individual examiner reliability suggests that experience is an
important factor in reliability.
Commercial Relationships: Stephanie Cox, None; Catherine
Vuong, None; Lisa Jones-Jordan, None; Kelly K. Nichols, None;
Jason J. Nichols, Vistakon (R), Vistakon (F), Alcon (R), Alcon (F),
Bausch and Lomb (R)
Support: NIH/NEI grant R01 EY015519, NIH/NEI T35 EY007088
Program Number: 976 Poster Board Number: B0281
Presentation Time: 1:00 PM - 2:45 PM
Matrix Metalloproteinases-8, -9 and Myeloperoxidase are
Elevated in the Tears of Patients with Ocular Cicatricial
Pemphigoid and Stevens-Johnson Syndrome
Samer N. Arafat1, 2, Ana M. Suelves3, Sandra J. Spurr-Michaud2,
James Chodosh1, C. Stephen Foster3, Claes H. Dohlman1, Ilene K.
Gipson2. 1Massachusetts Eye and Ear Infirmary, Boston, MA;
2
Schepens Eye Research Institute, Massachusetts Eye and Ear
Infirmary, Boston, MA; 3Massachusetts Eye Research and Surgery
Institution, Cambridge, MA.
Purpose: To determine levels of matrix metalloproteinases (MMPs),
tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and
inflammatory marker myeloperoxidase (MPO) in the tears of patients
with ocular cicatricial pemphigoid (OCP) and Stevens-Johnson
syndrome (SJS).
Methods: Tears were collected from 7 SJS, 38 OCP and 40 postcataract (control) eyes. 60μL of sterile balanced salt solution was
instilled on each eye and tears washes were recovered with a
micropipette. The tear concentrations of MMP-2, -3, -7, -8, -9 and 12 (ng/mL) in each eye were measured using a Fluorokine® Human
MMP MultiAnalyte Profiling (MAP) Kit. TIMP-1 and MPO
concentrations (ng/mL) were measured using a Fluorokine® human
Cardiac B MAP Kit. Both assays were run on a Bio-Rad Bio-Plex
analyzer powered by Luminex® technology. Total MMP activity was
measured fluorometrically using an OmniMMP™ RED fluorogenic
substrate. Values for all assays were standardized to the protein mass
loaded in each assay.
Results: The concentrations (ng/μg total protein) of MMP-8, -9 and
MPO were statistically higher in tears of patients with SJS (4.3 ± 2.0,
10.2 ± 4.5 and 6.7 ± 2.0, respectively) than in OCP (0.4 ± 0.2, 0.9 ±
0.4 and 1.5 ± 0.4, respectively), and both were significantly higher
than in control (0.01 ± 0.003, 0.06 ± 0.01 and 0.4 ± 0.1, respectively).
The ratio of MMP-8/TIMP-1 and MMP-9/TIMP-1 were statistically
higher in SJS (7.4 ± 3.4 and 16.2 ± 6.6, respectively) than in OCP
(1.1 ± 0.6 and 4.6 ± 2.5, respectively) and both were significantly
higher than in control (0.01 ± 0.003 and 0.05 ± 0.01, respectively).
The total MMP specific activity (RFU/min/μg protein) was higher in
SJS (25.02 ± 10.60) than in both OCP (4.75 ± 2.73) and control (1.25
± 0.87). Spearman rank correlation tests showed significant
correlations between MMP-8 and MMP-9, and between MMP-9 and
MPO across all groups. MMP-8 correlated with MPO in control and
OCP patients (p < 0.0001), and to a lesser extent with SJS (p =
0.066).
Conclusions: Since activated neutrophils are known to be a source of
MMP-8, -9 and MPO, the high levels of these enzymes in the tears of
SJS and OCP patients and the strong correlation between MMP-8,
MMP-9 with MPO suggest that inflammatory cells are the primary
source of the elevated enzymes. In addition to MMP-8 and MMP-9,
MPO was found to be a marker of inflammatory ocular surface
disease.
Commercial Relationships: Samer N. Arafat, None; Ana M.
Suelves, None; Sandra J. Spurr-Michaud, None; James Chodosh,
Alcon (C), Allergan (C), 3-V Biosciences (C), Novabay (C); C.
Stephen Foster, Abbott Medical Optics (C), Abbott Medical Optics
(F), Alcon Laboratories, Inc. (C), Alcon Laboratories, Inc. (F),
Allergan, Inc. (C), Allergan, Inc. (F), Eyegate Pharmaceuticals, Inc.
(I), Eyegate Pharmaceuticals, Inc. (F), IOP Opthalmics (C), Ista
Pharmaceuticals (C), Lux Biosciences, Inc. (C), Lux Biosciences,
Inc. (F), Novartis Pharmaceuticals Corporation (C), Novartis
Pharmaceuticals Corporation (F), XOMA Ltd (C); Claes H.
Dohlman, None; Ilene K. Gipson, None
Support: Boston Keratoprosthesis Fund (MEEI), NEI RO1 03306 to
IKG
Program Number: 977 Poster Board Number: B0282
Presentation Time: 1:00 PM - 2:45 PM
Autologous Serum Tears for Treatment of Dry Eye Syndrome in
Graft Versus Host Disease
Ryan J. Fante, Roni M. Shtein, Shahzad I. Mian, Munira Hussain.
Ophthalmology and Visual Sciences, UM Kellogg Eye Center, Ann
Arbor, MI.
Purpose: To evaluate the effectiveness of autologous serum tears for
treatment of severe dry eye syndrome (DES) in patients with Graft
Versus Host Disease (GVHD).
Methods: Retrospective review of 30 eyes of 15 patients with GVHD
and severe DES was performed at baseline and after initiating
treatment with autologous serum tears. Severity of ocular surface
disease was evaluated using the Schirmer test, fluorescein staining
and Ocular Surface Disease Index (OSDI). Paired t-tests were
performed to compare baseline assessment to post-treatment scores.
Results: The study population included 15 total patients, 7 female
and 8 male; the average age was 54 years (s.d =9, range 40-67).
Follow-up duration averaged 11 months (range 1-40), and 60% were
concurrently using the Prosthetic Replacement of Ocular Surface
Environment (PROSE) lens. Patients had an average of 3 blood
draws (s.d.=2, range: 1-8) for serum tears in this time period.
There was a statistically-significant improvement in OSDI scores at 1
month and final time-points compared to pre-treatment (baseline:
59.7 +/- 27.0; 1 month: 42.5 +/- 22.1, p=0.03; 3 month: 49.5 +/-37.0,
p=0.07; final: 40.1 +/- 26.7, p=<0.001). Schirmer score significantly
improved at 1 month compared to pre-treatment (baseline: 2.6 +/3.1; 1 month: 5.0 +/-4.0, p=0.01). Schirmer scores did not
significantly differ at 3 month and final time points (3 month: 4.7 +/5.9; final: 4.6 +/- 6.5). Fluorescein staining score did not significantly
differ from pre-treatment at any time points (baseline: 2.3 +/- 1.1; 1
month: 2.5 +/- 1.5; 3 month: 1.2 +/- 1.5; final: 1.9 +/- 1.3).
Conclusions: Autologous serum tears are an effective treatment for
DES in patients with GVHD. Additional studies are needed to
compare the effectiveness of autologous serum tears to other
treatments for patients with severe DES from GVHD.
Commercial Relationships: Ryan J. Fante, None; Roni M. Shtein,
None; Shahzad I. Mian, None; Munira Hussain, None
Program Number: 978 Poster Board Number: B0283
Presentation Time: 1:00 PM - 2:45 PM
Autologous Serum Tears May Result in Subclinical Changes in
Corneal Immunity: An In Vivo Confocal Microscopy Study
Ahmad Kheirkhah1, Shruti Aggarwal1, Bernardo M. Cavalcanti1,
Deborah Witkin1, Nadia Wong1, Monique Trinidad1, Candice
Williams1, Reza Dana2, Pedram Hamrah1, 2. 1Ocular Surface Imaging
Center, Department of Ophthalmology, Massachusetts Eye and Ear
Infirmary, Harvard Medical School, Boston, MA; 2Cornea and
Refractive Surgery Service, Massachusetts Eye and Ear Infirmary,
Harvard Medical School, Boston, MA.
Purpose: To evaluate the effect of autologous serum tears treatment
on corneal immune cells including dendritic cell (DC) density with
laser in vivo confocal microscopy (IVCM).
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Methods: This retrospective study included 23 patients who received
20% autologous serum tears (4-8 x/day), were on concurrent antiinflammatory treatment, and were followed with serial laser IVCM.
Only patients were included who did not have any change in antiinflammatory regimen during the follow-up period. Three IVCM
images of one randomly selected eye for each patient were analyzed
by two masked observers for DC density. Images were analyzed for
baseline, as well as 3 and 6 months after treatment with serum tears,
and compared to 23 age-matched control subjects. Student’s t-test
and ANOVA were used for analysis.
Results: Nineteen female and 4 males with a mean age of 53.3 ±
18.92 years (range, 19-97 years) were included. The indications for
using serum tears were dry eye disease (20 eyes) and keratoneuralgia
(3 eyes). At baseline, DC density was significantly higher in patients
with dry eye disease (55.8 ± 9.9 cells/mm2, P=0.01), but not in
keratoneuralgia (40.3 ± 18.4, P=0.37), compared to the control group
(19.1± 3.1). DC density increased from 53.8 ± 8.9 cells/mm2 before
treatment, to 103.1 ± 22.4 at 3 months (191% increase, P=0.04) and
to 165.1 ± 57.8 at 6 months (307% increase, P=0.06).
Conclusions: Treatment with autologous serum tears may be
associated with increased density of corneal epithelial DC in patients
with concurrent anti-inflammatory treatment, demonstrating changes
in corneal immunity. Additional prospective clinical trials are
required to confirm the data and determine clinical implications.
Commercial Relationships: Ahmad Kheirkhah, None; Shruti
Aggarwal, None; Bernardo M. Cavalcanti, None; Deborah
Witkin, None; Nadia Wong, None; Monique Trinidad, None;
Candice Williams, None; Reza Dana, Allergan (C), Alcon (C),
Bausch & Lomb (C), Eleven Bio (I), GSK (F), Novabay (C),
Revision Optics (C), Novaliq (C), RIgel (F); Pedram Hamrah, None
Support: NIH K08-EY020575, Research to Prevent Blindness
Career Development Award, Falk Medical Research Trust, New
England Corneal Transplant Research Fund
140 Cell and Molecular Biology and Stem Cells
Sunday, May 05, 2013 1:00 PM-2:45 PM
Exhibit Hall Poster Session
Program #/Board # Range: 979-1023/B0284-B0328
Organizing Section: Cornea
Program Number: 979 Poster Board Number: B0284
Presentation Time: 1:00 PM - 2:45 PM
Human Adipose-Derived Stem Cells Support the Growth of
Limbal Stem/progenitor Cells
Hua Mei, Martin N. Nakatsu, SHEYLA GONZALEZ, Elfren R.
Baclagon, Sophie X. Deng. Ophthalmology, Jules Stein Eye Institute,
UCLA, Los Angeles, CA.
Purpose: To examine whether human adipose-derived stem cells
(ASCs) could support the growth of human limbal stem/progenitor
cells (LSCs).
Methods: Three different culture methods were tested, including
culturing the LSCs directly on the feeder cells (regular method), a 3dimensional (D) culture system (sandwich method) and a 3-D culture
system coated with fibrin (fibrin method). Freshly isolated limbal
epithelial cells (LECs) in single cell suspension or cell sheets were
co-cultured with the feeder cells for 2 weeks. Cultured LECs were
examined for their cell morphology, proliferation rate, p63alphabright population and expressions of putative LSC markers, ABCG2,
ΔNp63, N-cadherin and keratin (K) 14, and the differentiation marker
K12. LECs cultured on 3T3-J2 feeder cells using the regular method
were used as a control.
Results: Single LECs cultured on ASCs had limited proliferation and
displayed differentiated morphology in all three culture methods.
When LECs were cultured as cell sheets, the sandwich method
supported the highest proliferation rate for ASCs, which was 4.0-fold
higher than the control (p<0.01). Compared to the control, cell sheets
cultured with ASCs expressed a similar mRNA level of ABCG2,
ΔNp63 and N-cadherin in all three culture methods, a significantly
lower level of K14 in sandwich and fibrin methods (decreased by
54% and 72%, respectively, p<0.05) and a significantly lower level
of K12 in the regular, sandwich and fibrin methods (decreased by
65%, 85% and 90%, respectively, p<0.05). LECs cultured with ASCs
were compact and small in size, and contained comparable
percentages of p63alpha-bright progenitor cells to the control in all
three culture methods (regular, 5%; sandwich, 5%; fibrin, 3%; 3T3-J2
control, 7%). The absolute number of p63alpha-bright cells produced
in the ASC sandwich method was 7-folds higher than that in the
control (p=0.051).
Conclusions: ASCs support the growth of LSCs in the form of cell
sheets but not single cell suspension. The sandwich method appears
to be a more favorable culture method to expand LSCs on ASCs than
the regular method.
Commercial Relationships: Hua Mei, None; Martin N. Nakatsu,
None; SHEYLA GONZALEZ, None; Elfren R. Baclagon, None;
Sophie X. Deng, None
Program Number: 980 Poster Board Number: B0285
Presentation Time: 1:00 PM - 2:45 PM
Periostin is associated with the properties of human corneal
epithelial progenitor cells
Yangluowa Qu, Cheng Li, Wei Li, Zuguo Liu. Eye Institute of
Xiamen University, Xiamen, China.
Purpose: Periostin functions as a ligand for integrins to support
adhesion and migration of epithelial cells. Periostin has been recently
known as a component of the extracellular matrix expressed by
fibroblasts in normal tissues and stroma of primary tumors. Periostin
over-expression was reported in several types of cancers. However,
little is known about periostin in human corneal epithelium. This
study was to explore the expression pattern and potential role of
periostin in maintaining the properties of human corneal epithelial
progenitor cells.
Methods: Fresh donor corneal limbal tissues were used to make
cryosections and isolate corneal and limbal epithelia. The primary
human corneal epithelial cells (HCECs) were generated from limbal
tissue explants. The mRNA expression was evaluated by reverse
transcription and quantitative real-time PCR, and the protein
production and localization were detected by immunofluorescent
staining and Western blot analysis. The expression and functional
role of periostin were further evaluated in primary HCECs at
different growth stages and in an in vitro wound healing model at
different time points
Results: Periostin protein was highly immunolocalized in the basal
cells of human limbal epithelium where corneal epithelial stem cells
locate. Periostin immunoreactivity was co-localized with epithelial
stem cell associated marker nuclear p63, but not co-localized with the
corneal epithelial differentiated marker Keratin 3. Periostin mRNA
was expressed higher in limbal epithelium than that in central corneal
epithelium. In primary HCECs, the mRNA expression and protein
production of periostin were much higher in the 50% confluent
cultures containing higher proliferative cells, than that in the 70% and
100% confluent cultures containing more differentiated cells. In an in
vitro wound healing model, periostin mRNA levels were significantly
and gradually up-regulated in 4-16 hours, and this up-regulation was
further confirmed at protein levels in 16-48 hours by Western blot
analysis
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Conclusions: These findings demonstrated that periostin is a novel
protein that is mainly expressed by basal layer of human limbal
epithelial cells and co-localized with p63. Periostin expression is
associated with higher proliferative capacity and less differentiation
conditions in HCECs. Periostin may have a potential role in
maintaining the properties of human corneal epithelial progenitor
cells
Commercial Relationships: Yangluowa Qu, None; Cheng Li,
None; Wei Li, None; Zuguo Liu, Bausch&Lomb (R), Allergern (R),
Alcon (R), Santen (R)
Support: supported by Chinese National key sencientific research
project 2013CB967003
Program Number: 981 Poster Board Number: B0286
Presentation Time: 1:00 PM - 2:45 PM
Differentiation Potential of Limbal Fibroblasts and Bone
Marrow Mesenchymal Stem Cells to Corneal Epithelial Cells
Kishore Reddy Katikireddy, Reza Dana, Ula V. Jurkunas. Schepens
Eye Research Institute and Massachusetts Eye and Ear, Department
of Ophthalmology, Harvard Medical School., Boston, MA.
Purpose: It has been shown that human limbal fibroblasts (LFs) and
bone marrow mesenchymal stem cells (BM MSCs) are multipotent.
Specifically, a side population of stage-specific embryonic antigen-4
(SSEA4)-positive LFs differentiates into a variety of cell types. The
present study compared the stem cell characteristics of SSEA4+ and
SSEA4- LFs to those of BM MSCs, and determined their potential to
differentiate into the corneal epithelial phenotype.
Methods: Human cadaveric limbal tissue (n=6) was treated with
dispase to remove the epithelium and endothelium. Single cell
suspensions were obtained by digesting the stroma with 0.025%
trypsin. Stem cell enrichment was performed by exposure of BM
MSCs and LF cells in KnockOut ESC/iPSC medium for 12-15 days.
LF and BM MSCs were sorted for SSEA4+ and SSEA4- cells by
Magnetic-Activated Cell Sorting. Cell doubling time (CDT), stem
cell (SC) marker analysis, and colony forming efficacy (CFE) were
performed on sorted LF and BM MSCs. Epithelial phenotype was
achieved using induction and differentiation media. Differentiated
cells were characterized for corneal cytokeratins (CKs).
Results: After separation, enriched LFs and BM MSCs, 97.4 ± 0.6%
and 93.5 ± 0.7% SSEA4+ subgroups were achieved, respectively.
The CDT of SSEA4+ LFs was 102 ± 1 hr and SSEA4- LFs was 58.2
± 1.5 hrs (p=0.002). CDT of SSEA4+ BM MSCs was 105 ± 1 hr. and
SSEA4- BM MSC was 56.3 ± 2 hrs (p=0.002). LF and BM MSC
subgroups were negative for pan-cytokeratin. After enrichment,
SSEA4+ cells showed the ability to form cell aggregates, while
SSEA4- cells were mostly adherent to the culture plates. The
transcript levels of SC markers OCT4, SOX2, Nanog and Rexo1
were higher in SSEA4+ than SSEA4- groups of LF and BM MSCs
(p<0.05). Upon induction and differentiation, both SSEA4+ LFs and
BM MSCs exhibited an epithelial morphology and positivity for
CK3, CK12, and CK8, with high CFE (p<0.02), whereas the SSEA4cells exhibited a fibroblast morphology and were negative for corneal
epithelial markers.
Conclusions: Although both LFs and BM MSCs express stem cell
markers and have some transdifferentiation potential, only SSEA4+
LFs clearly demonstrate the ability to differentiate into corneal
epithelial cell phenotype. These findings establish a potential
alternative source of corneal epithelial cells that can be harvested for
ocular surface reconstruction.
Commercial Relationships: Kishore Reddy Katikireddy, None;
Reza Dana, Allergan (C), Alcon (C), Bausch & Lomb (C), Eleven
Bio (I), GSK (F), Novabay (C), Revision Optics (C), Novaliq (C),
RIgel (F); Ula V. Jurkunas, 61/482,769 (P), Altheos (C)
Support: Massachusetts Lions Eye Research Fund (UJ), Cornea
Donor Research Fund (UJ and RD), Curing Kids Fund (UJ).
Program Number: 982 Poster Board Number: B0287
Presentation Time: 1:00 PM - 2:45 PM
The effect of hypoxia on the growth of limbal stem cells
Dean Hallam1, Christin Henein1, Satomi Miwa1, Sajjad Ahmad2,
Gabriele C. Saretzki1. 1Newcastle University, Newcastle-Upon-Tyne,
United Kingdom; 2University of Liverpool, Liverpool, United
Kingdom.
Purpose: This study aims to detail the effects of hypoxia (3%
oxygen) on the in vitro culture of human limbal stem cells. The
hypothesis is that hypoxic conditions may be beneficial to limbal
stem cell (LSC) growth, as these cells come from the neural ectoderm
lineage. In addition a number of stem cells are cultured in hypoxic
conditions such as embryonic, neural crest and hematopoietic stem
cells. It is thought that hypoxic conditions can shield stem cells from
the damaging effects of reactive oxygen species, limiting propagation
of DNA damage and retaining a more quiescent state. The successful
growth and expansion of limbal stem cells is important for the
clinical treatment of limbal stem cell deficiency (LSCD).
Methods: Extraction of human corneal epithelial cells was performed
used serial trypsinsation followed by expansion on 3T3 mouse
fibroblasts. Colony forming efficiency (CFE) assays were a
performed after 12 days. RT-Q-PCR was performed on primary cell
cultures to detect mRNA levels. Annexin V assay was used to
measure apoptosis, whilst DAPI was used to determine cell cycle
state. Metabolic activity was measured using a Seahorse XFS, which
detects changes in extracellular fluctuations in oxygen and protons.
Results: CFE assays showed a decreased number of cells and
colonies in hypoxia. Quantitative RT-PCR revealed that the
expression of positive LSC markers and telomerase genes was higher
in hypoxia. The expressions of differentiation markers were lower in
hypoxia. Flow cytometry showed a greater proportion of cells
cultured in hypoxia were in G0/G1phase (83.6%) compared to
normoxia (79.7%). The proportion of cells which were dead or
undergoing apoptosis was 22% in normoxia and 13.1% in hypoxia.
Metabolic data showed that cells grown in hypoxia had a higher basal
oxygen consumption and a higher oxygen capacity during the
experiment whilst cells in normoxia had a lower values.
Conclusions: We demonstrated that human corneal epithelium stem
cells cultured in hypoxic conditions may exhibit a quiescent stem cell
phenotype, which may be essential for maintaining stemness by
protecting cells from differentiation and apoptosis. Metabolic data
also suggests that cells grown in hypoxia are more metabolically
active and have a higher mitochondrial respiratory capacity than
those grown in normoxia. As 3% oxygen may be closer to
physiological oxygen levels and that these conditions may favour
mitochondrial biogenesis.
Commercial Relationships: Dean Hallam, None; Christin Henein,
None; Satomi Miwa, None; Sajjad Ahmad, None; Gabriele C.
Saretzki, None
Support: Biomedical Research Council
Program Number: 983 Poster Board Number: B0288
Presentation Time: 1:00 PM - 2:45 PM
Downregulation of master transcription factor, Pax6, contributes
to autoimmune-mediated keratinizing squamous metaplasia of
the ocular surface via the aberrant activation of basal progenitor
cells
Ying-Ting Chen, Nancy A. McNamara. Proctor Foundation, Dept
Ophthalmology, University of California, San Francisco, San
Francisco, CA.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Purpose: To study the role of ocular master regulator, Pax6, in the
dysregulation of keratinocyte progenitor cells (PCs) during T cellmediated keratoconjunctivitis sicca (KCS) with keratinizing
squamous metaplasia (SQM).
Methods: Aire KO mice and immunodeficient (scid) recipients of
Aire KO-derived autoreactive CD4+T cells were used as models of
autoimmune SQM. Proliferative activity was measured by the density
of Ki67+ basal cells, designated as the mitotic index (MI). The
relative length of S phase in the 4-phase cell cycle was obtained by
dividing the density of BrdU+ cells (labeled by a single BrdU
pulsing) by the MI. Activated PCs, transient amplifying cells (TACs),
postmitotic cells (PMCs), young terminally differentiated epithelial
cells (TDCs) and immune effector cells were labeled by continuous
BrdU pulsing for 7 days. Label-retaining cells including slow-cycling
SCs, long-lived PMC/TDCs and memory T cells were visualized
after a 21-day BrdU. Local adenoviral transfer of Pax6 was employed
to assess Pax6’s modulatory effects on PC’s self-renewal and
differentiation in Aire KO’s SQM tissue.
Results: Compared to wild type mice, the MI of p63+ PCs increased
3.1±1.2 fold in the limbus and cornea of Aire KO mice. Such an
increase in proliferation was accompanied by a 42% decrease in Pax6
transcripts, a 52% decrease in scid recipients of Aire KO-derived
CD4+ T, and an 83% decrease in clonal cells of Aire KO PCs
cultivated on 3T3 feeders (p < 0.05, n=5 per group). BrdU
incorporation kinetics revealed a 1.9-fold, 2.1-fold, and 0.5-fold
labeling index in Aire KO mice following 1-shot, 7-day pulse and 21day chase protocols, respectively (all p < 0.05 vs WT at 1-fold).
Together, these results suggested downregulation of Pax6 is
associated with an accelerated epithelial turnover and a relatively
shorter S phase in rapid cycling cells. Adenoviral induction of Pax6
in Aire KO basal PCs restored the corneal phenotype.
Conclusions: The downregulation of Pax6 in basal PCs during ocular
autoimmune SQM is mediated by PC- and limbal niche-reactive
CD4+ T effectors. Restoration of Pax6 reverses aberrant epidermallineage commitment suggests adjuvant Pax6 gene therapy may
compliment immunosuppressants as a novel therapeutic approach to
manage SQM disease in sicca patients
Commercial Relationships: Ying-Ting Chen, None; Nancy A.
McNamara, None
Support: R01 EY016203-06
Program Number: 984 Poster Board Number: B0289
Presentation Time: 1:00 PM - 2:45 PM
A Comparison Between Three Mouse Models of Corneal Limbal
Stem Cell Deficiency
Neda -. Afshar, Asadolah Movahedan, Ali R. Djalilian.
Ophthalmology and Visual science, University of Illinois at Chicago,
Chicago, IL.
Purpose: Creating a mouse model that best represents the corneal
limbal stem cells deficiency (LSCD)
Methods: Three different methods were used to create mouse models
of corneal LSCD in C57BL/6J mice (6 eyes per group). Group 1:
Complete mechanical removal of corneal epithelium with a blunt
scraper from limbus to limbus. Group 2: Complete mechanical
corneal epithelial removal followed by heat cauterization, 360
degrees all around the limbus. Group 3: Applying 0.1% sodium
hydroxide (NaOH) to the limbal area for 30 seconds using 2mm filter
paper discs, after removing the whole corneal epithelium with a blunt
scraper. Slit lamp examination was performed to evaluate the degree
of fluorescein staining and corneal neovascularization compared to
control eyes. Eyes were removed for whole mount immunostaining
for Cytokeratin 12 and Cytokeratin 8.
Results: All groups showed evidence of late fluorescein staining with
neovascularization consistent with limbal stem cell deficiency. The
results in group 1 showed more variability with varying degrees of
staining/neovascularization and gradual recovery of corneal type
epithelium by two months in some eyes. The results were less
variable in group 2 with more stable pattern of LSCD over time in all
eyes. Group 3 demonstrated the most severe phenotype with frequent
hemorrhage and significant opacification of the corneal stroma. Long
term studies with quantitative comparison is currently underway.
Conclusions: Mechanical corneal epithelial removal in combination
with thermal injury (group 2) provides a more reproducible method
(compared to mechanical removal alone) and a less destructive
method (compared to alkali injury) for developing corneal LSCD in
mice.
Commercial Relationships: Neda -. Afshar, None; Asadolah
Movahedan, None; Ali R. Djalilian, None
Support: Career development grant K08EY017561-A1 to A.R.D.,
Core grant EY01792 from the NIH, and by the Cless Family
Foundation. A.R.D. is the recipient of a Career Development Award
from Research to Prevent Blindness.
Program Number: 985 Poster Board Number: B0290
Presentation Time: 1:00 PM - 2:45 PM
Optimization of Ex Vivo Expansion of Limbal Epithelial
Progenitors by Maintaining Native Niche Cells on Denuded
Amniotic Membrane
Megha A. Mahabole, Szu-Yu Chen, Scheffer C. Tseng. R&D, Tissue
Tech Inc., Miami, FL.
Purpose: Transplantation of ex vivo expanded limbal epithelial
progenitor cells (LEPC) on denuded amniotic membrane (dAM) is an
alternative solution for treating corneal blindness due to limbal SC
deficiency. We reported isolation and expansion of limbal niche cells
(NCs) that express embryonic SC (ESC) and angiogenic markers in
serum-free modified ESC medium (MESCM). We question whether
the conventional ex vivo expansion protocol of LEPC on dAM can be
further optimized by maintaining limbal NCs by switching the culture
medium from supplemented hormonal epithelial medium (SHEM) to
MESCM.
Methods: A limbal cluster was obtained from each 1/12 piece of the
human corneosclera ring and was cut 1 mm from within and beyond
the anatomic limbus and further digested in 1 mg/ml collagenase A
for 18 h at 37 degrees celsius, transferred to dAM and cultured in
SHEM or MESCM. On Day 8-10, epithelial outgrowth sheets were
removed by dispase and subjected to real-time qPCR and
immunostaining for expression of corneal markers (p63α, pax6, K12)
and NC markers (FLK-1, NG2, PDGFR-B, CD31 and CD34). 1000
single cells were seeded on 6-well dish containing MMC-3T3 for 1214 days before rhodamine B staining.
Results: qPCR of epithelial outgrowth on dAM at Day 8 in SHEM
showed a significant loss of corneal and ESC markers when compare
to freshly collagenase-isolated limbal clusters, suggesting that the
conventional ex vivo expansion method was not optimized.
Nonetheless, the resultant monolayer sheets and cell size were
consistently smaller in MESCM than in SHEM on Day 8. qPCR
further confirmed significant upregulation of NC markers in
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
outgrowth sheets expanded in MESCM when compare to that in
SHEM on Day 10. Cytospin preparations further revealed a
significantly higher percentage of PCK-/ Vim+ cells found in
MESCM (14.8%, n= 1352) than SHEM (0.6%, n=1531, p<0.05).
qPCR and immunostaining showed a substantial higher expression of
corneal SC markers (K15, Bmi-1, Msi-1) in MESCM than in SHEM.
Although colony-forming efficiency was similar, the number of
holoclones was higher in MESCM than SHEM.
Conclusions: These data collectively suggest that MESCM is a better
culture medium for preservation of limbal native NCs to support
expansion of LEPC on dAM.
Commercial Relationships: Megha A. Mahabole, Tissue Tech Inc.
(E); Szu-Yu Chen, Tissue Tech Inc (F), Tissue Tech Inc (E), Tissue
Tech Inc (P); Scheffer C. Tseng, NIH, NEI (F), TissueTech, Inc. (F),
TissueTech, Inc. (E), TissueTech, Inc. (P)
Support: NIH, NEI, R01EY06819
Program Number: 986 Poster Board Number: B0291
Presentation Time: 1:00 PM - 2:45 PM
Self Assembly of the Limbal Epithelial Stem Cell Niche in Vitro
Hideyuki Miyashita, Satoru Yoshida, Tetsuya Kawakita, Kazuo
Tsubota, Shigeto Shimmura. Ophthalmology, Keio University School
of Medicine, Shinjyuku-ku, Japan.
Purpose: The corneal limbus contains cytokeratin 15 (K15) positive
basal epithelial cells and melanocytes that form units with basal
epithelial cells. We confirmed whether long-term cultivated epithelial
cell sheets can reconstruct melanocytes-epithelial cell units.
Methods: Human limbal epithelial cells were isolated from eyebank
eyes, seeded on fibrin-coated cell culture inserts, and 3 dimensional
co-cultured with feeder cells. Cells were fed with supplemented
hormonal epithelial medium with EGF or KGF and a ROCK inhibitor
Y-27632 at day 3, day 5, and every day after day 7 for up to 3
months. Cryosections and whole cell sheets were fixed with ice-cold
4% paraformaldehyde in PBS, and stained with melanocyte marker
MART-1 and epithelial precursor marker K15.
Results: MART-1 positive melanocytes were observed in whole
mounted epithelial cell sheets in all culture conditions.
Immunohistochemisrty showed that melanocytes were scattered as
MART-1 positive cells in the basal layer of cultured epithelium.
Melanocytes showed dendritic morphology, some of which were
proximal to K15 highly positive epithelial cell clusters. In sheets
derived from dense-pigmented limbus, melanin pigments were
observed in basal cells around the melanocytes.
Conclusions: Melanocytes and limbal epithelial cells can selfassemble melanin units in vitro, mimicking the limbal niche in vivo
Commercial Relationships: Hideyuki Miyashita, None; Satoru
Yoshida, None; Tetsuya Kawakita, None; Kazuo Tsubota,
AcuFocus, Inc (C), Allergan (F), Bausch Lomb Surgical (C),
Functional visual acuity meter (P), JiNS (P), Kissei (F), Kowa (F),
Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), Echo Denki (P),
Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPT Company (P);
Shigeto Shimmura, None
Support: Grant (H22-saisei-ippan-002) from Ministry of Health,
Labour and Welfare, Japan
Program Number: 987 Poster Board Number: B0292
Presentation Time: 1:00 PM - 2:45 PM
Activation of the Hedgehog pathway is involved in the expansion
of compound niches after corneal injury
Ahdeah Pajoohesh-Ganji, Sonali Ghosh, Gauri Tadvalkar, Mary Ann
Stepp. Anatomy & Regenerative Biol, George Washington Univ,
Washington, DC.
Purpose: The hedgehog (Hh) signaling pathway has been suggested
to regulate corneal homeostasis and is up-regulated during corneal reepithelialization. Sonic Hh interacts with its receptor Patched-1 on
cell surfaces to mediate the movement of Gli1/2 to the nucleus and
the activation of genes involved in cell proliferation and
differentiation. The corneal epithelium is maintained by stem cells
which are found at the limbus in higher numbers. We recently
showed that progenitor cells for both corneal epithelial and goblet
cells are found within compound niches (CNs) present at the limbal
region of the healthy BALB/c mouse cornea. CNs proliferate upon
wounding, and give rise to both corneal epithelial and goblet cells. If
wounds get close to the limbus, goblet cells that arise from the CN
are found on the central cornea, creating an unsuitable corneal
surface and preventing the appropriate passage of light. These studies
were initiated to investigate the role played by the Hh signaling
pathway in proliferation of CNs and generation of corneal goblet
cells during corneal homeostasis and in response to injury.
Methods: Adult BALB/c mice were wounded using a 2.5mm
trephine and a dulled blade. Mice were allowed to heal for different
time points after wounding: 1, 7, 14, 21, 28 days. Unwounded
corneas were used as control. After sacrifice, corneas were processed
for whole mount immunofluorescence to localize sonic hedgehog
(Shh) and Patched-1 as well as Muc5ac+ CNs. A minimum of 3
corneas were used for each time point and antibody combination
evaluated.
Results: Patched -1 is present in the cells that make up the CN in
unwounded corneas but Shh, the Patched-1 ligand, is absent.
However, 1 and 7 days after wounding, Patched-1 and Shh colocalize in CNs both at the limbus and on the central cornea in cells
that arise from the disaggregation of CNs. While many of these
Patched-1+Shh+ cell clusters are Muc5ac+, few are Muc5ac-.
Conclusions: These experiments indicate that activation of the Hh
signaling pathway is involved in the expansion and/or migration of
CNs that takes place after wounding. Hh signaling may regulate the
differentiation of CNs into goblet cells. If so, manipulating this
pathway may reduce the numbers of the goblet cell clusters formed
after wounding in the central cornea.
Commercial Relationships: Ahdeah Pajoohesh-Ganji, None;
Sonali Ghosh, None; Gauri Tadvalkar, None; Mary Ann Stepp,
None
Support: EYO08512 (MAS)
Program Number: 988 Poster Board Number: B0293
Presentation Time: 1:00 PM - 2:45 PM
Unraveling the limbal epithelial stem cell niche: miRs-103/107
help maintain E-cadherin-mediated cell-cell contacts
Julia V. Katsnelson1, 2, Jong Kook Park1, Wending Yang1, Han Peng1,
Robert M. Lavker1. 1Dermatology, Northwestern University,
Chicago, IL; 2Rush University College of Medicine, Chicago, IL.
Purpose: In an effort to understand the roles that microRNAs
(miRNAs) play in regulating corneal epithelial stem cells and their
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
transient amplifying (TA) progeny, we compared miRNA expression
patterns in the stem cell-enriched limbal epithelium versus the TA
cell-enriched corneal epithelium at several developmental stages.
Methods: Laser capture microdissection (LCM) was used to
precisely isolate fresh populations of limbal and corneal epithelium
from unfixed cryosections, thereby acquiring miRNA expression
profiles from relatively “quiescent” cells. LCM was performed on
mouse limbal and corneal epithelium from postnatal (PN) mice at
days: 3, 7, 14 and 28. Total RNA was isolated from triplicate samples
and miRNA expression profiles were obtained using qRT-PCR arrays
(Exiqon). We conducted loss-of-function experiments with miR-103
and -107 in cultured human limbal keratinocytes (HLEKs), in
conjunction with Northern, Western, and immunohistochemical
analyses. We used luciferase reporter assays in HeLa cells to identify
potential targets of miR-103 /107.
Results: miR-103 /107 were preferentially expressed in the limbal
epithelium at each of the developmental time points. Northern blot
analysis revealed higher expression of miR-103 /107 in HLEKs
versus human corneal epithelial keratinocytes. Treatment of HLEKs
with antagomirs to either miR-103 or miR-107, resulted in an initial
loss of cell-cell contacts, which yielded small, rounded cells
compared to control-treated (antago-124) HLEKs. Consistent with a
loss in cell-cell contact, E cadherin (E-cad) was no longer
immunohistochemically detectable at the cell-cell junctions, 3hr postantago treatment. A marked downregulation was noted in p120
catenin and E-cad protein, essential components of adherens
junctions. NEDD9, a non-catalytic scaffolding protein that negatively
regulates localization of E-cad and promotes its degradation, was
shown to be a target of miR-103/107.
Conclusions: Maintenance of cell-cell contact and communication
between stem cells and TA cells are crucial for stem cell niche
homeostasis. The positive role that miR-103/107 plays in E-cadherinmediated limbal epithelial cell-cell contacts via down-regulation of
NEDD9 helps stabilize this aspect of the stem cell niche and maintain
stem cell-TA cell integrity.
Commercial Relationships: Julia V. Katsnelson, None; Jong
Kook Park, None; Wending Yang, None; Han Peng, None; Robert
M. Lavker, None
Support: NIH Grants EY06769, EY019463
Program Number: 989 Poster Board Number: B0294
Presentation Time: 1:00 PM - 2:45 PM
Comparison of Different Limbal Epithelial Stem Cell Isolation
Methods to Improve the Epithelial Sheet Quality for
Transplantation
SHEYLA GONZALEZ, Martin N. Nakatsu, Hua Mei, Sophie X. Deng.
Jules Stein Eye Institute UCLA, Los Angeles, CA.
Purpose: To investigate different preparation methods of human
limbal stem cells (LSCs) for cultivation using dispase II, collagenase
A and the explant culture.
Methods: Limbal tissues were incubated with dispase II at 37°C for
2 hours (h) or with collagenase A at 37°C or 4°C for 2h or overnight
to obtain limbal epithelial cell sheets. Half of the cell sheets were
further incubated with trypsin to achieve single cell suspension. The
explant culture was done using a 2x2 mm limbal biopsy. Limbal
epithelial cells and limbal tissues were cultured on growth-arrested
3T3-J2 mouse cells in SHEM5 medium. The phenotype assessment
included stem/progenitor morphology and expression level of LSC
putative markers including ABCG2, p63α, N-cadherin and
cytokeratin (K) 14, the differentiation marker K12, and the stromal
cell markers vimentin (Vim) and α-smooth muscle actin by qRT-PCR
and immunohistochemistry.
Results: No LSC growth on 3T3-J2 cells was achieved when limbal
tissues were incubated with collagenase overnight at 37°C. Among
the other collagenase treatments tested, the most efficient was
incubation at 37°C for 2h in the incubator. This isolation method
yielded the highest K14+Vim- progenitor cell population (86.2%) and
a moderate K14-Vim+ stromal cell number (9.7%). The LSCs had the
best stem/progenitor-like morphology and the highest level of
putative LSC markers. Collagenase but not dispase isolated the entire
epithelial sheet with the underneath stromal cells. When the
phenotype of cultured LSCs was compared among all three culture
methods, all LSCs displayed a stem/progenitor-like morphology.
However, LSCs cultured from collagenase digestion had the highest
mRNA level of putative LSC markers and the highest percentage of
p63α bright cells (21.7% vs. 6% from explant and 12.7% from
dispase, P<0.05). Cultures from collagenase isolation and explant
yielded a higher total number of p63α bright cells (P=0.3) that was 4fold higher than that from dispase isolation, due to a higher cell
growth rate in explant. When LSCs were cultured as cell sheets, the
mRNA level of putative LSC markers was higher than that from as
single cell suspension.
Conclusions: Limbal stromal cells from the collagenase treatment
and in the explant culture support a more efficient expansion of LSCs
in vitro on 3T3 feeder cells. The presence of native LSC niche
appears to help maintaining the LSC phenotype.
Commercial Relationships: SHEYLA GONZALEZ, None;
Martin N. Nakatsu, None; Hua Mei, None; Sophie X. Deng, None
Support: CIRM grant TR2-01768 and NEI grant 5T32EY007026-35
Program Number: 990 Poster Board Number: B0295
Presentation Time: 1:00 PM - 2:45 PM
Thermolysin treated AM preserves the BM providing superior
expansion and preservation of cornea epithelial cells
Aaron Yeung, Andrew Hopkinson, Harminder S. Dua. Division of
Ophthalmology and Visual Sciences, University of Nottingham,
Nottingham, United Kingdom.
Purpose: : Amniotic Membrane (AM) has grown in popularity over
the years as a substrate for epithelial expansion and therefore a
potential treatment adjuvant in limbal stem cell deficiency. The AM
basement membrane (BM) provides the necessary structure for cell
attachment, and the AM itself is postulated to possess antiinflammatory and anti- scarring properties. Tenascin C (TNC) is a
multimodular glycoprotein that is part of the ECM located at the BM.
It has the potential to engage within the microenvironment of the
stem cell niches. TNC may have a role in maintaining corneal
epithelial stemness. Thermolysin treated AM is a standardized
method for denuding AM whilst preserving the BM. The aims of our
study were to investigate whether thermolysin treated AM could
provide a suitable microenvironment for expansion and preservation
of epithelial cells.
Methods: Primary cells from corneo-scleral explants were cultured
on intact AM and thermolysin treated AM. Relative quantification for
various “stem cell associated” markers was performed using both real
time PCR and immunofluorescence.
Results: Thermolysin treated AM demonstrated greater stratification
on primary cells with higher levels of TNC expression and
maintaining cells with greater p63 and ABCG2 expression and lower
DSG levels.
Conclusions: The AM could provide a suitable microenvironment
similar to a stem cell niche for expansion and preservation of
epithelial cells. Thermolysin treated AM preserves the BM and
therefore permits superior cell to BM adhesion. Preservation of the
AM BM may be the key to underlying cell interactions that maintain
stem cell “stemness”.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Commercial Relationships: Aaron Yeung, None; Andrew
Hopkinson, None; Harminder S. Dua, None
Program Number: 991 Poster Board Number: B0296
Presentation Time: 1:00 PM - 2:45 PM
Characterization of Biomaterial free Cell Sheets Cultured from
Human Oral Mucosal Epithelial Cells
Mee Kum Kim1, 2, Joo Hee Lee3, Ja Young Lee1, 2, Ah Young Koh2,
Yun Hee Kim3, Won Ryang Wee1, 2, Saewha Jeon3. 1Ophthalmology,
Seoul National University Hospital, Seoul, Republic of Korea;
2
Laboraory of Corneal Regenerative Medicine and Ocular
Immunology, Seoul Artificial Eye Center, Seoul, Republic of Korea;
3
Cutigen Research Institute, Tego Science Inc., Seoul, Republic of
Korea.
Purpose: To investigate the characteristics of biomaterial free cell
sheets cultured from oral mucosal epithelial cells both in vitro and in
a limbal deficient animal model.
Methods: Both human oral mucosal epithelial cells and limbal
epithelial cells were cultured to form cell sheets with or without
fibrin glue for 2 weeks. The resulting sheets were detached using a
buffer containing 1% dispase and transplanted to limbal deficient
rabbits which had been chemically injured with lamellar limbectomy.
The adhesion stability of these biomaterial-free cell sheets were
evaluated three days after transplantation. In parallel, Colony
Forming Efficiency(CFE) as well as the immunohistochemistry using
antibodies specific to proliferation and differentiation of epithelial
cells were performed to characterize the cell sheets.
Results: CFE in Human oral mucosal epithelial cells and limbal
epithelial cells were measured to be 57.5% and 14%, respectively.
K1, K3, and K4 were expressed in mucosal epithelial sheets while K3
and K12 were in limbal epithelial sheets. The cell sheets were
composed of three to six layers of stratified, well differentiated
mucosal epithelial cells with 87.5% viable cells present. The in vivo
adhesion stability of biomaterial-free cell sheets of oral mucosal
epithelial cells was comparable to that of fibrin-based counterpart.
Conclusions: Our results suggest that biomaterial free cell sheets of
oral mucosal epithelial cells can be a good candidate in the treatment
of limbal deficient diseases.
Commercial Relationships: Mee Kum Kim, None; Joo Hee Lee,
None; Ja Young Lee, None; Ah Young Koh, None; Yun Hee Kim,
None; Won Ryang Wee, None; Saewha Jeon, Tego Science Inc. (E)
Program Number: 992 Poster Board Number: B0297
Presentation Time: 1:00 PM - 2:45 PM
Human adipose-derived stem cells promote wound healing of
corneal epithelial cells in vitro
Ladan Espandar1, Tomas Blanco1, Rose Mathew1, Natalie A.
Afshari2, Bruce Bunnell3, Daniel R. Saban1. 1Ophthalmology, Duke
University, Durham, NC; 2University of California San Diego, San
Diego, CA; 3Tulane University, New Orleans, LA.
Purpose: Human adipose-derived stem cells (hASCs) have been
shown to promote wound healing in the skin, and may thus have
potential application for ocular surface regenerative therapy and
wound healing. The purpose of this study is to investigate the effect
of hASCs on corneal epithelial cells (CECs) using an in vitro wound
healing model.
Methods: An established in vitro epithelial wound-healing model
was utilized in which a 0.5 mm scratch was made on a monolayer of
confluent human corneal epithelial cells (hCECs). hASCs seeded on a
transwell membrane was added to determine their effect on epithelial
wound healing. Controls included scratched CECs without the
addition of hASCs, or mitomycin-C-treated scratched hCECs. Wound
closure was evaluated by time-lapse inverted microscopy for a
minimum of 24 hours. The average wound width and migration speed
at seven random areas were assessed and quantified digitally using
MetaMorph® software.
Results: Wound closure in the condition containing CECs alone took
as long as 18 hours, whereas the addition of hASCs decreased this
time significantly to 13 hours. Likewise, without hASCs the average
wound width (166.6 µm) was significantly higher than with hASCs
(138.7 µm, p<0.01). In addition, epithelial migration speed of CECs
alone was 57.7 µm/hr, while the addition of hASCs increased this to
70 µm/hr. No migration or wound closure was observed with the
mitomycin-C-treated hCECs control.
Conclusions: hASCs promote wound closure of corneal epithelial
cells in vitro. Therefore, future studies are warranted to examine the
therapeutic potential of hASCs in corneal epithelial wound healing.
Commercial Relationships: Ladan Espandar, None; Tomas
Blanco, None; Rose Mathew, None; Natalie A. Afshari, None;
Bruce Bunnell, None; Daniel R. Saban, Schepens Eye Res Inst,
Mass Eye and Ear, (P), Eleven Biotherapuetics (R)
Support: Duke small internal grant
Program Number: 993 Poster Board Number: B0298
Presentation Time: 1:00 PM - 2:45 PM
NGF inhibited apoptosis through phosphorylation of FOXO3a in
human corneal epithelial cells
Tingting Qian, Jiaxu Hong, JianJiang Xu. Ophthalmology, Eye &
Ear, Nose, Throat Hosp, Shanghai, China.
Purpose: To investigate the possible mechanism by which NGF
regulated the cell survival of human corneal epithelial cells.
Methods: Human corneal epithelial cells (HCECs) cultured in vitro
were identified by immunostaining for cytokeratin12, a biomarker for
corneal epithelium. Cells were stimulated with NGF at 25ng/ml for 1
hour. The cell apoptosis was analyzed by flow cytometry. Western
blotting was applied to examine the expression levels of p140trkA, pAkt, p-FOXO3a and Bim. Immunofluorescence was used to observe
the distribution of p-FOXO3a in the cells. Then, PI3K/Akt pathway
inhibitor LY294002 was treated to confirm the PI3K/Akt signaling
pathway by which NGF regulated the apoptosis of human corneal
epithelial cells. Furthermore, western blotting and RT-PCR was used
to check the expression of pro-apoptotic FoxO target Bim. Gene
reporter experiments were performed to examine the activity of Bim
promoter, which was dependent on a FoxO binding site.
Results: HCECs expressed cytokeratin12. The results of flow
cytometry analysis showed that the cell apoptosis was inhibited by
NGF. P140trkA, as well as the PI3K/Akt cascade, was activated
immediately cells were stimulated. Western blotting and
immunofluorescence analysis revealed that he forkhead transcription
factor FOXO3a, was phosphorylated and cytoplasm translocated by
the regulation of PI3K/Akt signaling pathway in HCECs.
Furthermore, both the mRNA and protein levels of Bim were
decreased dramatically after NGF treatment. Gene reporter
experiments demonstrated that in HCE cells inhibition of Bim
promoter was dependent on a FoxO binding site.
Conclusions: These data suggested that NGF regulated HCECs
survival via PI3K/Akt-regulated phosphorylation of FOXO3a in
human corneal epithelial cells, and that the phosphorylation of
FoxO3a was responsible for the transcriptional down-regulation of
pro-apoptotic FoxO target Bim.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
epithelium and the subependymal zone. Several reports have
identified links to Wnt signaling transducer beta-catenin, for which a
role in maintenance of limbal stem cells has been proposed. PPARG
is a nuclear receptor associated with differentiation pathways of
adipocytes and epithelial cells as well as growth inhibition of
carcinoma cells. Nuclear export of PPARG is mediated by
MAP2K1/MEK1, which invites to speculate about a possible
upstream activity of the MAPK-pathway in putative limbal
progenitors. Further research is warranted to elucidate the functional
involvement of SOX-9 and PPARG in corneal epithelial cell
differentiation, and to assess the usefulness of modulating their
activity in the context of cell therapy.
Commercial Relationships: Johannes Menzel-Severing, None;
Matthias Zenkel, None; Friedrich E. Kruse, None; Ursula
Schlotzer-Schrehardt, None
Commercial Relationships: Tingting Qian, None; Jiaxu Hong,
None; JianJiang Xu, None
Program Number: 994 Poster Board Number: B0299
Presentation Time: 1:00 PM - 2:45 PM
Transcription factor screening of limbal vs. corneal epithelium
identifies distinct patterns for SOX-9 and peroxisome
proliferator-activated receptor gamma (PPARG)
Johannes Menzel-Severing, Matthias Zenkel, Friedrich E. Kruse,
Ursula Schlotzer-Schrehardt. Department of Ophthalmology,
University of Erlangen-Nuremberg, Erlangen, Germany.
Purpose: To identify transcriptional regulators potentially involved
in limbal epithelial stem cell homeostasis and differentiation.
Methods: Limbal and central corneal epithelial specimens were
obtained from four human post-mortem donor eyes using laser
capture microdissection. RNA extracted from these specimens
underwent linear amplification (MessageAmp II, Ambion). Samples
were screened for differential expression of stem cell transcription
factor genes using quantitative real-time PCR arrays
(SABiosciences). Candidate genes were confirmed using specific
real-time PCR hydrolysis probe assays (Roche) and
immunohistochemistry of frozen corneal sections.
Results: Of 86 genes screened, nine appeared to be differentially
expressed, and were therefore investigated further (FOXP2,
HOXA11, KLF2, SOX9, STAT3, WRN, MYC, DACH1, PPARG).
Using individual assays, increased mRNA expression in limbal
specimens was confirmed for SOX9 and PPARG.
Immunohistochemistry showed nuclear localization of SOX-9 in
limbal basal cells, whereas no specific staining was seen in central
corneal epithelium. PPARG was detected within nuclei of basal
epithelial cells of conjunctiva and central cornea, while
perinuclear/cytoplasmic staining was observed in small, basal cell
clusters at the limbus.
Conclusions: Transcription factor SOX-9 has been suggested to
contribute to progenitor cell regulation in hair follicle, intestinal
Program Number: 995 Poster Board Number: B0300
Presentation Time: 1:00 PM - 2:45 PM
Wnt promotes proliferation of corneal epithelial progenitor cells
in xeno-feeder free cultivation
Kyung-Sun Na1, 2, Jeewon Mok2, Jungmook Lyu2, Choun-Ki Joo1, 2.
1
Department of Ophthalmology, The Catholic University of Korea,
Seoul, Republic of Korea; 2Catholic Institutes of Visual Science, The
Catholic University of Korea, Seoul, Republic of Korea.
Purpose: This study was to establish a simple, xeno-feeder free
method for cultivating human corneal limbal explants , and also to
explore the effect of Wnt signaling on epithelial progenitor cell
proliferation in this cultivation system in vitro and in vivo.
Methods: The limbal tissue explants from cadaveric donor was
cultured in Isocove’s Modified Dulbecco’s Medium (IMDM) and low
calcium Panserin 801 medium in 1:1 ratio. The outgrowing cells were
examined to characterize with flow cytometry,
immunohistochemistry, and real-time PCR (RT-PCR). SpragueDawley male rats after alkali injuries using 1N NaOH were used for
in vivo verification, after which cultivated epithelial sheets were
transplanted. Corneal opacity, re-epithelialization, and
neovasculazation was observed for 2 week, and the tissue sections
analyzed with hematoxylin and eosin stain (HE stain). Conditioned
media from L cells secreting Wnt-3a (Wnt3a-L-CM) was used in the
cultivation system, and morphological changes and gene expression
level were observed.
Results: There was migration of fibroblast like stromal cells from
limbal explants initially, and then, epithelial cells migrated and
grown on stromal cells as an autofeeder layer, which was revealed by
morphological and immunohistochemical methods. RT-PCR showed
that the expression of epithelial progenitor cells are more intense
compared to fresh limbal tissue. Side population (SP) cells were
detected 0.43 ± 0.04 % (n=5) of the primary culture. Flow cytometry
resulted 49.12% of E-cadherin, 40.44% of p63, and 44.55% of
ABCG2 identified in the cells from explants. Maintaining corneal
transparency without neovascularization was observed in rats after
cultivated epithelial sheets transplantation for 2 weeks. Predominant
increased tightly packed epithelial cells with Wnt3a-L-CM were
observed compare to the control CM. ABCG2, p63, Lef1, and CK3
was increased in Wnt3a-L-CM.
Conclusions: This explants culture system with combining media to
use stromal cells as autofeeder layer, showed to expand sufficient
limbal epithelial progenitor cells in vitro and to be transplanted
restoring transparency. Also, these findings demonstrated that Wnt
signaling play an important role in the proliferation of limbal
epithelial progenitor in the proposed cultivation system. This study
may have clinical impact on the expansion of corneal epithelial
progenitor cells for ocular surface reconstruction.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Commercial Relationships: Kyung-Sun Na, None; Jeewon Mok,
None; Jungmook Lyu, None; Choun-Ki Joo, None
Program Number: 996 Poster Board Number: B0301
Presentation Time: 1:00 PM - 2:45 PM
Human limbal epithelium expanded in a complex medium or
medium with human serum: a comparison of morphology and
cytokeratin expression
Meeta Pathak1, Kristiane Haug1, Aboulghassem Shahdadfar1, Eli
Gulliksen1, Magnus Roeger3, Liv Drolsum1, Jon K. Slettedal1, Bjorn
Nicolaissen1, Katerina Jirsova2. 1Centre for Eye Research, Dept. of
Ophthalmology, Oslo University Hospital, Oslo, Norway;
2
Laboratory of the Biology and Pathology of the Eye, General
Teaching Hospital and Charles University, Prague, Czech Republic;
3
Dept. of Pathology, Oslo University Hospital Ullevål, Oslo, Norway.
Purpose: We compared aspects of ultrastructural morphology and
the expression of selected cytokeratins in human limbal epithelium
expanded in a complex medium (COM), which contains various
recombinant growth factors, hormones, Cholera toxin and fetal
bovine serum (FBS), and culture medium with human serum (HS) as
the only growth supplement (HS).
Methods: Limbal biopsies retrieved from corneo-scleral rings were
placed with the epithelium facing down on the basement membrane
surface of samples of human amniotic membranes. The biopsies were
cultured in parallel in either COM or HS at 37°C, 5% CO2, and 95%
air. Culture medium was changed every 2-3 days and the epithelium
was expanded for 3 weeks. Specimens were examined by light
microscopy, transmission electron microscopy (TEM) and
immunohistochemistry. Expression of CKs 3, 7, 12, 14, and 19 was
evaluated using fluorescence microcopy, and intensity was graded on
a scale from 0 (negative) to 3 (strongly positive).
Results: By TEM, cytoplasmic density and distribution of
intermediate filaments showed a similar pattern in epithelial cells
expanded in both types of medium. In basal cells, the filaments were
loosely organized and inconspicuous apart from those associated with
intercellular junctions. Clusters of linear or wavy intermediate
filaments (tonofilaments) were encountered to a varying extent in the
cytoplasm of cells in the supra-basal layers.
By immunohistochemistry, expression of CK3 was not detected
(grade 0) in sections of expanded epithelium regardless of type of
medium. Both COM and HS supported the expression of CK 7, 12,
14 and 19, and grading of the samples for intensity of fluorescence
revealed a similar pattern in epithelium engineered in the two types
of medium. Intensity of fluorescence in sections of epithelium labeled
for CK12 and CK19 was evaluated as weak (grade 1), while the
intensity in sections of epithelium labeled for CK7 and CK 14 was
evaluated as strongly positive (grade 3).
Conclusions: Our findings indicate that COM and HS may equally
support the expression of selected CKs, suggesting similar degrees of
epithelial proliferation and differentiation. Further, a difference in
density of cytoplasmic intermediate filaments between basal and
supra-basal cells indicates a maintained gradient for epithelial
differentiation in both types of medium.
Commercial Relationships: Meeta Pathak, None; Kristiane Haug,
None; Aboulghassem Shahdadfar, None; Eli Gulliksen, None;
Magnus Roeger, None; Liv Drolsum, None; Jon K. Slettedal,
None; Bjorn Nicolaissen, None; Katerina Jirsova, None
Program Number: 997 Poster Board Number: B0302
Presentation Time: 1:00 PM - 2:45 PM
Comparative gene expression analysis of human cornea limbal
epithelial stem cells and differentiated corneal epithelium
Goran Petrovski1, 2, Reka Albert1, 2, Zoltan Vereb2, Morten C. Moe3,
Ole Kristoffer Olstad4, Andras Berta1, Laszlo Fesus2. 1Department of
Ophthalmology, University of Debrecen, Medical and Health Science
Center, Debrecen, Hungary; 2Stem Cells and Eye Research
Laboratory, Department of Biochemistry and Molecular Biology,
University of Debrecen, Medical and Health Science Center,
Debrecen, Hungary; 3Department of Ophthalmology, Oslo University
Hospital, Oslo, Norway; 4Department of Medical Biochemistry, Oslo
University Hospital, Oslo, Norway.
Purpose: Limbal epithelial stem cells (LESCs) are responsible for
corneal epithelium regeneration. Specific molecular markers for
LESCs have not been well defined. Our goal was to find new putative
markers for these cells and to identify associated molecular pathways.
Methods: Limbal tissue explants and central corneal epithelium were
harvested from cadavers (according to the Guidelines of the Helsinki
Declaration). The explants were cultured ex vivo and expanded into
LESCs in a human serum containing medium. Genome-wide
microarray analysis was performed using Affymetrix GeneChip
Human Gene 1.0 ST Array containing more than 28,000 gene
transcripts. Functional analysis using Ingenuity software was carried
out to identify pathways and molecules specific for LESCs.
Results: LESCs showed upregulated expression of 10 top molecules
(flavin containing monooxygenase (FMO) 1 and 2, fibronectin 1
(FN1), kallikrein (KLK) 6 and 7, transcobalamin 1 (TCN1),
semaphorin 3A (SEMA3A), annexin A3 (ANXA3), V-set domaincontaining T-cell activation inhibitor 1 (VTCN1) and heat shock
protein beta-8 (HSPB8), and downregulated expression of cartilage
acidic protein 1 (CRTAC1), alcohol dehydrogenase class 4 mu/sigma
chain (ADH7), hepatic leukemia factor (HLF), CD36, Doublecortin
domain containing 5 (DCDC5), Diacylglycerol kinase beta (DGKB),
protein prune homolog 2 (PRUNE2), anoctamin 4 (ANO4), death
associated protein-like 1 (DAPL1) and carbonic anhydrase (CA6)
compared to differentiated corneal epithelium (p<0.05). Cannonical
pathways specific for LESCs, including RAR activation, antigen
presentation and axonal guidance signaling could be identified
(p<0.001). A number of molecules functioning in cellular movement
(381), proliferation (567), development (552), death and survival
(520), and cell-to-cell signaling (290) were found with top biological
functions in LESCs (p<0.001). In addition, 5 upstream regulators
could be identified in LESCs (TGFB1, TNF, IFNG, ERBB2 and
OSM).
Conclusions: Gene and molecular pathways may provide more
specific understanding of the signaling molecules associated with
LESCs. Future better identification and use of LESCs in treatment of
ocular surface diseases and discovery of innovative therapies may be
aided by gene array technology.
Commercial Relationships: Goran Petrovski, None; Reka Albert,
None; Zoltan Vereb, None; Morten C. Moe, None; Ole Kristoffer
Olstad, None; Andras Berta, None; Laszlo Fesus, None
Support: Partial financial support was obtained from the TAMOP4.2.2-08/1/2008-0015 grant (Hungary); the Research Council of
Norway, the Blindemissionen IL, the Norwegian Association of the
Blind and Partially Sighted, the Faculty of Medicine University of
Oslo and Oslo University Hospital (Norway)
Program Number: 998 Poster Board Number: B0303
Presentation Time: 1:00 PM - 2:45 PM
A NEW RAPID METHOD OF DENUDING HUMAN
AMNIOTIC MEMBRANE FOR LIMBAL AND STEM CELL
CULTURE
Alexander V. Ljubimov1, 3, Dhruv Sareen1, Loren Ornelas1, Anais
Sahabian1, David M. Hemmati1, 3, Chantelle A. Ghiam1, 3, William J.
Brunken2, Yaron S. Rabinowitz1, Clive Svendsen1, Mehrnoosh
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Saghizadeh1. 1Regenerative Medicine Institute, Cedars-Sinai Medical
Center, Los Angeles, CA; 2Ophthalmology and Cell Biology, State
University of New York, Downstate Medical Center, Brooklyn, New
York, NY; 3University of California, Los Angeles, CA.
Purpose: Human amniotic membrane (HAM) is a common
substratum for culturing limbal cells for future transplantation. Best
results are obtained when amniotic epithelium is removed from
HAM. Most existing methods require long treatments and leave many
cells attached to HAM. The purpose was to develop a reliable method
of HAM denuding with effective and fast removal of amniotic
epithelium.
Methods: Fresh HAM was mechanically separated from chorion, and
cryopreserved in PBS with 10% DMSO. After thawing and washing,
HAM was de-epithelialized by different methods: soaking in 0.02%
EDTA in PBS at 37°C for 1 hour; EDTA followed by gentle scraping
with electric toothbrush; EDTA followed by scraping with n-heptanol
soaked cotton tip; 125 μg/ml thermolysin in PBS at 37°C for 9 min
followed by gentle mechanical scraping. Alternatively, HAM placed
in CellCrownTM inserts was rubbed on the epithelial side for 10-30
seconds with cotton tip soaked in 0.5 M NaOH and immediately
washed in PBS. Denuded OCT-embedded HAM was cryosectioned
and immunostained for typical components of limbal basement
membrane (BM), including laminin α2, γ1, and γ3 chains, α1/α2 type
IV collagen, perlecan, nidogen-2, and fibronectin. NaOH-denuded
HAM was used to culture human telomerase-immortalized corneal
epithelial cells, limbal cells from corneoscleral rims, and induced
pluripotent stem cells (iPSC) derived from limbal epithelial cells.
Cultured cells were checked for putative stem cell marker expression
(ΔNp63α, ABCG2, and keratins 14, 15, 17, and 19) by
immunostaining.
Results: Control HAM was positive for all BM markers except for
laminin α2 chain that gave weak and inconsistent staining. HAM decellularization with EDTA or EDTA with n-heptanol left out many
adherent epithelial cells; rubbing with toothbrush produced local
tears. Thermolysin and NaOH resulted in the best cell removal with
continuous staining for all BM markers tested. However,
thermolysin-treated HAM became fragile and could be easily
damaged during manipulation, which was not seen after NaOH
denuding. Corneal epithelial cell line, limbal cells, and limbalderived iPSC all grew well on NaOH-denuded HAM. Cultured cells,
especially limbal cells were positive for putative stem cell markers.
Conclusions: HAM de-cellularization with NaOH results in rapid
and thorough amniotic cell removal, and ensures excellent
preservation of HAM structure and robust stem cell growth on
denuded HAM.
Commercial Relationships: Alexander V. Ljubimov, None; Dhruv
Sareen, None; Loren Ornelas, None; Anais Sahabian, None;
David M. Hemmati, None; Chantelle A. Ghiam, None; William J.
Brunken, None; Yaron S. Rabinowitz, None; Clive Svendsen,
None; Mehrnoosh Saghizadeh, None
Support: NIH EY13431, CTSI grant UL 1RR033176, and
Regenerative Medicine Institute grants.
Program Number: 999 Poster Board Number: B0304
Presentation Time: 1:00 PM - 2:45 PM
Expansion of Human Corneal Epithelial Stem/Progenitor Cells in
Feeder-Free Explant Cultures
Sophie X. Deng, Martin N. Nakatsu, SHEYLA GONZALEZ, Hua Mei.
Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA.
Purpose: To present a feeder-free culturing method of human
corneal epithelial stem/progenitor cells in vitro.
Methods: Primary limbal epithelial cell (LEC) sheets isolated from
human sclerocorneal tissues were trypsinized to produce single LECs
and co-cultured with growth arrested 3T3-J2 feeder cells for 14 days.
Additionally, the outgrowth of LECs from limbal explant pieces was
also cultured for 14 days in the presence or absence of 3T3-J2 feeder.
The phenotype of the cultured LECs was assessed by their mRNA
expression level of putative stem cell markers and differentiation
marker by qRT-PCR and immunocytochemistry. The percentage of
p63 bright cells in each culture was assessed, and the cell
proliferation was evaluated by the Ki67 expression and cell number.
Results: We observed no significant difference in cultured LEC
morphology among each culturing method. The LEC growth rate
increased over 9-fold in NF explant cultures compared to 3T3-J2
explant cultures and the adjusted growth rate between NF cultures
and 3T3-J2 single LEC cultures had similar yields (p>0.05). Gene
expression of putative limbal stem cell markers, ABCG2 and ΔNp63
were elevated among NF cultures compared to the gold standard and
explants on 3T3-J2. We observed a 7.6-fold and 2.2-fold increase in
ABCG2 and ΔNp63 expression respectively when comparing NF
explant cultures to the gold standard, while there was only a 4.4-fold
and 1.4-fold increase in ABCG2 and ΔNp63 expression respectively
when comparing 3T3-J2 explant cultures to the gold standard.
However, we did observe an increase in K12 expression in NF
explant cultures when compared to the gold standard (6.2-fold).
There was a large increase in Ki67 proliferation (4.0-fold) when
compared to the gold standard. There was no difference in Ki67
expression between 3T3-J2 explant cultures and the gold standard.
Finally, examination of p63α expression in each condition reviewed
no discernable differences in the percentage of p63α bright cells
between the gold standard (9.0%) and NF explant cultures (10.0%),
but we did see a decrease in the 3T3-J2 explant cultures (6%).
Conclusions: 3T3 feeder cells may not be necessary for the growth
of stem/progenitor cell population in the primary explant culture. The
explants themselves may already contain niche factors that are
required for the viability of corneal stem cells.
Commercial Relationships: Sophie X. Deng, None; Martin N.
Nakatsu, None; SHEYLA GONZALEZ, None; Hua Mei, None
Support: CIRM Grant TR2-01768, NIH Grant EY021797
Program Number: 1000 Poster Board Number: B0305
Presentation Time: 1:00 PM - 2:45 PM
Sox9, a determinant of hair follicle stemness, identifies a subset of
murine basal corneal epithelial cells expressing putative stem cell
markers
Rachel Sartaj1, Aihong Liu1, Elaine Fuchs2, Mark Rosenblatt1.
1
Ophthalmology Department, Weill Cornell Medical College, New
York, NY; 2Laboratory of Mammalian Cell Biology and
Development, Rockefeller University, New York, NY.
Purpose: To localize the expression of Sox9 in the cornea and to
identify genes co-expressed with this determinant of hair follicle
stemness.
Methods: Antibodies against Sox9 were used to immunostain
corneal sections from wild-type mice, and the nuclear localization of
Sox9 determined by wide field and confocal microscopy.
Localization of Sox9 within the cornea epithelium was confirmed by
immunostaining for GFP in Sox9-eGFP mice expressing GFP under
the control of the sox9 promoter region. Corneal epithelial cells were
isolated from Sox9-egfp mice via sequential enzymatic treatments,
and the cell suspension used for FACS sorting to obtain purified
population of Sox9-expressing (GFP +) cells and Sox9 nonexpressing (GFP-) cells. RNA was separately isolated from GFP +
and GFP - populations and qRT-PCR performed to determine the
relative expression of putative corneal epithelial stem cell markers
ABCG2, p63 and N-cadherin (Cdh2) as well as the differentiation
markers keratin-12 (Krt12) and involucrin (Ivl).
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Results: In the mouse cornea, Sox9 and GFP proteins were located in
the basal cells of central and limbal cornea epithelium in postnatal
day 12 (P12) and adult mice (P70). The expression of Sox9 and GFP
proteins are restricted to the basal layer of the corneal epithelium at
P12 and increases to 2 layers as the Sox9 progeny divides from the
basal layer to one row above. Quantitative PCR experiments showed
a significantly higher relative expression of known corneal epithelial
stem cell markers in the Sox9- expressing cells obtained by FACS
sorting. We found that there was up regulation of ABCG2 (5 fold),
p63 (18 fold), and Cdh2 (3.6 fold), in the sox9 expressing cells. In
contrast, when we analyzed the corneal differentiation markers in
these cells, we found down regulation of Krt12 (5 fold) and Ivl (2.5
fold).
Conclusions: Sox9 may be a novel marker of corneal epithelial stem
cells given its localization to basal corneal epithelial layers an its coexpression with other putative stem cell markers. A possible role for
Sox9 in mediating corneal epithelial stemness could have utility in
the diagnosis and treatment of corneal limbal stem cell deficiency.
Commercial Relationships: Rachel Sartaj, None; Aihong Liu,
None; Elaine Fuchs, None; Mark Rosenblatt, None
Support: Starr Foundation Tri-Institutional Stem Cell Initiative,
NYSTEM, Research to Prevent Blindness
Program Number: 1001 Poster Board Number: B0306
Presentation Time: 1:00 PM - 2:45 PM
Limbal mesenchymal stromal cells (L-MSC) display
immunosuppressive properties across donor and species
boundaries
Damien G. Harkin1, 2, Laura J. Bray2, 1, Celena Heazlewood3, Kerry
Atkinson3. 1School of Biomedical Sciences, Queensland University of
Technology, Brisbane, QLD, Australia; 2Queensland Eye Institute,
Brisbane, QLD, Australia; 3Mater Medical Research Institute,
Brisbane, QLD, Australia.
Purpose: We have evaluated the immunosuppressive properties of LMSC with the view to using these cells in allogeneic cell therapies for
corneal disorders. We hypothesized that L-MSC cultures would
suppress T-cell activation, in a similar way to those established from
human bone marrow (BM-MSC).
Methods: MSC cultures were established from the limbal stroma of
cadaveric donor eye tissue (up to 1 week postmortem) using either
conventional serum-supplemented growth medium or a commercial
serum-free medium optimized for bone marrow derived MSC
(MesenCult-XF system). The MSC phenotype was examined by flow
cytometry according to current and emerging markers for human
MSC. Immunosuppressive properties were assessed using a mixed
lymphocyte reaction (MLR) assay, whereby the white cell fraction
from two immunologically incompatible blood donors are cultured
together in direct contact with growth arrested MSC. T-cell activation
(proliferation) was measured by uptake of tritiated thymidine. Human
L-MSC were tested in parallel with human BM-MSC and rabbit LMSC. Human and rabbit L-MSC were also tested for their ability to
stimulate the growth of limbal epithelial (LE) cells in colony
formation assays (for both human as well as rabbit LE cells).
Results: L-MSC cultures were >95% negative for CD34, CD45 and
HLA-DR and positive for CD73, CD90, CD105 and HLA-ABC.
Modest levels (30%) of CD146 expression were observed for L-MSC
cultures grown in serum-supplemented growth medium, but not those
grown in MesenCult-XF. All MSC cultures derived from both human
and rabbit tissue suppressed T-cell activation to varying degrees
according to culture technique and species (MesenCult-XF >> serumfed cultures, rabbit L-MSC >> human L-MSC). All L-MSC
stimulated colony formation by LE cells irrespectively of the
combination of cell species used.
Conclusions: L-MSC display immunosuppressive qualities, in
addition to their established non-immunogenic cell surface marker
profile, and stimulate LE cell growth in vitro across species
boundaries. These results support the potential use of allogeneic or
even xenogeneic L-MSC in the treatment of corneal disorders.
Commercial Relationships: Damien G. Harkin, None; Laura J.
Bray, None; Celena Heazlewood, None; Kerry Atkinson, Osiris
Therapeutics Inc (I), Mesoblast Ptl (C)
Support: Supported by NHMRC Project Grant No. 553038
Program Number: 1002 Poster Board Number: B0307
Presentation Time: 1:00 PM - 2:45 PM
EDC/NHS cross-linked amniotic membrane preferentially
preserves corneal epithelial progenitor cells by activating Wnt/β
catenin signaling
David H. Ma1, Hung-Chi Chen1, Jui-Yang Lai2, Kevin S. Ma1, LungKun Yeh1, Unique Yang3, Jessica Ma1. 1Ophthalmology, Chang Gung
Memorial Hospital, Taipei, Taiwan; 2Institute of Biochemical and
Biomedical Engineering, Chang Gung University, Taipei, Taiwan;
3
Cell Biology, University of California, Berkeley, Berkeley, CA.
Purpose: Previously, we have shown that EDC/NHS cross-linked
denuded amniotic membrane (CLDAM) is compatible for the growth
of human limbo-corneal epithelial (HLE) cells in vitro and in vivo
(Biomaterials, 2010, 31: 6647-6658), in this study we further
investigate whether CLDAM preferentially preserves HLE progenitor
cells and the underlying mechanism.
Methods: HLE cells expanded from explants were cultured on dish
(HLE/dish), on denuded AM (HLE/DAM) and on CLDAM
(HLE/CLDAM). When near confluency, cell density, BrdU label
retention, and colony formation assay (CFA) were analyzed.
Immunoconfocal microscopy, Western blot, and Q-PCR for keratin
12, connexin 43, ABCG2, deltaNp63α, β-catenin and TCF-4 were
performed. Finally, selective GSK3β inhibitors SB216763 or
SB415286 were added to HLE/dish cultures to evaluate CFA and
deltaNp63α expression.
Results: Compared with HLE/dish or HLE/DAM, HLE cells on
CLDAM were more compact in morphology, expressed higher level
of p63, ABCG2, and lower level of connexin 43 and keratin 12. CFA
was highest in HLE/CLDAM, so were the nuclear expression of βcatenin and TCF-4. Addition of GSK3-β inhibitors to HLE/dish
cultures not only increased CFE but also the expression of stem cell
marker p63.
Conclusions: CLDAM showed tendency to better preserve HLE
progenitor cells in vitro, and Wnt/βcatenin signaling may be
involved, possibly through the activation of p63.
Commercial Relationships: David H. Ma, None; Hung-Chi Chen,
None; Jui-Yang Lai, None; Kevin S. Ma, None; Lung-Kun Yeh,
None; Unique Yang, None; Jessica Ma, None
Support: NSC99-2314-B-182A-027-MY3
Program Number: 1003 Poster Board Number: B0308
Presentation Time: 1:00 PM - 2:45 PM
Immobilized HC-HA Preserves Limbal Niche Cell Phenotype to
Prevent Limbal Epithelial Progenitor Cells from Differentiation
Bo Han1, 2, Yingting Zhu1, Suzhen Zhang1, Scheffer C. Tseng1. 1Tissue
Tech, Miami, FL; 2Department of Ophthalmology, Union Hospital
Huazhong University of Science and Technology, Wuhan, China.
Purpose: We have recently reported the success of isolating limbal
niche cells (LNC) that can prevent limbal epithelial progenitor cells
(LEPC) from differentiation in a reunion assay in 3-dimentional (3D)
Matrigel. Amniotic membrane (AM) alone can help expand residual
limbal stem cells in vitro and in vivo. Because we have successfully
isolated heavy chain-hyaluronan complex (HC-HA) from AM, we
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
wonder whether HC-HA can preserve LNC to prevent LEPC from
differentiation.
Methods: LNC were cultured in MESCM on plastics, 3D Matrigel,
or immobilized HC-HA. qRT-PCR and immunostaining were used to
compare expression of markers that have been reported for LNC.
AMD 3100 was used to block SDF1/CXCR4 signaling. LEPC were
added to LNC aggregates formed on immobilized HC-HA in
MESCM and compared to the reunion assay in 3D Matrigel for 7
days. qRT-PCR, Western blotting, and immunostaining were used to
characterize expression of markers for LEPC and corneal epithelial
differentiation.
Results: LNC aggregated on 2D immobilized HC-HA at day 1 with
small and round cells and expressed 2- to 4-fold higher ESC markers
such as Nanog, Oct4, Rex1, and Sox2 at day 7 than plastic and 3D
Matrigel. Such aggregation was mediated by 3 to 4-fold higher
expression of SDF1 and CXCR4 and could be abolished by
AMD3100 added on day 0 but not day 4. Presumably because of high
expression of SDF1 and CXCR4, LNC attracted reunion with LEPC
on immobilized HC-HA, similar to our recent report that LNC
attracted reunion with LEPC in 3D Matrigel. We speculate that such
a reunion with LEPC will prevent LEPC from differentiation.
Conclusions: Limbal niche cells form aggregates on HC-HA via
SDF1/CXCR4 signaling, express higher levels of ESC markers, and
attract reunion with LEPC. Studies are underway to characterize
whether such reunion on immobilized HC-HA may prevent LEPC
from differentiation.
Commercial Relationships: Bo Han, Tissue Tech (F); Yingting
Zhu, Tissue Tech Inc (F), Tissue Tech Inc (E), Tissue Tech Inc (P);
Suzhen Zhang, TissueTech, Inc (E); Scheffer C. Tseng, NIH, NEI
(F), TissueTech, Inc. (F), TissueTech, Inc. (E), TissueTech, Inc. (P)
Support: NIH, NEI, RO1 EY06819
Program Number: 1004 Poster Board Number: B0309
Presentation Time: 1:00 PM - 2:45 PM
Comparison of Human and Mouse Feeders, Xeno-free and
Standard Media in Different Oxygen Tensions for Preservation
of Human Limbal Progenitor Cells during Culture
Kalliopi Stasi1, Mina Massaro-Giordano1, Jiayan Huang1, John
Gearhart2. 1Scheie Eye Institute, University of Pennsylvania,
Philadelphia, PA; 2Cell and Developmental Biology, Institute for
Regenerative Medicine, University of Pennsylvania, Philadelphia,
PA.
Purpose: To identify culture conditions that allow preservation of
limbal stem cells in culture with human feeders and xeno-free media
for potential clinical use.
Methods: Limbal epithelial cells were isolated from 82 cadaveral
donors or 29 cataract patients with trypsin or dispase/trypsin in 13
variations. Feeders used: 3T3-J2, adult or neonatal or fetal human
dermal fibroblasts, lung fibroblasts MRC-5 and human limbal
fibroblasts. Media used: Xeno media per Pellegrini group, MCBD
151 (Schotzer-Schrehardt), or SHEM (Tseng), and Xeno-free media
with Calcium 1.3mM, 1.05mM, 0.4mM and 0.1mM, and EGF 0, 2.5,
5, 10 or 20ng/ml, under O2 20%, 14% or 5%. Statistical analysis was
performed on outcomes: quantitative immunocytochemistry (Q-ICC)
for p63α (cytospins), RT-PCR for p63α, Δp63 and K12, Colony
Forming Efficiency (CFE), Holoclone Forming Efficiency,
Percentage of Aborted Colonies, and number of passages. ICC: p63α,
p63 (4A4), ABCG2, Bmi1, c/EBPδ, K12, K15 and MUC1 on
cytospins and coverslips. Statistical analysis: ANOVA/MANOVA
with SAS 9.2
Results: Isolation methods (cadaveral donors) evaluated for yield,
viability and CFE. Yield was correlated with dispase (+0.58, p=0)
and days in preservation (-0.52, p=0) and affected by trypsin (p=0)
and days in preservation (p=.0006), viability was affected by trypsin
(p=0.0269), and CFE was correlated with days in preservation (-0.47,
p=0) and yield (+0.43, p=.0004) and affected by donor age (p=.0001)
and trypsin (p=.04). Isolation method selected: dispase 2.4U/ml x2
hrs and TLE x10 min. Multivariate analysis of effect of variables on
number of passages (max 10) from 15 patients cultured on 3T3-J2
showed significant effect of donor age (0.032, p= 0.002) and medium
[SHEM (-2, p=0), Xeno-Free Ca0.01 with EGF10 (0.67, p=.04) or
EGF20 (1.67, p=0)], and no effect of O2 14% vs. 20%. ANOVA of
34 combinations of feeder, medium and O2 conditions was
significant for Q-ICC (p=.007) and CFE (p=.0002) but not significant
for age, gender, feeders, medium or O2 alone. MRC-5 feeders with
Xeno-free Calcium 0.01mM and EGF10ng/ml at 20% O2 were
effective in Q-ICC (x4.9 fold) and p63α PCR (x1.4 fold) compared to
baseline 3T3 feeders with Pellegrini medium.
Conclusions: Xeno-Free media and human feeders may effectively
preserve limbal progenitors in culture as measured with multiple
outcomes in head-to-head comparisons.
Commercial Relationships: Kalliopi Stasi, None; Mina MassaroGiordano, Tear Lab (R); Jiayan Huang, None; John Gearhart,
None
Support: NIH Grant K12EY015398, Pennsylvania State
0426/554248/8319, RPB-Unrestricted
Program Number: 1005 Poster Board Number: B0310
Presentation Time: 1:00 PM - 2:45 PM
Outcomes of Penetrating Keratoplasty after ex vivo Expanded
Autologous Limbal Stem Cell Transplantation in Humans
Oliver J. Baylis1, 2, Hardeep S. Mudhar4, Majlinda Lako1, 3,
Francisco C. Figueiredo2, 1. 1Institute of Genetic Medicine,
Newcastle University, Newcastle-upon-Tyne, United Kingdom;
2
Department of Ophthalmology, Royal Victoria Infirmary, Newcastle
Upon Tyne, United Kingdom; 3North East England Stem Cell
Institute, Newcastle University, Newcastle Upon Tyne, United
Kingdom; 4National Specialist Ophthalmic Pathology Service, Royal
Hallamshire Hospital, Sheffield, United Kingdom.
Purpose: To study the clinical and histological outcomes of patients
who received ex vivo expanded autologous limbal stem cells (LSC)
on human amniotic membrane (AM) followed by penetrating
keratoplasty (PK) for the treatment of unilateral total limbal stem cell
deficiency (LSCD).
Methods: Prospective, single-centre, noncomparative, interventional
case series. Participants: 5 consecutive patients with unilateral LSCD
were treated at the Department of Ophthalmology, Royal Victoria
Infirmary, Newcastle upon Tyne, UK between April 2006 and
November 2012. Intervention: HLA-matched PK was performed at
least 6 months after ex vivo expanded autologous LSC using an
animal and feeder free method combined with high dose topical
steroid. Outcome measures: LSC survival was assessed by slit lamp
biomicroscopy, histology of excised corneal buttons and post-PK
corneal impression cytology (IC) showing absence of goblet cells. PK
survival, best corrected visual acuity (BCVA), patient-reported
outcomes and complications were also recorded.
Results: All patients were male with a mean age of 52 (range 20-77).
Six PK were performed in 5 eyes of 5 patients, with 1 regraft. Mean
LSC and PK follow up was 63 months (range 35-78) and 28 months
(range 4-48) respectively. Postoperatively, satisfactory ocular surface
reconstruction was maintained in all eyes (100%), as confirmed by
IC. One patient was lost to follow-up 4 months after PK. At last
examination, BCVA improved in 4 eyes (≥6/36). Vision impairment
and pain scores improved in all patients (p<0.05). Complications: PK
rejection with subsequent failure occurred twice in 1 eye. Histology
of excised corneas after PK showed complete absence of goblet cells,
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
100% CK3 +ve corneal epithelial cells and presence of p63 +ve LSClike cells in the basal epithelium in all corneas. In addition, TEM
revealed a uniform stratified epithelium, typical of the normal central
cornea.
Conclusions: This study demonstrates that transplantation of ex vivo
expanded autologous LSC cultured on AM followed by PK is an
effective method of reconstructing the corneal surface and restoring
useful vision in patients with unilateral total LSCD. Despite HLAmatching PK and high dose topical steroid, graft rejection with
failure may still occur in this high risk group of patients. Further
studies with more patients and longer follow-up should be conducted.
Commercial Relationships: Oliver J. Baylis, None; Hardeep S.
Mudhar, None; Majlinda Lako, None; Francisco C. Figueiredo,
None
Support: MRC, Newcastle Healthcare Charity UK NIHR
Biomedical Research Centre for Ageing and Age-related disease and
the Newcastle upon Tyne NHS Hospitals trust
Program Number: 1006 Poster Board Number: B0311
Presentation Time: 1:00 PM - 2:45 PM
Integrating human epithelial stem cells and keratocytes into a
transparent, collagen based bioengineered cornea for disease
modelling
James W. Foster1, Ana Maria Ionescu1, Roanne R. Jones2, Ricardo
M. Gouveia1, Che J. Connon1. 1School of Chemistry, Food and
Pharmacy, University of Reading, Reading, United Kingdom;
2
Department of Optics, University of Granada, Granada, Spain.
Purpose: To demonstrate the integration of epithelial stem cells and
keratocytes into a single construct using defiend conditions. This will
allow the interplay between the two cell types that form the anterior
cornea to be investigated. Since the collagen constructs are intended
to replace the principal refractive and image forming component of
the eye, their optical quality was also studied to ensure the
functionality of these bioengineered tissues.
Methods: Epithelial stem cells and keratocytes were isolated from
cadaverous human tissue and cultivated in vitro. Keratocytes were
encapsualted within compressed collagen gels and allowed to
proliferate and differentiate for 7 days before addition of limbal
epithelial stem cells under differentiation media conditions.
Expression levels of specific keratocyte markers ALDH-1, Lumican,
Col5a1, and Keratocan were determined through PCR and
fluorescent microscopy. The optical quality of resulting constructs
was determined by Contrast Transfer Function (CTF), a simple
method for the accurate quantification of contrast between adjacent
objects. Several bar patterns of alternating white and black lines with
increasing frequency (2, 4, 6, 10, 20, 31, and 61 cycles/mm) were
displayed on a LCD and images were captured using a N90 Nikon
camera. The CTF and transparency levels were calculated using
adequate image analysis software (ImageJ). Secretion of ECM
components by keratocytes and epithelial cell behaviour were
examined over 3 weeks.
Results: The inclusion of cells within the gels increased transparency
by 10% to 66%, reaching a value sufficient for a collagen construct to
be used as a corneal replacement. The phenotype of the epithelial
stem cell was characterized by IHC, qPCR, and flow cytometry as
ABCG2- and ΔNP63-positive, and CK3-negative. The epithelial stem
cells differentiated to CK3-positive cells with accompanying
stratification (>4 cells thick). Keratocytes were shown to secrete
col5a1, lumican, and keratocan. Keratocyte organisation showed
increased orthogonal alignment within the gels.
Conclusions: Here we have recapitulated some of the complex
interplay between human epithelial and stromal cells within a defined
culture system. This resulted in corneal constructs with therapeutic
levels of transparency and the desirable cell phenotypes.
Commercial Relationships: James W. Foster, None; Ana Maria
Ionescu, None; Roanne R. Jones, None; Ricardo M. Gouveia,
None; Che J. Connon, None
Program Number: 1007 Poster Board Number: B0312
Presentation Time: 1:00 PM - 2:45 PM
Evaluation of Corneal Stem/Progenitor Cells in leptin deficient
mice
Hiroki Ueno1, Takaaki Hattori2, Yuta Kumagai1, Noboru Suzuki3,
Satoki Ueno1. 1Ophthalmology, St Marianna Univ School of Med,
Kawasaki, Japan; 2Ophthalmology, Tokyo Medical University,
Tokyo, Japan; 3Immunology and Medicine, St. Marianna Univ
School of Med, Kawasaki, Japan.
Purpose: Diabetic keratopathy (DK) remains difficult to be treated. It
can cause corneal persistent epithelial defects, suggesting a role of
corneal nerves in maintaining the corneal homeostasis. Leptin
deficient mice which are widely accepted as an animal model of mild
type-II diabetes mellitus (DM) have sensory nerve conduction
deficits, small sensory nerve fiber neuropathy,intra-epidermal sensory
nerve fiber loss. The purpose of this study is to investigate whether
putative corneal stem/progenitor cells are altered in obese mice and to
understand the pathogenesis in obesity and diabetes mice lacking the
leptin gene.
Methods: 12 week-old male mice with mild type II DM (C57 6JHam
ob/ob mice) were assessed by beta-III tubulin (neural marker)
immunostaining. Real-time polymerase chain reaction was performed
to quantify expression of ATP-binding cassette subfamily G member
2 (ABCG2), hairy enhancer of split 1 (Hes1), the low-affinity NGF
receptors (p75) as corneal progenitor cell markers. Keratin 19 and
Hes1 were assessed in type II DM mice and controls by
immunofluorescence microscopic studies.
Results: Beta-III tubulin expression detected with immunostaining
was decreased in the diabetic corneas. Corneal subbasal plexus of
nerve fibers with mild type II DM were preferentially thinner and had
fewer branches compared to the normal mice. Hes1 and Keratin 19
expression noted with immunostaining was diminished in corneas of
diabetes mellitus when compared with normal corneas. Similarly,
mRNA expression levels for Hes1 and p75 were decreased in corneas
with diabetes.
Conclusions: Our results suggest that corneal stem/progenitor cells
could be altered in animal model of obesity and mild type II diabetes
mellitus. Our data may provide novel evidence for the close
connection between innervation and maintaining corneal progenitor
cells and/or the stem cell niche in cases of mild type II diabetes
mellitus and leptin deficient.
Commercial Relationships: Hiroki Ueno, None; Takaaki Hattori,
None; Yuta Kumagai, None; Noboru Suzuki, None; Satoki Ueno,
None
Program Number: 1008 Poster Board Number: B0313
Presentation Time: 1:00 PM - 2:45 PM
Retinal Pigment Epithelial Cell Differentiation in Primary
Human Limbal Neurosphere Cultures
Samuel McLenachan1, 2, Dana Zhang1, 2, Fred K. Chen1, 2. 1Ocular
Tissue Engineering, Lions Eye Institute, Nedlands, WA, Australia;
2
Centre of Ophthalmology and Visual Science, University of Western
Australia, Perth, WA, Australia.
Purpose: The human corneoscleral limbus contains multipotent stem
cells that can be isolated and cultured for clinical applications, such
as the treatment of limbal stem cell deficiency. In culture, limbal
stem cells can be induced into the neural linage to produce cells
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
displaying the characteristics of photoreceptors. Here, we have
examined the hypothesis that retinal pigment epithelial (RPE) cells
can be produced from primary human limbal cultures.
Methods: Human corneoscleral rims were incubated with
collagenase to facilitate the removal of the limbal epithelium (LE).
LE was dissociated non-enzymatically and cultured in the presence of
EGF, FGF2 and Noggin to produce floating neurospheres (LiNS).
LiNS were plated on a geltrex matrix to form adherent colonies. In
parallel, primary limbal cells were cultured in keratinocyte media to
produce adherent LE cell monolayers. Primary LE and LiNS cultures
were examined by microscopy and immunocytochemical analysis.
Results: Two different populations of neurospheres were evident in
primary human LiNS cultures; pigmented and non-pigmented.
Comparison of primary keratinocyte and neurosphere cultures
revealed the predominance of non-pigmented LiNS in cultures with
stromal keratocyte contamination. Pigmented LiNS expressed the
ocular transcription factors PAX6, OTX2 and MITF, and contained
cells expressing the RPE specific markers RPE65 and ZO1.
Conclusions: Culture of human limbal epithelial stem cells in the
presence of EGF, FGF2 and Noggin leads to the induction of LiNS
containing pigmented cells expressing the RPE cell markers MITF,
RPE65 and ZOI.
Human limbal neurosphere
Human LiNS cells immunostained for RPE65 (green) and zonus
occludin-1 (red)
Commercial Relationships: Samuel McLenachan, None; Dana
Zhang, None; Fred K. Chen, None
Program Number: 1009 Poster Board Number: B0314
Presentation Time: 1:00 PM - 2:45 PM
Transpalntation of umbilical mesenchymal stem cells cures the
corneal defects of Mucopolysaccharidosis VII mice
Vivien J. Coulson-Thomas1, Bruce Caterson2, Chia-Yang Liu1,
Winston W. Kao1. 1Ophthalmology, University of Cincinnati,
Cincinnati, OH; 2Laboratory of Connective Tissue Biology, School of
Biosciences, Cardiff University, Cardiff, United Kingdom.
Purpose: Mucopolysaccharidoses (MPS) are a family of related
disorders caused by a mutation in one of the lysosomal
exoglycosidases required for the sequential degradation of
glycosaminoglycans (GAGs). MPS are progressive disorders in
which GAGs and their metabolic derivatives accumulate in
lysosomes compromising cellular activity and ultimately leading to
cell death. MPS VII, Sly syndrome, caused by a mutation in βglucuronidase, manifests as hepatomegaly, skeletal dysplasia, short
stature, corneal clouding and developmental delay, due to the
accumulation of heparan sulfate (HS), dermatan sulfate and
chondroitin 4,6-sulfate (CS). Current treatment regimens for MPS are
not effective for treating corneal clouding and mental development.
Methods: We hypothesized that umbilical mesenchymal stem cells
(UMSC) transplanted into the corneal stroma can participate in the
catabolism of GAGs, thus providing a means of cell therapy for MPS.
For such, human UMSC were intrastromally transplanted into
corneas of 1, 2 and 3 month-old MPS VII mice.
Results: UMSC transplantation restored the dendritic and hexagonal
morphology of host keratocytes and endothelial cells, respectively,
and in vivo confocal microscopy (HRTII) revealed reduced corneal
haze. Immunohistochemistry using antibodies against HS and CS
chains, as well as, LAMP2 revealed a decrease in GAG content and
both lysosomal number and size in the treated corneas to levels
similar to that of littermate controls. Labeling UMSC intracellular
compartments prior to transplantation revealed the distribution of
UMSC exosomes throughout the corneal stroma and endothelium. An
in vitro co-culture assay between skin fibroblasts isolated from
MPSVII mice and UMSC labeled with LysoSensor demonstrated that
neutral exosomes released by the UMSC are up taken by the
fibroblasts and proceed to fuse with the acidic lysosomes.
Conclusions: Therefore, transplanted UMSC participate in
extracellular GAG turnover and aid host keratocytes to metabolize
accumulated GAG, suggesting that UMSC could be a good
alternative for treating corneal defects associated with MPS and other
congenital metabolic disorders. Moreover, given the simplicity of the
treatment, we suggest it as prophylactic treatment upon diagnosis in
order to avoid the development of corneal clouding.
Commercial Relationships: Vivien J. Coulson-Thomas, None;
Bruce Caterson, Abcam, Cambridge, UK (I), CosmoBio, Japan (I);
Chia-Yang Liu, None; Winston W. Kao, None
Support: NIH/NEI RO1 EY021768, Research to Prevent Blindness,
and Ohio Lions eye Research Foundation
Program Number: 1010 Poster Board Number: B0315
Presentation Time: 1:00 PM - 2:45 PM
Dental Pulp: a Source of Stem Cells with Keratocyte Potential
Martha L. Funderburgh1, Fatima N. Syed-Picard2, Charles S. Sfeir2,
James L. Funderburgh1. 1Department of Ophthalmology, Univ of
Pittsburgh Sch of Med, Pittsburgh, PA; 2Center for Craniofacial
Regeneration, University of Pittsburgh, Pittsburgh, PA.
Purpose: Blindness due to corneal stromal opacity can be
successfully treated by allografts; however, inflammation or previous
graft rejections predict poor outcome for some allogenic tissue in the
stroma. These difficult cases may benefit from autologous stem cell
therapy or from autologous grafts of bioengineered tissue. Dental
pulp contains a potent stem cell population with immune-suppressive
properties, cells that could be the autologous stem cells ideal for
corneal regeneration. The purpose of this study was to explore the
potential for dental pulp stem cells (DPSC) to adopt a keratocyte
phenotype.
Methods: Dental pulp extracted from human molars was dispersed
with collagenase, cultured and used at passage 3. Cells expressing
CXCR4 protein were isolated using MACS technology. Expression
of stem cell genes was determined using flow cytometry and qPCR.
Differentiation to keratocytes was carried out on collagen gels or
aligned nanofiber substrata in serum-free medium containing FGF2
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
and ascorbate-2-phosphate.
Results: Cultured cells from dental pulp expressing the chemokine
receptor CXCR4 were isolated by immunoaffinity. CXCR4+ DPSC
cells had increased expression of pluripotent genes OCT4, NANOG,
and SOX2, neural crest marker p75NTR, and genes expressed by
corneal stromal stem cells ABCG2, KIT, SIX2, PAX6, and
NOTCH1. When the CXCR4+ cells were cultured on collagen gels in
medium that induced keratocyte differentiation, expression of stem
cells genes was downregulated and mRNAs for several keratocytespecific products were upregulated, including keratocan (KERA),
ALDH3A1, PTGDS and enzymes involved in keratan sulfate
synthesis beta-1,3, glucosaminyltransferase 7 (B3GNT7) and corneal
6-O-glucosaminyl-sulfotransferase 6 (CHST6). The expression levels
of these genes were almost identical to those in corneal stromal stem
cells, which under the same conditions produce a stroma-like
extracellular matrix.
Conclusions: CXCR4 appears to be a key cell-surface marker of
stem cells with potential to differentiate in the keratocyte lineage.
The availability and potency of stem cells from dental pulp makes
these cells an excellent candidate as a source of autologous stromal
cells for future bioengineering or cell-based therapies for stromal
blindness.
Commercial Relationships: Martha L. Funderburgh, None;
Fatima N. Syed-Picard, None; Charles S. Sfeir, None; James L.
Funderburgh, None
Support: NIH Grants EY016415 (JLF), P30-EY008098,
F31DE019753 (FS-P), Eye & Ear Foundation of Pittsburgh, Research
to Prevent Blindness
Program Number: 1011 Poster Board Number: B0316
Presentation Time: 1:00 PM - 2:45 PM
Corneal stroma derived MSCs maintain immunosuppression
with wound healing capacity in vitro
Zoltan Vereb1, Reka Albert1, 2, Morten C. Moe4, Laszlo Fesus1, Eva
Rajnavolgyi3, Andras Berta2, Goran Petrovski1, 2. 1Department of
Biochemistry and Molecular Biology, University of Debrecen,
Debrecen, Hungary; 2Department of Ophthalmology, University of
Debrecen, Debrecen, Hungary; 3Department of Immunology,
University of Debrecen, Debrecen, Hungary; 4Deparment of
Ophthalmology, Oslo University Hospital, Oslo, Norway.
Purpose: Mesenchymal stem cells (MSC) are the stromal cells of
bone marrow, but they can also be found in other tissues including
the cornea. Our goal was to isolate and cultivate human corneal
stroma MSC-like cells (CSMSCs) and study their role in immunity
and wound healing.
Methods: Corneal buttons were harvested from cadavers (according
to the Guidelines of the Helsinki Declaration). The isolated stromal
cells were cultured ex vivo in human serum containing medium. The
expression of well-known MSC, hematopoietic, endothelial markers
as well as high-end glycosylation products were measured by
fluorescent microscopy and FACS in comparison to bone marrow
derived MSCs. To investigate the stemness of CSMSCs, gene array
analysis and standardized in vitro differentiation assays were
performed. The immunosuppressive function of these cells was
studied by mitogen activated lymphocyte reaction in a co-culture
with CSMSCs. Proliferation was measured by BrDU incorporation
assay. To describe the immunophenotype of the CSMSCs, the cells
were activated by TLR ligands and pro-inflammatory cytokines and
the secreted cytokines measured by ELISA. ECIS based wound
healing assay was performed to test the regenerative potential of
these cells.
Results: The cells isolated from human corneal stroma grew as
monolayers in vitro and could be maintained in culture for more than
10 passages (n=6). According to the definition of the ISCT, the most
important MSC markers (CD73, CD90 and CD105) were highly
expressed on the surface of CSMSCs with absence of endothelial
(CD31, VEGFR2) or hematopoietic cell markers (CD34, CD45,
CD69, CD133). The CSMSCs were able to differentiate into fat, bone
and cartilage tissues showing the potency of the CSMSCs. These
cells could close wounds within 24 hrs in vitro. They could suppress
the proliferation of mitogen activated peripheral blood lymphocytes
and secrete suppressive cytokines upon pro-inflammatory activation,
therefore, strengthening their unique immunosuppressive phenotype
in inflammation.
Conclusions: We demonstrate a method for isolating and cultivating
MSC-like cells from human corneal stroma. The ex vivo data suggest
that these cells may have a role in wound healing and immunological
processes in the eye that can possibly be used in future treatments of
ocular diseases and corneal stroma injuries.
Commercial Relationships: Zoltan Vereb, None; Reka Albert,
None; Morten C. Moe, None; Laszlo Fesus, None; Eva
Rajnavolgyi, None; Andras Berta, None; Goran Petrovski, None
Program Number: 1012 Poster Board Number: B0317
Presentation Time: 1:00 PM - 2:45 PM
Engraftment and Survival of Human Umbilical Mesenchymal
Stem Cells in the Mouse Cornea: An Immunofluorescent
Computed Tomography Study
Behdad Kavianpour1, Geraint J. Parfitt1, Hongshan Liu2, Winston W.
Kao2, Donald J. Brown1, Yilu Xie1, Mikhail Geyfman1, James V.
Jester1, Jennifer Simpson1. 1Gavin Herbert Eye Institute, University
of California, Irvine, Irvine, CA; 2Department of Ophthalmology,
University of Cincinnati, Cincinnati, Ohio, OH.
Purpose: While the field of ocular regenerative medicine has
advanced dramatically, little is known about the engraftment and
migration characteristics of intra-stromal stem cell transplantation in
the cornea. To better understand intra-stromal stem cell engraftment
and migration, the purpose of this study was to use a novel imaging
modality (immunofluorescent computed tomography or ICT) to
localize and quantify DiO-labeled human umbilical mesenchymal
stem cells (hUMSCs) transplanted into mouse corneas.
Methods: hUMSCs were DiO-labeled and injected (20,000
cells/cornea) using a 33-gauge needle and Hamilton syringe into the
corneal stroma of twelve C57BL/6 mice. Cell survival following
injection through the Hamilton needle and syringe was assessed by
trypan blue exclusion. At two, four and twelve weeks post-injection,
mice were sacrificed and corneas were excised and fixed in 2% PFA.
The corneas were then dehydrated and embedded in butyl methyl
methacrylate and polymerized under UV light at 4C. Once
polymerized, corneas were serially sectioned (2µm thick), stained
with DAPI and imaged using a Leica DMI6000B. Corneal volumes
were then reconstructed using Amira and fiji software to quantify and
characterize the distribution of DiO-labeled hUMSCs.
Results: At 2 weeks, 594 DiO labeled hUMSCs were identified in
the stroma and migrated to occupy a volume of 1.23*107 μm3. At 4
weeks, 45 DiO labeled hUMSCs were detected in the stroma that
occupied a volume of 4.28*107 μm3. At 12 weeks, only 19 DiO
labeled hUMSCs in a stromal volume of 4.68*107 μm3 were
identified. This suggests that the number of engrafted DiO-labeled
hUMSC decreased over a 12-week period, although greater migration
was observed over time. Cells injected through the Hamilton syringe
showed cell viability averaging 11.66%.
Conclusions: ICT is a novel imaging modality that can track and
quantify stem cell engraftment and migration over time. In this
preliminary study, hUMSCs appear to migrate and occupy a larger
volume over time, however, total cell engraftment appeared to
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
markedly decline based on DiO labeling. This suggests that technique
modifications may be required to optimize intra-stromal stem cell
viability. Antibody staining for markers of human stem cell
transplantation and keratocyte differentiation may also aid our
understanding of corneal stem cell fate and verify loss of cells or
labeling.
Commercial Relationships: Behdad Kavianpour, None; Geraint
J. Parfitt, None; Hongshan Liu, None; Winston W. Kao, None;
Donald J. Brown, None; Yilu Xie, None; Mikhail Geyfman, None;
James V. Jester, None; Jennifer Simpson, None
Support: NIH Grant EY022365 Mesenchymal Stem Cell Therapy
For Corneal Cystinosis; Research to Prevent Blindness, Inc;
Discovery Eye Foundation; The Skirball Program in Molecular
Ophthalmology; The Cystinosis Research Foundation
Program Number: 1013 Poster Board Number: B0318
Presentation Time: 1:00 PM - 2:45 PM
Influence of Secreted Ly6/uPAR-Related Protein-1 (Slurp1) on
Corneal Stromal Fibroblast Proliferation, Interaction with the
Extracellular Matrix, and Migration
Shivalingappa K. Swamynathan1, 2, Sudha Swamynathan1.
1
Ophthalmology, Univ Pittsburgh Sch of Med, Pittsburgh, PA; 2Cell
Biology and Physiology, Univ Pittsburgh Sch of Med, Pittsburgh,
PA.
Purpose: Previously, we demonstrated that the secreted Ly6/uPARrelated protein-1 (Slurp1) is abundantly expressed in the cornea and
is downregulated in diverse pro-inflammatory conditions. Here, we
examine the effects of Slurp1 on corneal stromal fibroblast cell
proliferation, interaction with the extracellular matrix (ECM), and
migration, to understand the cellular basis of Slurp1 functions in the
cornea.
Methods: The effect of Slurp1 on corneal fibroblast behavior was
assessed in vitro by treating human telomerase reverse transcriptase
(hTERT)-immortalized mouse corneal stromal cell line MK/T1 with
histidine-tagged mouse Slurp1 (His-Slurp1) produced in E. coli and
partially purified by Ni ion resin column chromatography. Effect of
His-Slurp1 on MK/T1 cell (i) density was assessed by crystal violet
staining followed by measurement of absorbance at 590 nm, (ii)
interaction with the ECM was evaluated on cell culture plates coated
with different ECM components, and (iii) migration was assessed by
in vitro gap filling assays.
Results: Compared with the control, His-Slurp1-treated mouse
corneal stromal fibroblast MK/T1 cells (i) density increased at a
slower pace suggesting that Slurp1 inhibits cell proliferation, (ii)
adhered with lower affinity to collagen-I-, collagen-IV-, vitronectinor fibronectin-coated culture plates suggesting that Slurp1 affects
cell-matrix interaction, and (iii) migrated at a slower pace in gap
filling assays suggesting that Slurp1 inhibits cell migration.
Conclusions: Our results demonstrate that Slurp1 inhibits corneal
stromal fibroblast MK/T1 cell proliferation, cell-matrix adhesion and
migration, revealing the cellular basis for corneal functions of Slurp1.
These results are consistent with the decreased expression of Slurp1
in corneas exposed to pro-inflammatory conditions where the stromal
fibroblasts proliferate at a higher rate, and migrate rapidly.
Commercial Relationships: Shivalingappa K. Swamynathan,
None; Sudha Swamynathan, None
Support: Department of Ophthalmology, University of Pittsburgh
Start-up funds, Eye and Ear Foundation of Pittsburgh, Research to
Prevent Blindness and NIH core grant P30 EY08098
Program Number: 1014 Poster Board Number: B0319
Presentation Time: 1:00 PM - 2:45 PM
Stromal Cells derived from Amniotic Membrane are capable to
reestablish corneal opacity
Yonathan Garfias1, 2, Alejandro Navas1, Jessica Nieves-Hernández1,
Gibran A. Estua1, Rodrigo Bolaños-Jiménez1. 1Research Unit,
Institute of Ophthalmology, Mexico City, Mexico; 2Biochemistry,
Faculty of Medicine, Universidad Nacional Autónoma de México,
Mexico City, Mexico.
Purpose: Stromal mesenchymal stem cells are non-hematopoietic
derived cells found in the bone marrow stroma such as in many
stromal tissues. The amniotic membrane is an elastin and avascular
fetal membrane that is in contact to the fetus. A mature amniotic
membrane possesses 20-50 x 10 <6>mesenchymal cells. .
By the other hand, there are many corneal disorders that directly
affect the corneal limbus, driving inflammation, conjunctivalization
or neovascularization of the corneal tissue. The pronostic depends on
the injured area of the limbus where the corneal stem cells are
localized. Although, it has recently reported that the cells derived
from the amniotic membrane mesenchyma are source to
adipogenesis, chondrogenesis, osteogenesis and myogenesis, its
function as a source for regeneration of the ocular surface has not
been studied.
The aim of the present study is to determine the utility of these cells
to restablish the ocular surface in a chemical burn murine model.
Methods: Mesenchymal cells were obtained from a placenta using
dispase/collagenase method. The cells were cultured and
characterized by flow cytometry. Cellular transdifferentiation assays
were performed using conditioned media. A murine chemical burn
was performed in order to determine the efficacy of these cells to
restablish the corneal clarity. Corneal histology was performed to
identify the incorporation of human mesenchymal cells in the murine
cornea.
Results: The cells obtained from the amniotic membrane
mesenchyma were capable to attach to the plastic wells showing a
fibroblast-like morphology. These cells presented mesenchymal stem
cell markers such as CD29, CD73, CD44 and CD105, meanwhile,
they were negative to CD45 and HLA-DR. Interestingly, these cells
were capable to differentiate into neurons and chondrocytes. When
these cells were intracamerally injected in a mouse burn model, the
corneal opacity was significantly reduced in comparison to the
untreated cornea. When the histology of the cornea was performed, it
was evident that the human amniotic membrane cells were
incorporated to the mouse cornea, reestablishing the structure of the
corneal tissue.
Conclusions: The use of cells derived from the mesenchyma of the
amniotic membrane is an important cell source to be used in the
regenerative ophthalmology.
Commercial Relationships: Yonathan Garfias, Institute of
Ophthalmology (P); Alejandro Navas, None; Jessica NievesHernández, Institute of Ophthalmology (P); Gibran A. Estua,
None; Rodrigo Bolaños-Jiménez, None
Support: CONACYT 160286
Program Number: 1015 Poster Board Number: B0320
Presentation Time: 1:00 PM - 2:45 PM
Corneal endothelial cells derived from monkey iPS cells: a short
term evaluation
Shin Hatou1, Satoru Yoshida1, Kazunari Higa2, Hideyuki Miyashita1,
Emi Inagaki1, Erika Kimura3, Ryuhei Hayashi3, Kazuo Tsubota1,
Kohji Nishida3, Shigeto Shimmura1. 1Department of Ophthalmology,
Keio Univ School of Medicine, Shinjuku-ku, Japan; 2Department of
Ophthalmology, Tokyo Dental College Ichikawa General Hospital,
Ichikawa, Japan; 3Department of Ophthalmology, Osaka University,
Osaka, Japan.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Purpose: To evaluate the short term function of tissue-engineered
corneal endothelial cells (TECE cells) derived from monkey induced
pluripotent stem cells (iPS cells).
Methods: Cynomolgus monkey iPS cells were cultured in KSR
medium for 1 week and subsequently in N2 medium for 1 week,
supplemented with TGF-beta inhibitor and BMP inhibitor. The iPSderived neural crest cells were isolated as CD271 positive fraction by
cell sorter. Next these cells were proliferated with EGF and FGF2,
and subsequently medium was changed to an “endothelium-deriving
medium” including GSK-3beta inhibitor, retinoic acid and ROCK
inhibitor. These cells were dispersed on collagen sheet and TECE cell
sheets were obtained. The pump function attributable to Na,KATPase activity of TECE cell sheets was measured with an Ussing
chamber, and compared with that of human corneal endothelial cell
line (B4G12 cells). In vivo function of TECE was measured as
central corneal thickness of rabbit eyes transplanted with TECE cell
sheets for 8 days after surgery and compared with control eyes
deprived of endothelium.
Results: Hexagonal mosaic pattern monolayer TECE cells were
obtained. Pump function of TECE was 2.34±0.46 mV, whereas that
of B4G12 cells was 1.07±0.20 mV. The corneal thickness of TECE
transplanted rabbit eyes (589.25±164.8μm) maintained significant
lower corneal thickness than control eyes (1105.8±165.9μm)
throughout the post-operative period.
Conclusions: In vitro and short term in vivo function of monkey iPSderived TECE were observed. Further long-term in vivo evaluation of
TECE transplantation to monkey eyes is needed.
Commercial Relationships: Shin Hatou, None; Satoru Yoshida,
None; Kazunari Higa, None; Hideyuki Miyashita, None; Emi
Inagaki, None; Erika Kimura, None; Ryuhei Hayashi, None;
Kazuo Tsubota, AcuFocus, Inc (C), Allergan (F), Bausch Lomb
Surgical (C), Functional visual acuity meter (P), JiNS (P), Kissei (F),
Kowa (F), Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), Echo
Denki (P), Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPT
Company (P); Kohji Nishida, Alcon (C), Alcon (F), HOYA (F),
Senju (F), Pfizer (F), Santen (F), Osaka University (P); Shigeto
Shimmura, None
Support: Highway Program for realization of regenerative medicine
from Ministry of Education, Culture, Sports, Science and
Technology, Japan.
Program Number: 1016 Poster Board Number: B0321
Presentation Time: 1:00 PM - 2:45 PM
Bone Marrow-Derived Endothelial Progenitor Cells for
Treatment of Corneal Endothelial Dysfunction
Yao Fu, Chunyi Shao, Xianqun Fan. Department of Ophthalmology,
Ninth People’s Hospital, Medical School of Shanghai Jiaotong
University, Shanghai, China.
Purpose: To investigate the feasibility of inducing bone marrowderived endothelial progenitor cells (BEPC) to differentiate into
corneal endothelial cells (CEC) for the treatment of corneal
endothelial dysfunction.
Methods: BEPC were isolated from human fetal bone marrow, and
expression of Dil-Ac-LDL, UEA-1, CD133 and CD34 were
examined to identify the cells. BEPC were co-cultured with CEC for
10 days in a transwell system with conditioned medium from CEC,
and then cell transdifferentiation was examined by
immunocytofluorescence and electron microscopy. With a porcine
corneal acellular matrix as the carrier, the induced BEPC were
transplanted onto a cat’s cornea from which Descemet’s membrane
and the endothelium had been stripped.
Results: The induced BEPC resembled CEC in polygonal shape,
expressing aquaporin-1, tightly opposed cell junctions, and neurone-
specific enolase. Twenty-eight days after transplantation, the
transparency gradually returned to the corneas transplanted with the
induced BEPC on porcine corneal acellular matrix .
Conclusions: Human fetal BEPC could be induced into corneal
endothelial-like cells in vitro. Features of the induced BEPC
indicated that they may be useful for the treatment of corneal
endothelial dysfunction.
Commercial Relationships: Yao Fu, None; Chunyi Shao, None;
Xianqun Fan, None
Support: grant from the National Nature Science Foundation of
China (81000366)
Program Number: 1017 Poster Board Number: B0322
Presentation Time: 1:00 PM - 2:45 PM
Standardization of human corneal endothelial cell isolation and
the use of denuded amniotic membrane as a scaffold for human
corneal endothelial cells
Kalpana Suresh, Tanvi Khanna, Alan M. Punnoose, Sarah Kuruvilla,
Vishnu D. Narayanam, RAMYA RAVINDRAN, Varshini Varadaraj.
Ophthalmology, Sri Ramachandra Universiy, Chennai, India.
Purpose: To identify the best technique for complete denudation of
amniotic membrane.
To standardize the isolation of human corneal endothelial cells.
To use the denuded amniotic membrane as a scaffold for isolated
human corneal endothelial cells.
Methods: Human amniotic membrane denudation was carried out
using 1.2 units/ml of Dispase II at 37degree C for 60 minutes
followed by mechanical scraping and microscopic examination. This
was followed by isolation of corneal endothelial cells using human
donor cadaveric eyes unfit for surgical usage. Corneal endothelial and
descemet’s membrane sheets were peeled in a manner similar to
capsulorrhexis after corneoscleral button excision and enzymatically
digested with 2mg/ml of collagenase II solution at 37 degree C and 5
% CO2 for 2 hrs. Pre plating was done onto an uncoated culture ware
to separate any attached fibroblasts from the endothelial cells which
were then seeded onto denuded amniotic membrane in OptiMEM
media supplemented with human epidermal growth factor, fibroblast
growth nerve growth factor and bovine pituitary extract. The cells
were analyzed microscopically to assess if they maintained their
polygonal morphology and subjected to RT-PCR analysis for Keratin
3, neuron specific enolase, Vimentin and collagen VIII mRNA
markers.
Results: Microscopic examination of the denuded amniotic
membrane showed no epithelial cell remnants and an underlying
exposed stromal collagen. Peeling of the corneal endothelial and
descemet’s membrane gave sheets of ideal thickness exhibiting
typical cobblestone morphology. Enzymatic digestion of the
harvested corneal tissue left behind acellular descemet’s sheets with
the endothelial cells seen floating individually or in tightly packed
clusters with preplating aiding in a more fibroblast free endothelial
cell isolation. Microscopic evaluation showed that a few isolated cells
managed to scaffold onto the amniotic membrane and that they
manage to retain that adhesion during subsequent media
replacements.
Conclusions: Usage of Dispase-II for enzymatic digestion of
amniotic membrane yielded a complete denudation which acted as a
successful scaffold for harvested corneal endothelial cells. Further
studies can be done for endothelial cell proliferation serving as an in
vitro model for corneal tissue engineering studies.
Commercial Relationships: Kalpana Suresh, None; Tanvi
Khanna, None; Alan M. Punnoose, None; Sarah Kuruvilla, None;
Vishnu D. Narayanam, None; RAMYA RAVINDRAN, None;
Varshini Varadaraj, None
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Program Number: 1018 Poster Board Number: B0323
Presentation Time: 1:00 PM - 2:45 PM
Isolation and characterization of p75NTR positive and highproliferative corneal endothelial cells from the human corneal
endothelium
Susumu Hara, Ryuhei Hayashi, Tomofumi Kageyama, Motokazu
Tsujikawa, Kohji Nishida. Ophthalmology, Osaka University
Graduate Scool of Medicine, Suita, Japan.
Purpose: The corneal endothelium is believed to be developmentally
originated from periocular mesenchyme via neural crest. The human
corneal endothelial progenitor cells (HCEPs) have been investigated
because of their potential availability for the tissue regenerative
medicine. However, the existence and the properties of HCEPs have
not been elucidated yet. We attempted to isolate the HCEPs from the
human corneal endothelium by using the specified culture system and
p75 neurotrophin receptor (p75NTR).
Methods: The Descemet's membranes were stripped from the human
cornea, then, treated with a cell dissociated reagent. To isolate the
HCEPs, the endothelial cells were seeded on the dish coated with
laminin and cultured in the serum-free media containing basic
fibroblast growth factor. Expression of neural crest markers in the
isolated HCEPs was examined by real-time PCR and
immunostaining.
Results: The proliferating cells were appeared at around 14 days
after the seeding, exhibited a bipolar, spindle-shaped morphology,
similar to neural crest cells. Interestingly, the proliferating cells
expressed neural crest markers, p75NTR and Sox9. The colony
forming efficiency was approximately 0.31±0.04%, showed no
significant relation to donor ages. The proliferating cells were able to
undergo passage several times in younger donors below 60 years old,
and the proliferative capability was higher than that of human corneal
endothelial cells cultivated by the conventional method with fetal
bovine serum containing media.
Conclusions: We succeeded in the isolation of HCEPs which had
high p75NTR expression and proliferative capability.
Commercial Relationships: Susumu Hara, None; Ryuhei
Hayashi, None; Tomofumi Kageyama, None; Motokazu
Tsujikawa, Shionogi & Co. (C), Daiichi Sankyo Co. (F), Daiichi
Sankyo Co. (R), Santen Co. (R), AMO Co. (R); Kohji Nishida,
Alcon (C), Alcon (F), HOYA (F), Senju (F), Pfizer (F), Santen (F),
Osaka University (P)
Support: Grants-in-Aid for Scientific Research from the Ministry of
Health, Labor and Welfare and from National Institute of Biomedical
Innovation in Japan
Program Number: 1019 Poster Board Number: B0324
Presentation Time: 1:00 PM - 2:45 PM
Spatial transcriptome of human cornea using next generation
sequencer
Suguru Nakagawa1, Tomohiko Usui1, Hiroki Ueda2, Genta Nagae2,
Shogo Yamamoto2, Satoru Yamagami1, Hiroyuki Aburatani2, Shiro
Amano1. 1Ophthalmology, Univ of Tokyo Sch of Med, Tokyo, Japan;
2
Div of Genome Science, RCAST, University of Tokyo, RCAST,
University of Tokyo, Tokyo, Japan.
Purpose: Cornea consists of anatomically distinct three layers,
corneal epithelium (CEp), corneal stroma (CS), corneal endothelium
(CEn), that have different developmental lineages and molecular
functions. To reveal their transcriptional program, we performed
spatial gene expression analysis of human cornea using next
generation sequencer.
Methods: 100 ng of total RNA was extracted from the
macroscopically dissected tissue fractions; CEp, CS, CEn, limbal
epithelium (LEp) and conjunctiva (Cj) of the human donor
corneoscleral tissue. For each sample, approximately 150 million of
100-bp, paired-end reads were sequenced (HiSeq2000, Illumina) and
mapped against transcriptome database using BWA aligner. Quantity
of 86,817 transcripts was corrected by gene/exon length
(RPKM/FPKM), and then compared using regression analysis.
Results: 24,601 Transcript (28.3%) were expressed in common with
all cell fractions. Global comparison of each spatial transcriptome
showed that CS shows the closest pattern against CEn (correlation
coefficient (R^2); CEn vs. CS 0.844, vs. CEp 0.807, vs. LEp 0.811,
vs. Cj 0.821). We identified the candidate transcripts significantly
upregulated in CEn against other tissue fractions; CEp (n=1,760),
LEp (n=1,513), CS (n=1,482), Cj (n=1,322), respectively. As
commonly up-regulated transcripts in the CEn, we listed up more
than 300 coding genes in addition to previously reported CEnspecific markers such as COL8A2, CA2, SLC4A4, CDH2.
Conclusions: We successfully demonstrated the layer-specific
pattern of transcriptome and identified the novel up-regulated genes
in CEn. This resource information is useful for understanding the
spatial difference in transcriptional program to modulate cell
lineages.
Commercial Relationships: Suguru Nakagawa, None; Tomohiko
Usui, None; Hiroki Ueda, None; Genta Nagae, None; Shogo
Yamamoto, None; Satoru Yamagami, None; Hiroyuki Aburatani,
None; Shiro Amano, Topcon (P)
Program Number: 1020 Poster Board Number: B0325
Presentation Time: 1:00 PM - 2:45 PM
Human cornea proteome: Identification and quantitation of the
proteins of the three main layers including epithelium, stroma
and endothelium
Thomas Dyrlund1, Ebbe Toftgaard Poulsen1, Carsten Scavenius1,
Camilla Lund Nikolajsen1, Ida B. Thøgersen1, Henrik Vorum2, Jan J.
Enghild1. 1Department of Molecular Biology, University of Aarhus,
Aarhus C., Denmark; 2Department of Ophthalmology, Aalborg
Hospital, Aarhus University Hospital, Aalborg, Denmark.
Purpose: Diseases of the cornea are common and refer to conditions
like infections, injuries and genetic defects. Morphologically, many
corneal diseases affect only certain layers of the cornea and separate
analysis of the individual layers is therefore of interest to explore the
basic molecular mechanisms involved in corneal health and disease.
Methods: The three main layers including the epithelium, stroma and
endothelium of healthy human corneas were isolated and the proteins
were (i) separated by SDS-PAGE followed by in-gel trypsinization,
(ii) in-solution digested without prior protein separation or, (iii) insolution digested followed by cation exchange chromatography. The
resulting peptides were separated by LC-MS/MS and analysed on a
TripleTOF 5600 mass spectrometer. Proteins were identified in the
Swiss-Prot database using the Mascot algorithm and quantified using
Mascot Distiller. Data extraction and processing was done using MS
Data Miner.
Results: A total of 3250 unique Swiss-Prot annotated proteins were
identified in human corneas, 2737 in the epithelium, 1679 in the
stroma and 880 in the endothelial layer. Of these, 1787 proteins have
not previously been identified in the human cornea by mass
spectrometry. In total, 771 proteins were quantified, 157 based on insolution digestion and 770 based on SDS-PAGE separation followed
by in-gel digestion of excised gel pieces. Protein analysis revealed
that many of the identified proteins were human plasma proteins
involved in the complement system, coagulation and defence against
pathogen infections.
Conclusions: The separation of human corneas into the three main
layers combined with modern mass spectrometry provides new
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
insight into the proteins present in the individual layers and the
relative abundance in each layer. This provides a useful reference
dataset when exploring basic molecular mechanisms involved in
corneal diseases, many of which are restricted to a specific corneal
layer.
Commercial Relationships: Thomas Dyrlund, None; Ebbe
Toftgaard Poulsen, None; Carsten Scavenius, None; Camilla
Lund Nikolajsen, None; Ida B. Thøgersen, None; Henrik Vorum,
None; Jan J. Enghild, None
Support: R01 EY012712
Program Number: 1021 Poster Board Number: B0326
Presentation Time: 1:00 PM - 2:45 PM
Immunocytochemical analysis after treatment with
osmoprotective and oil containing lubricants in dry eye and
refractive surgery patients
Renata R. Loureiro, Rossen M. Hazarbassanov, Joyce L. Covre,
Priscila C. Cristovam, Jeison D. Barros, Jose A. Gomes.
Ophthalmology, UNIFESP, Sao Paulo, Brazil.
Purpose: To evaluate immunostaining patterns of inflammation and
osmoprotection markers after treatment with osmoprotective
lubricant compared to oil containing and non-osmoprotective
lubricants, in evaporative dysfunctional tear syndrome (EDTS) post
refractive surgery patients.
Methods: 45 patients (74,28 % female)(Mean age ± SD, 32.5
±10.35) were enrolled. Participants were randomized to receive
topical drops QID for the 1st month and BID for the following 2
months of Optive®, FreshTears®, (Allergan, Inc., Irvine,
California).They were divided into 2 groups, (A) 15 patients with
EDTS, (B) 30 patients without EDTS who were referred to either
LASIK (15) or PRK (15). In group A, 5 patients (10 eyes) were
treated with either Optive®, FreshTears® or Endura®, as well as 5
patients (10 eyes) from group B/PRK and 5 patients (10 eyes) from
group B/LASIK. All patients were submitted to the following tests
for EDTS diagnose: Ocular Surface Disease Index (OSDI), patient
symptomatology questionnaire, visual acuity (VA), biomicroscopy,
Schirmer I test without anesthesia, tear film osmolarity, fluorescein
break up time (FBUT), fluorescein and lissamine green 1% staining
(Oxford grading), impression cytology (IC) and
immunocytochemistry (ICC) for an inflammation marker (HLA-DR)
and L-carnitine, osmoprotective component.
Results: Pre-treatment and 3 month follow-up exams are completed
for both groups. ICC of conjunctiva samples showed 42.86%
positivity for HLA-DR staining, on group A and 20% for group
B/LASIK, 30% for PRK, before treatment (p=0.4896, χ2 test). There
was lower HLA-DR staining for EDTS patients treated with Optive®
and Endura® (28.11% and 35.6%). ICC for L-carnitine staining was
53.33% positive for A, 22% for LASIK and10% for PRK subgroup,
before treatment (p=0.041, χ2 test). L-carnitine ICC staining posttreatment showed high positivity for FreshTears® and Endura®
groups, in contrast to a lower staining for Optive® subgroup.
Conclusions: Conjunctival cells showed tendency of higher
expression of inflammation marker HLA-DR on EDTS patients, and
for L-carnitine as well, which could be reduced after osmoprotective
therapy. Those markers could be used to detect EDTS in early stage
and as prognostic tool for EDTS treatment.
Commercial Relationships: Renata R. Loureiro, None; Rossen M.
Hazarbassanov, None; Joyce L. Covre, None; Priscila C.
Cristovam, None; Jeison D. Barros, None; Jose A. Gomes,
Allergan (C), Pfizer (C), Genon (C), MSD (C)
Support: None in the Support field below
Clinical Trial: nct01741987
Program Number: 1022 Poster Board Number: B0327
Presentation Time: 1:00 PM - 2:45 PM
The Regeneration Potential of Mouse Lacrimal Gland Following
Duct Ligation Procedure
Ying Liu1, Tetsuya Kawakita1, Machiko Sugiyama1, Masatoshi
Hirayama1, Motoko Kawashima1, Yoko Ogawa1, Masataka Ito2,
Shigeto Shimmura1, Kazuo Tsubota1. 1Ophthalmology, Keio
University School of Medicine, Tokyo, Japan; 2National Defense
Medicine College, Saitama, Japan.
Purpose: To observe the regeneration and proliferation potential of
adult mouse lacrimal gland (LG) following duct-ligation (DL)
procedure and duct-ligation-release (DLR) procedure.
Methods: Adult mice were divided into two groups. One group was
subjected to the DL of the right LG, and glands were collected at day
0, 3, 7 and 14 after DL (n=3-5, at each time point). Another group
was subjected to the DLR for 7 days and the ligation was released at
day 7 of the right LG. Then the glands were collected at day 9, 12, 14
and 17 after the ligation was released. Tear production and the gland
weight were measured during the duct ligation / release (DL/R)
procedure. Tissues were investigated by Hematein & Eosin (H&E)
staining and immunohistochemistry.
Results: Tear secretion of DL and DLR group at day 3, 7, 9, 12 and
14, has significantly decreased compared with control (p<0.01, at
each time point). After DL/R the LG went through atrophy and the
weight of LG DL group at day 14, has significantly decreased
compared with control (p<0.01). And DLR group at day 9, 10 and
day 14 has significantly decreased compared with control (p<0.01,
p<0.05, p<0.01, respectively). The H&E staining showed the regular
acinar unit structures could not be observed, and the lumen of ducts
were expanded accompanied with the duct unit increased in the later
stage of DL/R. The immunohistochemistry of CD45 (a marker of
common lymphocyte) positive cells were increased after DL/R and
the immunohistochemistry of nestin (a marker of neuron stem cell)
showed the nestin-positive cells in both two groups.
Conclusions: Adult mouse LG has a strong regeneration potency
after atrophy as a result of inflammation.
Commercial Relationships: Ying Liu, None; Tetsuya Kawakita,
None; Machiko Sugiyama, None; Masatoshi Hirayama, None;
Motoko Kawashima, Santen Pharmaceutical Co., Ltd (F); Yoko
Ogawa, None; Masataka Ito, None; Shigeto Shimmura, None;
Kazuo Tsubota, AcuFocus, Inc (C), Allergan (F), Bausch Lomb
Surgical (C), Functional visual acuity meter (P), JiNS (P), Kissei (F),
Kowa (F), Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), Echo
Denki (P), Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPT
Company (P)
Program Number: 1023 Poster Board Number: B0328
Presentation Time: 1:00 PM - 2:45 PM
Prosthetic Replacement for the Ocular Surface Environment
(PROSE) Alters The Lacrimal Functional Unit
Yvonne Wang, Ryan M. St Clair, Michelle N. Lee, Kimberly C.
Sippel, Jessica Ciralsky, Priyanka Sood, Ana G. Alzaga Fernandez,
Christopher E. Starr, Mark Rosenblatt. Ophthalomogy, Weill Cornell
Medical College, New York, NY.
Purpose: To determine if PROSE wear causes changes in tear
production, corneal sensitivity and corneal nerve morphology.
Methods: All patients referred to Weill Cornell Ophthalmology for
PROSE treatment were considered for the study. At one visit prior to
PROSE device wear and at one visit after at least 30 days of
uninterrupted PROSE wear (for at least 6 hours per day), the
following measurements were taken: tear production using the
Schirmer’s test with anesthesia (n = 9 patients, 17 eyes), central
corneal sensitivity using a Cochet-Bonnet Aesthesiometer (n = 8
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
patients, 12 eyes), and corneal nerve morphology by confocal
microscopy (ConfoScan 4, Nidek). A single best image showing
nerves in the sub-basal nerve plexus (n = 6 patients, 8 eyes) and
stromal nerves (n = 4 patients, 5 eyes) was chosen from each
appointment. Images were analyzed for sub-basal epithelial nerve
fiber length and tortuosity and stromal nerve trunk thickness using
NeuroLucida Software (MBF Bioscience, Williston, VT). Sub-basal
epithelial nerve density was calculated by dividing the total length of
nerve fibers in each image by the area photographed. Tear
production, central corneal sensitivity, sub-basal epithelial nerve
density and tortuosity, and stromal nerve trunk thickness were
compared before PROSE wear and after uninterrupted PROSE wear
using a paired t-test.
Results: Tear production significantly decreased from 17.5 ± 5.2 mm
baseline to 13.3 ± 6.1 mm after at least 30 days of PROSE wear (p =
0.00098). In contrast, central corneal sensitivity increased
significantly from baseline (46.3 ± 6.4 mm) after at least 30 days of 6
hours or more of PROSE wear (53.8 ± 7.7 mm; p = 0.010). Neither
sub-basal epithelial tortuosity (1.075 ± 0.035 vs. 1.077 ± 0.049; p =
0.91), nor sub-basal nerve density (1711 ± 878 µm /mm2 vs. 1845 ±
707 µm/mm2; p = 0.70) were altered by PROSE wear. Stromal nerve
trunk thickness decreased significantly from an initial value of 8.5 ±
2.3 µm to 4.7 ± 1.9 µm after wear (p = 0.038).
Conclusions: Changes in tear production and corneal nerve
parameter suggest that PROSE treatment may affect elements of the
lacrimal functional unit. The PROSE device shields the ocular
surface from its normal environment and may allow for functional
changes in the neural output with a concomitant change in tear
production.
Commercial Relationships: Yvonne Wang, None; Ryan M. St
Clair, None; Michelle N. Lee, None; Kimberly C. Sippel, None;
Jessica Ciralsky, None; Priyanka Sood, None; Ana G. Alzaga
Fernandez, None; Christopher E. Starr, None; Mark Rosenblatt,
None
Support: This investigation was supported by grant UL1TR000457
of the Clinical and Translational Science Center at Weill Cornell
Medical College and Research to Prevent Blindness
220 Immunology, Allergy, Neovascularization
Monday, May 06, 2013 8:30 AM-10:15 AM
TCC 303 Paper Session
Program #/Board # Range: 1286-1292
Organizing Section: Cornea
Program Number: 1286
Presentation Time: 8:30 AM - 8:45 AM
Lens-derived Sema3A inhibits angioblast migration and
vascularization of the developing cornea
Peter Y. Lwigale, Chelsey McKenna. Biochemistry and Cell BiologyMS140, Rice University, Houston, TX.
Purpose: To determine the role of Sema3A during ocular
vasculogenesis and formation of the avascular cornea. Given that
angioblasts and ocular blood vessels in the periocular region express
Nrp1, a receptor for both Vegf (angiogenic factor) and Sema3A (antiangiogenic factor), we hypothesized that lens-derived Sema3A
prevents angioblast migration and vascularization of the developing
cornea.
Methods: We identified the localization of migratory angioblasts and
forming vasculature in the periocular region of Tg(tie1:H2B:eYFP)
transgenic quail. We examined the expression of Vegf and Sema3A
in the lens by immunohistochemistry and quantified their mRNA by
qPCR. We then blocked Sema3A signaling from the region of the
presumptive cornea by lens ablation or injection of Sema3A
inhibitory peptides. We also investigated whether addition of
Sema3A would inhibit Vegf-induced vascularization of the cornea.
Furthermore, we analyzed Nrp1(Sema-/-) mutant mice that lack
Sema/Nrp1 signaling for defects in corneal avascularity.
Results: Our results show that angioblasts do not migrate into the
region of the forming cornea located between the ectoderm and lens.
Both Sema3A and Vegf are present in the lens, but the levels of
Sema3A transcripts are significantly higher than Vegf during cornea
development. Inhibition of lens Sema3A resulted in ectopic
angioblast migration and vascularization of the forming cornea.
Addition of Sema3A protein inhibited Vegf-induced vascularization
of the cornea. We also observed ectopic angioblasts and vasculature
in corneas of Nrp1(Sema-/-) mutant embryos.
Conclusions: Together, our results clearly indicate that corneal
avascularity is established early during ocular development and that,
Sema3A signaling from the lens plays a crucial role in this process.
Commercial Relationships: Peter Y. Lwigale, None; Chelsey
McKenna, None
Support: NIH Grant EY018050 and EY022158
Program Number: 1287
Presentation Time: 8:45 AM - 9:00 AM
Cornea Intravital Multiphoton Visualization of the Resident
Mononuclear Phagocyte Network in Allergy
Tomas Blanco1, Matthew Kan2, Michael Gunn2, Daniel R. Saban1, 2.
1
Ophthalmology, Duke University School of Medicine, Durham, NC;
2
Department of Immunology, Duke University School of Medicine,
Durham, NC.
Purpose: The cornea houses an extensive network of resident
mononuclear phagocytes, including macrophages, Langerhan’s cells,
langerin+ dendritic cells (DC) and CD11b+ DC. Their function(s),
particularly independent of recruited inflammatory monocytes, is
poorly understood. A novel mouse line was established with a
CX3CR1-cre x ROSA26floxSTOPfloxGFP reporter system, wherein
all progeny derived from the macrophage-dendritic cell precursor
(MDP) lineage permanently express eGFP. We examined these
corneas via intravital multiphoton microscopy in a model of ocular
allergy, previously shown by our group to have corneal
manifestations
Methods: Transgenic mice were used and compared with
commercially available knock in CX3CR1 GFP/+. Mice were
immunized systemically with OVA (100 ug) + pertussis toxin (300
ng) + aluminum hydroxide (1 mg), or left naïve as a control. After 2
wk, mice were challenged with an eye drop of Texas Red-conjugated
(250 ug OVA). Microscopy was performed before and at various
indicated time-points after OVA challenge. A multiphoton
microscope (900 nm emission) was used at < 5% of the laser power.
3 separate (high efficiency non-descanned) detectors in the epi
position were used for 2nd harmonic generation, GFP, and Texas
Red.
Results: Before challenge, GFP+ cells were detectable within the
sub-basal plexus, and seemed to be interconnected via GFP+
membrane nanotubules (MNT). Within 1-3 hrs post challenge in
immunized mice, these sub-basal cells were found extending their
processes and making contact with OVA at the surface. Other
morphologically distinct GFP+ OVA+ cells appeared, which were
intraepithelial (i.e. anterior to the sub-basal plexus) and not
associated with MNT. In the stroma, GFP+ MNTs were not
detectable pre-challenge. GFP+ cells became OVA+ by 6-12 hrs post
challenge. This occurred despite the observation that ‘free form’
OVA seemingly could not penetrate the ocular surface. Interestingly,
however, GFP+ MNT became visible in the stroma and appeared to
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
extend upward toward the sub-basal plexus. All GFP+ cells were
largely undetectable in and around 24 hours, except for infiltrated
intraepithelial cells.
Conclusions: These data provide evidence to refute the notion that
resident mononuclear phagocytes in the cornea are immunologically
inert, as well as direct evidence to suggest that such cells are
interconnected and may contribute to shaping an adaptive immune
response.
Commercial Relationships: Tomas Blanco, None; Matthew Kan,
None; Michael Gunn, None; Daniel R. Saban, Schepens Eye Res
Inst, Mass Eye and Ear, (P), Eleven Biotherapuetics (R)
Support: R01EY021798
Program Number: 1288
Presentation Time: 9:00 AM - 9:15 AM
Intravital Multiphoton Microscopy of Corneas and Draining
Lymph Nodes Shows Increased Velocity of Dendritic Cells after
Corneal Transplantation and Directionality in Corneal Allografts
Takefumi Yamaguchi1, 2, Kai Hu1, 2, Deshea L. Harris1, 2, Pedram
Hamrah1, 2. 1Cornea Service and Schepens Eye Research Institute,
Massachusetts Eye and Ear Infirmary, Boston, MA; 2Harvard
Medical School, Boston, MA.
Purpose: The role of dendritic antigen presenting cells in corneal
transplantation has been firmly established. While their functional
characterization has thus far mainly relied on the analysis of ex vivo
studies, there remains a clear need to investigate their behavior in the
context of intact tissues in real-time. Here we evaluate in vivo
kinetics of dendritic cell (DC) in the cornea and submandibular
draining lymph nodes (dLN) after corneal transplantation and
trigeminal axotomy as a control for nerve damage model.
Methods: Corneal buttons from BALB/c (allogeneic) and C57BL/6
(syngeneic) mice were orthotopically grafted onto CD11c-GFP-DTR
(C57BL/6 background) recipients. CD11c-GFP DCs were imaged in
the graft, peripheral recipient cornea and dLN under anesthesia using
multiphoton microscopy 2 weeks after corneal transplantation and
trigeminal axotomy. The density, kinetics and speed of DCs were
calculated and 3D movies rendered using high performance 4D
imaging software (IMARIS).
Results: The density of CD11c-GFP+ cells in grafts and recipient
corneas significantly increased after corneal transplantation and
trigeminal axotomy compared to controls (p< 0.01). CD11c-GFP+
cells velocity in grafts significantly increased from 0.58 μm/min
(normal, central cornea) and 1.26 (axotomy) to 1.30 (syngeneic), and
1.80 (allogeneic, P<0.001). Velocity of CD11c-GFP+ cells in
peripheral recipient cornea significantly increased from 0.94 μm/min
(normal, peripheral cornea) and 1.22 (axotomy) to 2.31 (syngeneic),
and 2.36 (allogeneic, P<0.001). Velocity of CD11c-GFP+ cells in
dLN significantly increased from 2.01μm/min to 2.92 after allogeneic
corneal transplantation (P<0.001). The meandering index, an index
for directionality, significantly increasedin corneal allografts (p<0.01)
compared to isografts or axotomy.
Conclusions: These studies are the first to demonstrate long-term
migratory kinetics of corneal DCs after corneal transplantation in
both the cornea and submandibular dLNs through high-resolution
intravital multi-photon microscopy. DCs demonstrate increased
velocity and directionality in the cornea and dLN after allografts
compared to axotomy and isografts. Multiphoton microscopy can
potentially be a powerful tool to study the pathogenesis of ocular
diseases in real-time.
Commercial Relationships: Takefumi Yamaguchi, None; Kai Hu,
None; Deshea L. Harris, None; Pedram Hamrah, None
Support: NIH K08-EY020575 (PH), Research to Prevent Blindness
Career Development Award (PH), Fight for Sigh (PH),
Massachusetts Lions Eye Research Fund (PH), Japanese Eye Bank
(TY), Bausch Lomb Research Fellowship Award (TY), Uehara
Memorial Fellowship (TY)
Program Number: 1289
Presentation Time: 9:15 AM - 9:30 AM
The CCR7-CCL19/CCL21 Axis Mediates Enhanced AntigenPresenting Cell Trafficking In High-Risk Corneal
Transplantation
Jing Hua, William Stevenson, Thomas H. Dohlman, Narghes
Calcagno, Negar Pirmadjid, Zahra Sadrai, Sunil K. Chauhan, Daniel
R. Saban, Reza Dana. Schepens Eye Research Institute,
Massachusetts Eye and Ear Infirmary, Harvard University, Boston,
MA.
Purpose: High-risk (HR) corneal transplantation is associated with
ocular surface inflammation, antigen-presenting cell (APC)
maturation and a high rate of graft rejection. Mature APCs are
capable of migrating to the draining lymph nodes (LNs) and initiating
rejection. This study aimed to examine the magnitude, kinetics and
molecular mechanisms of APC trafficking in HR transplantation.
Methods: Corneas from congenic CD45.1 or eGFP transgenic mice
(C57Bl/6 background) were transplanted to naïve (low risk, LR) or
suture-induced neovascularized (HR) BALB/c recipient beds.
Ipsilateral draining LNs were excised at 4, 24, and 48h posttransplantation and cells were analyzed using FACS (CD45.1 + and
eGFP+ for donor-derived APCs, and YAe+ for alloantigen-bearing
recipient APCs). Quantitative PCR and ELISA were performed to
evaluate the factors involved in APC homing to draining LNs.
Secretory factors from LN explant cultures were assessed using
transwell migration assays.
Results: The CD45.1+, eGFP+ and YAe+ cells were detected in the
LNs as early as 4h post-transplantation. The frequency of YAe+ cells
in HR LNs was significantly higher than in LR at all time points (4h:
0.68±0.14% vs. 0.13±0.06, 24h: 0.70±0.09 vs. 0.32±0.01, and 48h:
0.64±0.06 vs. 0.30±0.05 of total cells, p<0.0001, two-way ANOVA,
Bonferroni post-test). CD45.1+ frequencies were significantly higher
in HR LNs than in LR LNs at 24h (1.09±0.12% vs. 0.78±0.01 of total
cells, p<0.0001) and eGFP + frequencies showed a similar trend. The
donor-derived APCs in HR LNs exhibited higher frequencies of
MHC IIhiCCR7+ cells than in LR LNs. CCR7 ligands (CCL19 and
CCL21) were significantly upregulated in the HR LNs at both mRNA
and protein levels. The supernatant of HR LN explant cultures
augmented mature APC migration, and neutralization of CCL19 and
CCL21 in transwell assays dramatically reduced migration of mature
APCs (0.8x104/well compared to 4.7x104, n=3, p<0.01).
Conclusions: Trafficking of both donor- and recipient-derived APCs
is enhanced in HR transplantation. Higher frequencies of mature
CCR7+ APCs as a result of enhanced CCL19 and CCL21 expression
in the draining LNs of HR recipients augment the trafficking of graft
site-derived APCs to the lymphoid tissues.
Commercial Relationships: Jing Hua, None; William Stevenson,
None; Thomas H. Dohlman, None; Narghes Calcagno, None;
Negar Pirmadjid, None; Zahra Sadrai, None; Sunil K. Chauhan,
None; Daniel R. Saban, Schepens Eye Res Inst, Mass Eye and Ear,
(P), Eleven Biotherapuetics (R); Reza Dana, Allergan (C), Alcon
(C), Bausch & Lomb (C), Eleven Bio (I), GSK (F), Novabay (C),
Revision Optics (C), Novaliq (C), RIgel (F)
Support: R01-EY20889 NIH
Program Number: 1290
Presentation Time: 9:30 AM - 9:45 AM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Understanding the Mechanism of Donor Bone Marrow Derived
Dendritic Cells in Promoting Corneal Allograft Survival in the
Rat
Thomas Ritter, Oliver Treacy, Aideen Ryan, Mourice Morcos,
Marese Cregg, Mikhail Nosov, Lisa O'Flynn. Medicine, Nt'l Univ of
Ireland, Galway, Galway, Ireland.
Purpose: To understand the mechanism of ex-vivo generated donor
bone marrow derived dendritic cells (BMDCs) on promoting corneal
allograft survival in the rat.
Methods: BMDCs were propagated from Dark Agouti (DA) rat bone
marrow precursor cells in complete medium supplemented with rat
GMCSF (5ng/ml) and IL-4 (5ng/ml). For glucocorticoid treatment of
BMDCs, dexamethasone (Dexa) (10-6M) was added to the culture. A
fully allogeneic rat corneal transplantation model (DA to LEW) was
used for in vivo studies. Day 10 donor BMDCs +/- Dexa were
harvested and 1x106cells/ml injected intravenously into recipients 7
days prior to corneal transplant surgery. Graft survival and
development of opacity, edema and neovascularisation were
monitored throughout the therapy. On the average day of rejection
the immune microenvironment (cell populations and cytokines
expressed) within the graft and the draining lymph nodes was
analysed. Alloantibody production was analysed for all experimental
groups by flow cytometry.
Results: Ex vivo generated BMDCs have a semi-mature phenotype
and can be treated with Dexa to maintain their immature phenotype
(reduction in MHC II, CD80 and CD86, n=5 p<0.05). These cells are
functional antigen presenting cells but have a reduced allostimulatory
capacity compared to control (control DCs: 32.23 ± 2.562%,
BMDCs: 4.450 ± 1.354% and Dexa BMDCs: 5.920 ± 0.24%). When
applied in vivo BMDC and Dexa BMDC significantly prolong
corneal allograft survival (MST> 30d, n=14 p<0.0004 and n=24
p<0.0001 resp.) compared to untreated allogeneic controls (MST 18d,
n=11). A significant reduction in the total number of graft infiltrating
cells was observed for treated groups (p<0.05 each group n=4). Both
treatments resulted in significant ratios of CD4+FoxP3+ cells to
CD4+CD25+ cells and increased indoleamine mRNA expression
within the graft (p<0.05 each group n=4). The same observation was
made at the level of the draining lymph nodes. Interestingly, the
alloantibody response (IgG1 and IgG2) was significantly different
between BMDCs (n=4) treated and Dexa BMDCs (n=6) treated
groups.
Conclusions: Our results demonstrate a significant therapeutic effect
of donor-derived BMDCs with and without glucocorticoid treatment
in corneal transplant survival. This represents a novel therapeutic
approach for the prevention of corneal allograft rejection.
Commercial Relationships: Thomas Ritter, None; Oliver Treacy,
None; Aideen Ryan, None; Mourice Morcos, None; Marese Cregg,
None; Mikhail Nosov, None; Lisa O'Flynn, None
Support: Science Foundation Ireland (SFI) 07/IN.1/B925
Program Number: 1291
Presentation Time: 9:45 AM - 10:00 AM
Influences on effector functions of monocyte-derived
macrophages in corneal allograft rejection
Thabo Lapp1, 3, Nandi Simpson1, Sarah Zaher2, 1, Benjamin Chain1,
Thomas Reinhard3, Mahdad Noursadeghi1, Frank Larkin2. 1Division
of Infection and Immunity, University College London, London,
United Kingdom; 2NIHR Biomedical Research Centre, Moorfields
Eye Hospital, London, United Kingdom; 3University Eye Hospital,
University Medical Centre Freiburg, Freiburg, Germany.
Purpose: To examine inhibition of recruitment of monocyte and
related macrophages and factors derived from these cells in the
context of corneal graft rejection.
Methods: Flow cytometry was used to examine monocyte
recruitment at the site of corneal graft rejection, and multiplex protein
assays to quantify pro-inflammatory mediators in this context. An
inflammatory milieu was modelled by stimulation of human
macrophages with lipopolysaccharide (LPS) or interferon (IFN)γ.
Transwell apparatus were used to model monocyte migration towards
inflammatory foci across an endothelial barrier and the effect of
targeting chemokine pathways was tested by depleting specific
chemokines or using neutralising antibodies to selected chemokine
receptors. The cytotoxic effect on human corneal endothelium was
tested by confocal microscopy enumeration of nuclei after ex vivo
exposure to inflammatory mediators and inflammatory monocytes.
Results: Aqueous humour samples showed a selective enrichment for
classical (CD14++/CD16-) monocytes during corneal graft rejection.
We established the repertoire of monocyte chemokines upregulated in
inflammatory responses and tested the effect of targeting these
pathways on monocyte recruitment. We show that depletion of
individual chemokines only partially attenuates monocyte
recruitment, and that targeting of chemokine receptors may be more
effective. Confocal microscopy showed that macrophage derived
inflammatory milieus in this model had significant cytopathic effect
on human corneal endothelial cells.
Conclusions: We have built on the evidence from human and rodent
studies supporting a role for monocyte recruitment in the
immunopathogenesis of corneal rejection. We have developed human
experimental models to identify candidates for therapeutic targeting
to inhibit monocyte recruitment and cytopathic mechanisms for
corneal endothelial injury during corneal allograft rejection. These
models may lead to novel immunotherapeutic strategies to modulate
the pathogenesis of rejection and prolong the survival of corneal
allografts.
Commercial Relationships: Thabo Lapp, None; Nandi Simpson,
None; Sarah Zaher, None; Benjamin Chain, None; Thomas
Reinhard, None; Mahdad Noursadeghi, None; Frank Larkin,
None
Support: Gertrud Kusen Foundation (Germany), Rosetrees Trust
(United Kingdom), NIHR Biomedical Centre for Ophthalmology,
Moorfields Eye Hospital & UCL Institute of Ophthalmology
Program Number: 1292
Presentation Time: 10:00 AM - 10:15 AM
MMP12 Regulation of Corneal Inflammation
Matilda F. Chan1, Jeffrey Lin1, Neeraj Ramakrishnan1, Zena Werb2.
1
Ophthalmology/Proctor Foundation, Univ of California-San
Francisco, San Francisco, CA; 2Anatomy, University of California,
San Francisco, San Francisco, CA.
Purpose: An excessive accumulation of immune cells or a delay in
the clearance of immune cells can result in corneal fibrosis and loss
of corneal clarity. The underlying mechanisms that determine the
kinetics of immune cell recruitment and clearance following corneal
injury are not fully understood. Recent data has demonstrated that
matrix metalloproteinases (MMPs) may have important regulatory
roles in inflammation. We tested the hypothesis that MMP12
significantly contributes to the recruitment of inflammatory cells
following corneal injury.
Methods: Alkaline burn injuries created by topical application of
0.1N NaOH were performed on corneas of WT and MMP12-/- mice.
Corneas of Mmp12-/- and WT mice were collected 6 days after
injury. Macrophage recruitment was assessed by whole mount
technique. CCL2 expression levels were analyzed using qPCR, a
protein array, and ELISA assay. The effect of blocking CCL2 on
macrophage recruitment was evaluated by whole mount assay. The
expression levels of macrophage M1 and M2 markers were measured
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
using qPCR analysis.
Results: After injury, macrophage accumulation was significantly
increased in MMP12-/- mice compared with WT mice. Analysis of
the expression levels of several CCL chemokines demonstrated
significantly elevated RNA and protein levels of CCL2 in Mmp12-/corneas compared with WT corneas. The trafficking of macrophages
into the central cornea following injury was significantly reduced by
subconjunctival injections of CCL2 blocking antibodies. The
expression of a macrophage M1 marker (TNF-α) was increased in
injured corneas of Mmp12-/- mice while expression of a macrophage
M2 marker (CD23) was reduced.
Conclusions: Excessive accumulation of macrophages following
corneal injury and an M1 macrophage phenotype favor a fibrotic
response to corneal injury. MMP12 appears to protect against a
fibrotic response to injury by negatively regulating CCL2 expression,
decreasing macrophage accumulation, and by promoting a tissue
reparative M2 macrophage phenotype.
Commercial Relationships: Matilda F. Chan, None; Jeffrey Lin,
None; Neeraj Ramakrishnan, None; Zena Werb, None
Support: NIH Grant K08EY018858
necessary to validate these results for in vivo human corneal tissue.
Additionally, safety aspects at high intensities must be investigated.
Stiffness increase of all treatment groups compared to control group.
237 Corneal Cross-linking and Biomechanics
Monday, May 06, 2013 8:30 AM-10:15 AM
Exhibit Hall Poster Session
Program #/Board # Range: 1611-1645/D0246-D0280
Organizing Section: Cornea
Program Number: 1611 Poster Board Number: D0246
Presentation Time: 8:30 AM - 10:15 AM
The efficacy of corneal cross-linking shows a sudden decrease
with very high intensity UV-light and short treatment time
Jeremy Wernli1, 3, Silvia Schumacher1, 3, Eberhard Spoerl2, Michael
C. Mrochen1, 3. 1IROC Science to Innovation AG, Zurich,
Switzerland; 2Ophthalmology, Carl Gustav Carus University Hospital
Dresden, Dresden, Germany; 3IROC Innocross AG, Zurich,
Switzerland.
Purpose: Standard treatment in case of progressive keratectasia is
UV-triggered corneal cross-linking. For irradiances larger than 10
mW/cm2 and treatment times below 10 min the scientific proof of a
biomechanical strengthening effect is insufficient. The authors
investigated the biomechanical strengthening of ex-vivo corneal
tissue treated with irradiances between 3 mW/cm2 and 90 mW/cm2
and illumination times from 30 minutes to 1 minute, respectively.
Methods: 100 porcine eyes received riboflavin+UV treatment
(constant irradiation dose of 5.4 J/cm2) with different intensities and
illumination times and were randomly assigned into 10 groups. A
control group (80 eyes) was not irradiated but underwent the same
treatment otherwise. Young’s modulus at 10% strain was determined
for each strip after uniaxial stress-strain measurement. A KruskallWallis test was used for statistical analysis.
Results: A statistically significant difference (α=0.01) was found
between the median value of Young’s modulus of the treatment
groups up to 45 mW/cm2 (illumination times from 30 min to 2 min)
compared to the control group. There was no statistically significant
difference between the treatment groups from 50 mW/cm2 up to 90
mW/cm2 (illumination times of less than 2 min) and the control
group.
Conclusions: The ex vivo results of corneal cross-linking performed
in porcine corneas show that the Bunsen-Roscoe reciprocity law is
only valid for illumination intensities up to 40 to 50 mW/cm2 and
illumination times of more than 2 min. Further experiments are
Young’s modulus at 10% strain of the control and different treatment
groups.
Commercial Relationships: Jeremy Wernli, IROC Innocross AG
(C); Silvia Schumacher, IROC Innocross AG (C); Eberhard
Spoerl, None; Michael C. Mrochen, IROC Innocross AG (I)
Program Number: 1612 Poster Board Number: D0247
Presentation Time: 8:30 AM - 10:15 AM
Corneal Biomechanical Properties after UV Cross-linking in the
Rabbit
Michael D. Twa1, Jiasong Li2, Ravi Kiran Manapuram2, Floredes M.
Menodiado2, Salavat Aglyamov3, Stanislav Emelianov3, Kirill Larin2.
1
College of Optometry, University of Houston, Houston, TX;
2
Biomedical Engineering, University of Houston, Houston, TX;
3
Biomedical Engineering, University of Texas, Austin, TX.
Purpose: Elasticity imaging has been applied in other areas of
medicine and more recently used to characterize the structural
properties of ocular tissues. An OCT-based elastography method was
developed and measurements were performed in rabbit corneal tissue
before and after UV-riboflavin corneal cross-linking (CXL).
Methods: Corneal elastography measurements were performed using
a Phase Stabilized Swept Source Optical Coherence Elastography
(PhS-SSOCE) with a sensitivity of ~10 nm along with air-pulse tissue
stimulation. Surface wave propagation was measured over a 6x6mm
area before and after UV-riboflavin corneal cross-linking. Tissue
properties (Young's modulus, surface wave propagation speed and
surface wave amplitude) were measured.
Results: Treatment resulted in a measurable increase corneal
stiffness confirmed by mechanical extensiometry (before CXL:
E=1.32±0.39MPa; after CXL: E=2.34±0.91MPa). Surface wave
amplitude and velocity was greatest near the excitation position
(Amplitude= 993nm and Velocity=0.8±0.09m/s) and decreased from
this point to the apex. Following cross-linking surface wave
amplitudes decreased (141nm) and wave velocity increased (8.2±5.6
m/s).
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Conclusions: Ocular elastography can be performed using a
combination of phase-sensitive OCT and air pulse stimulation. This
method can detect low amplitude tissue excitation, which can be used
to quantify corneal stiffness.
Commercial Relationships: Michael D. Twa, None; Jiasong Li,
None; Ravi Kiran Manapuram, Bioptigen Inc., (E); Floredes M.
Menodiado, None; Salavat Aglyamov, None; Stanislav Emelianov,
None; Kirill Larin, None
Support: EY022362
Program Number: 1613 Poster Board Number: D0248
Presentation Time: 8:30 AM - 10:15 AM
The Rigidity of Corneas before and after Corneal Cross-linking as measured by Corvis® ST
Sashia Bak-Nielsen, Iben Bach Pedersen, Anders Ivarsen, Jesper
Hjortdal. Ophthalmology, Aarhus University Hospital, Aarhus,
Denmark.
Purpose: Corneal Cross-linking (CXL) for treating keratoconus has
been shown to stiffen the cornea in vitro and clinical studies have
documented that progression of keratoconus is halted in most cases.
The Corvis ST from Oculus dynamically measures corneal
deformation by Scheimpflug imaging during an air puff. The pattern
of deformation theoretically depends on the intraocular pressure, the
corneal thickness and the material properties of the cornea. The
purpose of this study was to measure the possible stiffening effect of
CXL on patients with keratoconus, comparing groups with and
without a previous CXL procedure.
Methods: Thirty-seven keratoconus patients were included
- 19 with untreated keratoconus
- 18 with keratoconus treated with CXL 4-50 months previously
(median 27 months)
Furthermore 31 healthy subjects were included as a control group.
Apart from being measured with Corvis ST the subjects underwent a
full ophthalmic examination including Pentacam topography. The
Pentacam measurements were used to stage the patients into 4 groups
based on the severity of the keratoconus. The stiffness of the cornea
was evaluated by the radius of corneal concavity at the maximum
deformation (DR) as calculated by the Corvis ST.
Results: There was no significant difference in DR between the
untreated keratoconus group and the CXL treated group although DR
appeared marginally larger in eyes that had underwent CXL (unpaired t-test, p>0.05, Table 1). Increasing keratoconus severity
(Grades 1-4) had only a small and insignificant influence on DR
(ANOVA, p>0.05). DR in untreated and CXL treated keratoconus
groups was significantly smaller compared with normal eyes.
Conclusions: Eyes with keratoconus have a smaller DR as measured
by the Corvis ST, but the DR was similar in untreated and CXL
treated eyes. As the effect of CXL mainly is exerted in the anterior
part of the corneal stroma, it can be speculated that DR is insensible
to changes in corneal material properties in the anterior stroma.
Further studies of corneal deformation parameters in the same
patients before and after CXL are needed to further evaluate the
Corvis ST and the effect of CXL.
Table 1: Mean DR and 95% Confidence interval (CI) for keratoconus
eyes (grade 1-4), CXL treated eyes (grade 1-4) and normal eyes
Commercial Relationships: Sashia Bak-Nielsen, None; Iben Bach
Pedersen, None; Anders Ivarsen, None; Jesper Hjortdal, Carl
Zeiss Meditec (R)
Program Number: 1614 Poster Board Number: D0249
Presentation Time: 8:30 AM - 10:15 AM
Association of Ambient Solar Radiation with Biomechanical
Properties of the Cornea In an elderly population: The Alienor
Study
Cedric Schweitzer1, Cecile Delcourt2, Florence Malet1, Melanie Le
Goff2, Jean-Francois Korobelnik1, 2, Marie B. Rougier1, Marie-Noelle
Delyfer1, 2, Jean Francois Dartigues2, Pascale Barberger-gateau2,
Joseph Colin1. 1Ophthalmology, University Hospital Pellegrin,
Bordeaux, France; 2INSERM, ISPED, Bordeaux university,
bordeaux, France.
Purpose: To analyze the association of ambient solar radiation (SR)
on biomechanical properties of the cornea in adult patients.
Methods: The ALIENOR (Antioxydants, Lipides Essentiels,
Nutrition and maladies OculaiRes) Study is a population-based
epidemiological study on age-related eye diseases. In 2009-2010, 625
subjects, aged 75 years or more, had an eye examination, including
intraocular pressure, central corneal thickness (CCT) measurements,
and an evaluation of the biomechanical properties of the cornea using
the ocular response analyser® (ORA®, reichert inc., USA). Mean
lifetime ambient SR was estimated using residential history. Global
ambient annual SR (a measure of solar energy including all
wavelengths) was estimated using astronomic formulas and the
statistics of sunshine hours at each location. Then, for each
participant, average annual ambient SR was estimated by weighting
annual ambient solar radiation at each location by the time spent at
that location. Participants were classified in 3 groups (Group 1:
<458.53 kJ/cm2, Group 2: 458.53-474.74 kJ/cm2 (reference), Group
3: >474.74 kJ/cm2). Corneal hysteresis (CH), corneal resistance
factor (CRF), corneal compensated intraocular pressure (IOPcc),
Goldmann correlated intraocular pressure (IOPg) and CCT
parameters were analyzed between SR groups, using mixed linear
regression models taking into account data from both eyes and their
intra-individual correlations.
Results: After adjustment for age and gender, there was a significant
association of CH and CRF values with SR higher than 474.74
kJ/cm2 (CH: -0.39mmHg, 95% confidence interval (CI): -0.75;-0.03,
p=0.03/ CRF: -0.38mmHg, 95% CI: -0.74; -0.03, p=0.035), whereas
there was no significant association of CH and CRF values with SR
lower than 458.53 kJ/cm2 (CH: 0.01mmHg, 95% CI: -0.34; 0.36,
p=0.96/ CRF: 0.002mmHg, 95% CI: -0.35; 0.35, p=0.99). The CCT
was not significantly associated with SR ( SR<458.53: CCT:-2.48µm,
95%CI: -11.93;6.97, p=0.61/ SR> 474.74: CCT: -4.25µm, 95%CI: 13.86;5.35, p=0.38).
Conclusions: CH and CRF were significantly lower in subjects
exposed to high ambient solar radiation. Ambient UV exposure might
induce histological changes of the cornea that influence its
biomechanical properties.
Commercial Relationships: Cedric Schweitzer, None; Cecile
Delcourt, Laboratoires Théa (F), Novartis (C), Bausch+Lomb (C);
Florence Malet, None; Melanie Le Goff, None; Jean-Francois
Korobelnik, Alcon (C), Allergan (C), Bayer (C), Carl Zeiss Meditec
(C), Novartis (C), Thea (F); Marie B. Rougier, THEA (C),
Bausch&Lomb (C), Allergan (C), Kemin (C); Marie-Noelle Delyfer,
Thea Laboratories (F); Jean Francois Dartigues, Novartis (F),
IPSEN (F); Pascale Barberger-gateau, Danone (F), Vifor Pharma
(C); Joseph Colin, Alcon (C), Abbott (C), AdditionTechnology (C)
Support: Laboratoires Théa, Fondation Voir et Entendre
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Program Number: 1615 Poster Board Number: D0250
Presentation Time: 8:30 AM - 10:15 AM
Effect of UVA-Rb cross-linking on through-thickness strains in
canine corneas
Joel Palko1, Xueliang Pan2, Jun Liu3, 4. 1Wexner Medical Center,
Ohio State University, Columbus, OH; 2Center for Biostatistics, Ohio
State University, Columbus, OH; 3Deprtment of Biomedical
Engineering, Ohio State University, Columbus, OH; 4Department of
Ophthalmology, Ohio State University, Columbus, OH.
Purpose: Monitoring the mechanical changes of the cornea before
and after corneal cross linking (CXL) provides valuable insight into
CXL mechanisms and may help provide more personalized treatment
plans for this therapy in the prevention of progressive keratoconus.
The purpose of this study was to measure through-thickness strains in
the cornea at physiologic IOPs before and after CXL using noninvasive ultrasound.
Methods: The anterior 3/4 of paired canine corneoscleral shells
including a CXL treated group (n=6) and a control group (n=6) were
mounted to a pressurization chamber within 10hrs of euthaniasia. The
CXL group completed a standard clinical CXL protocol using
riboflavin (Rb solution and UVA radiation (370nm, irradiance
3mW/cm2). Control eyes were given an identical Rb treatment
without UVA irradiation. Cornea ultrasound scans (at 55 MHz) along
the nasal-temporal (NT) and superior-inferior (SI) cross-sections
were obtained before and after treatment as IOP was gradually
increased from 5 mmHg to 45mmHg. Strain tracking was performed
using a previously validated method (Tang & Liu, J. Biomech. Engrg
2012, 134(9)). Mean radial compressive strains and tangential tensile
strains were calculated for the anterior, middle, and posterior one
thirds of the cornea thickness in the nasal-temporal (NT) and
superior-inferior (SI) directions. Mean strains at IOPs of 10, 20, and
30mmHg were compared between the CXL and control groups using
mixed linear models with repeated measures.
Results: Statistically significant reductions in tensile and
compressive strains were found in the SI orientation at all three IOPs
and all three layers in the CXL group (all p<0.05), with the exception
of radial strains in the posterior third of the cornea. Similar mean
strain reductions were found in the NT direction. The changes in the
mean strains were small and not significant in the control group
(p>0.05). The anterior third appeared to have larger tensile strain
reduction than the posterior layer in the CXL group.
Conclusions: Ultrasound strain tracking revealed that the Rb-UVA
CXL procedure significantly reduced corneal strains (i.e., stiffened
the cornea) during physiologic IOP elevation with more pronounced
effects observed in the anterior cornea. The ability to measure and
monitor cornea strains may provide insight into the biomechanical
effects of CXL and better define its role as a treatment for certain
ocular disorders.
Commercial Relationships: Joel Palko, None; Xueliang Pan,
None; Jun Liu, None
Support: NIHRO1EY020929 (JL), Ohio State University College of
Medicine (JP)
Program Number: 1616 Poster Board Number: D0251
Presentation Time: 8:30 AM - 10:15 AM
In Vivo Evaluation of Corneal Biomechanical Properties After
Corneal Collagen Cross-linking Therapy
Raksha Urs1, Harriet Lloyd1, Ronald H. Silverman1, 2.
1
Ophthalmology, Columbia University Medical Center, New York,
NY; 2Frederic L. Lizzi Center for Biomedical Engineering, Riverside
Research Institute, New York, NY.
Purpose: Collagen cross-linking therapy (CXL) is emerging as a
treatment option for keratoconus. This procedure strengthens the
biomechanical properties of the cornea by cross-linking the collagen
bonds. However, biomechanical tests, to evaluate CXL outcome,
have been performed only on ex vivo tissue. In vivo, the efficacy of
the treatment is verified by assessing vision quality. The objective of
this project is to demonstrate an in vivo technique to determine
difference in biomechanical strength of the cornea after CXL.
Methods: CXL procedure was performed on the right eyes of 6
rabbits. The left eyes were used as controls. Acoustic Radiation Force
(ARF) was used to assess corneal stiffness in vivo, once before
treatment (Baseline BL) and weekly for four weeks after treatment
(W1-W4). Cornea was exposed to ARF using a single element
transducer (25 MHz central frequency; 6 mm aperture; 18 mm focal
length; Panametrics V324-SU). The beam sequence consisted of 20
pushing tonebursts of 400 μs duration (80% duty cycle). Imaging
impulses were interleaved in the dead time to allow the same
transducer to acquire radiofrequency data during the push mode to
image corneal displacement. Acoustic power levels were within
FDA-specified levels for ophthalmic safety. Displacement of the
front and back surfaces of the cornea were used to determine the
change in corneal thickness and strain. ARF induced strain was fit to
the Kelvin-Voigt model to determine the elastic modulus. The
average moduli were calculated for the six rabbits, for each of the
five time points (BL, W1-W4).
Results: At the end of four weeks, ARF measurements showed an
increase of average elastic modulus by 33% in the treated eye, and
3% in the control eye. Paired t-tests revealed statistically significant
differences between treated and untreated eyes from W1-W4
(p=0.0005, 0.04, 0.0007, 0.006). There was no significant difference
between right and left eyes before treatment (p=0.95).
Conclusions: Our findings demonstrate statistically significant
differences in stiffness between control and CXL-treated rabbit
corneas in vivo based on axial stress/strain measurements obtained
using ARF. The capacity to non-invasively monitor corneal stiffness
offers the potential for clinical monitoring of CXL. Longer term
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
studies will be required to elucidate the effectiveness of this noninvasive technique.
Commercial Relationships: Raksha Urs, None; Harriet Lloyd,
None; Ronald H. Silverman, None
Support: AIUM EER; NIH Grant EY021529; Research to Prevent
Blindness
Program Number: 1617 Poster Board Number: D0252
Presentation Time: 8:30 AM - 10:15 AM
Long Term Follow-Up of Corneal Topographic Indices Following
Collagen Crosslinking In Eyes with Keratoconus
Erick Hernández-Bogantes, Lihteh Wu, David Flikier. Instituto de
Cirugía Ocular, San José, Costa Rica.
Purpose: To compare the pre-treatment and 5 year post-treatment
visual acuity and corneal topographic indices in eyes with
keratoconus that were treated with corneal collagen crosslinking.
Methods: An observational, uncontrolled, retrospective study of 7
patients (12 eyes) with keratoconus that were treated with epitheliumoff corneal collagen crosslinking between March and December of
2007 was conducted. Pre-treatment and 5 year post-treatment anterior
corneal surface (keratoconus index, index of vertical asymmetry,
index of surface variance, central keratoconus index, minimum radius
of curvature, index of height asymmetry, index of height
decentration) and normalized Belin-Ambrosio indices (front
elevation, back elevation, pachymetric progression, corneal thinnest
point and the total analysis) were obtained by the Pentacam
topographer, Oculus Inc. (Wetzlar, Germany). The uncorrected visual
acuity and best corrected visual acuity were also compared.
Results: The 5 year follow-up showed that both central keratoconus
index (P= 0.0452) and best corrected visual acuity (P= 0.0313)
improved following corneal collagen crosslinking. At 5 years of
follow-up, there were no statistically significant differences between
the pre-operative and post-operative values of either the other
anterior surface variables or the normalized Berlin-Ambrosio indices.
Conclusions: Even though the surface of the cornea does not appear
to be dramatically altered after five years of corneal collagen
crosslinking, improvement in visual acuity is obtained and remains
constant.
Commercial Relationships: Erick Hernández-Bogantes, None;
Lihteh Wu, Heidelberg Engineering (R); David Flikier, None
Program Number: 1618 Poster Board Number: D0253
Presentation Time: 8:30 AM - 10:15 AM
Comparison of corneal changes between standard and
transepithelial riboflavin-UVA crosslinking method using
multiphoton microscopy and second harmonic imaging
Praveena Gupta1, Best Anyama2, Kevin M. Wells2, Massoud
Motamedi3, 1, Bernard F. Godley1, Gracie Vargas3. 1Ophthalmology
& Visual Sciences, Univ of Texas Medical Branch, Galveston, TX;
2
School of Medicine, University of Texas medical Branch,
Galveston, TX; 3Center for Biomedical Engineering, Univ of Texas
Medical Branch, Galveston, TX.
Purpose: Corneal crosslinking procedures are offered as a treatment
for keratoconus and corneal ectasic disorders. This study was
undertaken to investigate the comparative stromal changes after the
UVA-crosslinking on a riboflavin-debrided and a riboflavintransepithelial treated porcine cornea using multiphoton microscopy
and second harmonic generation signals.
Methods: Fifteen fresh pig eyes were treated using either the
standard method of riboflavin-UVA crosslinking or the
transepithelial method (riboflavin TRIS-EDTA) for 30 min at
irradiance of 3mW/cm2. All corneas were then stained with a cell
death marker and processed for non-invasive multiphoton
microscopy and second harmonic signal imaging. Data collected
were analyzed using image J.
Results: Standard CXL treatment resulted in severe loss of the
classic interwoven collagen architecture all the way up to 300 uM
depths, whereas, similar changes were noted only up to 150 uM depth
in the TE method of CXL. Significantly higher numbers of dead
keratocytes were counted at all the depths in the standard CXL
exposed eyes in contrast to fewer keratocyte deaths that were more
pronounced only in the anterior stroma in the TE-CXL treated
corneas (p<0.05). However, in both the treatment groups decreasing
number of dead keratocytes were noted in deeper stroma than in the
anterior stroma, suggesting the role of riboflavin for crosslinking.
Conclusions: This is the first study that shows direct comparison
between the standard and the transepithelial (RicrolinTE, Sooft Italia)
method of riboflavin UVA-crosslinking. We have shown structural
alterations between the cross-linked corneal stroma in the two
currently used method of treatment. This information will be useful
in clinical decision making when choosing the treatment strategy on
diseased thin corneas.
Commercial Relationships: Praveena Gupta, None; Best Anyama,
None; Kevin M. Wells, None; Massoud Motamedi, None; Bernard
F. Godley, None; Gracie Vargas, None
Support: Unrestricted RPB grant to the Department
Program Number: 1619 Poster Board Number: D0254
Presentation Time: 8:30 AM - 10:15 AM
Biaxial Biomechanical Study of UVA-RF Corneal Cross-linking
William A. Eddington, Marc D. Friedman, Evan A. Sherr, David
Muller. Avedro, Waltham, MA.
Purpose: To evaluate a method of biaxial extensometry applied to
corneal flaps created with a femtosecond laser system for
determining changes induced by various UVA-RF based corneal
cross-linking protocols.
Methods: Fresh whole porcine globes were obtained <24 hours
postmortem in saline on ice from Sioux-preme (Sioux City, IA). Eyes
were brought to 37oC, epithelium was removed with a dull blade,
intraocular pressure of 15mmHg was applied using a water column,
and corneal thickness readings were recorded using an ultrasonic
pachymeter (DGH Technology, Exton, PA) Eyes were separated into
three groups containing 7 eyes each. Eyes were then soaked in a
solution consisting of 0.1%RF and 0.85% saline for 20 minutes. One
group was set aside (Control) and the other two groups were placed
under UVA light at 365nm with an irradiance of 3mW/cm2 for a total
UVA dose of 5.4J/cm2 and 2.7J/cm2 for groups 2 and 3 respectively.
The eyes were then placed under an Intralase femtosecond laser
system (Abbot Medical Optics, Santa Ana, CA) and 200um thick
corneal flaps were cut from the anterior center of the cornea. Corneal
flaps were removed and attached to a biaxial extensometer (CellScale
Biotester 5000, Waterloo, ON) using a 5mm wide attachment
mechanism (Figure 1) and submerged in a 37oC saline bath. Flaps
were stretched from a relaxed state at a rate of 4um/s until a load of
5N or sample failure was reached.
Results: Stress strain curves can be generated and stiffness values of
each group were calculated at various displacements. Statistically
significant results (P<.05) can be observed between groups.
Conclusions: Using a thin corneal flap allows for the detection of
changes of the mechanical properties of corneal tissue induced by
both large and small changes in the cross-linking procedure. This
method is potentially able to detect changes in corneal mechanics
induced with UVA-RF cross-linking routines with varying riboflavin
concentration, UVA irradiance, total UVA energy, UVA application
patterns, and ambient atmospheres.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
will be expanded to include an anisotropic, fiber-dependent
hyperelastic material formulation.
Figure 1: Corneal flap attached to biaxial extensometer.
Commercial Relationships: William A. Eddington, Avedro Inc.
(E); Marc D. Friedman, Avedro Inc (E); Evan A. Sherr, Avedro,
Inc. (E); David Muller, Avedro Inc (E)
Program Number: 1620 Poster Board Number: D0255
Presentation Time: 8:30 AM - 10:15 AM
Finite Element Analysis of Treatment of Corneal Astigmatism
with Collagen Crosslinking
IBRAHIM SEVEN1, 2, Abhijit Sinha Roy1, William J. Dupps1, 2.
1
Ophthalmology, Cleveland Clinic Cole Eye Inst, Cleveland, OH;
2
Biomedical Engineering, Cleveland Clinic Lerner Research Institute,
Cleveland, OH.
Purpose: To test the hypothesis that selective collagen crosslinking
can alter corneal astigmatism and to assess the effects of treatment
orientation and pattern.
Methods: 3D geometry of a patient with corneal astigmatism was
measured by a Scheimpflug tomography system (Pentacam v.1.61).
Elevation data was fit and extrapolated with 12th order Zernike
polynomials routine which was coded in Python2.7. The extrapolated
data was imported into Solidworks (ver. 2011). The geometry was
meshed with hexahedral elements by Trugrid (v. 2.3.3). Corneal
biomechanical properties were defined as nonlinear, isotropic and
incompressible. The intra-ocular pressure was assumed as 15 mmHg.
Finite element analyses were performed using Abaqus (v. 6.11).
Since the in vivo (or pre-operation) geometry was pre-stressed by
intra-ocular pressure, the geometry was solved for the no-load
condition and pre-stresses were calculated. We simulated 4 different
crosslinking treatment patterns with a stiffening factor of 2 and
effective depth of 300 um. The treatment was simulated with a bowtie pattern oriented on the steep astigmatism axis (pattern1), a bow-tie
pattern oriented on the flat astigmatism axis (pattern2), a fan pattern
(pattern3), and a central ellipse pattern oriented on the flat
astigmatism axis (pattern4). The corneal anterior surface coordinates
were extracted at the end of the each simulation and the anterior
surface axial curvature was calculated. The refractive index was as
assumed as 1.3375.
Results: Keratometry (Sim K) values in the preoperative eye (in
vivo) were 44.85/46.22@78. The SimK value changed to
44.06/46.62@90, 44.83/45.88@45, 44.76/46.27@84,
44.64/45.64@51 with pattern1, pattern2, pattern3, and pattern4
treatment, respectively. The astigmatism value was 1.37in the preoperation stage. The value changed to 2.56, 1.05, 1.51, and 1.00 with
pattern1, pattern2, pattern3, and pattern4 treatment, respectively.
Conclusions: In this pilot study, simulated patterned collagen
crosslinking had an effect on corneal astigmatism. Treatments on the
steep axis increased astigmatism, while treatments orthogonal to the
steep axis decreased astigmatism. Additional clinical geometries with
regular and irregular astigmatism will be investigated and modeling
Figure 1 - Comparison of different treatment patterns
Commercial Relationships: IBRAHIM SEVEN, None; Abhijit
Sinha Roy, Carl Zeiss Meditec (F), Cleveland Clinic Innovations (P),
Topcon Inc. (F); William J. Dupps, Zeimer (C), Topcon (F), Avedro
(F), Carl Zeiss Meditec (F), Cleveland Clinic Innovations (P)
Program Number: 1621 Poster Board Number: D0256
Presentation Time: 8:30 AM - 10:15 AM
Corneal geometric stress factor to evaluate response to corneal
collagen cross-linking in keratoconus
Riccardo Vinciguerra1, 2, Cynthia J. Roberts3, Ashraf M. Mahmoud3,
Claudio Azzolini2, Paolo Vinciguerra1. 1Opthalmology, Humanitas
Clinical and Research Center, Milan, Italy; 2Dept. of Surgical and
Morphological Sciences, University of Insubria, Circolo Hospital,
Varese, Italy; 3Ophthalmology and Biomedical Engineering, The
Ohio State University, Columbus, OH.
Purpose: To evaluate corneal stress distribution based purely on
geometry without consideration of loading via intraocular pressure,
pre and post corneal collagen cross-linking (CXL) with a new
tomographic parameter, “Corneal Geometric Stress Factor”.
Methods: Tomographic data from four hundred and eighty subjects
(323 right eyes and 340 left eyes) were collected retrospectively from
Istituto Clinico Humanitas (Rozzano, Italy) with up to 70 months preCXL and 60 months post-CXL. Pentacam U12 files (Oculus
Optikgerate GmbH, Wetzlar, Germany) were transferred to The Ohio
State University and processed independently using custom software.
Corneal Geometric Stress Factor (CGSF) was calculated at
corresponding points from curvature and pachymetric maps to create
a CGSF map. CGSF evaluates the cornea’s contribution to Hoop
stress without considering the applied load (intraocular pressure) and
it can be expressed as the radius of curvature over twice of the
thickness (CGSF=R/2t). Cone Location and Magnitude Index
(CLMI) and Flat zone Location and Magnitude Index (FLMI) were
applied to the CGSF map to obtain maximum stress and minimum
stress and to calculate the level of asymmetry in the stress pattern.
Pre and post CXL regression analyses were performed.
Results: Regression analysis showed a significant (p<0.0001)
positive correlation of asymmetry stress distribution before CXL
demonstrating increasing asymmetry in the stress pattern, and a
significant negative correlation after CXL (p=0.0001) demonstrating
a pattern of reducing asymmetry over time. Maximum and minimum
stress factors similarly had positive correlation before CXL
(p<0.0001), indicating increasing stress over time and negative
correlation after CXL (p<0.0001), indicating decreasing stress over
time.
Conclusions: Biomechanical analysis shows CXL is able not only to
stop the progression of the disease, but even to reverse the cycle of
biomechanical decompensation of keratoconic corneas. These
findings can be explained by the fact that the decrease of thickness
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
and curvature associated with CXL treatment induces a consequent
reduction in both minimum and maximum stress as well as a
reduction of asymmetry in the stress distribution. IOP can modify the
stress magnitude, but not change the pattern demonstrated.
Commercial Relationships: Riccardo Vinciguerra, None; Cynthia
J. Roberts, Oculus Optikgerate GmbH (C), Ziemer Ophthalmic
Systems AG (C), Sooft Italia (R), Carl Zeiss Meditec (F); Ashraf M.
Mahmoud, None; Claudio Azzolini, None; Paolo Vinciguerra,
SOOFT Italia (C), Oculus Optikgerate GmbH (C), NIDEK, Japan
(C), Schwind (C)
Program Number: 1622 Poster Board Number: D0257
Presentation Time: 8:30 AM - 10:15 AM
Evaluation of the riboflavin and Ultraviolet light effect on
keratocytes cultivated in vitro
Joyce L. Covre, Priscila C. Cristovam, Renata R. Loureiro, Rossen
M. Hazarbassanov, Mauro S. Campos, Élcio H. Sato, Jose A. Gomes.
Ophthalmology, UNIFESP, Sao Paulo, Brazil.
Purpose: Evaluate the riboflavin and ultraviolet light effect on
human keratocytes cultivated in vitro.
Methods: Keratocytes were obtained from human corneal rims
remnants of tissue previously used in corneal transplants at the
Department of Ophthalmology of UNIFESP/EPM, and cultured in
DMEM/F12 medium with FBS until confluence. The cell cultures
were characterized by immunofluorescence with antibodies to K3
(epithelial marker), Thy 1(fibroblast marker), α-SMA
(myofibroblast), Lumican and Keratocan (keratocyte markers). The
corneal stromal cells were covered with collagen (200µL and 500
µL) and 0.1% of riboflavin and were exposed to ultraviolet light
(UV) for 30 minutes. After 24 hours, cytotoxicity was determined by
MTT assay and cell viability was quantified by means of dye Hoechst
33342/Propidium Iodide.
Results: All cell cultures reached confluence around 20 days.
Immunofluorescence demonstrated positive expression of keratocyte
markers (Lumican and Keratocan) and negative expression of
epithelial (K3), fibroblast (Thy 1) and myofibroblast (alpha-SMA)
markers. After riboflavin and UV light exposure, cultivated cells
without collagen layer presented higher cytotoxicity with MTT(one
way ANOVA, p<.0001) analysis and lower rates of apoptosis and
necrosis.
Conclusions: Keratocytes cultures were successfully obtained and
characterized by immunofluorescence to Lumican and Keratocan.
Collagen proved protective effects against UV light exposure.
Commercial Relationships: Joyce L. Covre, None; Priscila C.
Cristovam, None; Renata R. Loureiro, None; Rossen M.
Hazarbassanov, None; Mauro S. Campos, None; Élcio H. Sato,
None; Jose A. Gomes, Allergan (C), Pfizer (C), Genon (C), MSD (C)
Support: None in the Support field below
Program Number: 1623 Poster Board Number: D0258
Presentation Time: 8:30 AM - 10:15 AM
Long term evaluation of corneal permeability following crosslinking in a live animal model
Jay M. Stewart, Ricardo Lamy, Elliot Chan. Ophthalmology, Univ of
California-San Francisco, San Francisco, CA.
Purpose: To evaluate the corneal permeability in rabbits
approximately one year after corneal cross-linking (CXL).
Methods: New Zealand white rabbits were used for the study.
Control eyes were left unoperated. In cross-linked eyes, the Dresden
CXL protocol was performed, including corneal epithelial removal,
administration of topical riboflavin 0.1% for 30 minutes, and corneal
irradiation with ultraviolet-A at a dose of 5.4 J/cm2. Between 11 and
12 months later, in vivo corneal permeability was evaluated by
quantifying pupillary constriction during a 30-minute period
following topical application of 100 micrograms of pilocarpine.
Results: In control eyes, pupillary diameter decreased by a mean of
2.75 mm (n=6), while in CXL eyes the decrease was 2.25 mm
(n=12), suggesting that the corneal changes induced by CXL resulted
in reduced permeation of pilocarpine into the anterior chamber.
However, this difference did not reach statistical significance, likely
due to the small sample size.
Conclusions: We have previously reported that CXL reduces corneal
permeability, measured both in vivo and ex vivo. The current results
suggest that these effects are long-lasting.
Commercial Relationships: Jay M. Stewart, None; Ricardo
Lamy, None; Elliot Chan, None
Support: Research to Prevent Blindness; That Man May See, Inc.
Program Number: 1624 Poster Board Number: D0259
Presentation Time: 8:30 AM - 10:15 AM
Modulation of matrix stiffness throughout corneal wound healing
following phototherapeutic keratectomy
Sara M. Thomasy1, Vijaykrishna K. Raghuanthan1, Peter C. Strom1,
Jasmyne C. Sermeno1, Paul Russell1, Christopher J. Murphy1, 2.
1
Surgical and Radiological Sciences, School of Veterinary Medicine,
University of California, Davis, Davis, CA; 2Ophthalmology &
Vision Science, School of Medicine, University of California, Davis,
Davis, CA.
Purpose: While many attributes of corneal stromal wound healing
have been well characterized, the alterations in the biophysical
attributes of the corneal wound itself have not been adequately
investigated. Corneal stromal haze is a relatively common
complication following phototherapeutic keratectomy (PTK) and
compromises corneal transparency and visual acuity. We
hypothesized that the elastic modulus, a measure of stiffness, would
increase following PTK and correlate with the appearance of stromal
haze. The purpose of this study was to determine the elastic modulus
of the corneal stroma following PTK in rabbits using atomic force
microscopy (AFM).
Methods: Following corneal epithelial debridement, New Zealand
white rabbits underwent PTK (6 mm diameter, 100 μm deep) on the
right eye (OD). Corneal wound healing was monitored with digital
slit lamp biomicroscopy, ultrasonic pachymetry and spectral-domain
optical coherence tomography (SD-OCT). Rabbits were euthanized at
days 1, 3, 7, and 21. Immediately following euthanasia, the corneal
epithelium was removed from both eyes (OU) and the left eye (OS)
received a PTK similar to OD. An 8 mm central corneal button was
harvested OU and AFM was performed.
Results: Mean ± SD elastic modulus of the anterior corneal stroma
OS was 0.62 ± 0.29 kPa. At days 1 and 3, elastic modulus was 2.5fold greater OD versus OS and correlated with an increase in central
corneal thickness from corneal edema. At day 7, elastic modulus was
8-fold greater OD in comparison to OS and correlated with the
appearance of stromal haze as well as corneal re-epithelialization. At
day 21, stromal haze increased in density with elastic modulus
remaining 8-fold greater OD versus OS.
Conclusions: Elastic modulus of the anterior corneal stroma is
dramatically altered following PTK and correlates initially with the
development of edema and later with formation of stromal haze. With
emerging engineering approaches for modulating corneal
biomechanics such as crosslinking with riboflavin and hyaluronidase
to stiffen or soften the stroma, respectively, it is imperative to better
understand the long-term consequences of changing the biophysical
characteristics of the corneal stroma.
Commercial Relationships: Sara M. Thomasy, None;
Vijaykrishna K. Raghuanthan, None; Peter C. Strom, None;
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Jasmyne C. Sermeno, None; Paul Russell, None; Christopher J.
Murphy, Ocular Services On Demand (I), Ocular Services On
Demand (C), Platypus Technologies LLC (I), Imbed LLC (I), EyeKor
LLC (I), Allergan (C), Genentech (C), Sarcode (C), Covance (C)
Support: NIH KO8EY021142 (SMT), NIH R01EY019970 (CJM),
and Research to Prevent Blindness (UC Davis)
Program Number: 1625 Poster Board Number: D0260
Presentation Time: 8:30 AM - 10:15 AM
Comparison of Biomechanical and Tomographic Data in
Subclinical Keratoconus
Paolo Vinciguerra1, Renato Ambrosio3, Mario R. Romano1, Isaac C.
Ramos3, Claudio Azzolini2, Silvia Trazza1, Riccardo Vinciguerra1, 2.
1
Ophthalmology, Istituto Clinico Humanitas, Milan, Italy; 2Dept. of
Surgical and Morphological Sciences, University of Insubria, Circolo
Hospital,, Varese, Italy; 3Instituto de Olhos Renato Ambrósio, Rio De
Janeiro, Brazil.
Purpose: To evaluate the tomographic and biomechanical corneal
changes in subclinical keratoconus (KC).
Methods: Five patients with very asymmetric KC were
retrospectively compared with fellow eyes without tomographic
evidences of KC. Tomographic and biomechanical data were
respectively obtained with Pentacam and Corvis ST (Oculus
Optikgerate GmbH, Wetzlar, Germany).
The eyes without tomographic evidences of KC were also compared
with five healthy subjects pachymetry- and intra ocular pressure
(IOP)-matched.
From Pentacam analysis we considered: minimal pachymetry, single
and total deviation values(Dv) from Belin-Ambrosio Enhanced
Ectasia Display (BAD), and all topometric indexes obtained in the
topometric map.
From Corvis ST analysis we considered: time (t1-2), length (l1-2)
and velocity (v1-2) of first and second applanation, time (tC), peak
distance (pC), radius (rC) and deformation amplitude (dC) of highest
concavity, IOP and pachymetry
Results: Comparison between KC and the fellow eyes revealed
significant differences in single and total Dv evaluated (p<0.05)
except for Dv of average pachymetric progression index and
deviation of minimum thickness. Similarly topometric values showed
a significant difference (p<0.05) between KC and fellow eyes in
selected indexes.
Corvis ST analysis indicated non significant difference between KC
and fellow eyes (p>0.05) in all parameters, showing that fellow eyes
had the same pathological biomechanical behavior of the eyes with
manifest disease, whereas tomographic analysis didn’t show any
significant pathological changes.
Comparison between KC patients’ “healthy” eyes and control group
showed only scattered significant difference in topometric indexes
and in tomographic data in BAD Dv; however the overall total Dv
difference was not significant. Conversely biomechanical data
revealed significant differences in t1-2 (p<0.01) , v1 (p=0.002), tC
(p<0.001), rC (p<0.001), and dC (p<0.001) between KC patients and
control group.
Conclusions: Biomechanical analysis with Corvis ST is able to show
significant differences between normal eyes and subclinical
keratoconus when normal tomographic data show normality or only
small abnormalities. In conclusion Corvis ST could be a valid aid in
the screening for the risk of ectasia in refractive surgery patients and
for early diagnosis of keratoconus.
Commercial Relationships: Paolo Vinciguerra, SOOFT Italia (C),
Oculus Optikgerate GmbH (C), NIDEK, Japan (C), Schwind (C);
Renato Ambrosio, Oculus (C), Alcon (C), Allergan (C), AMO (C),
Mediphacos (C), Bausch & Lomb (C), Pfeizer (C); Mario R.
Romano, Bausch and Lomb (C); Isaac C. Ramos, None; Claudio
Azzolini, None; Silvia Trazza, None; Riccardo Vinciguerra, None
Program Number: 1626 Poster Board Number: D0261
Presentation Time: 8:30 AM - 10:15 AM
Correlation of Biomechanic Parameters Measured by Corvis ST
(Oculus®) and by Ocular Response Analyzer (ORA, Reichert®)
Michael Haustein, Eberhard Spoerl, Lutz E. Pillunat. Dept
Ophthalmology, University of Dresden, Dresden, Germany.
Purpose: To compare a newer device (CorVis ST, Oculus Inc.®)
with the standard device (Ocular Response Analyzer (ORA,
Reicherts®) in measuring in-vivo corneal biomechanical properties.
Additionally, multiple regression analysis is used to evaluate
potential influence factors (e.g. IOP, K-value, corneal thickness
(CCT), axial length (AL), anterior chamber depth (ACD)).
Methods: 65 eyes of 65 normal subjects were included in this
prospective, randomized, observational study. Measurements by
ORA, Corvis, Pentacam and IOL-master were randomly taken in all
participants. Biomechanical parameters were taken by ORA: corneal
hysteresis (CH) and corneal resistant factor (CRF) and by CorVis ST:
deformation amplitude (DA), radius (R), and wing-distance (WD).
Intraocular pressure was measured by GAT, ORA and Corvis.
Pentacam (CCT, ACD) and IOL-Master (AL, K-value) were used to
quantify anatomical status. SSPS®: T-test, intaraclass correlation
coefficient, Pearmans correlation and multiple linear regression
analysis were used for statistics.
Results: CH correlates very poor to all parameters of CorVis ST
(DA, R, WD), r<0.1. Also, there is no significant correlation to CCT,
ACD, and AL. CRF shows a significant correlation to CorVis ST
parameters (DA: p=0.02; r=-0.35/R:p=0.016; r=0.36) and to CCT by
Pentacam (p=0.001; r=0.526). However, there is no significant
correlation to WD, AL, and ACD. DA depends negatively on CRF
(p=0.02; r=-0.348), IOP (p<0.001; r=-0.611) and CCT (p=0.011; r=0.442), but only IOP is a significant influence factor of DA in
multiple linear regression analysis (p=0.026; r=0.75) [CRF: p=0.402;
CCT: p=0.731].
Conclusions: CorVis ST and ORA are two useful devices to measure
corneal biomechanics in vivo. As known, CH represents viscoelastic
properties of corneal matrix and CRF dumping properties of the
cornea. Therefore, CRF is a degree of elastic resistance.
Due to lack of correlation between CH and CorVis parameters and
also significant correlation between CRF and DA CorVis measures
more corneal dumping properties, probably corneal stiffness or
rigidity.
However, DA depends on IOP, CRF, and CCT. Therefore, our results
support the biomechanical model/equation of Friedenwald: F=
(E*CCT*b*DA)/l + IOP*10^(K*V)*A
Commercial Relationships: Michael Haustein, None; Eberhard
Spoerl, None; Lutz E. Pillunat, None
Program Number: 1627 Poster Board Number: D0262
Presentation Time: 8:30 AM - 10:15 AM
The correlation between ocular response analyzer keratoconus
match index and subjective topographic assessment using the
Orbscan to screen for keratoconus
Ryan A. Vasan, Ryan M. St Clair, Syed A. Hussnain, Ana G. Alzaga
Fernandez, Christopher E. Starr. Ophthalmology, Weill Cornell
Medical College, New York, NY.
Purpose: To evaluate the correlation between the Keratoconus Match
Index (KMI) of the Ocular Response Analyzer (ORA) and cornea
fellowship-trained graders using the Orbscan topographer when
evaluating patients for keratoconus (KCN)
Methods: This was a retrospective study including patients over the
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
age of 18 who underwent both Orbscan and ORA testing. The ORA
KMI software compares the corneal biomechanical properties of the
examined eye to a normative database and assigns the eye to one of
five categories: normal, KCN suspect, mild KCN, moderate KCN,
and severe KCN. Three masked MD observers (graders A, B, and C),
all of whom are cornea fellowship trained, examined the Orbscan
topography of each of the eyes, and classified each eye into the same
5 categories. The agreement between observers’ classification and
classification by KMI, as well as agreement between the individual
observers, was calculated using overall agreement and free-marginal
kappa statistic.
Results: The KMI software identified 26 eyes as normal, compared
to 16, 32, and 28 eyes for graders A, B, and C. 16 eyes were
classified as “KCN suspect” by KMI, compared to 14, 3, and 7 eyes
by graders A, B, and C respectively. 3 eyes were classified as “mild
KCN” by KMI, compared to 7, 5, and 6 eyes by graders A, B, and C,
respectively. 1 eye was classified as “moderate KCN” by KMI,
compared to 5, 4, and 3 eyes by graders A, B, and C, respectively. No
eyes were classified as “severe KCN” by KMI, compared to 4, 2, and
2 eyes by graders A, B, and C, respectively.
The three graders agreed with each other 55% of the time, with a
free-marginal kappa of 0.448 (moderate agreement). Grader A and
KMI agreed 30% overall, with a free-marginal kappa of 0.130 (slight
agreement). Grader B and KMI agreed 46% overall, with a freemarginal kappa of 0.321 (fair agreement). Grader C and the KMI
agreed 46% overall, with a free-marginal kappa of 0.321. KMI
agreed with all 3 graders 13% of the time and with at least one grader
63% of the time. The KMI did not agree with any grader 37% of the
time.
Conclusions: There is only slight to fair agreement between
observers and the KMI, compared to moderate inter-observer
agreement. The KMI alone is not well correlated with the subjective
interpretation of topography, and should be used in conjunction with
other modalities to evaluate a possible keratoconus suspect.
Commercial Relationships: Ryan A. Vasan, None; Ryan M. St
Clair, None; Syed A. Hussnain, None; Ana G. Alzaga Fernandez,
None; Christopher E. Starr, None
Program Number: 1628 Poster Board Number: D0263
Presentation Time: 8:30 AM - 10:15 AM
Investigation of corneal vibration during air puff deformation
using numerical approaches with clinical validation
Zhaolong Han1, Cynthia J. Roberts2. 1Department of Civil
Engineering, School of Naval Architecture, Ocean and Civil
Engineering, Shanghai Jiao Tong University, Shanghai, China;
2
Department of Ophthalmology and Department of Biomedical
Engineering,The Ohio State University, Columbus, OH.
Purpose: To investigate cornea dynamics under air pulse
deformation and relate vibration amplitude to corneal elasticity.
Methods: The phenomenon of in vivo cornea dynamic vibration
under air pulse deformation is predicted using a nonlinear dynamic
model based on a simplified kinematic differential equation
describing corneal motion, y(t), as a function of equivalent external
forces, corneal mass (m), damping constant (c) representing the
viscoelastic capacity of the cornea, and the coefficient of corneal
elasticity, (k). Corneal vibration is validated clinically with two
keratoconic subjects and two normal subjects greater than 50 years of
age with acquired measurements using the CorVis ST, a Scheimpflug
system capturing approximately 140 images of a single horizontal
corneal meridian during a 30ms air puff at greater than 4,000
frames/s, as well as the Ocular Response Analyzer (ORA) to provide
corneal compensated intraocular pressure (IOPcc). In addition a 3D
finite element (FE) cornea model is constructed in the environment of
ANSYS 11.0 to simulate the results produced.
Results: Modeling predicts greater vibration amplitude with lower
coefficient of elasticity, as seen in Figure (a), which is validated with
clinical measurements. Keratoconic subjects demonstrated greater
vibration amplitude than older normal subjects. An example of
vibration data from one keratoconic subject is shown in Figure (b)
which represents corneal surface motion of a single point through the
time course of the air puff. Also shown are two meshes from finite
element modeling with high and low elasticities in Figure (c), which
demonstrate greater depth of deformation with lower elastic modulus
at consistent IOP.
Conclusions: Both numerical methods and clinical measurements
show that softer corneas demonstrate larger vibration amplitude and
greater depth of deformation compared to stiffer corneas. Future
work will investigate the influence of IOP on corneal elasticity and
vibration amplitude, as well as increase the complexity of the
numerical modeling.
Commercial Relationships: Zhaolong Han, None; Cynthia J.
Roberts, Oculus Optikgerate GmbH (C), Ziemer Ophthalmic
Systems AG (C), Sooft Italia (R), Carl Zeiss Meditec (F)
Program Number: 1629 Poster Board Number: D0264
Presentation Time: 8:30 AM - 10:15 AM
Modeling corneal response to an air puff using deformation data
to derive Young’s modulus
Kimberly M. Metzler1, Cynthia J. Roberts1, 2, Steven M. Whitaker3,
Michael J. Lawrence3, Jennifer E. Malik1, Jeffrey P. Bons3.
1
Biomedical Engineering, Ohio State University, Columbus, OH;
2
Opthalmology, Ohio State University, Columbus, OH; 3Mechanical
and Aerospace Engineering, Ohio State University, Columbus, OH.
Purpose: To create and validate a computational model that
describes the deformation characteristics of human donor corneas
mounted in an artificial anterior chamber in response to an air puff.
Methods: A 2-dimensional COMSOL Multiphysics model of an air
jet impinging on a human donor cornea mounted in an artificial
anterior chamber was created. The physical air jet was generated by
the CorVis ST, a device used clinically to evaluate deformation
response in living corneas. This air jet was characterized with hot
wire anemometry to acquire spatial flow velocity data. The hot wire
was placed at the jet exit on the CorVis, and then moved outward
with micrometer control to distances of 3, 6, 9, 12, 15, 20, and 25 mm
along the centerline. The duration of the hot wire anemometry
recordings lasted 40 ms. Preliminary data of the temporal profile
shows that the peak velocity along the centerline during the air puff
as it exits the device (distance x = 0) is over 100 m/s. At distances
between 9 and 12 mm from nozzle of the CorVis ST, the peak
velocity reaches above 90 m/s. Accordingly, the model inlet velocity
representing the CorVis ST was set at 100 m/s. Corneal dimensions
were modeled by constructing an ellipse inside an 8mm sphere that
was sectioned to have a width of 12 mm. The cornea section was
mounted onto a rigid body within the model, representing the
Barron’s Artificial Anterior Chamber. Intraocular pressure was
manipulated to be 10, 20, 30, 40, and 50 mmHg. Deformation data
from a donor corneal-scleral rim mounted on an artificial anterior
chamber at these pressures was used to validate the model. The
model was run iteratively at each pressure to determine the Young’s
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
modulus required to produce experimentally determined
deformations.
Results: Maximum deformation amplitude for the model was
matched to experimental deformation data within 0.01% error. The
Young’s moduli were 1.569, 1.740, 1.899, 2.099, and 2.250 MPa for
pressures of 10, 20, 30, 40, and 50 mmHg, respectively.
Conclusions: This model supports the known relationship that as
IOP increases, the cornea will become stiffer. Future studies will
include developing a 3D model as well as modeling the whole globe.
Image shows the model deformation at 10 mmHg intraocular
pressure.
Image shows the model deformation at 50 mmHg intraocular
pressure.
Commercial Relationships: Kimberly M. Metzler, None; Cynthia
J. Roberts, Oculus Optikgerate GmbH (C), Ziemer Ophthalmic
Systems AG (C), Sooft Italia (R), Carl Zeiss Meditec (F); Steven M.
Whitaker, None; Michael J. Lawrence, None; Jennifer E. Malik,
None; Jeffrey P. Bons, None
Support: Ohio Lions Eye Research Foundation
Program Number: 1630 Poster Board Number: D0265
Presentation Time: 8:30 AM - 10:15 AM
OCT-Vibrography: A Novel Non-Contact Method to Estimate
Corneal Biomechanical Properties
Sabine Kling1, Ernest Chang2, Giuliano Scarcelli2, Nandor Bekesi1,
Seok H. Yun2, Susana Marcos1. 1Instituto de Optica, Consejo
Superior de Invest Cientificas, Madrid, Spain; 2Wellman Center,
Massachusetts General Hospital, Boston, MA.
Purpose: Corneal biomechanics are key for diagnosing corneal
pathologies and evaluating treatments that alter corneal geometry or
stiffness. Most methods to measure corneal biomechanics are
destructive, while in-vivo techniques (e.g. air-puff imaging) are
biased by corneal geometry and IOP. We developed a new technique
to determine corneal material parameters, while reducing current
prevalent restrictions.
Methods: Sound excitation (100-110 db) together with phase
sensitive OCT was used to measure the profile of the natural
frequencies (range 200-1000 Hz) in corneal tissue. The technique was
tested in-vitro on 6 freshly enucleated pig and bovine eyes, using
corneal flaps, corneal buttons and whole eyes. Different conditions
were tested (virgin, riboflavin-dextran instillation, cross-linking
CXL) to determine the effect of corneal rigidity and hydration.
Changes in corneal stiffness were determined by the shift of natural
frequencies and viscoelastic behavior by the phase lag between sound
wave and corneal oscillation. Finite element (FE) models were built
to simulate the experimental observations.
Results: We found an experimental shift in the first natural frequency
of 101.25±67.1 Hz between the anterior flaps of virgin and CXL
corneas; no significant shift was observed for posterior flaps (12.5±32.0 Hz). Corneal buttons and globes confirmed a frequency
shift after cross-linking. The phase lag was sensitive to the tested
frequency and to corneal fixation (δ(flap)=4.06, δ (button)=6.19 and
δ(globe)= 5.93 at 355Hz). FE-models predicted the first natural
frequency to be strongly correlated with corneal stiffness
(ΔE=+1.4MPa, Δf(globe)=+100Hz, Δf(button)=+250Hz,
Δf(flap)=+150Hz). Natural frequencies of higher modes were also
sensitive to IOP, corneal curvature, thickness and density.
Conclusions: OCT vibrography is a promising non-invasive
technique for the estimation of corneal biomechanical properties,
allowing the separation of corneal stiffness from other parameters.
Shift of the first resonance frequency with increased corneal rigidity.
Commercial Relationships: Sabine Kling, None; Ernest Chang,
None; Giuliano Scarcelli, massachusetts general hospital (P);
Nandor Bekesi, None; Seok H. Yun, Massachusetts General
Hospital (P); Susana Marcos, Essilor (F), PCT/ES2012/070185 (P)
Support: FIS2011-25637; ERC-2011 AdG-294099
Program Number: 1631 Poster Board Number: D0266
Presentation Time: 8:30 AM - 10:15 AM
Regional Variation of Biomechanical Properties of Intact Eye
Globes
Ahmed Elsheikh1, 2, Charles Whitford1, Akram Joda1, Ahmed Abass1,
Fangjun Bao4, Paolo Rama3. 1Engineering, University of Liverpool,
Liverpool, United Kingdom; 2National Institute for Health Research
Biomedical Research Centre at Moorfields Eye Hospital NHS
Foundation Trust and UCL Institute of Ophthalmology, London,
United Kingdom; 3Ophthalmology Department, San Raffaelle
Scientific Institute, Milan, Italy; 4Eye Hospital, Wenzhou Medical
College, Wenzhou, China.
Purpose: To determine the regional variation in biomechanical
properties across the whole surface of the cornea and sclera of human
and porcine eyes
Methods: Four human eyes and eight porcine eyes were tested using
a new inflation test method to determine the regional variation of
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
their stress-strain behavior. New techniques have been developed to
allow the testing of intact eye globes, prevent degradation and
swelling, remove internal components, and simulate the eye's support
by the surrounding soft tissue. The specimens were covered with a
fine-particle speckle pattern and the behavior monitored using
spatially oriented cameras. Finite element models that closely
represent eye topography were constructed (Fig 1) and the
deformation across eye surface used to determine regional variation
of corneal and scleral stiffness within an inverse modeling procedure.
Results: The results revealed consistent stiffness variation trends in
both human and porcine eyes. The stiffness (measured using tangent
modulus) was highest in the limbal region and reduced gradually
across scleral surface until the posterior pole region and the area
surrounding the optic nerve, where the lowest stiffness was recorded,
Fig 2 (P<0.05 in porcine eyes, no statistical analysis was possible in
human eyes due to the small number tested). Within the cornea, the
stiffness in the central 6mm diameter region was higher than the
surrounding peripheral region (by 32±12%, P<0.05).
Conclusions: Testing intact eye globes allowed reliable comparisons
between the behavior within the cornea and sclera. It also enabled
quantifying the behavior of the anterior sclera, which acted as the
clamped area in earlier tests on corneal buttons and scleral spheres.
The tests revealed gradual stiffness reductions from the limbus to the
posterior pole and from central to peripheral cornea. The results can
help improve the accuracy of predictive modeling of ocular
biomechanical performance and ocular response to surgical
procedures. Future ocular biomechanics testing is expected to
concentrate on whole eye globes due to the superiority and reliability
of the results obtained in spite of the much more demanding and time
consuming testing procedure compared to testing corneas and scleras
separately.
Fig 1 Numerical model used to determine material properties of a
porcine eye
Fig 2 Average regional variation of tangent modulus over the cornea
and sclera
Commercial Relationships: Ahmed Elsheikh, None; Charles
Whitford, None; Akram Joda, None; Ahmed Abass, None;
Fangjun Bao, None; Paolo Rama, None
Support: UK Engineering & Physical Sciences Research Council
grant EP/H052046/1
Program Number: 1632 Poster Board Number: D0267
Presentation Time: 8:30 AM - 10:15 AM
Effect of Different Hydration Media on Ex Vivo Corneal
Elasticity Measurements
Janice Dias, Noel M. Ziebarth. Biomedical Engineering, University
of Miami, Coral Gables, FL.
Purpose: To determine the effect of various hydration media (PBS,
BSS, and 15% Dextran) on ex vivo corneal elasticity.
Methods: Experiments were conducted on fifteen porcine eyes (5
eyes each for PBS, BSS, and 15% Dextran; <3 days postmortem).
The eyes were retrieved from an abattoir, placed in a bag filled with
saline, and shipped to the laboratory overnight. Upon arrival, the
epithelium was removed using a cotton swab, and the cornea was
excised with a generous scleral rim and placed in 20% Dextran
overnight to restore the cornea to physiological levels (average:
500µm). With the thickness restored, the cornea was mounted onto a
custom chamber and immersed in a hydration medium (PBS, BSS, or
15% Dextran) for elasticity testing. While maintained in each
medium, corneal elasticity measurements were performed for 2
hours; measurements were conducted at 5-minute intervals for the
first 30 minutes and then at 15-minute intervals for the remaining 90
minutes. A custom-built Atomic Force Microscope designed for the
mechanical testing of ophthalmic tissues was used to perform the
elasticity testing. Tips of traditional AFM cantilevers modified with
glass microbeads (diameter: 60-75µm) were used to ensure tissuelevel mechanical response. Young’s modulus was calculated using
the Hertz model for a spherical indenter. Corneal thickness
measurements were taken with a pachymeter before and after
elasticity testing.
Results: The hydration medium used affects the stability of corneal
thickness and elasticity measurements over time. After 2 hours, the
corneas in PBS and BSS had swelled by 81.6% and 75.7%,
respectively; the thickness of corneas in 15% Dextran decreased by
14%. On average, Young’s modulus of elasticity increased 168kPa,
103kPa, and 29kPa with time for corneas maintained in PBS, BSS,
and 15% Dextran, respectively. There appears to be a relationship
between the corneal swelling and the resultant elasticity; greater
edema resulted in higher values of the Young’s modulus (stiffer
cornea). As the cornea swells, the presence of the limbus is
hypothesized to prevent peripheral diffusion for water escape,
creating a stiffening effect.
Conclusions: The maintenance of corneal hydration is important for
stable and accurate characterization of corneal biomechanics over
time. 15% Dextran is suggested to be an effective hydration media in
maintaining both corneal thickness and elasticity over time.
Commercial Relationships: Janice Dias, None; Noel M. Ziebarth,
None
Support: NIH Ruth L. Kirschstein National Research Service
Awards for Individual Predoctoral Fellowships to Promote Diversity
in Health-Related Research (1F31EY021714); UNCF/MERCK
Graduate Science Research Dissertation Fellowship
Program Number: 1633 Poster Board Number: D0268
Presentation Time: 8:30 AM - 10:15 AM
Comparison of Biomechanical Effects of Small Incision Lenticule
Extraction (SMILE) and Laser in situ Keratomileusis (LASIK):
A Finite Element Analysis Study
Abhijit Sinha Roy1, William J. Dupps1, 3, Cynthia J. Roberts2.
1
Ophthalmology, Cleveland Clinic Cole Eye Institute, Cleveland,
OH; 2Department of Ophthalmology and Department of Biomedical
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Engineering, The Ohio State University, Columbus, OH;
3
Department of Biomedical Engineering, Cleveland Clinic Lerner
Research Institute, Cleveland, OH.
Purpose: LASIK creates a flap in the anterior corneal stroma,
followed by ablation of the exposed stroma with an excimer laser. A
newer procedure, small incision lenticule extraction (SMILE), does
not create a flap and uses a femtosecond laser to remove a lenticule
having a shape corresponding to the programmed refractive
correction. It is hypothesized that since a flap is avoided in SMILE,
the mechanical stresses in a post-SMILE cornea more closely
approximate those of an idealized cornea with unaltered material
properties than in a comparable LASIK correction.
Methods: Finite element models of myopic surgery using patientspecific corneal geometry and an anisotropic, collagen fiberdependent formulation were constructed for LASIK and SMILE.
Surgical parameters, the magnitude of myopic correction, flap
thickness in LASIK and depth of lenticule creation in SMILE were
varied. Further, since the corneal stroma is generally stiffer in the
anterior region, two sets of models with uniform and depth-dependent
material properties were constructed. A geometry-matched model
with unaltered material properties from the preoperative state but
with post-operative geometry (including thickness) was used for
comparing the magnitude and distribution of von Mises stresses in
SMILE and LASIK models.
Results: We observed the stress distribution in the post-SMILE
simulations to be similar to that of the geometry-matched model since
much of the stiffer anterior stroma is preserved. In contrast, LASIK
consistently reduced the stress in the flap and increased the stress in
the residual stromal bed compared to the postoperative geometrymatched model. Further, increase in the thickness of the flap or depth
of lenticule creation resulted in a greater amount of increase in the
stress in residual stromal bed of the LASIK model compared to the
SMILE model.
Conclusions: SMILE may present less biomechanical risk to the
residual bed of susceptible corneas than comparable corrections
involving LASIK flaps. Corrections at greater stromal depths in
SMILE may be possible with less relative risk of ectasia than a
comparable LASIK correction.
Commercial Relationships: Abhijit Sinha Roy, Carl Zeiss Meditec
(F), Cleveland Clinic Innovations (P), Topcon Inc. (F); William J.
Dupps, Zeimer (C), Topcon (F), Avedro (F), Carl Zeiss Meditec (F),
Cleveland Clinic Innovations (P); Cynthia J. Roberts, Oculus
Optikgerate GmbH (C), Ziemer Ophthalmic Systems AG (C), Sooft
Italia (R), Carl Zeiss Meditec (F)
Program Number: 1634 Poster Board Number: D0269
Presentation Time: 8:30 AM - 10:15 AM
Biomechanical properties of the cornea and graft after
Descemet's stripping endothelial keratoplasty
Saima Qureshi1, Ninita H. Brown1, Naazli Shaikh2, 1.
1
Ophthalmology, Howard University Hospital, Washington, DC;
2
Ophthalmology, Veterans Affairs Medical Center, Orlando, FL.
Purpose: To measure the biomechanical properties of the cornea and
graft after Descemet’s stripping endothelial keratoplasty (DSEK)
using the ocular response analyzer (ORA) and to evaluate the
correlation between corneal compensated intraocular pressure
(IOPcc) and Goldmann applanated intraocular pressure (IOP GAT).
Methods: A cross-sectional study of 7 eyes of 7 patients with Fuch’s
endothelial dystrophy who underwent DSEK with at least 6 months
of follow up. Corneal hysteresis (CH), cornea resistance factor
(CRF), cornea compensated intraocular pressure (IOPcc) were
measured using the ORA. At the same visit, IOP GAT was obtained.
A comparison of post-DSEK ORA parameters to those of nondiseased corneas and the correlation of IOPcc to IOP GAT were
investigated.
Results: Mean patient age was 71.85±11.48 years. The mean follow
up period was 25.71±12.72 months. Mean CH and CRF were
9.90±1.40 mmHg and 9.61±1.16mmHg, respectively. The difference
of CH and CRF of post-DSEK corneas were not found to be
significant when compared to CH and CRF values of non-diseased
corneas1 (p value= 0.5745, unpaired t test). Cornea-compensated
intraocular pressure was not found to be significantly different when
compared with IOP GAT (p value= 0.3731, paired t test).
Conclusions: The biomechanical properties of the cornea and graft
after DSEK are found to be within the range of values of nondiseased corneas. The difference between IOPcc and IOP GAT after
DSEK was not found to be significant.
1
Values for non-diseased corneas: CH=10.25±1.6, n=100;
CRF=10.25±1.85, n=100. Source: Montard R, Kopito R, Touzeau O,
Allouch C, Letaief I, Borderie V, Laroche L. Ocular response
analyzer: feasibility study and correlation with normal eyes. J Fr
Ophtalmol. 2007 Dec.30(10):978-84.
Commercial Relationships: Saima Qureshi, None; Ninita H.
Brown, None; Naazli Shaikh, None
Program Number: 1635 Poster Board Number: D0270
Presentation Time: 8:30 AM - 10:15 AM
Influence of Pregnancy on the Corneal Biomechanical Properties
Roo Min Jun, Ga Eun Cho, Kyu-Ryong Choi. Dept of
Ophthalmology, School of Medicine, Ewha Womans University,
Seoul, Republic of Korea.
Purpose: To determine the difference of corneal biomechanical
Properties and central corneal thickness (CCT) between pregnant and
non-pregnant women.
Methods: This is a prospective case control study. Twenty six nonpregnant women and twenty six pregnant women with no ocular
pathology or systemic diseases were recruited. Corneal hysteresis
(CH) and the corneal resistance factor (CRF) were measured with the
Ocular Response Analyzer and the CCT was measured with an
ultrasonic pachymeter.
Results: Twenty six eyes of 26 non-pregnant women (30.3 ± 3.8,
range 23-38) and 26 eyes of 26 pregnant women (31.5 ± 2.9, range
24-38) were included. CCT was not different between two groups
(Non-pregnant vs. pregnant: 553.8 ± 26.3vs. 547.5 ± 29.4, P > 0.05).
The CH was slightly lower in pregnant group but it was not
statistically significant (11.1 ± 1.7 vs. 10.7 ± 1.6, P > 0.05). On the
other hand, the CRF was statistically lower in the pregnant group
(10.2 ± 1.7 vs. 9.2 ± 1.4, P = 0.03).
Conclusions: The CCT and CH were not statistically different
between pregnant and non-pregnant women. The CRF was
significantly lower in the pregnant group. Change of hormone may
influence the corneal biomechanical properties in pregnant women.
Commercial Relationships: Roo Min Jun, None; Ga Eun Cho,
None; Kyu-Ryong Choi, None
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Program Number: 1636 Poster Board Number: D0271
Presentation Time: 8:30 AM - 10:15 AM
Elastic modulus of keratocytes and myofibroblasts differ
regardless of substratum stiffness
Vijay K. Raghuanthan1, Peter C. Strom1, Sara M. Thomasy1, Paul
Russell1, Christopher J. Murphy1, 2. 1Dept of Surgical and
Radiological Sciences, University of California Davis, Davis, CA;
2
Dept of Ophthalmology and Vision Science, University of California
Davis, Davis, CA.
Purpose: Injury to the corneal stroma initiates a cascade of events
resulting in the differentiation of quiescent keratocytes (K) to
activated fibroblasts (F) and myofibroblasts (M). This transformation
is critical for appropriate corneal wound healing and its dysregulation
can result in scar and haze formation. Transforming growth factor-β1
(TGFβ1) is critical in differentiating keratocytes and fibroblasts to
myofibroblasts. We investigated the influence of substratum stiffness
on TGFβ1 induced K-F-M transition and measured the elastic
modulus of the different cell types.
Methods: Primary rabbit corneal keratocytes were isolated and
cultured in serum free media. Cells were then cultured on
polyacrylamide substrates of differing stiffness (5 or 25 kPa ) or on
tissue culture plastic (TCP; >1GPa) in serum free media in the
absence or presence (10 ng/ml) of TGFβ1. Expression of α-smooth
muscle actin (αSMA) was determined by qPCR and
immunocytochemistry (ICC) to confirm myofibroblast
transformation. Keratocytes and myofibroblasts were visually
identified by differences in cell morphology. Keratocytes had a
characteristically stellate morphology while the myofibroblasts were
flatter and more spread out. Elastic moduli of keratocytes and
myofibroblasts on the various substrates were measured by atomic
force microscopy (AFM).
Results: Overall, interaction with softer substrates inhibited genesis
of the myofibroblast phenotype. In the presence of TGFβ1,<10%, 4050% and 90% of cells displayed a myofibroblast phenotype on 5 kPa
gels, 25 kPa gels and TCP, respectively. The elastic modulus of
myofibroblasts (3.5 ± 1 kPa) was significantly greater in comparison
to keratocytes (1.3 ± 0.3 kPa) regardless of the underlying substratum
stiffness.
Conclusions: Substratum stiffness profoundly influences the number
of cells transitioning from keratocytes to myofibroblasts in the
presence of TGFβ1. Softer gels, approximating values for stiffness
found in vivo, stabilize the corneal stromal cell phenotype.
Myofibroblasts are markedly stiffer than keratocytes irrespective of
the underlying substratum stiffness. This is possibly due to increased
actin cytoskeletal architecture in a myofibroblast. In aggregate,
results suggest that substratum stiffness modulates the molecular
mechanism by which cell differentiation occurs but not the intrinsic
cytoskeletal properties that define the myofibroblast phenotype.
Commercial Relationships: Vijay K. Raghuanthan, None; Peter
C. Strom, None; Sara M. Thomasy, None; Paul Russell, None;
Christopher J. Murphy, Ocular Services On Demand (I), Ocular
Services On Demand (C), Platypus Technologies LLC (I), Imbed
LLC (I), EyeKor LLC (I), Allergan (C), Genentech (C), Sarcode (C),
Covance (C)
Support: EY019970, P30EY12576
Program Number: 1637 Poster Board Number: D0272
Presentation Time: 8:30 AM - 10:15 AM
Corneal biomechanical properties among healthy Chinese,
Indian and Caucasian: A pilot study
Yin Zhi Wong, Andrew K. Lam. School of Optometry, The Hong
Kong Polytechnic University, Hong Kong SAR, Hong Kong.
Purpose: Chinese have high prevalence of myopia than other ethnic
groups. Lower corneal hysteresis was reported in Chinese with longer
axial length. This study compared corneal biomechanical properties
in healthy Chinese, Indian and Caucasian.
Methods: One hundred and forty-eight normal subjects (age range
from 9-31 years old) were enrolled in this study (Chinese= 93,
Indian= 27, Caucasian= 28). Corneal hysteresis (CH) and corneal
resistance factor (CRF) were obtained using the Ocular Response
Analyzer. Axial length (AL) was measured using optical biometry
while central corneal thickness (CCT) was obtained using noncontact specular microscopy. Due to the limitation of the settings, AL
was measured in 97 subjects while CCT was only measured in 51
subjects. Both eyes of each subject were used for analysis. The
associations between CH and CRF with age, gender, AL, corneal
curvature and CCT were evaluated.
Results: All ethnic groups had similar corneal curvature and CCT
(p> 0.05). Chinese subjects had significantly longer AL
(24.05±1.11mm) compared with Indian subjects (23.59±0.94mm) and
Caucasian subjects (23.47±0.66mm). Indian subjects had
significantly higher CH (11.81±1.33mmHg) than Chinese subjects
(11.25±1.37mmHg) and Caucasian subjects (11.34±1.76mmHg).
Indian subjects also had significantly higher CRF
(11.86±1.87mmHg) than Chinese (11.15±1.48mmHg) and Caucasian
subjects (10.71±1.87mmHg). Age had negative association with CH
and CRF (p< 0.0001). CH and CRF were significantly higher in
female than in male (p< 0.0001). There was significantly negative
association between AL and CH (p= 0.001), but no significant
association was found between AL and CRF (p> 0.05). CH and CRF
were significantly increased with CCT (p< 0.0001). After adjusting
for age, gender, CCT, AL and corneal curvature, there were still
significant ethnical difference in CH (p< 0.05) and CRF (p< 0.01).
Conclusions: Different corneal biomechanical properties were found
in Chinese, Indian and Caucasian. A longitudinal study is needed to
determine the role of CH in the axial elongation.
Commercial Relationships: Yin Zhi Wong, None; Andrew K.
Lam, None
Program Number: 1638 Poster Board Number: D0273
Presentation Time: 8:30 AM - 10:15 AM
Novel Corneal Biomechanical Parameters in Myopes vs
Emmetropes
Rachel Lee1, Robert Chang1, Ian Y. Wong2, Jimmy S. Lai2, Jacky W.
Lee2, Kuldev Singh1. 1Stanford University School of Medicine,
Stanford, CA; 2Hong Kong University School of Medicine, Hong
Kong, Hong Kong.
Purpose: While population-based studies have shown that myopia is
a risk factor for glaucoma, the underlying basis of this correlation is
unknown. We aim to identify novel corneal biomechanical
parameters that differentiate myopic from normal eyes using a novel
technology.
Methods: This prospective, cross-sectional study of 80 subjects with
varying degrees of myopia and 62 emmetropies was conducted at
Queen Mary Hospital in Hong Kong. The Corvis ST device (Oculus,
Wetzlar, Germany), which couples a pneumotonometer with a high
speed Scheimpflug camera, was used to measure IOP, CCT, and new
biomechanical parameters including corneal deformation amplitude,
inward and outward applanation velocity, and highest concavity
peak-to-peak distance. The right eye of all subjects and controls
underwent Corvis analysis. Myopes were subsequently categorized as
being high (vision correction at least -9.0 D), moderate (-6.0 D to -9.0
D), or mild (-3.0 D to -6.0 D) myopes. Exclusion criteria included
known corneal disease, intraocular surgery within three months
preceding the study, or prior history of refractive surgery.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Results: Significant findings included differences in outward
applanation velocity (myope: -0.38±0.08 m/s; control: -0.33±0.05
m/s; P=3.2E-5) and peak-to-peak distance (myope: 2.43±0.24 mm;
control: 2.32±0.20 mm; P=3.6E-3). Interestingly, high (n=19), but
not moderate (n=25) or low (n=36), myopes exhibited statistically
significant differences in these two corneal biomechanical parameters
as compared to controls.
There was moderate correlation between IOP, CCT and visual acuity
with outward applanation velocity (R=0.43; 0.34; 0.50, respectively),
and with peak-to-peak distance (R=0.40; 0.15; 0.32, respectively). No
correlation was found between age and outward applanation velocity
(R=0.060) or peak-to-peak distance (R=0.039). Average IOP in
myopes and emmetropes were 15.2±2.2 mm Hg and 14.9±2.1 mm
Hg, respectively (P=4.0 E-1); average CCT in myopes and
emmetropes was 554±34 μm and 554±39 μm, respectively (P=9.5 E1).
Conclusions: Myopes had a greater mean outward applanation
velocity and greater peak-to-peak distance at highest concavity than
emmetropic controls. In particular, high myopes demonstrated a
corneal biomechanical profile distinct from that of emmetropes.
Increased corneal deformability in high myopes, also found in
glaucoma patients from another study, may indicate a relationship
between high myopia and glaucoma.
Commercial Relationships: Rachel Lee, None; Robert Chang,
None; Ian Y. Wong, bayer (C); Jimmy S. Lai, Pfizer (R), Allergan
(R), Alcon (R); Jacky W. Lee, Allergan (F), Alcon (F), AMO (F);
Kuldev Singh, Alcon (C), Allergan (C), Santen (C), Bausch and
Lomb (C), Transcend (C), Ivantis (C), Sucampo (C), iScience (C)
Support: Stanford School of Medicine- Traveling Scholars Grant
Program Number: 1639 Poster Board Number: D0274
Presentation Time: 8:30 AM - 10:15 AM
The relationship between anterior segment biometry and corneal
biomechanics in myopia
Hetal Buckhurst1, Bernard Gilmartin2, Robert Cubbidge2, Manbir
Nagra2, Nicola S. Logan2. 1School of Health Professions, Plymouth
University, Plymouth, United Kingdom; 2School of Life and Health
Sciences, Aston University, Birmingham, United Kingdom.
Purpose: The Reichert Ocular Response analyser (ORA) provides
data on corneal biomechanics via measures of corneal hysteresis
(CH), corneal resistance factor (CRF) and additional waveform
parameters (AWPs). Utilising the Scheimplug principle the Pentacam
(Oculus) provides data on the biometry of the cornea and anterior
segment (AS). Given the premise that there is correspondence
between the biomechanical and biometric properties of the AS we
examine the nature of the correspondence and whether it is altered in
myopia.
Methods: Data were collected from adults 18-40 yrs of British-White
and British-South-Asian descent [MSE (D) 20 non-myopes (≥-0.50)
0.64±1.38; 22 myopes (<-0.50) -6.24±4.28]. The Pentacam was used
to measure minimum corneal thickness (CT), central CT (CCT),
peripheral CT, anterior chamber volume (ACV) and depth (ACD),
corneal volume (CV), anterior and posterior best fit sphere (BFS) and
front (FS) and back surface (BS) keratometry flat (Kf) and steep (Ks).
The ORA was used to record CH, CRF and 37 AWPs. Refractive
error (Rx) and axial length were measured using autorefraction and
the Zeiss IOLMaster. Correlations between Pentacam and ORA
parameters were tested using Pearson’s correlation coefficient.
Multiple linear regression was used to determine the level of
association between Pentacam and ORA data. One-way repeated
measures ANOVAs tested differences in Pentacam and ORA
parameters for Rx, ethnicity and age.
Results: No significant effect of Rx, age and ethnicity was found for
the Pentacam and ORA metrics. CV was associated with CH
(p=0.006), explaining 17.7% of its variability, whereas CRF was
related to CCT (p<0.001) explaining 29.1% of its variability. Several
AWPs were found to be associated with 7 Pentacam parameters
(Anterior BFS, ACD, ACV, FSKs, FSKf, CCT and inferior CT, all
p<0.05) e.g. respectively anterior BFS explained 19.1%, 25.8%,
23.1% and 13.6% of the variability of P1area, P2area, P2area1 and
Dive2 and ACD explained 15.9%, 14.2%, 10.4% and 11.4% of the
variability of Aspect1, Aspect21, Uslope11 and Dslope21.
Conclusions: The study demonstrates significant correspondence
between ORA and Pentacam parameters that is limited to specific
combinations of AWPs and biometric parameters. The
correspondence was shown to be independent of Rx. Of interest
would be investigation of the relationship between anterior biometric
and biomechanical properties in myopia in young developing eyes.
Commercial Relationships: Hetal Buckhurst, None; Bernard
Gilmartin, None; Robert Cubbidge, None; Manbir Nagra, None;
Nicola S. Logan, None
Program Number: 1640 Poster Board Number: D0275
Presentation Time: 8:30 AM - 10:15 AM
Comparison of biomechanical properties of cornea in diabetic
and nondiabetic primary open angle glaucoma patients
Faruk Ozturk1, Serkan Akkaya2, Ertugrul Can3. 1Ulucanlar Eye
Hospital, Ankara, Turkey; 2Yildirim Beyazit Hospital, Ankara,
Turkey; 3Ondokuz Mayis University, Samsun, Turkey.
Purpose: To investigate corneal biomechanical properties in primary
open angle
glaucoma patients with and without diabetes mellitus (DM), and to
evaluate the effect
of metabolic control and duration of DM on biomechanical properties
of cornea.
Methods: A total of 101 eyes of 101 primary open-angle glaucoma
(POAG)
patients (60 with diabetes and 41 without diabetes) were recruited in
this prospective
study. After a complete ophthalmic examination, corneal hysteresis
(CH) and corneal
resistance factor (CRF) of the patients were measured by the ocular
response
analyzer (ORA). Central corneal thickness (CCT) measurements
were measured with
ultrasound pachymeter. Glucolysed haemoglobine (HbA1c)
measurements of the
patients were made on the same day as ophthalmic assessments were
performed.
Results: CRF measurements in diabetic group were found to be
statistically
significant compared to control group (p=0,01). There were no
statistically significant
differences between two groups regarding CCT and CH
measurements (p=0,37 for
CCT and p=0,11 for CH). In diabetic group, the correlation analyses
showed that CH
and CRF were not significantly correlated with HbA1c levels and
duration of diabetes.
Conclusions: Significant increase in CRF measurements in diabetic
POAG
patients and absence of any statistically significant difference in CCT
measurements
suggest that there would be some corneal biomechanical changes
related to
increased corneal stiffness which is depicted by the increase of CRF
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
values parallel
to failure of corneal hydration. It was found that duration and
metabolic control of
diabetes did not change the biomechanical properties of cornea.
Commercial Relationships: Faruk Ozturk, None; Serkan Akkaya,
None; Ertugrul Can, None
Program Number: 1641 Poster Board Number: D0276
Presentation Time: 8:30 AM - 10:15 AM
Collagen Macrostructure and Corneal Shape: Lessons from
Different Species
Moritz Winkler1, Yilu Xie2, Tiffany Yuen1, Golroxan Shoa1, Robert
Hueter3, Kathy K. Svoboda4, Christopher J. Murphy5, Donald J.
Brown2, James V. Jester2, 1. 1Biomedical Engineering, University of
California, Irvine, Irvine, CA; 2Gavin Herbert Eye Institute,
University of California, Irvine, Irvine, CA; 3Mote Marine
Laboratory, Sarasota, FL; 4Biomedical Sciences, Texas A&M Health
Science Center, Dallas, TX; 5Department of Surgical and
Radiological Sciences, University of California, Davis, Davis, CA.
Purpose: The cornea plays a critical role both as a protective window
to the eye and as a refractive lens. In aquatic vertebrates, it provides
little refractive power, while in terrestrial vertebrates corneal shape
needs to be precisely controlled to project a focused image on the
retina. Little is known about the changes in the structural
organization of corneal collagen during evolution. The purpose of
this study was to begin to characterize the macrostructural
organization of corneal collagen in divergent species in order to
uncover basic mechanisms controlling corneal shape.
Methods: Eyes from various species (fish, shark, birds, mammals)
were fixed under pressure using paraformaldehyde to control postmortem swelling. Serial full-width (limbus to limbus) cross-sections
(250μm thick) were cut using a vibratome. Sections were imaged
using nonlinear optical high resolution macroscopy (NLO-HRMac)
of second harmonic generated (SHG) signals. 3-D images were
rendered using Amira software, and collagen fiber structures were
quantified with custom-written ImageJ macros.
Results: In aquatic vertebrates stromal collagen macrostructure
consisted of simplified layers (stacks) of fibers that extended from
limbus to limbus as continuous sheets of collagen, much like
‘plywood’ in construction. Adjacent sheets were rotated 87°, forming
orthogonal plies, with successive layers showing a continual rotation
of over 360°. In birds collagen sheets were organized into distinct
fibers with a uniform branching and fusing pattern similar to that of
chicken wire and presented a honeycomb appearance. Fibers in the
same plane appeared to extend from limbus to limbus, and successive
layers showed a 270° rotation through 2/3 stromal depth, very similar
yet distinct from fish and shark. By contrast, collagen fiber
organization in mammals was irregular with varying degrees of
branching depending on the species (human > dog > cat > rabbit).
Mammals also lacked orthogonal rotational, nor were fibers
constrained to extend from limbus to limbus within the same plane.
Conclusions: We have previously shown in the human cornea that
collagen fiber branching and interconnectivity is associated with
tissue rigidity. In this study, fiber branching was detected in corneas
from terrestrial vertebrates suggesting that branching and increased
corneal rigidity may play a role in the evolutionary adaptation of the
cornea from a protective window to a refractive lens.
Commercial Relationships: Moritz Winkler, None; Yilu Xie,
None; Tiffany Yuen, None; Golroxan Shoa, None; Robert Hueter,
None; Kathy K. Svoboda, None; Christopher J. Murphy, Ocular
Services On Demand (I), Ocular Services On Demand (C), Platypus
Technologies LLC (I), Imbed LLC (I), EyeKor LLC (I), Allergan
(C), Genentech (C), Sarcode (C), Covance (C); Donald J. Brown,
None; James V. Jester, None
Support: NIH Grant EY018665, Research to Prevent Blindness, Inc.,
Discovery Eye Foundation, and the Skirball Program in Molecular
Ophthalmology
Program Number: 1642 Poster Board Number: D0277
Presentation Time: 8:30 AM - 10:15 AM
Inter- and Intra-Lamellar Slippage of Collagen Fibrils as a
Potential Mechanism of Keratoconus Progression
Michael Koster1, Craig Boote2, Keith M. Meek2, Priscilla G. Fowler3,
Christopher A. Girkin3, Guenther Meschke1, Rafael Grytz3. 1Institue
for Structural Mechanics, Ruhr University Bochum, Bochum,
Germany; 2School of Optometry and Vision Sciences, Cardiff
University, Cardiff, United Kingdom; 3Ophthalmology, University of
Alabama at Birmingham, Birmingham, AL.
Purpose: To assess if inter- and intra-lamellar slippage of collagen
fibrils may lead to progressive cone formation in keratoconus.
Methods: A generic finite element model of the human eye was
generated that incorporates the micro-architecture of collagen fibrils
in the corneo-scleral shell. Inter- and intra-lamellar slippage was
simulated through residual strains of collagen fibrils using a
microstructure-based constitutive formulation. Progressive inter- and
intra-lamellar slippage was imposed to an eccentric, 4-mm-diameter
area of the cornea while the model was subjected to normal IOP (15
mmHg). Topographic results were compared to clinical observation
of a keratoconus patient with an eccentric cone.
Results: Increasing inter- and intra-lamellar slippage led to
progressive cone formation of the cornea. The results were in good
agreement with topographic observation of keratoconus patients with
eccentric cone.
Conclusions: The numerical results support the assumption that
inter- and intra-lamellar slippage of collagen fibrils may be the
underlying mechanism that leads to progressive cone formation in
keratoconus.
Numerical simulation of keratoconus progression showing the
development of an eccentric cone due to inter- and intra-lamellar
slippage of corneal collagen fibrils.
Commercial Relationships: Michael Koster, None; Craig Boote,
None; Keith M. Meek, None; Priscilla G. Fowler, None;
Christopher A. Girkin, SOLX (F), Heidelberg Engineering (F);
Guenther Meschke, None; Rafael Grytz, None
Support: EyeSight Foundation of Alabama; Research to Prevent
Blindness Physician-Scientist Award
Program Number: 1643 Poster Board Number: D0278
Presentation Time: 8:30 AM - 10:15 AM
Clear Corneal Incision: Sealability of the Manual Versus LensAR
laser generated Full Thickness Incision
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
E. Valas Teuma1, Liz Dumanoir1, Aissatou Barry1, Gary Gray1, G.
Brock Magruder2, Steve Bott1. 1R&D, LensAR Inc, Orlando, FL;
2
LaserVue, Orlando, FL.
Purpose: The human cornea has the ability to self-seal after
penetrating incision wounds. The purpose of this work is to compare
the sealability of clear corneal incisions (CCIs) created by manual
means to those generated by the LensAR Laser System - fs 3D (LLSfs 3D).
Methods: A total of 22 human donor globes were used for this
experiment. The laser CCIs, were performed using the LLS-fs 3D.
Standard three-plane CCIs were used for both the manual and laser
CCIs. Post incisions, a high resolution Fourier domain optical
coherence tomography (OCT) system was used to measure the
incision geometry. After the OCT measurements, the sealability of
the CCIs was measured using a digital pressure gauge. The Seidel test
was utilized to detect a leak of the aqueous onto the cornea. A
computer-controlled stepper motor is used to push the plunger into
the eye. The IOP reported by the pressure gauge is recorded at the
first sign of leakage. If the IOP reaches 500 mmHg without leakage,
the test is considered complete.
Results: One tailed t-tests of the sealability of the manual versus
laser CCIs, indicate that the mean IOP at which leakage occurred was
the statistically the same for the two cases (p = 0.061). However,
variability of IOP at leakage for the laser was significantly less than
for the manual (±28 mmHg vs. ±94 mmHg). Regarding the measured
incision geometries, the geometric parameters measured for the laser
incisions were always at least as good in accuracy and precision as
the manual and in several cases was superior. For example, a two
tailed t-test of the mid-tunnel depth location versus target (50%)
indicates that the laser incision was statistically significantly closer to
the target value than the manual incisions (mean 46% vs. 33%,
p=0.26).
Conclusions: The IOP at leakage of laser versus manual CCIs were
statistically the same. However, an F-test of the variance of the IOP
elevation at which leakage occurred showed that the manual method
produced incisions with statistically higher variance than those of the
laser CCIs. OCT measurements showed that the mean value of the
location of the mid-plane of the three-plane CCI was placed
significantly closer to the target placement for the laser versus the
manual CCIs. Overall, the testing showed that the manual and laser
methods are statistically equivalent in sealability but that the laser
method produces more consistent wound geometry.
Commercial Relationships: E. Valas Teuma, Lensar Inc (E); Liz
Dumanoir, None; Aissatou Barry, None; Gary Gray, LensAR (E);
G. Brock Magruder, LensAR (C); Steve Bott, None
Program Number: 1644 Poster Board Number: D0279
Presentation Time: 8:30 AM - 10:15 AM
Effect of Intraocular Pressure on Speed-of-Sound and Thickness
in Ex Vivo Cornea in Intact Globes
Harriet Lloyd1, Mara Berganovsky1, Ronald H. Silverman1, 2, Raksha
Urs1. 1Ophthalmology, Columbia University Medical Center, New
York, NY; 2Frederic L. Lizzi Center for Biomedical Engineering,
Riverside Research, New York, NY.
Purpose: Ultrasound is regarded as the ‘gold standard’ for the
determination of corneal thickness, but standard methods for
measuring this parameter have required determination on excised
corneas, which is non-physiologic. We developed a means for
measurement of speed-of-sound in intact globes. Our objective was to
determine the effect of intraocular pressure (IOP) on corneal speed of
sound and thickness.
Methods: We acquired high-resolution ultrasound data on four ex
vivo pig corneas. The eyeball was mounted in a custom apparatus,
which included a sharpened, thin, flat metal surface that was inserted
across the anterior chamber. An 18 gauge needle attached to a saline
drip bag was inserted through the optic nerve. IOP was raised and
lowered by raising or lowering the saline bag and monitored with a
digital pressure gauge attached to the IV line. The eye and apparatus
was submerged in 20% dextran solution. Data of the cornea and of
the metal surface on either side of the globe were obtained using a
single-element focused transducer with a center frequency of 35
MHz. Pulse/echo ultrasound data was acquired at a 400 MHz sample
rate. We measured the shift in the metal flat echo compared to its
expected, interpolated position, and from this and the speed-of-sound
in aqueous and the dextran solution solved for the speed-of-sound
and thickness of the cornea.
Results: The speed-of-sound averaged over all cases showed
relatively little dependence on intraocular pressure. At 0 mm the
speed-of-sound averaged Hg 1556 m/s, at 40 mm Hg it was 1560 m/s
and upon return to 0 mmHg it was 1548 m/s. These differences were
not statistically significant. The thickness of the cornea at 0mmHg
measured 1.01 mm, at 40 mmHg it was 0.91 mm, and it recovered to
0.96mm as the pressure was gradually reduced to 0mmHg.
Conclusions: IOP had little effect on the speed-of-sound in the
cornea, indicating that ultrasound pachymeters would not be required
to be recalibrated to compensate for IOP. However, as pressure
increased, the cornea stretched and became thinner, recovering
gradually and partially as IOP was decreased.
Commercial Relationships: Harriet Lloyd, None; Mara
Berganovsky, None; Ronald H. Silverman, None; Raksha Urs,
None
Support: NIH Grant EY021529; AIUM EER; Research to Prevent
Blindness
Program Number: 1645 Poster Board Number: D0280
Presentation Time: 8:30 AM - 10:15 AM
Change of corneal curvature under the open eye condition and
the slightly closed eye condition
Yuko Shibata1, Hiroshi Uozato1, 2, Masakazu Hirota1, Takushi
Kawamorita1, 2. 1Ophthalmology & Visual Sciences, Kitasato Univ
Graduate School, Sagamihara, Japan; 2Orthoptics and Visual
Sciences, Kitasato University, Sagamihara, Japan.
Purpose: To assess the influence of squinted eyes, we measured
corneal curvature under the condition of widely opened eye and the
condition of slightly closed eye.
Methods: 39 eyes of 20 healthy subjects (age 21 - 42 yrs)
participated in this study. Corneal curvatures of central zone (1 - 4
mm as a diameter) were measured and front photo images of the
eyelids and eye were obtained at the same time by an anterior
segment imaging analyzer (Galilei, Ziemer Ophthalmic Systems,
Port, Switzerland) under both a condition with the eye opened widely
and a condition with the eye closed slightly in which subjects opened
their eyes narrowly with consciousness.
Results: The group mean size of slightly closed eye was 67.0 ± 12.2
% of that of widely opened eye condition (Range: 45.9 - 89.5%). The
mean SimK value average of widely opened eye was 43.59 ± 1.39 D
and that of slightly closed eye was 43.44 ± 1.57 D, the mean flat
SimK value of widely opened eye was 42.82 ± 1.32 D and that of
slightly closed eyes was 42.35 ± 1.84 D and the mean steep SimK
value of widely opened eye was 44.36 ± 1.53 D and that of slightly
closed eye was 44.52 ± 1.69 D. Significant differences were found in
flat SimK value and steep SimK value between two conditions
(Wilcoxon signed-ranks test, p < 0.05) but there was no significant
difference in SimK value average. The mean anterior instantaneous
curvature Mean K of widely opened eye was 43.03 ±1.30 D and that
of slightly closed eye was 42.98 ± 1.59 D, the mean anterior
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
instantaneous curvature Flat K of opened eye was 42.23 ± 1.28 D and
that of slightly closed eye was 41.83 ± 1.63 D and the mean anterior
instantaneous curvature Steep K of opened eye was 43.82 ± 1.42 D
and that of slightly closed eye was 44.13 ± 1.91 D. There was no
significant difference in anterior instantaneous curvature values of
two conditions (Wilcoxon signed-ranks test, p > 0.05).
Conclusions: Data obtained in this study shows that in the slightly
closed eye condition, cornea curvature of central zone had a little
change to become more astigmatic. It was suggested that even in the
slightly closed eye condition, corneal curvature was affected by the
eyelids and tear film’s distribution.
Commercial Relationships: Yuko Shibata, None; Hiroshi Uozato,
None; Masakazu Hirota, None; Takushi Kawamorita, None
238 Corneal Endothelium
Monday, May 06, 2013 8:30 AM-10:15 AM
Exhibit Hall Poster Session
Program #/Board # Range: 1646-1698/D0281-D0333
Organizing Section: Cornea
Contributing Section(s): Visual Neuroscience
Program Number: 1646 Poster Board Number: D0281
Presentation Time: 8:30 AM - 10:15 AM
Cyclosporin A inhibits cell death of Corneal Endothelial Cells by
protecting michondrial membrane potential
Toshinari Funaki, Kaori Ohtomo, Masahiro Yamaguchi, Akira
Matsuda, Akira Murakami. Ophthalmology, Juntendo Univ School of
Medicine, Bunkyo-ku, Japan.
Purpose: The number of corneal endothelial cells (CECs) decreases
with age because corneal endothelium does not divide in vivo. After
corneal transplantation, the endothelial cell density on donor corneas
rapidly declines by acute intraoperative trauma. Therefore it is
important to protect CECs during surgery for the survival of the
allograft.
The corneal endothelium are mitochondria-rich cells, indicate that
CECs are metabolically active. It is also reported that cell death is
associated with reduced functioning of mitochondria.
In glia cell and cardiomyocyte, Cyclosporin A (CsA) suppress cell
death by inhibition of the permeability transition pore (PTP) which
lead to stabilization of mitochondrial membrane potential as well as
immunosuppressive effect.
The purpose of this study was to investigate whether CsA inhibit cell
death in human cultured corneal endothelial cells (hCECs) and mouse
corneas.
Methods: HCECs were stimulated with H2O2(0~1000nM) for 1
hour. And whole mount cornea, endothelial cell side up from
C57BL/6 were stored DMEM serum free at 4C and then subjected to
various concentrations of H2O2 (0-1000M) for 1 hour with/without
various concentration CsA. Apoptosis were detected using Caspase
3/7 (Invitrogen). Mitochondria in corneal endothelial cells were
labeled by the addition of MitoTracker Red CMXRos to the corneal
cups (125 nM final concentration; Invitrogen) and maintaining for 30
minutes in a 37C in a 5% CO2 incubater.
Results: 1) Oxidative stress induced cell death was observed in a
concentration-dependent manner in hCECs.
2) CsA decreased cell death by about 10%, and suppressed the
reduction of mitochondrial membrane potential at 100nM in hCECs.
Only the highest concentration of CsA (1000nM) showed
cytotoxicity in hCECs.
3) CsA suppressed the reduction of mitochondrial membrane
potential at 100nM in whole mount mouse corneas.
Conclusions: We conclude that CsA suppress the reduction of
mitochondrial membrane potential from oxidative stress, and inhibit
cell death in corneal endothelial cells.
Commercial Relationships: Toshinari Funaki, None; Kaori
Ohtomo, None; Masahiro Yamaguchi, None; Akira Matsuda,
None; Akira Murakami, SEED(Japan) JP4855782 (P),
SEED(Japan) JP5132958 (P)
Support: Grant-in-Aid for Young Scientists B 23792005
Program Number: 1647 Poster Board Number: D0282
Presentation Time: 8:30 AM - 10:15 AM
Success Isolation of Human Corneal Endothelial Cells for
Clinical Use
Jin San Choi1, 3, Matthew Giegengack1, 2, Eun Young Kim4, Min
Jeong Kim4, Ralph D'Agostino5, Gilson Khang4, Shay Soker1. 1Wake
Forest Institute for Regenerative Medicine, Wake Forest University
Health Sciences, Winston-Salem, NC; 2Department of
Ophthalmology, Wake Forest School of Medicine, Winston-Salem,
NC; 3Oular Systems, INC, Winston-Salem, NC; 4BIN Fusion
Technology, Chonbuk National University, Jeonju, Republic of
Korea; 5Public Health Sciences-Department of Biostatistics, Wake
Forest School of Medicine, Winston-Salem, NC.
Purpose: Corneal transplantation is a common transplant procedure
to improve visual acuity by replacing the opaque or distorted host
tissue by clear healthy donor tissue. However, its clinical utility is
limited due to a lack of donor supply with high quality corneas.
Bioengineered neo-corneas, created using an expandable population
of human donor-derived corneal endothelial cells (HCECs), could
address this shortage. Thus, the objective of this study was to
evaluate HCEC sourcing with various isolation methods, including
enzymatic digestion, culture medium components, and adhesive
proteins.
Methods: HCECs were obtained from corneas with various aged
donors after endothelial keratoplasty. Under a dissection microscope,
the Descemet’s membrane, including the attached corneal
endothelium was stripped from the stroma and the cells were isolated
and expanded by explant culture and the use of enzymatic digestion
with enzyme such as collagenase II, dispase, or trypsin. In order to
improve the initial cell attachment, tissue culture plates were coated
with collagen IV, fibronectin, or fibronectin-collagen combination
coating mix (FNC) before cell plating.
Results: In results, HCECs were successfully isolated from 32%
(86/269) of donor corneas. Donor age and isolation method
influenced the characteristics of the resulting in vitro HCEC culture.
Under all conditions tested, FNC-coated plates showed higher quality
cultures than other coatings tested.
Conclusions: The results suggest that donor and isolation method
characteristics are the two factors that most directly affect the quality
of the resulting HCEC in vitro culture. These factors should guide the
clinical expansion of HCECs for the generation of bioengineered
neo-corneas.
Commercial Relationships: Jin San Choi, Ocular Systems, INC
(E); Matthew Giegengack, None; Eun Young Kim, None; Min
Jeong Kim, None; Ralph D'Agostino, None; Gilson Khang, None;
Shay Soker, None
Support: Ocular Systems,INC
Program Number: 1648 Poster Board Number: D0283
Presentation Time: 8:30 AM - 10:15 AM
A cell therapy approach to address corneal endothelial
dysfunction
Karen Alvarez-Delfin1, Noelia J. Kunzevitzky1, 2, Alejandra D.
Weisman1, Richard M. Merkhofer1, Jeffrey L. Goldberg1, 3. 1Bascom
Palmer Eye Institute, Interdisciplinary Stem Cell Institute, University
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
of Miami, Miami, FL; 2Emmetrope Ophthalmics, Coral Gables, FL;
3
Shiley Eye Center, University of California San Diego, San Diego,
CA.
Purpose: The endothelium is the innermost layer of the cornea and is
responsible for maintaining transparency. Human corneal endothelial
cell (HCEC) density gradually decreases with age, and their proper
function is affected by genetic diseases, such as Fuchs’ dystrophy, or
surgical trauma, ultimately leading to vision impairment. Such
endothelial dysfunction is an indication for corneal transplantation,
although there is a global shortage of tissue donors. To overcome the
current poor donor availability, here we isolate, expand, and
characterize HCECs in vitro as a first step toward cell therapy.
Methods: HCECs were isolated from cadaveric donor corneas and
cultured following the method described by Zhu and Joyce (2004). In
order to improve cell morphology and proliferation rate, different
media additives and culture dish coatings were assayed. Cellular
identity was assessed by morphology, RT-PCR, and
immunohistochemistry. Viability was measured by Trypan blue
staining after cells were stored at room temperature, 4oC or
cryopreserved in liquid N2.
Results: Cultured HCECs often demonstrated characteristic
hexagonal-like morphology for 2-3 passages, reaching passage 8 in
one culture. Time to reach confluence was highly influenced by
donor age, with youngest donors exhibiting higher proliferative rates.
Donor disease also affected culture quality. Overnight storage of
HCECs at 4oC dramatically reduced viability, while cryopreservation
maintained higher viability. Immunohistochemistry showed that
cultured HCECs were positive for zonula occludens-1(ZO-1) and
Na+K+ ATPase, common markers for HCEC. Fibroblastic-like
morphologic conversion positive for the marker α-smooth muscle
actin (α-SMA) was a common feature at late passages, appearing
earlier in poor quality cultures.
Conclusions: The in vitro expansion of HCEC from donor corneas
yields a number of suitable cells that could help treat patients
otherwise in need of cornea transplantation. Ongoing experiments are
addressing the ability of these cells to integrate into a host cornea and
restore endothelial function.
Commercial Relationships: Karen Alvarez-Delfin, None; Noelia
J. Kunzevitzky, None; Alejandra D. Weisman, None; Richard M.
Merkhofer, None; Jeffrey L. Goldberg, None
Program Number: 1649 Poster Board Number: D0284
Presentation Time: 8:30 AM - 10:15 AM
Comparison of early corneal peripherial endothelial cell loss
following femtosecond laser - assisted cataract surgery and
conventional phacoemulsification
Gábor L. Sándor, Ágnes I. Takács, Kinga Kranitz, Eva Juhasz, Illes
Kovacs, Zoltan Z. Nagy. Department of Ophthalmology, Semmelweis
University, Budapest, Hungary.
Purpose: To compare early corneal peripherial endothelial cell loss
after femtosecond laser - assisted cataract surgery and conventional
phacoemulsification, using non-contact specular microscopy.
Methods: In each group, 15 eyes (15 patients) underwent cataract
surgery using either femtosecond - laser assisted (Alcon LenSx laser
system, femtolaser group) or conventional phacoemulsification
(phaco group). All operations were performed by the same surgeon
with the same phaco machine (Infiniti, Alcon). Biometry was
performed using Lenstar LS 900 (Haag-Streit AG) non-contact
optical low-coherence reflectometer. Scheimpflug camera (Pentacam
HR, Oculus Optikgerate GmbH) was used to measure nucleus density
(Pentacam Nucleus Staging; PNS). Endothelial cell density was
measured by Specular Microscope NSP-9900 (Konan Nonco Robo)
preoperatively and 1 month after surgery by a semiautomated masked
manner. The measurements were performed at four identical points
on the peripherial area of the cornea. The mean value was used as the
average cell density of periphery.
For comparison of independent variables Mann Whitney U-test, and
for comparison of dependent variables Wilcoxon-test was used.
Results: There was no statistically significant difference in
demographics, PNS, axial lenght, phaco energy and effective phaco
time between the two study groups. We found no significant
difference in preoperative cell density in the two groups (femtolaser
group: 2898±168/mm2, phaco group: 2799±219/mm2, p=0,1776). 1
month after the surgery the cell density was slightly lower in the
phaco group (2696±233/mm2) than in the femtolaser group
(2833±140/mm2), but the difference was not statistically significant
(p=0,0887). The peripherial endothelial cell loss was significant in
both the phaco group (4%, p=0,004) and the femtolaser group (2%,
p=0,005).
Conclusions: Results of this study suggest that femtosecond laserassisted cataract surgery does not significantly differ in early corneal
peripherial endothelial cell loss from manual phacoemulsification.
There is a smaller tendency of decrease in peripherial endothelial cell
count after femtosecond laser-assisted cataract surgery compared
with conventional phacoemulsification, however the difference is not
statistically significant.
Commercial Relationships: Gábor L. Sándor, None; Ágnes I.
Takács, None; Kinga Kranitz, None; Eva Juhasz, None; Illes
Kovacs, None; Zoltan Z. Nagy, Alcon-LenSx Inc. (C)
Program Number: 1650 Poster Board Number: D0285
Presentation Time: 8:30 AM - 10:15 AM
Effect of glaucoma tube-shunt position on corneal thickness and
endothelial cell density
Euna Koo1, Jing Hou1, Ying Han1, Jeremy D. Keenan1, 2, Robert L.
Stamper1, Bennie H. Jeng1, 2. 1Ophthalmology, University of
California San Francisco, San Francisco, CA; 2Proctor Foundation,
University of California San Francisco, San Francisco, CA.
Purpose: To investigate post-operative changes in corneal thickness
and endothelial cell density (ECD) after tube-shunt implantation
Methods: Twenty-eight eyes of 24 glaucoma patients with
superotemporal tube-shunts, but without prior corneal transplantation
or previous tube-shunt placement, were evaluated at University of
California, San Francisco. Superotemporal (ST), central, and
inferonasal (IN) ECD and corneal thickness were measured by noncontact specular microscopy (CellChek XL™ Specular Microscope,
Irvine, CA) and ultrasound pachymetry (DGH™ Pachette 2, Exton,
PA), respectively. The angle of the tube relative to the cornea, tube
length, and distance between the tip of the tube and cornea, were
measured with anterior segment optical coherence tomography
(Visante™ OCT Anterior Segment Imaging, Dublin, CA). Linear
regression analyses were used to assess effects of tube position on
ECD and corneal thickness.
Results: The average time lag from tube implantation to time of
cornea and anterior segment measurements was 45±40.4 months
(range 1-156 months). Mean ST, central, and IN ECD (cells/mm2)
were 1649.7±800.0, 1940.1±862.0, and 1963.6±856.7, respectively.
Mean ST, central, and IN corneal thicknesses (μ) were 640.6±103.6,
536.3±86.8, and 650.8±83.6, respectively. ST ECD was lower than
central ECD (P=0.0001) and IN ECD (P=0.003, paired t-test). There
was no difference in ECD between central and IN cornea (P=0.97).
ST and IN cornea were thicker than central cornea (P<0.0001, paired
t-test), but ST and IN thickness were comparable. For each additional
degree in angle of tube away from the cornea, there were 21.6 (-12.155.2, 95% CI) more ST endothelial cells, though this was not
significant (P=0.20). There was no relationship between ST ECD and
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
tube length (P=0.30) or tube-corneal distance (P=0.80).
Conclusions: The presence of a tube shunt drainage device in the
anterior chamber causes significant local endothelial cell loss
compared to central cornea or cornea furthest away from the tube.
Endothelial cells near the tube may be better preserved with tubes
angled further away from the cornea, but a larger study is needed to
answer this question.
Commercial Relationships: Euna Koo, None; Jing Hou, None;
Ying Han, None; Jeremy D. Keenan, None; Robert L. Stamper,
Transcend (C), Genentech (C); Bennie H. Jeng, None
Program Number: 1651 Poster Board Number: D0286
Presentation Time: 8:30 AM - 10:15 AM
Human Cytomegalovirus-mediated inflammatory responses of
corneal endothelial cells
Michiko Kandori1, Dai Miyazaki1, Keiko Yakura1, Yumiko Noguchi1,
Yukimi Yamamoto1, Yoshitsugu Inoue1, Tatsuo Suzutani2.
1
Ophthalmology, Tottori University, Yonago, Japan; 2Microbiology,
Fukushima Medical University, Fukushima, Japan.
Purpose: To determine whether human cytomegalovirus (CMV) can
infect corneal endothelial cells, and characterize inflammatory
responses of corneal endothelial cells after infection.
Methods: Clinical isolate of CMV was purified by density gradient
and adsorbed to immortalized human corneal endothelial cells
(HCEn) at a multiplicity of infection 0, 0.01 or 0.1. The infectivity of
CMV into HCEn cells was analyzed by the induction of immediateearly, early, and late genes of CMV, and inflammatory cytokine
induction by real-time RT-PCR. Inflammatory responses of HCEn
cells were characterized by protein array analysis of the supernatants.
Results: Representative viral genes, including IE-1, UL-83, and
glycoprotein B, were sequentially induced after CMV infection.
CMV infection characteristically stimulated induction of IL-6 and
IDO1 (indoleamine 2,3-dioxygenase 1) mRNA by HCEn cells.
Protein array analysis showed significant induction of inflammatory
mediators, immune regulators, or neurotrophic factors , including
BDNF, BLC, GDNF, PIGF, MCP-1, EGF, TGF-β1, PDGF-BB, IL16, Leptin, MCSF, MCP-4, IP-10, IL-6, IL-13, IL-5, IL-15, GCP-2,
FGF-9, IFN-γ, RANTES, Ckβ8-1, Eotaxin, MIP-3α, VEGF.
Conclusions: CMV infects corneal endothelial cells, and provokes
anti-viral responses, together with induction of immune regulatory
mediators or neurotrophic factors. These results may give us clues to
elucidate the pathogenesis of CMV endotheliitis.
Commercial Relationships: Michiko Kandori, None; Dai
Miyazaki, None; Keiko Yakura, None; Yumiko Noguchi, None;
Yukimi Yamamoto, None; Yoshitsugu Inoue, None; Tatsuo
Suzutani, None
Program Number: 1652 Poster Board Number: D0287
Presentation Time: 8:30 AM - 10:15 AM
Engineering of Human Corneal Endothelial Grafts
Yingting Zhu, Bo Han, Szu-Yu Chen, Scheffer C. Tseng. Research,
Ocular Surface Ctr and Tissue Tech, Miami, FL.
Purpose: We have reported that knockdown by p120 siRNA
selectively activates p120-catenin/Kaiso signaling and successfully
expands contact-inhibited HCEC monolayers to an average size of
2.1 ± 0.3 mm in diameter from 1/8 of the descemet membrane
stripped from the corneoscleral rim. Herein, we would like to show
how incorporation of other means might further expand the
monolayer size.
Methods: HCEC monolayers derived from 1/8 stripped descemet
membrane and cultured to 7 days in SHEM were treated with
different concentrations of p120 siRNA weekly with or without 100
nM Kaiso siRNA or 5 µg/ml nocodazole, a microtubule disrupting
agent, for up to 5 weeks. Before termination, cells were further
treated with 10 µM BrdU for 4 h. Immunostaining was performed to
monitor cytolocalization of F-actin, α-catenin, β-catenin, p120
catenin, N-cadherin, ZO-1, Na+/K+-ATPase, and BrdU labeling.
Results: Consistent with our recent report, p120 siRNA knockdown
uniquely promoted proliferation of contact-inhibited HCEC by
nuclear translocation of p120 catenin to relieve repression by nuclear
Kaiso. Such proliferation was further enhanced to achieve an average
monolayer size of 4.1 ± 0.3 mm in diameter (n=3, p<0.05) when the
concentration of p120 siRNA was increased from 40 nM to 100 nM
and the duration was prolonged from 2 to 5 weeks. Additional
knockdown by 100 nM Kaiso siRNA, but not treatment of 5 µg/ml
norcodazole, further synergistically promoted the monolayer size to
5.0 ± 0.4 mm in diameter (n=3, p<0.05). Proliferating HCEC still
maintained a hexagonal shape, an comparable in vivo density, and a
normal expression pattern of F-actin, α-catenin, β-catenin, Ncadherin, ZO-1, and Na+/K+-ATPase. Furthermore, downregulation
of p120 catenin at the cell junction was completely reversed after
withdrawal of p120 siRNA for 1 week.
Conclusions: This new strategy of perturbing contact inhibition by
selective activation of p120 catenin-Kaiso signaling without
disrupting adherent junction can be exploited to engineer surgical
grafts containing normal human corneal endothelial cells to meet a
global corneal shortage and for endothelial keratoplasties.
Commercial Relationships: Yingting Zhu, Tissue Tech Inc (F),
Tissue Tech Inc (E), Tissue Tech Inc (P); Bo Han, Tissue Tech (F);
Szu-Yu Chen, Tissue Tech Inc (F), Tissue Tech Inc (E), Tissue Tech
Inc (P); Scheffer C. Tseng, NIH, NEI (F), TissueTech, Inc. (F),
TissueTech, Inc. (E), TissueTech, Inc. (P)
Support: NIH EY 022502
Program Number: 1653 Poster Board Number: D0288
Presentation Time: 8:30 AM - 10:15 AM
Human Corneal Endothelial Cells Cytotoxicity Study Using a
Custom Chamber which Controls Temperature and Oxygen
Levels
Radha Pertaub, Marc D. Friedman, David Muller. R&D, Avedro
Inc, Waltham, MA.
Purpose: To investigate the feasibility of using a custom-designed
chamber that controls the temperature and oxygen levels to mimic an
in vivo environment when measuring the cytotoxic level of
Riboflavin (RF) and UVA on human corneal endothelial cells.
Methods: A custom chamber is designed to hold two 96-well cell
culture plates in which cells can be irradiated from below using two
UVA sources (KXL™, Avedro, Waltham, MA) under the plates. Gas
composition is controlled by adjusting nitrogen and oxygen gas flow
rates into the chamber. Oxygen level is maintained at 10% (±20%)
using a calibrated oxygen meter (GAXT-X-DL-2, Honeywell,
Calgary, Canada). Bubbling the inlet gas mixture through a pre-inlet
in-line water chamber helps maintain a high humidity (>75%) in the
chamber. A thin heating sheet is adhered to an aluminum plate that
serves as lid and covers the plates while maintaining the cells at a
temperature of 37°C (± 20%). This lid is lined with a black flocked
material that serves the dual purpose of absorbing any UVA out of
the wells as well as acting as a black body radiator for a
homogeneous heat source. Black walled cell culture plates are seeded
with cells in every other column and row to minimize UVA crosscontamination. Human corneal endothelium cells are obtained from
the Leibniz Institute DSMZ-German Collection of Microorganisms
and Cell Cultures, in Braunschweig, Germany. Treatment cells are
dosed with 0.01%, 0.02% or 0.04% RF in Phosphate Buffered Saline
(PBS). They are then irradiated with either 3 mW/cm2 or 30 mW/cm2
for varying lengths of time for an increasing energy dosage.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Results: The optical density of the cells is measured via an MTT
assay and compared to controls (cells in PBS alone) to obtain the cell
viability after treatment with UVA-RF combination. Dose response
curves are plotted against exposure time for low and high irradiances
and the time required to get 50% cell viability (=EC50) is estimated
using a non-linear regression model and compared for high and low
irradiance treatments. Results show cell viability decreasing as a
function of increasing energy dose applied.
Conclusions: This custom chamber allows for the comparison of
cytotoxicity levels of UVA-RF combination on human corneal
endothelial cells while controlling the gaseous composition and
temperature of the chamber close to in vivo conditions.
Commercial Relationships: Radha Pertaub, Avedro Inc (E); Marc
D. Friedman, Avedro Inc (E); David Muller, Avedro Inc (E)
Program Number: 1654 Poster Board Number: D0289
Presentation Time: 8:30 AM - 10:15 AM
A Nonsynthetic, Biological Carrier for Cultivated Human
Corneal Endothelial Cells (HCECs) for potential therapeutic
purposes
Jesintha Navaratnam, Eli Gulliksen, Kristine Ustgaard-Andersen,
Jon K. Slettedal, Liv Drolsum, Bjorn Nicolaissen, Aboulghassem
Shahdadfar. Center for Eye Research, Oslo University Hospital,
Oslo, Norway.
Purpose: The aim of our ongoing study is to establish a carrier for
HCECs for therapeutic purposes. In the present study we investigate
the feasibility of using nonsynthetic, biological carrier for cultivated
HCECs.
Methods: Descemet’s membrane with the attached endothelial cells
was carefully dissected from human corneas in small strips. One part
harvested as non-cultured cells and the other cultivated in corneal
endothelial cell growth medium for 6 weeks at 37 degree Celsius
with 5% CO2 in a humidified atmosphere and the medium was
changed every 2-3 days. The cultivated HCECs were seeded on
acellular, nonsynthetic carrier and cultivated for further 3 weeks in
corneal endothelial cell expansion medium that was changed every 23 days. Cultivated HCECs on nonsynthetic carrier and non-cultured
HCEC were comparatively analyzed by qRT-PCR, electron
microscopy (EM) and immunohistochemistry.
Results: In our study the cultivated HCECs seeded on nonsynthetic
carrier formed a stable monolayer. Our results show that the
cultivated HCECs seeded on nonsynthetic carrier and the noncultured HCECs are functional and express stem cell markers when
analyzed by qRT-PCR. The expression levels of markers associated
with neural crest, stem cells and corneal endothelial function (SNAI1,
SNAI2, SOX9, NES, ZO-1, CX43, VADC2 and VADC3) are higher
in cultivated HCECs seeded on nonsynthetic carrier compared to
non-cultured HCECs. The structure of cultivated HCECs seeded on
nonsynthetic carrier on transmission EM is very similar compared to
ex vivo HCECs.
Conclusions: The preliminary results show that nonsynthetic carrier
used in this study can potentially be used in therapeutic purposes in
the future.
Commercial Relationships: Jesintha Navaratnam, None; Eli
Gulliksen, None; Kristine Ustgaard-Andersen, None; Jon K.
Slettedal, None; Liv Drolsum, None; Bjorn Nicolaissen, None;
Aboulghassem Shahdadfar, None
Program Number: 1655 Poster Board Number: D0290
Presentation Time: 8:30 AM - 10:15 AM
Differences in corneal endothelial abnormalities in the central
and intermediate zones in Fuchs’ corneal dystrophy
Hisataka Fujimoto, Takeshi Soma, Yoshinori Oie, Shizuka Koh,
Motokazu Tsujikawa, Naoyuki Maeda, Kohji Nishida. Osaka
University, Suita, Osaka, Japan.
Purpose: Fuchs’ corneal dystrophy is a progressive corneal
endothelial dystrophy that causes irreversible endothelial change and
bullous keratopathy. To investigate the regional differences in the
abnormality, we examined the corneal endothelium at multiple sites,
including the periphery.
Methods: 17 eyes of 9 patients (6 women and 3 men; 30-80 years
old) with Fuchs’ corneal dystrophy were studied at Osaka University
Hospital. We used a newly developed non-contact specular
microscope (NIDEK CEM-530) to examine the intermediate zone 27
degrees in the periphery from the corneal center in addition to the
center and the peripheral 5 degrees from the center. We measured the
size of the degenerative area with guttata and the cellular density in
the residual intact area.
Results: The abnormal areas in the central zone (center and 5 degrees
from the center) represented 65.4 ± 33.7% of the total area, and those
in the 27 degrees of the peripheral zone represented 27.1 ± 34.2% of
the total area. The results differed significantly (P < 0.001). The
abnormal areas in the 27 degrees of the peripheral zone of 16 of 17
eyes were significantly (P< 0.05) larger than those in the center. In
the 6 points 27 degrees of the peripheral zones, the abnormal areas
inferiorly were significantly (P < 0.001) larger than superiorly, by the
usage of z score analysis
Conclusions: In Fuchs’ corneal dystrophy, the corneal endothelial
cells degenerate more rapidly in the central zone than the
intermediate zone. This finding might offer new information to
facilitate an understanding of the disease mechanisms.
Commercial Relationships: Hisataka Fujimoto, None; Takeshi
Soma, HOYA corporation (R), Santen Pharmaceutical Co., Ltd (F),
Otsuka Pharmaceutical Co., Ltd (F); Yoshinori Oie, Santen (F),
HOYA (F); Shizuka Koh, Santen, Inc. (R), Johnson & Johnson (R),
Topcon (R), Otsuka Pharmaceutical Co. (R); Motokazu Tsujikawa,
Shionogi & Co. (C), Daiichi Sankyo Co. (F), Daiichi Sankyo Co. (R),
Santen Co. (R), AMO Co. (R); Naoyuki Maeda, Topcon (F), Santen
(R), Otsuka (R), Oculus (R), HOYA (R); Kohji Nishida, Alcon (C),
Alcon (F), HOYA (F), Senju (F), Pfizer (F), Santen (F), Osaka
University (P)
Program Number: 1656 Poster Board Number: D0291
Presentation Time: 8:30 AM - 10:15 AM
Development and Characterization of Decellularized Human
Corneal Stroma as a Scaffold for Tissue Engineering
Radhika Tandon1, Sujata Mohanty2, Himi Singh1, Deepika Gupta3,
Seema Sen4, Seema Kashyap4, Amit K. Dinda5, Manjeet Jassal3,
Ashwini K. Agrawal3. 1Department of Ophthalmology, Dr. Rajendra
Prasad Centre for Ophthalmic Sciences, All India Institute of Medical
Sciences, New Delhi, India; 2Stem Cell Facility, All India Institute of
Medical Sciences, New Delhi, India; 3SMITA Research Labs,
Department of Textile, Indian Institute of Technology, New Delhi,
India; 4Department of Ocular Pathology,Dr. Rajendra Prasad Centre
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
for Ophthalmic Sciences, All India Institute of Medical Sciences,
New Delhi, India; 5Department of Pathology, All India Institute of
Medical Sciences, New Delhi, India.
Purpose: To evaluate the potential use of decellularized human
corneas as a scaffold for cultivating human corneal endothelial cells.
Methods: Human corneal tissues (N=20) not suitable for
transplantation were used. Corneal endothelial cells were isolated by
explant culture method. For preparation of the scaffold, corneal
epithelium and endothelium were removed mechanically and
remaining corneal stroma was decellularized using enzymatic
method.The resulting acellular matrices were then subjected to
Haematoxylin-Eosin (H&E) staining to visualize cellular remnants;
quantitative analysis to determine the DNA content; Scanning
Electron Microscopy (SEM) for collagen fibril morphology; Alcian
blue staining to analyse extracellular matrix (ECM);
Immunohistochemistry for structural proteins collagen type I, II, IV,
fibronectin; and cytotoxicity assay of decellularized cornea using 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. Tensile
strength of corneal tissues &Light transmission ratios was estimated
using uniaxial load testing equipment &UV-visible
spectrophotometer. Native cornea served as control for all the
experiments. 10,000 human corneal endothelial cells were cultured
on decellularized scaffold for 14 days and then analyzed using SEM,
histology and Immunocytochemistry for ZO-1, and Na+ /K+ATPase.
Results: H&E staining demonstrated efficient elimination of cellular
components. Alcian blue confirmed good preservation of the
extracellular matrix and major structural proteins collagen type I, II
IV & fibronectin were retained. The amount of DNA in
decellularized cornea was 32+ 7.27ng/mg whereas in native cornea it
was 133+8.3ng/mg(p<0.05).The tensile strain at break was 38.8% &
42.4%;Young’s Modulus was 0.10 MPa& 0.14MPa and light
transmittance was 24.5% &22.7% in decelluarized and native corneas
respectively. In vitro cytotoxicity assays excluded the presence of
soluble toxins. Corneal endothelial cells could be efficiently cultured
and expanded on the acellular matrix. The SEM & Alizarin red
staining of cultured cells over decelluarized stroma showed surface
covered with a uniform monolayer of cells and they also expressed
functional markers Na+ /K+-ATPase & ZO-1.
Conclusions: Decellularized corneal stroma retains biomechanical
and functional properties to support endothelial cell proliferation and
expansion.
Commercial Relationships: Radhika Tandon, None; Sujata
Mohanty, None; Himi Singh, None; Deepika Gupta, None; Seema
Sen, None; Seema Kashyap, None; Amit K. Dinda, None; Manjeet
Jassal, None; Ashwini K. Agrawal, None
Support: Indian Council of Medical Reserach (IR382/2012)
Program Number: 1657 Poster Board Number: D0292
Presentation Time: 8:30 AM - 10:15 AM
Stemness Characteristics of Cultured Human Corneal
Endothelial Cells in Various Media
Young Joo Shin1, Eui Sang Chung2, Tae-Young Chung2, Joon-Young
Hyon3, 4, Won Ryang Wee3. 1Ophthalmology, Hallym University
College of Medicine, Seoul, Republic of Korea; 2Department of
Ophthalmology, Samsung Medical Center, Sungkyunkwan
University School of Medicine, Seoul, Republic of Korea;
3
Department of Ophthalmology, Seoul National University College
of Medicine, Seoul, Republic of Korea; 4Department of
Ophthalmology, Seoul National University Bundang Hospital,
Seongnam, Republic of Korea.
Purpose: To investigate the stemness characteristics of cultured
human corneal endothelial cells (HCEC) in various media.
Methods: HCECs were cultured in media A, B, BE, and E. The
morphology of cells was evaluated by inverted microscopy.
Immunofluorescence staining of zo-1, nestin, GFAP and COL8A2
was performed. Cell proliferation rate was evaluated with cell
counting kit -8 (CCK-8) assay.
Results: A few cultured cells were stained with nestin and almost
cells expressed COL8A2. The cell cultured in media A expressed
GFAP more. Cell shape became fibroblast-like in media A and
became mosaic pattern in media B and BE. Fibroblast-like cell shape
in media A was recovered to be mosaic pattern in media BE. Cell
proliferation rate was higher in media A and BE.
Conclusions: Cultured HCEC showed the stem cell markers
including nestin and GFAP. The expression of GFAP was different
according to media.
Commercial Relationships: Young Joo Shin, None; Eui Sang
Chung, None; Tae-Young Chung, None; Joon-Young Hyon, None;
Won Ryang Wee, None
Support: This study was supported by the Korea Science and
Engineering Foundation (KOSEF) grant (2012R1A1A2040118)
funded by the Korea government (MEST).
Program Number: 1658 Poster Board Number: D0293
Presentation Time: 8:30 AM - 10:15 AM
CD147 Knockdown Decreases Corneal Lactate Transport and
Endothelial Cell Viability
Shimin Li, Tracy T. Nguyen, Joseph A. Bonanno. School of
Optometry, Indiana University, Bloomington, IN.
Purpose: The glycolytic cornea produces large quantities of lactate.
Simultaneous export of catabolic lactate is crucial for the
maintenance of corneal health, hydration and transparency. The
corneal endothelium plays a pivotal role in lactate efflux through
transmembrane monocarboxylate transporters (MCTs). Previously,
we found that CD147, a multifunctional glycoprotein, is required to
sustain the expression of basolateral MCT1 and MCT4. Reports show
that CD147 is also involved in regulating cell apoptosis (Chen H,
Hum Reprod. 27(6):1568-76, 2012). In this study, we asked if CD147
plays a role in lactate transport and corneal endothelial cell viability.
Methods: Fresh rabbit corneas were cultured ex vivo and transfected
with 100 nM of CD147 siRNA or scrambled-sequence siRNA using
HiperFect transfection reagent. 72 hours post-transfection, the
endothelial monolayer was peeled and mounted in a double-sided
perfusion chamber. Lactate induced cell acidification (LIA) was
measured in response to 30 mM lactate in bicarbonate-free Ringer. In
situ cell death elicited by CD147 knockdown was assessed using
TUNEL assay.
Results: Steady state endothelial pHi was significantly lower in
CD147 knockdown (KD) corneas than control corneas (6.92 vs. 7.09,
p<0.02, n=9). When the apical surface was perfused with lactate, the
rate and amount of LIA were not significantly different in the KD
corneas compared to the control corneas (p<0.06 and p<0.40,
respectively). However, when lactate was perfused on the basolateral
surface, the rate and amount of LIA in the KD corneas was 2.9 and
2.1 fold less, respectively, in the KD corneas than the control corneas
(p<0.01 and p<0.03), consistent with the basolateral location of
CD147, MCT1 and MCT4. TUNEL positive cell counts were 1.5% +
0.002 in control corneas and 19.2% + 0.008 in KD corneas
respectively (p<0.01, n=6).
Conclusions: CD147 plays a crucial role in the transport of lactate
across the corneal endothelium by regulating the expression and
function of MCT1 and MCT4. CD147 is also involved in corneal
endothelial cell viability.
Commercial Relationships: Shimin Li, None; Tracy T. Nguyen,
None; Joseph A. Bonanno, None
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Support: EY08834
Program Number: 1659 Poster Board Number: D0294
Presentation Time: 8:30 AM - 10:15 AM
Disappearance and reappearance of cilia of corneal endothelium
preserved in corneal preservation media
Hidetoshi Tanioka, Katsuhiko Shinomiya, Satoshi Kawasaki, Shigeru
Kinoshita. Ophthalmology, Kyoto Prefectural Univ of Med, Kyoto,
Japan.
Purpose: It is known that the primary cilia of cells are present in
corneal endothelium. However, the primary cilia of donor corneal
endothelium disappear when preserved in corneal preservation media.
This study investigated the histological change of the cilia of corneal
endothelium preserved in corneal preservation media.
Methods: This study involved corneas obtained from Japanese white
rabbits. The corneas were preserved in Optisol-GS (Bausch and
Lomb, Rochester, NY) corneal preservation media at 4°C for 0, 1 and
7 days. The cornea preserved for 7 days was incubated at 37°C in
culture media (CnT-20; CELLnTEC Advanced Cell Systems AG,
Bern, Switzerland) for 2 days. Corneal endothelia of these corneas
were assessed by immunohistochemistry with Anti-Acetylated alpha
Tubulin antibody and scanning electron microscopy (SEM).
Results: Immediately after isolation, long process was observed by
SEM on the corneal endothelium, and it showed positive for primary
cilia by Anti-Acetylated alpha Tubulin antibody. No primary cilia
were observed on the corneal endothelium preserved for 1 or 7 days.
After 7-days preservation, primary cilia were observed on the
endothelium that underwent the 2-day incubation at 37°C.
Conclusions: The findings of this study using rabbit corneas suggest
that the primary cilia of the corneal endothelium of human donor
corneas for transplantation also disappear during preserved in
preservation media and reappear after transplantation, and show that
the existence of primary cilia is correlated with the viability of
corneal endothelium.
Commercial Relationships: Hidetoshi Tanioka, None; Katsuhiko
Shinomiya, None; Satoshi Kawasaki, None; Shigeru Kinoshita,
Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P), Otsuka
Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)
Support: Grant-in-Aid for Scientific Research (KAKENHI)
Program Number: 1660 Poster Board Number: D0295
Presentation Time: 8:30 AM - 10:15 AM
Endothelial loss and pachymetric change in patients undergoing
penetrating keratoplasty with 3 years of follow
Elisa D. Alegria, Regina Velasco, Oscar Baca, Alejandro Babayan.
Cornea, Hosp Nuestra Senora De La Luz IAP, Mexico, Mexico.
Purpose: Identify endothelial loss and pachymetric changes in
patients operated penetrating keratoplasty at follow up 3 years
Methods: Pre surgical endothelial count was obtained by the reports
of the LionsEye Bank Institute in Tampa, Florida.
A non contact specular microscopy with a microscope Topcon SP2000P in the operated was made in the operated eye. Specular
microscopy was taken only with the image captured by auto focus.E
Endothelial cel density was calculated by identify at minimum of 30
cels . Specular microscope automatically calculates and displays the
results. We also performed and compare it with ultrasonic
pachymetry Accutome Accupach V in the last visit.
Results: We analize 20 eyes.with 3 years of follow-up. Endothelial
loss is greater than 50% of initial basal count representing a p> 0002.
We observe the values of pre-surgical pachymetric compared against
the post surgical have a grat difference
Conclusions: Endothelial loss of patients undergoing penetrating
keratoplasty with follow at three years is more than 50%. The
thickness pachymetric also shows significant variation
Commercial Relationships: Elisa D. Alegria, None; Regina
Velasco, None; Oscar Baca, None; Alejandro Babayan, None
Program Number: 1661 Poster Board Number: D0296
Presentation Time: 8:30 AM - 10:15 AM
The Resting Potential of Rat`s Corneal Endothelial Cells
Nassim S. Calixto1, Vinicius V. Oliveira2, Renata Fleming2, Sebastiao
Cronemberger1, Adalmir Dantas2. 1Ophthalmology, Federal Univ of
Minas Gerais, Belo Horizonte, Brazil; 2Ophthalmology, Federal
University of Rio de Janeiro, Rio de Janeiro, Brazil.
Purpose: The mechanism whereby the cornea maintains its
transparency is not known. This study aims to register the presence
and to quantify the resting potential (RP) of endothelial corneal cells.
To the best of our knowledge this seems to be the first work to
systematically measure and quantify the RP of endothelial cells of the
cornea.
Methods: We performed 30 experiments on corneal preparations of
Sprague-Dawley rats (n=30). Immediately after decapitation the
eyeballs were removed and sectioned near the limbus. The corneas
were transferred to a chamber and infused with Balanced Salt
Solution (BSS) driven by a peristaltic pump in order to maintain the
BSS flowing at a rate from 0.8 to 0.85 ml/min. The temperature in the
chamber was set at 30 °C by means of a thermostatic bath. BSS is
composed of (mmol/l): Na+ 160.0; Cl- 130.0; -HCO3 25.0; K+ 5.0; H2PO4 3.0; Mg++ 1.0; Ca++ 1.0; Glucose 5.0, presenting a pH = 7.4
and 305 mOsm/Kg. The presence or absence of electrical signal was
detected by recording its voltage variations through two pore
electrodes delicately introduced into the cells and connected to a
Grass polygraph. The statistical analysis was made by Graph Pad
Prism 6.0.
Results: In the 30 experiments (30 eyes), the RP presented a mean of
40.60 with a standard deviation of 7.05 and a standard error of 1.29.
The graphics below illustrate the findings.
Conclusions: Although many studies are still needed to understand
the role of the endothelium in the transparency of the cornea, our
results demonstrate:
1. the presence of the resting potential in the corneal endothelium
cells
2. a mean of 40.60±7.05 mV for the resting potential of the rat’s
corneal endothelial cells.
This graph shows the mean value at the center line, the standard
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
deviation (SD) at the boxes, and the maximum and minimum values.
Commercial Relationships: Nassim S. Calixto, None; Vinicius V.
Oliveira, None; Renata Fleming, None; Sebastiao Cronemberger,
None; Adalmir Dantas, None
Program Number: 1662 Poster Board Number: D0297
Presentation Time: 8:30 AM - 10:15 AM
Wnt5a enhances cell migration through regulation of Cdc42 and
RhoA pathway in human corneal endothelial cells
JeongGoo Lee1, Daniel Sand1, 2, J M. Heur1, 2. 1Ophthalmology,
University of Southern California, Los Angeles, CA; 2Doheny Eye
Institute, Los Angeles, CA.
Purpose: Wnt5a activates β-catenin-independent pathways for
regulation of cellular functions that play crucial roles in wound
healing process in cornea including cell migration and polarity.
Elucidation of Wnt5a signaling may help identify potential
therapeutic targets for enhanced corneal wound healing; however,
Wnt5a signaling in corneal endothelial cells (CEC) has not been well
characterized.
Methods: Expression and/or activation of Frizzled-5, Cdc42, Rac1
were analyzed by immunoblotting. Scratch-induced directional
migration assay was employed to measure migratory rates. Activation
of Cdc42 and Rac1 were determined by GTP pull-down assay. RhoA
activity was measured using RhoA specific G-LISA assay kit.
Results: Stimulation of human CECs with Wnt5a alone resulted in
50% enhancement in cell migration as measured by scratch induced
migration assay and this effect could be completely blocked by
ML141 (Cdc42 inhibitor). Co-treatment of both Wnt5a and Y27632
(inhibitor of Rho-associated kinase) resulted in a 62% enhancement
of migration. The synergistic effects of Wnt5a and Y27632 on human
CECs migration were partially blocked by ML141 and this, in turn,
was completely abolished by treatment with both ML141 and RhoA
activator. Under these conditions, activation of both Cdc42 and Rac1
and inactivation of RhoA were observed in the Wnt5a treated cells.
We further confirmed that activated Cdc42 negatively regulates
RhoA activity and this regulation plays important role in endothelial
migration.
Conclusions: These findings suggest that Wnt5A mediated migration
in human CEC is mediated by activation of Cdc42 and inactivation of
RhoA.
Commercial Relationships: JeongGoo Lee, None; Daniel Sand,
None; J M. Heur, None
Support: Baxter Foundation, Research to Prevent Blindness, and
NIH Core Grant EY03040
Program Number: 1663 Poster Board Number: D0298
Presentation Time: 8:30 AM - 10:15 AM
Influence Of Hydrodynamic Culture Conditions On The
Expression Of Cell Junctions Of Tissue-Engineered Human
Corneal Endothelium
Olivier Roy1, Isabelle Brunette3, 4, Stephanie Proulx1, 2. 1LOEX/CUO
- Recherche, Centre de recherche du CHU, Quebec, QC, Canada;
2
Ophtalmologie, Université Laval, Quebec, QC, Canada; 3Centre de
recherche HMR, Montréal, QC, Canada; 4Ophtalmologie, Université
de Montréal, Montréal, QC, Canada.
Purpose: This study was undertaken in order to evaluate the
influence of fluid flow and pressure on the expression of cell
junctions of tissue-engineered corneal endothelium in vitro.
Methods: Cultured human corneal endothelial cells were seeded and
cultured on a previously devitalized corneal stroma, then either left in
standard culture conditions (static cultures) or placed in an artificial
anterior chamber with a perfusion rate of of 5 ul/min and a
hydrostatic pressure of 18 mm Hg (hydrodynamic cultures). A higher
perfusion rate than normal (2.6 ul/min) was chosen in order to
maintain a constant perfusion pressure and allow for the additional
leak of perfusate through the trabecular meshwork into the dish. After
4-6 days, corneas were photographed then fixed in 3.7%
formaldehyde for histology, 2.5% glutaraldehyde for transmission
electron microscopy or 4% paraformaldehyde for
immunofluorescence staining of F-actin and the tight junction protein
ZO-1. Controls consisted of devitalized corneas that were not seeded
with corneal endothelial cells, and were processed in the same
manner.
Results: Macroscopically, corneas maintained in hydrodynamic
cultures were more transparent than the static-cultured corneas. The
corneal stromas in the histology cross-sections were thinner in the
hydrodynamic-cultured corneas than in the static-cultured corneas.
The mean (±SD) collagen spacing, calculated using transmission
electron microscopy images, was 27 ±4 nm when corneas were under
hydrodynamic culture conditions whereas spacing was 46 ±9 nm
when corneas were left in static culture conditions. Control
devitalized corneas with no endothelium had a mean collagen spacing
of 32 ±6 nm when cultured under the same hydrodynamic conditions.
Hydrodynamic-cultured corneal endothelium expressed higher
amounts of ZO-1 protein, as assessed by immunostaining.
Conclusions: This study shows that corneal endothelial cells respond
to an anterior chamber flow and pressure by improving cell junction
expression in vitro. Ultimately, studying the effect of hydrodynamic
culture will enable an improved understanding of morphogenesis and
cell junction formation of the corneal endothelium.
Commercial Relationships: Olivier Roy, None; Isabelle Brunette,
None; Stephanie Proulx, None
Support: NSERC, FRQS Vision Research Network
Program Number: 1664 Poster Board Number: D0299
Presentation Time: 8:30 AM - 10:15 AM
The Quebec Corneal Cell Bank: Update on Culture Success of
Pathologic Human Corneal Endothelial Cells (2009-2012)
Mathieu Theriault1, Olivier Roy1, Olivier Rochette-Drouin1, MarieClaude Perron3, Isabelle Brunette3, 4, Stephanie Proulx1, 2.
1
LOEX/CUO - Recherche, Centre de recherche du CHU, Quebec,
QC, Canada; 2Ophtalmologie, Université Laval, Quebec, QC,
Canada; 3Centre de recherche HMR, Montréal, QC, Canada;
4
Ophtalmologie, Université de Montréal, Montréal, QC, Canada.
Purpose: The purpose of this study was to assess the feasibility of
initiating primary cultures of corneal endothelial cells from patients
suffering from various corneal diseases. We also evaluated which
conditions yielded the best results for culture.
Methods: Consenting patients undergoing penetrating keratoplasty or
Descemet’s stripping automated endothelial keratoplasty were
enrolled in this study. The cornea (or Descemet’s membrane),
removed at the time of surgery, was sent to the laboratory. Upon
receipt, specimens were photographed, endothelial cells were isolated
and cultured, and Descemet’s membranes were fixed in 3.7%
formaldehyde for histology. Data collected included diagnosis, age
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
and sex of the donor, time spent in Optisol, culture success and
number of cells obtained after a primary culture. Control healthy Eye
Bank corneas were processed in a similar manner.
Results: A total of 194 specimens were obtained between August
2009 and November 2012. Pathologies included Fuchs corneal
endothelial dystrophy (FECD; n=93), pseudophakic bullous
keratoplathy (PBK; n=33), FECD+PBK (n=4) and other corneal
disorders (n=64). Overall, 56 of the 194 diseased specimens (FECD
n=37; PBK n=3; FECD+PBK n=2; other n=14) and all of the 29
healthy corneas successfully initiated an endothelial cell culture.
Among the diseased specimens, the mean(±SD) donor age was 66.5
±11.9 years for successful cultures and 68.9 ±15.2 years for the
unsuccessful cultures. Time spent in Optisol was 2.7 ±1.2 and 3.2
±2.6 days for successful and unsuccessful cultures, respectively. The
diseased specimens yielded 120 000 ±86 000 cells and the healthy
corneas 122 000 ±55 000 cells.
Conclusions: This study shows that successful corneal endothelial
cell culture can be generated despite various corneal diseases. FECD
allowed the highest success rate. Once culture was initiated, a similar
number of cells was obtained from FECD, BPK and healthy
specimens. Patient sex and age, and the time spent by the specimen in
Optisol did not influence success rate. The Quebec Corneal Cell
Bank will become a useful tool for the study of various corneal
endotheliopathies.
Commercial Relationships: Mathieu Theriault, None; Olivier
Roy, None; Olivier Rochette-Drouin, None; Marie-Claude
Perron, None; Isabelle Brunette, None; Stephanie Proulx, None
Support: CIHR, FRQS Vision Health Research Network
Program Number: 1665 Poster Board Number: D0300
Presentation Time: 8:30 AM - 10:15 AM
Perception of Cornea and Glaucoma Subspecialists Regarding
Prevalence of Corneal Decompensation with Ex-Press Shunt
Placement
Shalin Shah1, Ngo Yen1, Thompson W. Hilary2, Jayne S. Weiss1.
1
Department of Ophthalmology, Louisiana State University Eye
Center, LSU School of Medicine, LSU Health Sciences Center, New
Orleans, LA; 2Biostatistics Section, School of Public Health,
Louisiana State University Health Sciences Center, New Orleans,
LA.
Purpose: Corneal decompensation is a recognized complication
associated with anterior chamber insertion of Ahmed, Baerveldt and
Molteno (ABM) shunts. By comparison, there are no publications
addressing corneal decompensation after Ex-Press shunt placement.
The purpose of this study was to assess the prevalence and onset of
corneal decompensation with ABM shunts and Ex-Press shunts as
perceived by cornea and glaucoma specialists.
Methods: A survey was distributed to members of the Cornea
Society and Glaucoma Society with questions about frequency and
onset of complications after anterior chamber placement of ABM and
Ex-Press shunts. The individual was requested to rank the following
side effects in order of perceived prevalence: chronic hypotony,
corneal decompensation, endophthalmitis, infection, malignant
glaucoma, pthisis bulbi, and retinal detachment. Time of onset to
corneal decompensation in Ex-Press shunts was compared to that of
the ABM group.
Results: 17 glaucoma subspecialists and 22 cornea subspecialists
participated. Corneal decompensation was listed as the most
prevalent of the seven possible complications by both subgroups (chi
square < .001). 65.0% of cornea subspecialists and 84.6% of
glaucoma subspecialists reported the risk of corneal decompensation
to be higher with uncomplicated ABM placement than with Ex-Press
shunts (chi square <0.001). The two subgroups also agreed the onset
to decompensation was less than two years in the ABM group (57.1%
cornea participants, 68.4% glaucoma participants) compared to
greater than 2 years in the Ex-Press shunt group (72.7 % cornea
respondents, 85.7% glaucoma respondents) (chi square <0.001).
53.8% of cornea respondents and 58.3% of glaucoma respondents
reported onset in the Ex-Press shunt group was greater than 5 years.
Conclusions: The results of the survey show a statistically significant
agreement among both glaucoma and cornea subspecialists that
corneal decompensation is the most prevalent complication of those
queried. They also agree decompensation is more prevalent and has a
shortened onset in ABM shunts than in Ex-Press shunts. Although
there is little literature on corneal decompensation associated with
Ex-Press shunts, this initial survey suggests the complication may
occur less frequently and is delayed with Ex-Press shunts, thus
warranting further investigation.
Commercial Relationships: Shalin Shah, None; Ngo Yen, None;
Thompson W. Hilary, None; Jayne S. Weiss, None
Support: Lions Eye Foundation, Research to Prevent Blindness
Program Number: 1666 Poster Board Number: D0301
Presentation Time: 8:30 AM - 10:15 AM
Biosafety of chitosan and collagen vitrigel membranes in the
corneal endothelium of young New Zealand Rabbits
Guillermo Mendoza1, Judith Zavala1, Marcos Garza-Madrid1, 2,
Alejandro Tamez1, Angel Zavala-Pompa1, Gabriela Brito3, Jorge A.
Cortés_Ramirez3, Jorge E. Valdez1, Jennifer Elisseeff2.
1
Ophthalmology Research Chair, Tecnológico de Monterrey,
Monterrey, Mexico; 2Translational Tissue Engineering Center,
Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore,
MD; 3Cátedra de Dispositivos Biomédicos, Tecnológico de
Monterrey, Monterrey, Mexico.
Purpose: To compare the biosafety of chitosan and collagen vitrigel
membranes as scaffolds in corneal endothelium bioengineering.
Methods: Six 3 mo New Zealand rabbits were anesthetized and
central corneal endothelium damage was induced in both eyes by
using a cryoprobe. Seven mm diameter biomembranes were
introduced in one eye through a peripheral corneal port. The
contralateral eye was used as control. Four rabbits received chitosan
biomembranes and two rabbits received collagen vitrigels. Animals
were sacrificed after one week. Both corneas were excised and
preserved in 10% formalin for histopathologic analysis with H&E
staining.
Results: The rabbits receiving chitosan-based biomembranes
developed sclerocorneal-limbus vascular congestion, corneal opacity,
and purulent exudates in the 48 h post-implant. On gross
examination, white and friable matter of 2-3mm diameter was
observed partially occupying the anterior chamber. Microscopic
analysis showed a profuse and abundant inflammatory process
(exudative phase) around the biomembrane, mainly composed of
polymorphonuclear leukocytes, piocytes, cellular debris, and
macrophages. The corneas were invaded by this inflammatory
process, with thickening caused by polymorphonuclear infiltrates,
edema and fragments of karyorrhexis. The corneas of rabbits treated
with collagen-based biomembranes remained transparent and showed
no changes after one week. Microscopic examination showed little
signs of surgical manipulation and no inflammatory alterations. The
control eyes retain their transparency and developed neither
macroscopic alterations nor histological inflammation.
Conclusions: Chitosan-based biomembranes are not suitable for in
vivo corneal endothelial bioengineering. Collagen vitrigels allowed
damaged corneas of young rabbits to retain their transparent quality
with no further complications. Collagen vitrigels are biocompatible
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
and good candidates as scaffolds for corneal endothelial cell
transplantation.
Rabbit corneas implanted with biomembranes. (A) Chitosan
membrane caused purulent exudate, vascular congestion, and corneal
opacity. (B) Collagen vitrigel remained transparent.
The control (A) and Vitrigel (B) biomambrane implanted eyes
retained transparency with no histological inflammation. Eyes
implanted with Chitosan biomembrane (C), developed inflammatory
exudative phase process around the biomembrane and corneal
thickening.
Commercial Relationships: Guillermo Mendoza, None; Judith
Zavala, None; Marcos Garza-Madrid, None; Alejandro Tamez,
None; Angel Zavala-Pompa, None; Gabriela Brito, None; Jorge A.
Cortés_Ramirez, IMPI (P); Jorge E. Valdez, None; Jennifer
Elisseeff, Collagen Vitrigel (P)
Program Number: 1667 Poster Board Number: D0302
Presentation Time: 8:30 AM - 10:15 AM
Examination of Endothelial Cell Count in HIV-Negative and
Positive Donors
John A. Gonzales1, David C. Gritz1, Patrick Gore2, Roy S. Chuck1.
1
Department of Ophthalmology & Visual Sciences, Montefiore
Medical Center, New York, NY; 2Lions Eye Institute, Saint
Petersburg, FL.
Purpose: To describe endothelial cell counts in corneal donors with
human immunodeficiency virus (HIV) compared to those without
HIV infection.
Methods: This is a retrospective, cohort comparison study drawn
from the records of a large regional eye bank. Corneas that were
procured which were later found to belong to a donor who was HIVpositive during the period from 2008-2012 were compared to corneas
that were procured from donors who were HIV-negative from
January 1, 2012-July 2, 2012. Donor corneas were classified as
having HIV-1/2 based on serologic evidence obtained from either the
presence of HIV antibodies, a positive viral nucleic acid
amplification test (NAT), or both.
Demographic data (age, gender, race) was also collected from the
same database for both cases and control. Phakic status was collected
at the time of slitlamp examination, which also reported in the
database for cases and controls.
Demographic data was compared between cases and controls using
Chi-squared and t-tests for categorical and continuous variables,
respectively. A generalized linear model was built to include all the
demographic/categorical variables (including HIV status) as well as
the continuous dependent variable (endothelial cell count).
Results: The donor cornea database during the target period included
endothelial cell counts in 1214 people: 20 HIV-positive cases and
1194 HIV-negative controls. The mean endothelial cell count in the
HIV-positive cases was 2608/mm2 while HIV-negative controls had
a mean endothelial cell count of 2621/mm2 (standard deviation 333
and 432, respectively). There was no statistically significant
difference in endothelial cell count in donors with or without HIV
infection after controlling for age (p=0.89). Donors of African and
Asian descent in the HIV-negative group had lower endothelial cell
counts compared to Caucasians (as the reference group).
Conclusions: Comparison of donor cornea endothelial cell counts
between HIV-negative and HIV-positive individuals has not been
previously described. We did not find a statistically significant
difference in endothelial cell counts due to HIV-infection status.
Since the patients were not known to be HIV-positive according to
their medical records and interview with family members, it may be
that the infection was relatively early in these patients.
Commercial Relationships: John A. Gonzales, None; David C.
Gritz, None; Patrick Gore, None; Roy S. Chuck, None
Program Number: 1668 Poster Board Number: D0303
Presentation Time: 8:30 AM - 10:15 AM
Morphological Complexity of Mouse Corneal Endothelial Cells
Revealed by Mosaic Analysis
Dennis M. Defoe, Whitney J. Rich, Theresa A. Harrison. Biomedical
Sciences, East Tennessee State University, Johnson City, TN.
Purpose: In a previous investigation, we examined how individual
corneal endothelial cells with distinct p27 gene mutations differ in
their proliferative behavior (Defoe et al., ARVO 2011). As a result of
these experiments, we noticed unusual structural features of the cells
that required further analysis. In the present study, we have begun to
examine the detailed morphology of single cells in situ, after marking
them by high cytosolic expression of green or red fluorescent proteins
(GFP or RFP).
Methods: To visualize small numbers of widely distributed cells
filled with fluorescent label, we used mosaic analysis with double
markers (MADM; Zong et al., 2005). For MADM, two mouse lines,
each with reciprocally chimeric transgenes consisting of partial
coding sequences for GFP and RFP, separated by an intronembedded LoxP site, were interbred with an Hprt-Cre-expressing
strain. Following the limited occurrence of Cre-mediated
interchromosomal recombination during mitosis, functional GFP and
RFP were reconstituted and each expressed in one of the two
daughter cells. Corneas from MADM mice were fixed, flat-mounted
and visualized by fluorescence confocal microscopy.
Results: Individual cells filled with GFP or RFP appear multipolar,
with many tapered pseudopodial processes radiating from their cell
body. Such extensions are indicative of a complex and highly
elaborate plasma membrane. Examination of rare cases where red and
green cells lie directly adjacent to one another reveals that processes
of the two cells undergo extensive interdigitation. Because of this
overlap, individual cells are observed to cover a greater area than
might be expected if their boundaries were mutually exclusive.
Conclusions: Our results give a picture of corneal endothelial cell
morphology very different from the polygonal outlines observed after
staining for actin filaments or intercellular junction proteins. The data
may help explain the discontinuous tight junction pattern seen in both
light and electron micrographs. More importantly, they suggest that
cell density alone may be an insufficient indicator of the overall
condition of the endothelium in health and disease.
Commercial Relationships: Dennis M. Defoe, None; Whitney J.
Rich, None; Theresa A. Harrison, None
Program Number: 1669 Poster Board Number: D0304
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Presentation Time: 8:30 AM - 10:15 AM
Systematic and individual differences in donor cornea
Endothelial Cell Density (ECD) measurements with specular
microscopy vs. sucrose light microscopy
Bart T. Van Dooren1, 2, Ilse J. Claerhout3, 4, Paul G. Mulder5,
Elisabeth Pels6. 1Opththalmology, Amphia Hospital, Breda,
Netherlands; 2Ophthalmology, Erasmus Medical Center, Rotterdam,
Netherlands; 3Cornea Bank, Ghent University Hospital, Ghent,
Belgium; 4Ophthalmology, Ghent University Hospital, Ghent,
Belgium; 5Amphia Academy, Amphia Hospital, Breda, Netherlands;
6
Cornea Bank, Euro Tissue Bank, Beverwijk, Netherlands.
Purpose: To evaluate systematic differences in ECD measurements
between non-contact specular microscopy, and sucrose assisted light
microscopy, in organ cultured human donor corneas in the Ghent
University Hospital Cornea Bank.
Methods: Measurements obtained between 1997 and 2011 in 1016
corneas from 551 donors were analyzed. Only donor corneas with
both specular microscopy EDCs and sucrose-assisted light
microscopy ECDs were included.
In 193 corneas, Topcon SP1000 specular microscopy ECDs were
compared to sucrose ECDs. In 167 corneas. Topcon SP2000P
(Topcon Corp, Tokyo, Japan) ECDs were compared to sucrose
ECDs. A manual counting technique using calibrated graticules on
printed photographs was used for SP1000 and Sucrose ECDs, and a
center counting method on the instrument itself was used for
SP2000P ECDs. Bland-Altman plots and estimates were used for
analysis, based on a linear mixed model analysis allowing for paired
corneas.
Results: Results are shown in figures 1 and 2.
SP1000 ECDs had a mean difference of 254.5 cells/mm2 (lower)
with sucrose ECDs, the limits of agreement (horizontal black lines)
were + 274.3 and - 783.2.
SP2000P ECDs had a mean difference of 71.2 cells/mm2 (lower)
with sucrose ECDs, with limits of agreement: + 805.2 and -947.6.
Unequal SD’s of two paired measurements cause a correlation
between the sum and the mean of those two measurements (Pitman’s
test). Hence the negative slope in the SP1000-sucrose ECD difference
vs. mean regression line , and positive slope in the SP2000-sucrose
ECD difference vs. mean regression line (sloped red lines)
Conclusions: Substantial systematic differences and huge individual
differences exist between ECDs obtained with different measurement
methods.
Specular microscopy in donor eyes resulted in only a minority of
cases in usable ECDs, and because of this and larger endothelial cell
counting sample sizes, sucrose ECDs remain the gold standard in
ECD determination in organ cultured donor corneas.
Erroneous magnification calibration in SP1000 was shown to result
in a larger systematic difference with sucrose ECD.
The small systematic error between SP2000P ECDs and sucrose
ECDs indicate that comparison of donor ECDs to in-vivo ECDs may
be justifiable, on the condition that instruments are calibrated
correctly. Huge individual measurement differences may occur.
Commercial Relationships: Bart T. Van Dooren, None; Ilse J.
Claerhout, None; Paul G. Mulder, None; Elisabeth Pels, None
Program Number: 1670 Poster Board Number: D0305
Presentation Time: 8:30 AM - 10:15 AM
Oxidative Stress Causes Mitochondrial Dysfunction in Human
Corneal Endothelial Cells
Thore Schmedt1, 2, Cecily E. Hamill1, 2, Yuming Chen1, 2, Ula V.
Jurkunas1, 2. 1Schepens Eye Research Inst, Boston, MA;
2
Massachusetts Eye and Ear, Boston, MA.
Purpose: Human corneal endothelial cells (HCEnCs) form a single
monolayer of hexagonal cells that are non-proliferative in vivo and
enter rapid cellular senescence in vitro, rendering them of limited use
in the study of endothelial cell biology. Introduction of telomerase
(hTERT) has been shown to extend the life span of HCEnCs, but it is
unclear whether hTERT expression protects mitochondria against
oxidative stress. The purpose of this study was to investigate the
effect of hTERT on mitochondrial integrity and apoptosis in response
to the pro-oxidant menadione in HCEnCs.
Methods: Highly uniform subpopulations of HCEnCs that exhibited
increased proliferative capacity were isolated from a 21-year-old
donor (HCEnC-21), and hTERT was introduced creating the HCEnC21T cell line. Cells were grown to confluence and treated with 25 and
100 µM of menadione sodium bisulfite for 1 hour. Non-treated cells
served as controls. Cell viability was measured by Trypan blue
exclusion using an automatic cell counter. Mitochondrial integrity
was determined using the JC-1 dye and red-to-green fluorescence
ratios to detect depolarized (damaged) mitochondria.
Results: Mitochondrial polarization of both cell lines decreased with
increasing concentrations of menadione. At baseline, the fluorescence
ratios of in HCEnC-21T and HCEnC-21 were 2.63±0.41 and
3.01±0.31, respectively. At 25 µM, the ratios dropped to 1.32±0.27 in
HCEnC-21T and 1.91±0.04 in HCEnC-21. This is an average
decrease of 49.9% (p=0.04) in HCEnC-21T and 36.5% (p=0.014) in
HCEnC-21. Cell viability tended to decrease, but was not
significantly lower after treatment with 25 µM. At 100 µM, the
fluorescence ratios were found to be 0.73±0.07 in HCEnC-21T and
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
0.76±0.09 in HCEnC-21. This corresponds to an average decrease of
72.3% (p<0.001) in HCEnC-21T and 74.6% (p<0.001) in HCEnC-21.
Similarly, cell viability was reduced by 26.2% (p=0.0014) in
HCEnC-21T and 14.9% (p=0.01) in HCEnC-21. There were no
significant differences between HCEnC-21T and HCEnC-21 at any
dosage.
Conclusions: Oxidative stress induced loss of mitochondrial integrity
in a dose-dependent manner, which preceded cell death in both
HCEnC-21T and HCEnC-21. Telomerase overexpression did not
augment resistance to the pro-oxidant in HCEnC-21T as opposed to
HCEnC-21. Both cell lines provide suitable models for the study of
HCEnC biology and endothelial disease pathogenesis.
Commercial Relationships: Thore Schmedt, None; Cecily E.
Hamill, None; Yuming Chen, None; Ula V. Jurkunas, 61/482,769
(P), Altheos (C)
Support: This work was supported by the National Institutes of
Health/National Eye Institute R01EY020581, a Massachusetts Lions
Eye Research Fund Grant and a Research to Prevent Blindness Grant
to UVJ.
Program Number: 1671 Poster Board Number: D0306
Presentation Time: 8:30 AM - 10:15 AM
Theoretical and Clinical Method to quantify the Corneal Suction
Pressure from normal corneas during the lifetime
Andre Heck, Fernando C. Abib. Federal University of Paraná,
Curitiba, Brazil.
Purpose: To demonstrate a methodology to quantify the Corneal
Suction Pressure (CSP) from normal corneas during the lifetime.
Methods: The Corneal Suction Pressure (CSP) is defined as the
corneal volume that oncotically works through on the summation of
the lateral membrane; the CSP is a volumetric unity (µm3) for 1µm
of the lateral membrane between 2 contiguous cells. To calculate the
CSP in normal corneas will be used clinical results from corneal
specular microscopy (CSM) with reliability indexes <0.05, and
Pentakan. The endothelial cell density (ECD) and the average of the
endothelial cell area (A) are routinely calculated by CSM. The ECD
and A of the corneal endothelial cell (CEC) changes during the
lifetime. The model will consider all possible ECD (4000 - 500
cells/mm2) and A (250 - 2000 µm2) values during the lifetime. The
Corneal Volume (CV) of the 10mm of the corneal diameter is
calculated by Pentakan. To demonstrate the theoretical model used to
quantify the CSP in corneas, the used range of the CV is from 40 to
70 mm2. Considering that the water flows through the lateral
membrane of the CEC, the study will calculate the summation of the
perimeter (P) of the estimated total population of the CEC (ΣCEC
Perimeter) with the predominant cell morphology - hexagonal. The P
of the CEC is calculated by the formula PCEC = 6sCEC and A =
6sCEC2√3/4 . The summation of CEC perimeter will be calculated
by the formula: ΣCEC Perimeter = 2πrh.ECD.6sCEC. The model
cornea data: radius of the corneal inner surface (r) = 6.93; corneal
sagital depth (h) = 3.06; side of the CEC (sCEC); π=3.14159. CSP is
defined as the CV/ 0.5ΣCEC Perimeter. The results of the CSP will
be present in a table with the corresponding ECD and A values and a
graphic model.
Results: The calculated values of the CSP can be able to represent
the smaller volume (µm3) of cornea which is correlated functionally
with 1 µm of the neighboring lateral cell membrane, which represents
the endothelial barrier and endothelial pump to maintain the corneal
trasnparency. The calculated CSP values for different corneal
volumes is presented in the Table and its behavior in the Graphic 1.
Conclusions: The used methodology was able to calculate the
Corneal Suction Pressure (CSP) from normal corneas with different
ECD and volumes during the lifetime with results of CSM and
Pentacan in clinical situations.
Commercial Relationships: Andre Heck, None; Fernando C.
Abib, Fernando C Abib (P)
Program Number: 1672 Poster Board Number: D0307
Presentation Time: 8:30 AM - 10:15 AM
Descemet Membrane Endothelial Keratoplasty (DMEK): Large
Descemetorhexis To Reduce Rebubbling Does Not Cause
Postoperative Peripheral Corneal Edema
Theofilos Tourtas, Julia M. Wessel, Bjoern O. Bachmann, Ursula
Schlotzer-Schrehardt, Friedrich E. Kruse. Department of
Ophthalmology, University of Erlangen-Nuremberg, Erlangen,
Germany.
Purpose: While DMEK renders better visual visual acuity than
DSAEK, it is associated with higher rebubbling rate because of
postoperative partial graft detachment. One possibility to reduce
rebubbling rate is to create a larger descemetorhexis which avoids an
overlap between the donor's descemet membrane and the implanted
DMEK graft thus decreasing graft detachment. However, the
presence of denuded stroma without descemet membrane or
endothelium after large descemetorhexis might cause stromal edema.
To evaluate the incidence of stromal edema in the early postoperative
phase, peripheral corneal thickness was compared between patients
undergoing DMEK with two different techniques.
Methods: A single-center, retrospective, consecutive case series of
30 patients undergoing DMEK for Fuchs endothelial dystrophy with
two different techniques: Based on intraoperative drawings and
postoperative slit-lamp examinations patients were divided into two
groups. In group A (n= 16 eyes) diameter of descemetorhexis was
approx. 10 mm, resulting in a 1-mm zone of denuded stroma. In
group B (n= 14 eyes) diameter was approx. 6 mm, resulting in a 1mm zone of overlapping. To assess corneal edema, peripheral corneal
thickness was measured by Scheimpflug imaging (Pentacam; Oculus,
Wetzlar, Germany) and anterior segment OCT in the 8 mm zone
within a 2-month follow-up.
Results: Preoperative peripheral corneal thickness in group A was
731 ± 63 µm and in group B 706 ± 48 µm (P=.241). Two months
after surgery, peripheral corneal thickness was 702 ± 45 µm and 678
± 48 µm in groups A and B (P=.179), respectively. Peripheral corneal
thickness was not significantly different between both groups, 2
months after surgery. Rebubbling rate was 12.5% in group A and
21.4% in group B.
Conclusions: DMEK with larger descemetorhexis leaving a small
zone of denuded stroma cause significantly enhanced graft adhesion
without increasing the incidence of peripheral corneal edema.
Commercial Relationships: Theofilos Tourtas, None; Julia M.
Wessel, None; Bjoern O. Bachmann, None; Ursula SchlotzerSchrehardt, None; Friedrich E. Kruse, None
Program Number: 1673 Poster Board Number: D0308
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Presentation Time: 8:30 AM - 10:15 AM
Objective Assessment of the Corneal Endothelium in Fuchs
Endothelial Dystrophy
Jay W. McLaren, Lori A. Bachman, Sanjay V. Patel. Ophthalmology,
Mayo Clinic, Rochester, MN.
Purpose: Assessment of the corneal endothelium in early stages of
Fuchs endothelial dystrophy is subject to sampling errors because of
the non-uniform distribution of guttae. In this study we developed a
standardized method of endothelial assessment and examined the
predictive relationship between the effective endothelial cell density
(ECD) and severity of Fuchs dystrophy based on subjective grades.
We also examined the relationship between effective ECD and
anterior corneal structural changes (backscattered light).
Methods: The corneal endothelium of 51 eyes from 30 patients, with
varying degrees of Fuchs endothelial dystrophy, was examined by
using confocal microscopy (ConfoScan 4, Nidek Technologies) with
a 20x non-contact objective. In 2 to 3 images that sampled the central
endothelium, local contiguous cell density was determined by using a
variable frame method (2 to 177 cells in 1 to 10 patches, depending
on the distribution of guttae). The effective ECD was the product of
the local cell density and the ratio of the sample area not covered by
guttae to total sample area, which was determined by image analysis.
The severity of the disease in each eye was assessed during slit-lamp
examination by two examiners based on a modified Krachmer grade
(1-6). In 55 eyes with Fuchs dystrophy from a second group of 30
patients, the clinical grade was predicted from the effective ECD and
the regression coefficients from the first group, and compared to the
subjective clinical grade assigned by one examiner. The relationship
between effective ECD and backscatter from the anterior stroma,
based on brightness of confocal images (40x objective, ConfoScan 4)
in the second group, was examined by Pearson regression.
Significance was determined by using generalized estimating
equation models.
Results: The effective ECD decreased linearly with subjective grade
(r=-0.93, p<0.001). The grade predicted from the effective ECD
differed from the subjective clinical grade by -0.1 ± 0.8 grade (mean
± SD). Mean backscatter from the anterior stroma was inversely
correlated with the effective ECD (r=-0.59, p<0.001).
Conclusions: The effective ECD in confocal images provides an
objective means of assessing severity of Fuchs dystrophy and
correlates with structural changes in the anterior stroma. It is
sensitive to changes in early stages of the disease, before clinical
edema appears, and might be a useful tool in clinical studies.
Commercial Relationships: Jay W. McLaren, None; Lori A.
Bachman, None; Sanjay V. Patel, None
Support: Research to Prevent Blindness, and Mayo Foundation
Program Number: 1674 Poster Board Number: D0309
Presentation Time: 8:30 AM - 10:15 AM
Density dependency of successful in vitro cultures of human
corneal endothelial cells using a dual media approach
Gary S. Peh1, Heng-Pei Ang1, Khadijah Adnan1, Xin-Yi Seah1,
Benjamin L. George1, Jodhbir S. Mehta2, 3. 1Ocular Tissue Eng &
Stem Cell Group, Singapore Eye Research Institute, Singapore,
Singapore; 2Corneal and External Eye Disease Service, Singapore
National Eye Centre, Singapore, Singapore; 3Department of Clinical
Sciences, Duke-NUS Graduate Medical School, Singapore,
Singapore.
Purpose: This study describes the importance of seeding density for
in vitro expansion of isolated primary human corneal endothelial
cells (CECs) propagated using a novel dual media culture system.
Methods: Primary human CECs isolated from paired donor corneas
were propagated using a two media culture system. A ‘proliferative’
medium was utilized in the expansion of CECs. Upon reaching ~90%
confluence, a ‘maintenance’ medium was used to stabilized and
retain the unique cellular morphology of expanded human CECs, as
depicted in Figure 1. Human CECs established using this culture
approach was expanded to the second passage (P2) to obtain
sufficient cells for the study. Subsequently, CECs were dissociated
and seeded at four densities: 2,500 cells per cm2 (‘LOW’); 5,000 cells
per cm2 (‘MID’); 10,000 cells per cm2 (‘HIGH’); and 20,000 cells per
cm2 (‘HIGHx2’). Cultures of these densities were analyzed for their
propensity to proliferate, and were subjected to morphometric
analyses comparing cell sizes, coefficient of variance, as well as cell
circularity when they became confluent.
Results: At the two lower seeding densities, plated human CECs
were more proliferative than cells seeded at higher densities, but this
observation was not statistically significant. Morphometric analyses
showed that cells at lower seeding densities were significantly larger
in size, heterogeneous in shape and less circular (fibroblastic-like) at
Day 10, and continued to be hypertrophic after one month in culture.
Comparatively, CECs seeded at the two higher densities were more
homogeneous at confluence, and were also morphologically more
compact and circular. Potentially, at the optimal seeding density of
10,000 cells per cm2, it was possible to obtain up to 2.5 x 107 cells at
the third passage from cultivated CECs established from a pair of
donor corneas. More importantly, these cultivated CECs retain their
unique cellular morphology.
Conclusions: Our results demonstrated a density dependency in the
culture of primary human CECs; sub-optimal seeding density results
in a decrease in cell saturation density, as well as a potential loss of
the proliferative potential of cultivated human CECs. As such, we
propose a seeding density of not less than 10,000 cells per cm2 for
regular passaging of primary human CECs.
Commercial Relationships: Gary S. Peh, Singapore Eye Research
Institute (P); Heng-Pei Ang, None; Khadijah Adnan, None; Xin-Yi
Seah, None; Benjamin L. George, None; Jodhbir S. Mehta, None
Support: National Research Foundation Translational and Clinical
Research Programme Grant, Singapore (R621/42/2008), and
Biomedical Research Council, Translational Clinical Research
Partnership Grant, Singapore (TCR0101673)
Program Number: 1675 Poster Board Number: D0310
Presentation Time: 8:30 AM - 10:15 AM
SLC4A11 is an EIPA-sensitive Na+-Dependent pHi Regulator
Diego G. Ogando1, Supriya S. Jalimarada1, Eranga N. Vithana2,
Joseph A. Bonanno1. 1School of Optometry, Indiana University,
Bloomington, IN; 2Singapore Eye Research Institute, Singapore,
Singapore.
Purpose: Mutations in SLC4A11 are associated with corneal
endothelial dystrophies. In a previous report (ARVO 2012 #6005) we
observed that SLC4A11 acts as a Na+:OH- cotransporter or a Na+: H+
exchanger but does not transport Na+:HCO3-. In the present study we
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
further tested these two hypotheses and a third one that SLC4A11
acts as a Na+: B(OH)4- (Borate) cotransporter.
Methods: HEK293 cells were transfected with phCMV2-HASLC4A11 (T) or empty vector (C). Membrane surface expression
was confirmed by immunofluorescence and membrane protein
isolation. Forty eight hours post-transfection the cells were loaded
with BCECF or SBFI dyes to measure pHi and Na+i in CO2/HCO3- rich (BR) or HCO3- -free (BF) Ringer.
Results: We previously observed that the BF to BR induced pH i
change (acidification and slow recovery) was not significantly
different between C and T cells. This transition was accompanied by
a slow Na+ influx (C: 0.882±0.187 vs T: 0.897±0.152 mM Na+/min,
p=0.951). A Na+-free pulse in BR produced a similar rate of
acidification in T and C (C: 0.042±0.014 vs T: 0.048±0.006 pH i/min,
p=0.741). Further, the rate of alkalinization upon Na+-addition in BR
was not significantly different (C: 0.131±0.029 vs T: 0.155±0.015
pHi/min, p=0.477). These results indicate that SLC4A11 does not
transport Na+: HCO3-. We previously reported that T cells recover
from NH4Cl pulse in BF 2.6 times faster than C cells. This recovery
was sensitive to the amiloride analogue EIPA (1 µM). A Na+-free
pulse in BF induces a faster acidification in T respect to C (C:
0.048±0.004 vs T: 0.074±0.009 pH i/min, p=0.039). Likewise, the rate
of alkalinization recovery upon Na+-addition in BF is higher in T (C:
0.151±0.023 vs T: 0.267±0.044 pH i/min, p=0.036). This recovery is
completely inhibited by EIPA 1 µM in C and T cells. These results
are consistent with SLC4A11 acting as a Na+: OH- cotransporter or a
Na+:H+ exchanger (NHE), however they do not rule out SLC4A11
being an activator of endogenous NHEs as the recovery in presence
of EIPA 1 µM is not significantly different between C and T. In T
cells the presence of Borate 10 mM did not affect the Na+-free
induced acidification rate (T: 0.097±0.018 vs T+Borate: 0.079±0.015
pHi/min, p=0.465); inconsistent with SLC4A11 being a Na+: Borate
transporter.
Conclusions: (1) SLC411 does not provide Na+:HCO3- or Na+:
Borate transport; (2) SLC4A11 transports Na+:OH-; or (3) it is not an
ion transporter per se but an activator of NHEs.
Commercial Relationships: Diego G. Ogando, None; Supriya S.
Jalimarada, None; Eranga N. Vithana, None; Joseph A. Bonanno,
None
Support: NIH grant EY008834
Program Number: 1676 Poster Board Number: D0311
Presentation Time: 8:30 AM - 10:15 AM
Effects of the Reliability Index called Sample Error of the corneal
specular microscopy on the repeatability of the results of the
examinations
Fernando C. Abib1, 2, Richard Y. Hida3, Ricadro Holzchuh3.
1
Anatomy, Federal University of Parana, Curitiba, Brazil; 2Cornea,
Clinica de Olhos Dr. Fernando Abib, Curitiba, Brazil;
3
Ophthalmology, University of Sao Paulo, Sao Paulo, Brazil.
Purpose: To know the effects of the reliability index, called Sample
Error (SE), over the repeatability of the specular microscopy
examinations with different number of the counted endothelial cells.
Methods: Transversal study of 122 endothelial examinations of 61
patients with cataract (63.97 ± 8.15 years old). The Reliability Index
called SE was calculated by a specific software (Cells Analyzer
(CA), Technicall, Brazil) developed to specular microscopy and
confocal microscopy. The Reliability Index of 95% and cut-off
Sample Error of 5% were used). Each examination of Konan noncontact NONCON ROBO® SP-8000 Specular Microscope was
submitted to standard cell counting, even at the same image,
according with the studied group: G40: 40 cells counted, G100: 100
cells counted; G150: 150 cells counted, and GCA: the counted cell
number was higher than the determined cell number by the Cells
Analyzer software in as many different images as necessary.
Reliability index interpretation - 1. The planned SE 5% > the
calculated SE: The endothelial examination will be statistically
representative of the corneal endothelial cell population; 2. The
planned SE 5% < calculated SE: The endothelial examination will
not be statistically representative of the corneal endothelial cell
population. The SE will be calculated and presented to G40, G100,
G150 and GCA. The behavior of the range of the 95% confidence
interval to ECD, A, CV and HEX, for each eye, comparing G40G100, G40-G150, and G40-GCA, will be presented.
Results: ECD average was 2,395.37±294.34 cells/mm2, A average
423.64±51.09 µm2, CV average 0.40±0.04 and HEX average
54.77±4.19%.
The G40 SE was 0.157±0.031, G100 SE was 0.093±0.024, G150 SE
was 0.075±0.010, and GCA SE was 0.037±0.005. The increase of the
marked cells decreases the SE and decrease the range of confidence
interval (right and left eyes respectively): G40-G100 - ECD: 37.00%
and 39.79%, A: 38.62% and 43.66%, CV:27.27% and 46.15%, HEX:
36.74% and 40.46%; G40-G150 - ECD: 50.89% and 49.87%, A:
52.05% and 53.33%, CV: 45.45% and 53.84%, HEX: 51.87% and
52.96%; G40-GCA - ECD: 75.79% and 77.39%, A: 75.95% and
77.37%, CV: 72.72% and 76.92%, HEX 75.93% and 76.71%.
Conclusions: The Reliability Index called Sample Error from the
Cells Analyser tutorial method improved close 75% the repeatability
of all results of the specular microscopy: ECD, A, CV and HEX.
Commercial Relationships: Fernando C. Abib, Fernando C Abib
(P); Richard Y. Hida, None; Ricadro Holzchuh, None
Program Number: 1677 Poster Board Number: D0312
Presentation Time: 8:30 AM - 10:15 AM
Comparing Quantitative and Qualitative Indices of the Donated
Corneas Maintained in Optisol GS with Those Kept in Eusol C
Mozhgan Rezaei Kanavi1, 2, Mohammad Ali Javadi1, 3, Tahereh
Chamani3, Pejman Fahim3. 1Ophthalmic Research Center, Shahid
Beheshti University of Medical Sciences, Tehran, Islamic Republic
of Iran; 2Department of Ophthalmology and Visual Sciences,
University of Wisconsin, Madison, WI; 3Central Eye Bank of Iran,
Tehran, Islamic Republic of Iran.
Purpose: This study was conducted to compare the quantitative and
qualitative indices of the donated corneas maintained in Optisol GS
with those kept in Eusol C storage media.
Methods: In an ante-grade single blind study, two corneas from each
donor with a death to preservation time of less than 30 hours and with
a minimum of “an apparent good cornea rating” were maintained in
corneal storage media; randomly one in Optisol GS and the other in
Eusol C. Slit-lamp biomicroscopic and specular microscopic
examinations were performed on days 1 and 7. The results of the
qualitative and quantitative indices and the final cornea rating were
recorded. Statistical analyses were performed to evaluate any
differences between the two media.
Results: 180 corneas from 90 donors with an age range of 29.3±22.4
years were entered into the study: 90 corneas in Optisol GS and the
other 90 in Eusol C. No specular images were obtainable on day 7 in
5 of the corneas maintained in Optisol GS and 4 of those corneas in
Eusol C, but based on slit-lamp biomicroscopic examinations they
were rated fair. The endothelial cell density of the corneas preserved
in Optisol GS on both day 1 and day 7 were significantly higher than
those maintained in Eusol C (P=0.007 and P=0.046 respectively).
Significant endothelial cell vacuolation was observed by day 7 but
more frequently seen in the corneas preserved in Eusol C than those
in Optisol GS (P=0.014). As the maintenance time of the donated
corneas increased there was no significant difference noted between
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
the cornea rating, endothelial pleomorphism, polymegathism and
mean cell area, stromal edema and Descemet’s folds of the two
groups.
Conclusions: This study shows the superiority of Optisol GS over
Eusol C in terms of maintaining a higher endothelial cell density and
a lower rate of endothelial vacuolation when the maintenance time
increases. Such superiority may be due to the presence of some
antioxidant compounds in the Optisol GS. However, it seems that
Eusol C can still be a suitable choice for the eye banks that have a
high turnover rate of corneas with a short preservation time before
transplantation.
Commercial Relationships: Mozhgan Rezaei Kanavi, None;
Mohammad Ali Javadi, None; Tahereh Chamani, None; Pejman
Fahim, None
Program Number: 1678 Poster Board Number: D0313
Presentation Time: 8:30 AM - 10:15 AM
Clinical Evaluation of a Novel Method to Generate Precut Tissue
for Descemet Membrane Endothelial Keratoplasty (DMEK)
Bjoern O. Bachmann1, Ursula Schlotzer-Schrehardt1, Martin
Boergel2, Friedrich E. Kruse1. 1Ophthalmology, University Erlangen,
Erlangen, Germany; 2German society for tissue transplantation,
Hannover, Germany.
Purpose: Descemet Membrane Endothelial Keratoplasty (DMEK) is
a novel procedure for transplantation of corneal endothelium
resulting in unsurpassed improvement of postoperative visual acuity.
A central issue regarding this technique is the complexity of graft
preparation which hinders its wide spread use. Therefore, the
evaluation of precut tissue for DMEK is essential for making this
technique amenable to a larger group of surgeons.
Methods: Precut preparation of organ cultured donor corneas was
preformed as previously described resulting in incompletely stripped
Descemet’s membranes where the central part was still attached to
the corneal stroma. Subsequent culture for up to 5 days was followed
by the use of the precut grafts for DMEK in patients with Fuchs’
endothelial dystrophy. Visual acuity, central corneal thickness
(Pentacam®, Oculus) and endothelial cell density (CellCheck
XLTM, Konan) were retrospectively analyzed during the early
postoperative period.
Results: Stripping was successfully completed in all grafts directly
prior to surgery. The average visual acuity (logMAR) improved from
0.49 +/- 0.13 preoperatively to 0.23 +/- 0.12 one month after DMEK.
Central preoperative corneal thickness decreased from 667 +/- 31µm
preoperatively to 555 +/- 60 µm at 1 week and 484 +/- 8 µm 4 weeks
after DMEK. Average endothelial cell density was 2496 +/- 271 cells
/mm2 before and 2275 +/- 247 cells/mm2 3 days after precut
preparation (2 days before DMEK). One month after DMEK
endothelial cell density further decreased to 1817 +/- 111 cell/mm2.
Conclusions: Precut preparation of DMEK tissue, generated by
incomplete Descemet stripping, leads to minor endothelial cell loss
during subsequent culture. The use of precut tissue for DMEK results
in a fast visual recovery, only minor additional endothelial cell loss
and a rapid decrease of corneal thickness.
Commercial Relationships: Bjoern O. Bachmann, None; Ursula
Schlotzer-Schrehardt, None; Martin Boergel, None; Friedrich E.
Kruse, None
Program Number: 1679 Poster Board Number: D0314
Presentation Time: 8:30 AM - 10:15 AM
Changes in Anterior Corneal Haze with Severity of Fuchs
Endothelial Dystrophy
Sejal Amin, Jay W. McLaren, Keith H. Baratz, Sanjay V. Patel.
Ophthalmology, Mayo Clinic, Rochester, MN.
Purpose: In corneas requiring endothelial keratoplasty for Fuchs
endothelial corneal dystrophy (FECD), haze (corneal backscatter)
from the anterior cornea is higher than normal and remains higher
than normal after keratoplasty. In this study, we examined the
changes in anterior corneal haze over a range of severity of FECD.
Methods: In a cross-sectional study, 95 corneas of 70 patients with
FECD (mean age, 67 years; range, 41-87 years) and 52 normal
corneas of 28 controls (mean age, 44 years; range, 21-77 years) were
examined by slit-lamp and confocal microscopy. Clinical grade of
FECD was determined by slit-lamp examination based on the
presence and extent of guttae, and the presence or absence of
clinically evident edema (modified Krachmer grades 1-6). FECD was
categorized as mild (grades 1-2), moderate (grades 3-4), or advanced
(grades 5-6). Corneas of control subjects were devoid of any central
guttae (grade 0). Corneal haze, measured from the reflected light
intensity profile of confocal microscopy (ConfoScan 4, Nidek
Technologies) images, was standardized to reflectivity from a known
concentration of a turbidity standard. Anterior corneal haze was
defined as the mean reflectivity in a 10-percentile range of stromal
thickness centered at the anterior stromal boundary. Haze was
compared between severities of FECD and normal by using
generalized estimating equation (GEE) models to account for any
correlation between fellow eyes of the same subject. The correlation
between anterior haze and grade was illustrated by the Pearson
correlation coefficient, and the significance was determined by GEE
models.
Results: Anterior corneal haze in FECD (1940 ± 700 scatter units
[SU], n=95) was higher than normal controls (1185 ± 229 SU, n=52,
p<0.001). Anterior corneal haze was higher in advanced (2176 ± 692
SU, n=67) than in moderate (1445 ± 260 SU, n=14, p<0.001) or mild
(1302 ± 236 SU, n=14, p<0.001) FECD, and higher in moderate
FECD than in controls (p=0.003). For all eyes, anterior corneal haze
was correlated with grade (r=0.68, p<0.001, n=147).
Conclusions: The increase in anterior corneal haze, which is
associated with disability glare, begins early in the course of FECD
and prior to the onset of clinically evident edema. Whether anterior
corneal haze in early grades of FECD is related to the presence of
chronic sub-clinical edema or to changes affecting the optical quality
of the cornea is uncertain.
Commercial Relationships: Sejal Amin, None; Jay W. McLaren,
None; Keith H. Baratz, Assessing the likelihood of developing
Fuchs Corneal Dystrophy (P); Sanjay V. Patel, None
Support: Research to Prevent Blindness; Mayo Clinic CTSA
NIH/NCATS UL1 TR000135; Mayo Foundation
Program Number: 1680 Poster Board Number: D0315
Presentation Time: 8:30 AM - 10:15 AM
Cell Line of Fuchs' Corneal Dystrophy Produces an Abnormal
Extracellular Matrix
Leona Ho1, Naoki Okumura1, EunDuck P. Kay1, Kenta Yamasaki1,
Satoshi Kawasaki2, Theofilos Tourtas3, Ursula SchlotzerSchrehardt3, Friedrich E. Kruse3, Shigeru Kinoshita2, Noriko
Koizumi1. 1Biomedical Engineering, Doshisha University, Kyoto,
Japan; 2Ophthalmology, Kyoto Prefectural University of Medicine,
Kyoto, Japan; 3Ophthalmology, University of Erlangen-Nürnberg,
Erlangen, Germany.
Purpose: Abnormal deposition of extracellular matrix (ECM), such
as corneal guttae, causes visual disturbance in patients with Fuchs’
endothelial corneal dystrophy (FECD), a chronic corneal disease that
is known to cause blindness. To date, no pharmaceutical intervention
is available for the treatment of patients afflicted with FEDC. The
purpose of this study was to establish a cellular in vitro model that
can be used for the potential development of a therapy to treat
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
patients with FECD.
Methods: Human corneal endothelial cells (HCECs) were cultured
from endothelial-Descemet membrane lamellae obtained from 3
FECD patients during Descemet's membrane endothelial keratoplasty
(DMEK) after obtaining informed written consent from each patient.
HCECs cultured from 3 individual normal donor corneas were used
as a control. The FECD-derived HCECs and the control HCECs were
then immortalized by SV40 and hTERT to produce iFECD and
iHCEC cell lines, respectively. The gene expression levels of ECM
components were then analyzed by TaqMan® real-time PCR. To
elucidate ECM production, iFECD and iHCEC cells were cultured in
a Transwell® culture system, and ECM deposition was then analyzed
by hematoxylin-eosin (HE) staining and immunohistochemistry after
2 weeks.
Results: Real-time PCR revealed a significantly increased production
of type I collagen, type IV collagen, and fibronectin (p<0.05) in
iFECD cells in comparison to iHCEC cells. In addition, the gene
expression of ZEB2, an epithelial to mesenchymal transition
transcription factor, was also found to be significantly increased
(p<0.01) in iFEDC in comparison to iHCEC cell lines. HE staining
showed that a significant thicker ECM was produced by iFECD cells
than by iHCEC cells (6.65±0.82μm and 3.14±0.64μm, respectively)
(p<0.01). Immunostaining also showed a greater intensity of staining
for ECM proteins, such as collagen and fibronectin, in iFECD cells.
Conclusions: We established a cellular model of FECD associated
with an abnormal production of ECM. These findings may prove
valuable as a research tool that can be used for the potential
development of a novel treatment option for patients with FECD.
Commercial Relationships: Leona Ho, None; Naoki Okumura,
None; EunDuck P. Kay, None; Kenta Yamasaki, None; Satoshi
Kawasaki, None; Theofilos Tourtas, None; Ursula SchlotzerSchrehardt, None; Friedrich E. Kruse, None; Shigeru Kinoshita,
Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P), Otsuka
Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R); Noriko
Koizumi, None
Support: Grant-in-Aid for JSPS Fellows by Japan Society for the
Promotion of Science
Program Number: 1681 Poster Board Number: D0316
Presentation Time: 8:30 AM - 10:15 AM
Evaluation of Novel Porcine Atelocollagen Vitrigel Membrane
with Curvature as Corneal Endothelial Cell Carrier
Junko Yoshida1, Seiichi Yokoo1, Satoru Yamagami1, Shiro Amano1,
Ayumi Oshikata2, Chika Okamoto2, Toshiaki Takezawa2. 1Department
of Ophthalmology, The University of Tokyo, Graduate School of
Medicine, Tokyo, Japan; 2Division of Animal Sciences, National
Institute of Agrobiological Sciences, Tsukuba, Japan.
Purpose: To evaluate the newly invented porcine atelocollagen
vitrigel membrane (Vitrigel) as a corneal endothelial cell carrier for
corneal transplants.
Methods: We implanted flat Vitrigel sheets into rabbit corneas by
three methods; on the front surface, in the mid stroma and onto the
back of corneas, in order to see the biocompatibility and the
transparency of Vitrigel (n=6). Postoperative evaluation was done by
slit lamp examination and photographs were taken at 1 month and 4
months later. Hematoxilin and Eosin Staining was done at
postoperative month 4. Curved Vitrigel was implanted onto the
posterior surface of rabbit cornea comparing to flat Vitrigel in order
to see whether it fits to the curvature of the cornea(n=4). Cell
culturing methods on curved Vitrigel sheet were also determined
using immortal human corneal endothelial cells.
Results: Vitrigel kept its transparency and there was no obvious
precipitates and inflammation in rabbit eyes during whole
observation period. While flat Vitrigel did not fit to the corneal
curvature, curved Vitrigel (radius of curvature 10mm, diameter 6
mm) attached to the posterior surface of corneas. We successfully
cultivated human corneal endothelial cells on the curved Vitrigel
sheet with cell count 3100 cells/mm2 by a method which utilizes 12
well plate and Teflon O-ring.
Conclusions: We invented novel porcine atelocollagen vitrigel
membrane with curvature, which was biocompatible and transparent
in rabbit eyes. Also a culturing method of human corneal endothelial
cells on curved Vitrigel was developed.
Commercial Relationships: Junko Yoshida, None; Seiichi Yokoo,
None; Satoru Yamagami, None; Shiro Amano, Topcon (P); Ayumi
Oshikata, KANTO CHEMICAL CO., INC. (P); Chika Okamoto,
None; Toshiaki Takezawa, KANTO CHEMICAL CO.,INC., Japan
(P)
Program Number: 1682 Poster Board Number: D0317
Presentation Time: 8:30 AM - 10:15 AM
Prosthetic Replacement of Ocular Surface Ecosystem (PROSE)
Device Wear Results in Decreased Endothelial Cell Density and
Decreased Endothelial Pleomorphism
Ryan M. St Clair, Yvonne Wang, Christopher E. Starr, Michelle N.
Lee, Ana G. Alzaga Fernandez, Kimberly C. Sippel, Jessica Ciralsky,
Mark Rosenblatt, Priyanka Sood. Ophthalmology, Weill Cornell
Medical College, New York, NY.
Purpose: To investigate the effect of the Prosthetic Replacement of
Ocular Surface Ecosystem (PROSE) device on endothelial cell
density and morphology.
Methods: This was a prospective study, including patients referred
for fitting with the PROSE device at Weill Cornell Medical College
who achieved more than 6 hours of wear time in at least one eye for
at least 30 days. Patients with epithelial defects, baseline endothelial
cell counts below 1500 cells per square millimeter, or active
intraocular inflammation were excluded. 8 patients (14 eyes) were
included in the study. Each patient had confocal microscopy images
of the corneal endothelium taken prior to PROSE wear, and at each 2
week follow up visit after wearing the device for longer than 6 hours
each day (Confoscan 4, Nidek). Endothelial cell density,
polymegatheism, and pleomorphism were calculated using NAVIS
software before and after PROSE wear and compared using a twotailed paired t test. Correlation between percent change in endothelial
cell density and days of PROSE wear for longer than 6 hours was
assessed using Pearson’s test for linear correlation.
Results: Prior to initiation of PROSE wear mean endothelial cell
count was 2499 +/-371 cells/mm2, which significantly decreased to
2336 +/- 365 cells/mm2 following at least 30 days of 6 hours or more
of daily PROSE wear (p=0.019). Similarly, this amount of wear
caused the percentage of hexagonal endothelial cells to decrease from
a baseline of 54.25% +/- 9.09 to 45.97% +/- 7.98 with device wear
(p=0.014). There was a significant correlation between days of
PROSE wear for more than 6 hours and percent change in endothelial
cell density (r=-0.39, p=0.0098). However, an index of
polymegatheism was not significantly different before or after
PROSE wear (37.05 +/- 9.4 vs. 38.34 +/- 6.59, p=0.41).
Conclusions: Use of the PROSE device is correlated with decreasing
endothelial cell density and increased endothelial cell pleomorphism.
Increased pleomorphism is considered a marker for endothelial cell
stress, and has been associated with contact lens wear. However,
decreased endothelial cell density has not been previously reported in
connection with use of the PROSE device. Endothelial cell loss or
stress may cause corneal edema in PROSE wearers.
Commercial Relationships: Ryan M. St Clair, None; Yvonne
Wang, None; Christopher E. Starr, None; Michelle N. Lee, None;
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Ana G. Alzaga Fernandez, None; Kimberly C. Sippel, None;
Jessica Ciralsky, None; Mark Rosenblatt, None; Priyanka Sood,
None
Support: Research to Prevent Blindness
Program Number: 1683 Poster Board Number: D0318
Presentation Time: 8:30 AM - 10:15 AM
Clinical Manifestation and Effect of Ganciclovir Therapy for
Cytomegalovirus Corneal Endotheliitis
Tsutomu Inatomi1, Noriko Koizumi2, Yuichi Ohashi3, Yoshitsugu
Inoue4, Manabu Mochizuki5, Kohji Nishida6. 1Ophthalmology, Kyoto
Prefectural Univ of Med, Kamigyo-Ku, Japan; 2Biomedical
Engineering Faculty of Life and Medical Sciences, Doshisha
University, Kyoto, Japan; 3Ophthalmology, Ehime University School
of Medicine, Ehime, Japan; 4Ophthalmology, Tottori University,
Faculty of Medicine, Tottori, Japan; 5Ophthalmology, Tokyo Medical
and Dental University Graduate School, Tokyo, Japan;
6
Ophthalmology, Osaka University Graduate School of Medicine,
Osaka, Japan.
Purpose: To report the findings of an epidemiological survey
conducted in Japan regarding the clinical manifestations and the
effect of ganciclovir antiviral therapy for the treatment of patients
with cytomegalovirus (CMV) corneal endotheliitis.
Methods: An epidemiological survey was conducted in Japan and
the clinical manifestations of 109 eyes of 106 cases diagnosed as
CMV corneal endotheliitis were analyzed retrospectively.
Prospective, multicenter clinical treatment was performed for 7 eyes
of 7 cases. The mean patient age was 67.4±10.4 years, and the mean
follow-up period was 12 months. The combined therapy of systemic
ganciclovir (5mg/kg, 2 times daily) with topical 0.5% ganciclovir
instillation was applied for 2 weeks following the tapering of topical
instillation. Clinical evaluation was performed by use of real-time
polymerase chain reaction (PCR) and slit-lamp examination.
Results: Of the total 106 cases, 85 cases (80.2%) were male and 21
cases (19.8%) were female. Coin-shaped lesions and linear keratic
precipitates (KPs) were observed in 70.6% and 8.3% of the cases,
respectively. Clinical manifestations included corneal edema
(73.4%), endothelial loss (81.7%), iritis (67.9%), and elevation of
intraocular pressure (66.1%). In the prospective study, the average
CMV viral load in aqueous humor before treatment was 8.55x104
copies/ml (range: 8.3x102 - 1.55x105 copies/ml). Coin-shaped
lesions or linear KPs were detected in 100% and 29% of the cases,
respectively, and 85.7% of the cases showed sustained virologic
response after the initial combined therapy. All cases showed
negative by PCR with additional topical ganciclovir therapy, but 3
cases (42.8%) showed the elevation of intraocular pression and 1 case
(14.5%) showed the recurrence. Mean endothelial cell density (CED)
(cell/mm2 ) at before and 1-month post treatment was 1160±365 and
1048±440, respectively. No significant reduction of CED and no
adverse effects were observed.
Conclusions: Corneal endotheliitis with coin-shaped lesions or linear
KPs, similar to the clinical rejection line, are major clinical
manifestations in CMV corneal endotheliitis. The combined therapy
of systemic and topical ganciclovir instillation is safe and effective
for the treatment of patients with CMV corneal endotheliitis.
Commercial Relationships: Tsutomu Inatomi, None; Noriko
Koizumi, None; Yuichi Ohashi, None; Yoshitsugu Inoue, None;
Manabu Mochizuki, Santen (F), Senju (F), Ohtsuka (F), DaiichiSankyo (F), Mitsubishi-Tanabe (F), AMO Japan (F), Alcon Japan
(F); Kohji Nishida, Alcon (C), Alcon (F), HOYA (F), Senju (F),
Pfizer (F), Santen (F), Osaka University (P)
Support: Research on Measures for Intractable Diseases 074
Program Number: 1684 Poster Board Number: D0319
Presentation Time: 8:30 AM - 10:15 AM
Expansion of corneal endothelial cells using biomimetic
engineered substrates
Rachelle Palchesko1, 3, James L. Funderburgh2, 3, Adam W.
Feinberg1, 3. 1Biomedical Engineering, Carnegie Mellon University,
Pittsburgh, PA; 2Ophthalmology, University of Pittsburgh,
Pittsburgh, PA; 3Louis J Fox Center for Vision Restoration,
University of Pittsburgh, Pittsburgh, PA.
Purpose: Corneal endothelial cells (CECs) are non-proliferative in
vivo with minimal proliferation in vitro, making expansion of these
cells for therapeutic application difficult. Our previous work has
shown that culturing cells on a biomimetic substrate that mimics the
mechanical and biochemical properties of Descemet’s membrane
enables the expansion of CECs >3000-fold compared to 139-fold on
tissue culture polystyrene (TCPS). Here, we demonstrate that the
biomimetic substrate also maintains CEC phenotype and prevents
transition to senescence or a fibroblast-like phenotype.
Methods: CECs were isolated from bovine corneas and cultured on
one of three different surfaces: the biomimetic substrate consisting of
collagen type IV coated polydimethylsiloxane soft elastomer (COL4PDMS) and two controls, TCPS and collagen type IV coated TCPS
(COL4-TCPS). CECs were cultured for 8 passages and
immunofluorescently labeled at passages 1, 5, and 8 for fibronectin
(FN), zonal occludins (ZO-1) and F-actin to analyze changes in
extracellular matrix production, cell-cell coupling and polygonal
morphology. At these same time points qRT-PCR was used to
quantify mRNA expression of COL4A2, COL8A1, and SLC4A4 as
CEC markers and COL3A1 as the fibroblastic gene marker.
Results: CECs cultured on the COL4-PDMS produced short,
immature FN fibrils at all time points where as CECs on TCPS and
COL4-TCPS produced large fibrils at passages 5 and 8 similar to
those produced by fibroblasts. On COL4-PDMS CECs grew at higher
density, had more continuous ZO-1 staining and maintained a
polygonal morphology. Gene expression followed a similar pattern
with fibroblast associated FN and COL3A1 genes higher on TCPS
and COL4-TCPS and CEC associated COL8A1 and SLC4A4 genes
maintained at levels comparable to CECs in vivo on COL4-PDMS.
Conclusions: We have demonstrated that a biomimetic substrate
which recapitulates the mechanical stiffness and collagen type IV
composition of Descemet’s membrane significantly enhances
expansion and maintains phenotypic stability of CECs in vitro
compared to TCPS and COL4-TCPS controls. Current efforts are
focused on extending this system to the expansion of human CECs to
enhance its clinical relevance. The ability to expand CECs is crucial
to obtain the cell numbers necessary for bioengineering a corneal
endothelium suitable for implantation, a future goal of this research
project.
Commercial Relationships: Rachelle Palchesko, None; James L.
Funderburgh, None; Adam W. Feinberg, Carnegie Mellon
University (P)
Support: This work was supported by the Ocular Tissue Engineering
and Regenerative Ophthalmology funding through the Louis J. Fox
Center for Vision Restoration, National Institutes of Health CORE
Grant P30 EY008098, EY09368 (to JLF), the Eye and Ear
Foundation of Pittsburgh, PA and an unrestricted Grant from
Research to Prevent Blindness, New York, NY
Program Number: 1685 Poster Board Number: D0320
Presentation Time: 8:30 AM - 10:15 AM
Meganuclease Targeting HSV-1 Protects Against Corneal
Endothelitis ex-vivo and in-vivo
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Eric E. Gabison1, 2, Marc Labetoulle4, Marine Gailledrat5, Jose A.
Sahel3, Benoit Chapelier3. 1Cornea Department, Fondation A. de
Rothschild, Paris, France; 2Ophthalmology Department, Hôpital
Bichat, Paris, France; 3Institut de la vision, Paris, France;
4
Ophthalmology Department, Hôpital du Kremlin Bicêtre, Paris,
France; 5Cellectis, Paris, France.
Purpose: Herpetic Keratitis is a leading cause of decreased best
corrected visual acuity in developed and developing countries. The
aim of this study is to assess the antiviral property of a meganuclease
targeting HSV in the prevention of HSV endothelitis ex-vivo and in
vivo.
Methods: Normal rabbit corneas were placed in organ culture and
transduced by a recombinant adeno-associated virus (rAAV)
allowing constitutive expression of meganucleases targeting HSV-1
genome or of a non-coding sequence. These organs were then
submitted to infection by recombinant HSV-1 F(1) virus equipped
with a LacZ expressing cassette at M.O.I. 0.001 to 0.1%. Infection
rates for plaques or cells in endothelium were established by
immunostaining of envelope protein gD or X-gal staining after the
end of first or second lytic cycle. Additionnaly, a rabbit model of
corneal HSV endothelitis was developed. Intracamerular injection of
HSV F(1) were performed in rabbit eyes 2 days following intravitreal
injection of steroid. Corneal edema, keratic precipitates, ocular
inflammation and infection rate for plaques or cells were analyzed in
this model.
Results: Meganuclease targeting the ICP0 gene which encodes an E3
ubiquitin ligase involved in viral reactivation and replication did not
change infection rates in the present organ culture model, but reduced
the average size of plaques in endothelium with a decrease of 2746%. Conversely, the meganuclease directed against the major capsid
protein UL19 lowered the number and size of plaques, both being
reduced by half at M.O.I. 0.001%. Consequently, the expression of a
meganuclease in endothelium, evidenced by RT-PCR, could either
reduce infectious particle production or induce cell resistance to
HSV-1. In vivo, experiments demonstrated a 30% decreased in
endothelial plaque formation. The rate of corneal edema and keratic
precipitates was reduced in Megnuclease treated eyes as compared to
controles.
Conclusions: Our organ culture and in vivo model of herpetic
endothelial infection are reproducible and efficient to quantify viral
proliferative capacity. Meganuclease transduction confers a
significant inhibition of viral pathogenic effect. Meganuclease gene
therapy targeting HSV-1 DNA may be an effective treatment to
protect against HSV endothelitis
Commercial Relationships: Eric E. Gabison, None; Marc
Labetoulle, None; Marine Gailledrat, Cellectis SA (E); Jose A.
Sahel, UPMC/Essilor (P), Second Sight (F); Benoit Chapelier, None
Support: OSEO ACTIVE GRANT FRANCE
Program Number: 1686 Poster Board Number: D0321
Presentation Time: 8:30 AM - 10:15 AM
Comparison of Endothelial Cell Density at the Central and
Peripheral Regions in a DSAEK Graft
Hiroko Nakagawa, Tsutomu Inatomi, Shigeru Kinoshita. Kyoto
Prefectural Univ of Med, Kyoto, Japan.
Purpose: To analyze endothelial remodeling, including the wound
healing, at the host/graft junction after Descemet’s Stripping
Automated Endothelial Keratoplasty (DSAEK), and compare the
change of corneal endothelium at the central and peripheral regions
of the graft using wide-field specular microscopy.
Methods: This study involved 10 eyes of 10 patients (mean age: 72
years) treated by DSAEK using internationally shipped precut donor
corneas. None of the patients had ocular complications prior to
surgery, and DSAEK was performed via the pull-through technique
using a Busin glide. Wide-field contact specular microscopy was
used to evaluate the alteration of endothelium at the central and
temporal peripheral corneal regions (approximately 1mm inside from
the graft edge) at 1, 6, and 12 months after surgery. Endothelial cell
density (ECD), coefficient of variation (CV), and the frequency of
hexagonal cells (6A) were analyzed to elucidate the post-DSAEK
endothelial remodeling pattern.
Results: Mean regional endothelial cell density (cells/mm2±SD) in
the center / periphery at 1, 6, and 12 months postoperative were
2286±409 / 1893±432, 2152±464 / 1550±284, and 2138±466 /
1152±378, respectively. Peripheral ECD was statistically lower than
central ECD at each follow-up time-point (p<0.05). At 12 months
postoperative, peripheral ECD was 38.8±11.6% lower than the
central ECD. ECD reduction rates of the central and peripheral
regions from 1 to 12 months postoperative were 6.9±7.6% and
29.2±14.7%, respectively. ECD reduction rates of the periphery were
statistically higher than of the center (p<0.01). The 6A(%) of the
central/peripheral region at 1, 6, and 12 months postoperative were
47.1/46.5, 56.0/55.0, and 58.4/57.1, respectively. CV of the
central/peripheral region at 1, 6, and 12 months postoperative were
0.31/0.33, 0.29/0.30, and 0.33/0.29, respectively.
Conclusions: Regional differences of ECD were found in the
DSAEK graft. Endothelial cell loss appeared in the peripheral region
of the graft and extended to the central region during the endothelial
remodeling. Wound healing in the host/graft junction may affect the
long-term survival of transplanted endothelium.
Commercial Relationships: Hiroko Nakagawa, None; Tsutomu
Inatomi, None; Shigeru Kinoshita, Senju Pharmaceutical Co (P),
Santen Pharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon
(R), AMO (R), HOYA (R)
Program Number: 1687 Poster Board Number: D0322
Presentation Time: 8:30 AM - 10:15 AM
Age-Dependent Differential Gene Expression in Human Corneal
Endothelium
Cynthia Wang, Ricardo F. Frausto, Anthony J. Aldave. Jules Stein
Eye Institute, Los Angeles, CA.
Purpose: To determine gene expression in the corneal endothelium
of healthy children and adults to provide insight into the normal ageassociated changes in corneal endothelial cell gene expression.
Methods: Total RNA was isolated from corneal endothelium
obtained from four pediatric (≤11 years old) and four adult (≥53
years old) donor eye bank corneas. The RNA samples were processed
and hybridized to the Affymetrix GeneChip 1.1ST array. Analysis of
the raw intensity values contained within CEL files was performed
using the gene expression module within the Partek Genomics Suite
software. A list of the most significant differentially expressed genes
between pediatric and adult corneal endothelium was obtained using
a p-value and fold-change cutoff of 0.01 and 2, respectively.
Results: Principal component analysis (PCA) of our expression
dataset demonstrated two distinct populations of samples in PCA1
with each being comprised of either the pediatric or adult samples,
while hierarchical clustering showed a strong relationship within
samples originating from the same age group. Differential gene
expression analysis identified ten significantly regulated genes, of
which seven encode for protein: ITGBL1, PREX2, DIO2, DLL4,
C9orf131, HIST1H3A, and TNFAIP3.
Conclusions: The identification of several differentially expressed
genes in pediatric and adult corneal endothelial cells suggests that
changes in gene transcription continue well after birth. After
validating the results of this study, we plan to investigate the role of
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
each significantly differentially expressed gene in normal and
abnormal corneal endothelial cell function.
Commercial Relationships: Cynthia Wang, None; Ricardo F.
Frausto, None; Anthony J. Aldave, Alcon (R), Allergan (R), NIH
(F), Bausch + Lomb (C), Allergan (C)
Support: RPB
Program Number: 1688 Poster Board Number: D0323
Presentation Time: 8:30 AM - 10:15 AM
ROCK inhibitor enhances adhesion and wound healing on
human corneal endothelial cells
Michael J. Nicolas1, Aurélien Pipparelli1, Yvan Arsenijevic1, Gilles
Thuret2, Philippe Gain2, Francois Majo1. 1Ophtalmology, JulesGonin eye hospital, Lausanne, Switzerland; 2Ophtalmology,
University of St Etienne, St Etienne, France.
Purpose: Recently it was reported that the ROCK inhibitor Y-27632
promotes adhesion, inhibits apoptosis, increases the number of
proliferating monkey corneal endothelial cells in vitro and enhance
corneal endothelial wound healing both in vitro and in vivo. Here, we
proposed to evaluate the effects of ROCK inhibitor on HCEC either
in vitro or ex vivo, firstly to assess the potential of this compound to
increase the number of corneal graft available for the clinic and
secondly to validate the previous results obtained in animal models, a
step required before potential clinical application.
Methods: Using organ culture human cornea (N=34), the effect of
ROCK inhibitor was evaluated either in vitro or ex vivo. Toxicity,
endothelial cell density, cell proliferation, apoptosis, cell
morphometry, adhesion and wound healing process were evaluated
by live/dead assay standard cell counting method, EdU labelling,
Ki67, Caspase3, Zo-1 and Actin immunostaining.
Results: In our study, we demonstrated for the first time in human
endothelial cells ex vivo and in vitro, that ROCK inhibitor did not
induce any toxicity effect and did not modulate metabolism activity.
Compared to animal model, ROCK inhibitor treatment did not induce
human endothelial cell proliferation. However, ROCK inhibitor
significantly enhances corneal endothelial cell adhesion and wound
healing.
Conclusions: These results strongly suggest that ROCK inhibitor is a
promising and safe compound to improve the treatment of corneal
endothelial dysfunction in human. ROCK inhibitor could be a
potential therapeutic strategy in order to improve adhesion of
transplanted human cultured endothelial cells. Furthermore, ROCK
inhibitor treatment can increase in human the closure of endothelial
cell defect.
Commercial Relationships: Michael J. Nicolas, None; Aurélien
Pipparelli, None; Yvan Arsenijevic, None; Gilles Thuret, None;
Philippe Gain, None; Francois Majo, None
Program Number: 1689 Poster Board Number: D0324
Presentation Time: 8:30 AM - 10:15 AM
Controlled Release of a Rho Kinase (ROCK)-Selective Inhibitor
with Polylactic Acid Microspheres
Sho Koda1, 2, Takashi Saito2, Junji Kitano1, Naoki Okumura1, 3,
Shigeru Kinoshita3, Yasuhiko Tabata2, Noriko Koizumi1, 3.
1
Biomedical Engineering, Doshisha University, Kyotanabe, Japan;
2
Biomaterials, Kyoto University, Kyoto, Japan; 3Ophthalmology,
Kyoto Prefectural University of Medicine, Kyoto, Japan.
Purpose: We previously reported that the Rho kinase (ROCK)
inhibitor Y-27632 promoted the wound healing of corneal endothelial
cells in a primate model. Thus, Y-27632 shows promise as a drug that
can be used for the clinical treatment of corneal endothelial defects.
The purpose of this present study was to design polylactic acid (PLA)
microspheres that can be used for the controlled release of Y-27632
in the anterior chamber.
Methods: PLA microspheres incorporating Y-27632 (Y-27632-PLA)
were prepared via a double-emulsion [(water in oil) in water] solvent
evaporation method. The in vitro release test of Y-27632-PLA was
performed in a phosphate-buffered solution (pH7.4) at 37°C.The
amount of Y-27632 released was determined by use of highperformance liquid chromatography (HPLC). To evaluate the toxicity
of PLA microspheres without Y-27632 after they were injected into
the anterior chamber of rabbit eyes, slit-lamp examinations, and
corneal thickness and intraocular pressure measurements were
performed up to14 days after injection, followed by
immunohistochemical analysis.
Results: Y-27632 was released from PLA microspheres over 28
days. The percent of Y-27632 released was 70.0, 77.9, and 98.2% at
7, 14, and 28 days, respectively. The release pattern of Y-27632
could be changed by altering the molecular weight of the PLA that
was used. In all eyes injected with the PLA microspheres, normal
corneal thickness was observed, with no inflammation or elevation of
intraocular pressure. The corneal endothelial cell density and the
expression of functional protein were found to be normal.
Conclusions: The findings of this study demonstrate that PLA
microspheres are promising candidates for the controlled release of
Y-27632. The Y-27632-PLA is applicable as a pharmaceutical agent
for the treatment of corneal endothelial dysfunction.
Commercial Relationships: Sho Koda, None; Takashi Saito,
None; Junji Kitano, None; Naoki Okumura, None; Shigeru
Kinoshita, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co
(P), Otsuka Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA
(R); Yasuhiko Tabata, None; Noriko Koizumi, None
Support: The Funding Program for Next Generation World-Leading
Researchers from the Cabinet Office in Japan ( LS117) The
Adaptable and Seamless Technology Transfer Program through
Target-driven R&D (AS2314212G)
Program Number: 1690 Poster Board Number: D0325
Presentation Time: 8:30 AM - 10:15 AM
Rho-kinase inhibitor enhances corneal endothelial cell
proliferation via p27 degradation
Ryohei Numata1, 2, Naoki Okumura1, 2, EunDuck P. Kay1, Makiko
Nakahara1, Shinichiro Nakano1, Morio Ueno2, Shigeru Kinoshita2,
Noriko Koizumi1. 1Biomedical Engineering, Doshisha University,
Kyotanabe, Japan; 2Ophthalmology, Kyoto Prefectural University of
Medicine, Kyoto, Japan.
Purpose: We previously reported that Rho kinase (ROCK) inhibitor
promotes wound healing of corneal endothelial cells in an animal
model and showed the possibility of the clinical application for
corneal endothelial deficient condition. The purpose of this study is
to determine the mechanism by which ROCK inhibitor promotes cell
proliferation in corneal endothelial cells.
Methods: Cultivate monkey corneal endothelial cells (MCECs) were
used for this study. Cell proliferation was analyzed by two methods
in the absence or presence of 10 μM the selective ROCK inhibitor
(Y-27632): BrdU ELISA assay to determine the incorporation of
BrdU into the newly synthesized DNA and CellTiter Glo assay to
measure the numbers of viable cells. Activation of Akt and
expression of Cdc25A and p27 were analyzed by western blotting.
Results: Incorporation of BrdU into the newly synthesized DNA was
doubled in the cells treated with ROCK inhibitor when compared to
that of the control cells. Likewise, the viable cell numbers of MCECs
treated with the inhibitor for 24 h or 48 h were 50% greater than the
cell numbers in the absence of ROCK inhibitor. In addition to the
increased cell numbers, the ROCK inhibitor promoted cell adhesion.
To elucidate the action of ROCK inhibitor in MCECs with or without
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
ROCK inhibitor, phosphorylation of Akt was analyzed for 1 h up to
24 h in serum-containing medium: in the absence of ROCK inhibitor,
phosphorylation of Akt was gradually increased to 12 h. Unlike the
control pattern, phosphorylation of Akt reached maximum at 1 h
following treatment of cells with ROCK inhibitor, after which the
level of phosphorylated Akt was gradually decreased. The expression
of Cdc25A, which activates Cdk2 that phosphorylates p27, was
observed during the early time periods in the presence of ROCK
inhibitor, while the control cells showed a late expression pattern of
Cdc25A. Similarly, the amount of p27 was greatly reduced from 1 h
following treatment of cells with ROCK inhibitor, whereas the
control cells maintained high levels of p27 up to 12 h.
Conclusions: The findings of the study demonstrate that ROCK
inhibitor activates PI 3-kinase signaling which subsequently
promotes degradation of p27 via Cdc25A pathway, thus leading to
cell proliferation of CECs. These data suggest that the ROCK might
be a useful pharmaceutical agent for corneal endothelial disease.
Commercial Relationships: Ryohei Numata, None; Naoki
Okumura, None; EunDuck P. Kay, None; Makiko Nakahara,
None; Shinichiro Nakano, None; Morio Ueno, None; Shigeru
Kinoshita, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co
(P), Otsuka Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA
(R); Noriko Koizumi, None
Support: The Adaptable and Seamless Technology Transfer
Program through Target-driven R&D (AS2314212G),The Funding
Program for Next Generation World-Leading Researchers from the
Cabinet Office in Japan ( LS117)
Program Number: 1691 Poster Board Number: D0326
Presentation Time: 8:30 AM - 10:15 AM
Proliferation Propensity of Cultured Human Corneal Endothelial
Cells and Their Plasticity Dictated by Culture
Microenvironments
Munetoyo Toda1, Kana Nakata1, kazuko asada1, Michio Hagiya1,
Morio Ueno1, Naoki Okumura2, Noriko Koizumi2, Junji Hamuro1,
Shigeru Kinoshita1. 1Department of Ophthalmology, Kyoto
Prefectural University of Medicine, Kyoto, Japan; 2Department of
Biomedical Engineering, Doshisha University, Kyoto, Japan.
Purpose: It is well known that human corneal endothelium cells
(HCECs) have poor proliferative ability under in vitro culture
conditions. The difficulty of cultivating HCECs hampers a detailed
analysis of their proliferation propensity. To detail molecular
mechanisms underlying this impaired HCEC proliferation, we
attempted to clarify the presence and proliferation propensity of
functionally heterogeneous subpopulations in cultured HCECs.
Methods: The proliferative properties of cultured HCECs were
evaluated by BrdU assay and carboxyfluorescein succinimidyl ester
(CFSE) dye dilution assay. To investigate if cultured HCECs contain
subpopulations with distinct metabolic requirements, HCECs were
stained with MitoTracker® Red (Life Technologies Corp., Carlsbad,
CA) to evaluate their mitochondria content. Flow cytometry (FCM)
using several surface markers was performed to characterize the
subpopulations.
Results: Cell percentages in the G1, S, and G2/M phase of the cell
cycle were determined by FCM using BrdU and 7-AAD. The
percentage of cells in the S- and G2/M-phase was about 40%, and
about 60% of the HCECs were arrested in the G1-phase. CFSE assay
detected 2 subpopulations with different proliferative properties. One
divided 7 times in 8 days of cultivation, while the other stopped cell
division at 3 times. We theorize that the latter entered premature cell
senescence at the early stage of cultivation, resulting in cell-cycle
arrest. These results suggest that each subpopulation has unique
metabolic turnover rates and energy requirements, as it is theorized
that poor proliferation is tied to energy from mitochondria, not from
glucose metabolism. Moreover, FMC using MitoTracker® Red
detected different mitochondrial content in the cultured HCECs. Each
subpopulation was stained with several cell surface markers selected
by global analysis, and we tried to characterize candidate markers for
the high-proliferative population.
Conclusions: These findings show that cultured HCECs have
different subpopulations and provide the possibility of establishing an
effective method for culturing HCECs containing an enriched
subpopulation with high proliferation ability.
Commercial Relationships: Munetoyo Toda, None; Kana Nakata,
None; kazuko asada, None; Michio Hagiya, JCR Pharmaceuticals
Co., Ltd (E); Morio Ueno, None; Naoki Okumura, None; Noriko
Koizumi, None; Junji Hamuro, None; Shigeru Kinoshita, Senju
Pharmaceutical Co (P), Santen Pharmaceutical Co (P), Otsuka
Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)
Program Number: 1692 Poster Board Number: D0327
Presentation Time: 8:30 AM - 10:15 AM
Cell-injection Therapy Using Rho Kinase Inhibitor in a Corneal
Endothelial Dysfunction Rabbit Model
Junji Kitano1, Naoki Okumura1, 2, EunDuck P. Kay1, Morio Ueno2,
Junji Hamuro2, Shigeru Kinoshita2, Noriko Koizumi1, 2. 1Biomedical
Engineering, Doshisha University, Kyotanabe, Japan;
2
Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,
Japan.
Purpose: To investigate feasibility of corneal endothelial
reconstruction by a cell-injection therapy using cultivated rabbit
corneal endothelial cells (RCECs) in a corneal endothelial
dysfunction rabbit model and to determine the optimum cell numbers
for the cell-injection therapy.
Methods: Rabbit corneal endothelium was denuded by intensive
mechanical scraping. Cultivated RCECs in the presence of 100μM of
Rho kinase (ROCK) inhibitor, Y-27632, were injected into the
anterior chamber of the host animals at three concentrations (2x10 5
cells, 5.0x105cells, or 1.0x106cells). The eyes of each animal were
kept in the face-down position for 3 hours. Slit-lamp examinations,
corneal thickness- and intraocular pressure- measurements, and
immunohistochemical analysis were performed for up to 14 days.
Results: All eyes received cell-injection therapy showed improved
corneal clarity; corneal clarity and corneal thickness recovered the
fastest rate in the host animals that received cultivated RCECs at
1.0x106cell numbers. In all animal groups, corneal endothelium
demonstrated the characteristic contact-inhibited monolayer with
polygonal cells that express the functional endothelial phenotypic
proteins, ZO-1 and Na+/K+-ATPase. When the endothelial cell
density of the host animals was measured, the animal that received a
cell injection at 1.0x106 cells demonstrated significant high cell
density with 3296.6±365.1 cells/mm2, while the other two animal
groups showed 1432.0±200.8 cells/mm2 (injected cell numbers:
2.0x105) and 2252.2±204.6 cells/mm2 (injected cell numbers: 5.0x105
cells). None of the eyes of the experimental animals showed elevated
intraocular pressure or immunological rejection.
Conclusions: The findings provide evidence that the cell-injection
therapy using appropriate cell numbers with ROCK inhibitor enables
corneal endothelial regeneration by tissue engineering technique and
may be a useful clinical alternative for corneal transplantation.
Commercial Relationships: Junji Kitano, None; Naoki Okumura,
None; EunDuck P. Kay, None; Morio Ueno, None; Junji Hamuro,
None; Shigeru Kinoshita, Senju Pharmaceutical Co (P), Santen
Pharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon (R),
AMO (R), HOYA (R); Noriko Koizumi, None
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Support: The Funding Program for Next Generation World-Leading
Researchers from the Cabinet Office in Japan ( LS117), The
Highway Program for realization of regenerative medicine
Program Number: 1693 Poster Board Number: D0328
Presentation Time: 8:30 AM - 10:15 AM
Effect of ROCK inhibiter on Apoptosis in Corneal Endothelial
Cells
Ai Odajima1, 2, Naoki Okumura1, 2, EunDuck P. Kay1, Wen Chen1,
Morio Ueno2, Shigeru Kinoshita2, Noriko Koizumi1. 1Department of
Biomedical Engineering, Faculty of Life and Medical Sciences,
Doshisha University, Kyotanabe, Japan; 2Department of
Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,
Japan.
Purpose: Apoptosis is reportedly involved in pathological condition
of corneal endothelium such as Fuchs dystrophy or post keratoplasty.
The purpose of this study is to investigate the feasibility of Rho
kinase (ROCK) inhibitorfor modulating apoptosis in corneal
endothelial cells.
Methods: Monkey corneal endothelial cells (MCECs) were cultured
and were then treated by UV radiation. Cell death was evaluated by
trypan blue staining.Apoptosis was evaluated by the quantity of the
released cytochrome c and caspase 3 (western blotting), activation
level of caspase 3 (CaspaseGlo 3/7 Assay), DNA flagmentation
(TUNEL). As an ex vivo apoptosis model, rabbit corneal tissue was
treated by either UV (100 J/m2) or H2O2 (100 µM). Then, apoptotic
cells were evaluated by Annexin Vimmunostaining after 24 hours.
Results: MCECs were exposed to UV radiation ranging from 10 to
1000J/m2, there was cell death in a dose-response manner; substantial
cell death was observed from 50 J/m2.UV radiation caused massive
release of cytochrome c into the cytoplasm; and cleavage of caspase
3 was increased with UV radiation. TUNEL positive DNA
fragmentationwas detected in the UV radiated cells.ROCK inhibitor
significantly decreased UV induced Annexin V-positive apoptotic
corneal endothelial cellsin rabbit corneal tissue compared to the
control (2.1±0.8% and10.9±3.3%, respectively)(p<0.01).
Likewise,ROCK inhibitor significantly decreased H2O2 induced
apoptotic cells compared to the control (2.3±1.1%
and11.6±1.0%,respectively)(p<0.01).
Conclusions: The findings of the present study indicate that UV
radiation induces apoptosis in CECs and that ROCK inhibiter could
protect corneal endotheliumfrom the apoptosis leading to cell death.
Modulation of corneal endothelial apoptosis by ROCK inhibitor
might be a useful therapeutic approach for corneal endothelial
disease.
Commercial Relationships: Ai Odajima, None; Naoki Okumura,
None; EunDuck P. Kay, None; Wen Chen, None; Morio Ueno,
None; Shigeru Kinoshita, Senju Pharmaceutical Co (P), Santen
Pharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon (R),
AMO (R), HOYA (R); Noriko Koizumi, None
Support: The Adaptable and Seamless Technology Transfer
Program through Target-driven R&D (AS2314212G),The Funding
Program for Next Generation World-Leading Researchers from the
Cabinet Office in Japan ( LS117)
Program Number: 1694 Poster Board Number: D0329
Presentation Time: 8:30 AM - 10:15 AM
Rho kinase inhibitor promotes cell adhesion of corneal
endothelial cells through inhibiting phosphorylation of MLC
Yuki Tsujimoto1, 2, Naoki Okumura1, 2, EunDuck P. Kay1, Ryohei
Numata1, Shigeru Kinoshita2, Noriko Koizumi1. 1Biomed Eng,
Doshisha University, Kyotanabe, Japan; 2Ophthalmology, Kyoto
Prefectural University of Medicine, Kyoto, Japan.
Purpose: We previously reported that Rho kinase (ROCK) inhibitor
promoted cell adhesion of cultivated corneal endothelial cells, and
enabled cell injection therapy combined with ROCK inhibitor in
cornea endothelial dysfunction animal model. The purpose of this
study is to determine the mechanism how ROCK inhibitor promotes
corneal endothelial cell adhesion on the substrate.
Methods: Cultivate monkey corneal endothelial cells (MCECs) were
used for this study. To examine the involvement of Rho/ROCK
signaling in cell adhesion, RhoA activity and phosphorylation of
MLC in MCECs cultured on non-adhesion or on adhesion culture
plate was evaluated by G-LISA RhoA activation assay and western
blotting. Next, to elucidate the effect of ROCK inhibitor on cell
adhesion, phosphorylation of MLC, FAK, and paxillin of the MCECs
seeded with or without ROCK inhibitor (Y-27632) was evaluated by
western blotting. The expression of actin and vinculin was also
determined by immunostaining. Then, adhere cell numbers of the
MCECs seeded with either C3 (RhoA inhibitor) or blebbistatin (MLC
inhibitor) were evaluated by CellTiter Glo assay.
Results: The activity of RhoA and phosphorylation of MLC was
higher in MCECs cultured on non-adhesion culture plate compared to
on adhesion culture plate. In MCECs seeded with ROCK inhibitor,
phosphorylation of MLC was suppressed, and phosphorylation of
FAK and paxillin was promoted compared to the control. ROCK
inhibitor also promoted actin spreading and the expression of
vinculin. Adhered cell numbers were significantly enhanced both by
C3 and blebbistatin compared to the control (125.4±4.7%,
172.3±1.7%, respectively) (p<0.05).
Conclusions: Our findings indicate that ROCK inhibitor activates
focal adhesion proteins through phosphorylation of MLC by
inhibiting Rho/ROCK signaling pathway. Modulation of cell
adhesion property on the substrate by ROCK inhibitor might provide
a new tool for cell based tissue engineering therapy.
Commercial Relationships: Yuki Tsujimoto, None; Naoki
Okumura, None; EunDuck P. Kay, None; Ryohei Numata, None;
Shigeru Kinoshita, Senju Pharmaceutical Co (P), Santen
Pharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon (R),
AMO (R), HOYA (R); Noriko Koizumi, None
Support: The Funding Program for Next Generation World-Leading
Researchers from the Cabinet Office in Japan ( LS117),The Highway
Program for realization of regenerative medicine
Program Number: 1695 Poster Board Number: D0330
Presentation Time: 8:30 AM - 10:15 AM
Quantitative Assessment of Endothelial Cell Loss of DMEK
Prepared Grafts by Eye Bank Technicians
Jeffrey D. Holiman1, Julia Talajic2, David Davis-Boozer1, 2, Mark A.
Terry2. 1Lions VisionGift, Portland, OR; 2Devers Eye Institute,
Portland, OR.
Purpose: To quantitatively assess the endothelial cell loss that occurs
during pre-stripping of Descemet’s membrane (DM) by eye bank
technicians in preparation for Descemet’s membrane endothelial
keratoplasty (DMEK) surgery.
Methods: Four research corneas with endothelium suitable for
transplant were selected for DMEK preparation. Each donor
corneoscleral rim was mounted on a trephine and DM was scored ~
1.0mm central to Schwalbe’s line. Cornea stained with trypan blue to
visualize endothelial damage prior to stripping and to enhance
visibility of free edge of DM. DM was peeled > 90 % and returned to
native position. DM was trephinated, saturated with Calcein AM
stain and unfolded on slide utilizing Healon. Adobe Photoshop was
used to assess endothelial cell loss (ECL) following florescent
photomicroscopy.
Results: Of the four corneas attempted for DMEK preparation, four
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
were successful. The DMEK grafts revealed a mean ECL of 17.5%
(range 14 to 21%).
Conclusions: Corneas can be prepared for DMEK surgery by eye
bank technicians and post-stripping microscopy can be performed to
assess graft quality. Additionally, the risk of tissue wastage is
transferred while conserving costly operating room time. Increasing
demand for DMEK prepared tissue warrants further investigations
regarding optimization of preparation techniques.
Commercial Relationships: Jeffrey D. Holiman, None; Julia
Talajic, None; David Davis-Boozer, None; Mark A. Terry, Bausch
and Lomb Surgical (R), Alcon (R), Optovue (C)
Program Number: 1696 Poster Board Number: D0331
Presentation Time: 8:30 AM - 10:15 AM
Interleukin-1β enhances cell migration through AP-1 and NF-κB
pathway dependent FGF2 expression in human corneal
endothelial cells
Daniel Sand, JeongGoo Lee, J M. Heur. Department of
Ophthalmology, Keck School of Medicine of the University of
Southern California, Los Angeles, CA.
Purpose: To investigate IL-1β signaling in migration of human
corneal endothelial cells (CECs).
Methods: Cell migration was measured using scratch-induced
directional migration assay. Expression and/or activation of IRAK, PI
3-kinase, p38, IKK, IκB, NF-κB, and FGF-2 were analyzed by
immunoblotting. AP-1 and NF-κB activities were measured by AP-1
and NF-κB ELISA assay kit, respectively. Binding of AP-1 and NFκB to the promoter region of the FGF-2 gene was determined by
chromatin immunoprecipitation.
Results: Stimulation of human CECs with IL-1β activated expression
of FGF2 and resulted in enhanced cell migration. This, in turn, was
abolished by treatment with either IL-1 receptor antagonist or SU5402, a pan FGF inhibitor. PI 3-kinase or IRAK 1/4 antagonists
demonstrated that IRAK 1/4 activates PI 3-kinase, which in turn
phosphorylates p38 and IKK α/β, leading to FGF2 expression
through activation of AP-1 and NF-κB in human CECs. Treatment of
IL-1β stimulated human CECs, with either AP-1 or NF-κB
antagonists, decreased FGF2 expression and resulted in reduced IL1β enhanced cell migration. Co-treatment of IL-1β stimulated human
CEC with both inhibitors completely blocked FGF2 expression and
IL-1β enhanced cell migration. Chromatin immunoprecipitation
assays demonstrated that AP-1 and NF-κB directly bind to the FGF2
promoter following IL-1β stimulation.
Conclusions: The results show that binding of IL-1β to its receptor in
human CEC leads to parallel activation of AP-1 and NF-κB
pathways, leading, in turn, to FGF2 expression and enhanced cell
migration.
Commercial Relationships: Daniel Sand, None; JeongGoo Lee,
None; J M. Heur, None
Support: Baxter Foundation, RPB, NIH Core Grant EY03040
Program Number: 1697 Poster Board Number: D0332
Presentation Time: 8:30 AM - 10:15 AM
The roles of TWEAK in human corneal endothelial cells
Masahiro Yamaguchi1, Nobuyuki Ebihara1, Toshinari Funaki1, Akira
Murakami1, Satoru Yamagami2. 1Ophthalmology, Juntendo
University, Bunkyo-ku, Japan; 2Ophthalmology, Tokyo University,
Tokyo, Japan.
Purpose: Tumor necrosis factor-like weak inducer of apoptosis
(TWEAK), a member of TNF superfamily, has several roles
including angiogenesis, differentiation control, chemokine
production, and so on. We previously reported the effectiveness of
TWEAK in corneal keratocyte (Ebihara et al, Exp Eye Res, 2009),
and in retinal pigment epithelial cell (Ebihara et al, Curr Eye Res,
2009). The roles of TWEAK in corneal endothelial cells was
examined this time.
Methods: (1) The expression of Fn14, receptor of TWEAK, was
examined with human corneal endothelial cells by FACS. (2) Protein
array and ELISA were conducted to measure TWEAK concentration
in human aqueous humor. (3) The chemokine expression in cultured
human corneal endothelial cells (cHCECs) was examined with
TWEAK or TWEAK/TFG-β. (4) Cell adhesion, proliferation, and
migration of cHCECs were investigated with adhesion, proliferation
and in vitro scratch assays, in addition of TWEAK or TWEAK/TGFβ.
Results: (1)Fn14 receptor was expressed on corneal endothelial cells.
(2) Soluble TWEAK was slightly detected in human aqueous humor.
(3) TWEAK stimulated the expression of RANTES, IL-8, MCP-1 in
cHCECs. TGF-β inhibited previous expression, but TGF-β with
TWEAK lost the inhibition. (4) TWEAK promoted migration of
cHCECs.
Conclusions: There are possibilities that an adequate concentration
of TWEAK in aqueous humor may maintain constancy of corneal
endothelial cells and that excessive expression of TWEAK may
induce inflammation.
Commercial Relationships: Masahiro Yamaguchi, None;
Nobuyuki Ebihara, None; Toshinari Funaki, None; Akira
Murakami, SEED(Japan) JP4855782 (P), SEED(Japan) JP5132958
(P); Satoru Yamagami, None
Program Number: 1698 Poster Board Number: D0333
Presentation Time: 8:30 AM - 10:15 AM
The transparency transcriptome: gene expression profile of
human corneal endothelial cells
Noelia J. Kunzevitzky1, 2, Karen Alvarez-Delfin1, Richard M.
Merkhofer1, Alejandra D. Weisman1, Jeffrey L. Goldberg1, 3. 1Bascom
Palmer Eye Institute & Interdisciplinary Stem Cell Institute,
University of Miami, Miami, FL; 2Emmetrope Ophthalmics, Coral
Gables, FL; 3Shiley Eye Center, University of California San Diego,
San Diego, CA.
Purpose: The corneal endothelium is a monolayer on the inner
cornea whose main function is to maintain corneal transparency. Loss
of function due to dystrophy, trauma or genetic abnormalities leads to
swelling, pain and loss of vision. Current treatment options include
DSAEK or penetrating keratoplasty—both limited by the shortage of
donor corneas. Injection of cultured human corneal endothelial cells
(HCECs) could provide a less invasive and readily available solution.
The protocol for purifying and expanding HCECs in vitro from
cadaveric corneas has been extensively described, but the criteria for
identifying these cells are not definitive. In order to better define the
HCECs’ identity and to ask basic cell biology questions about these
cells, we analyzed the gene expression profile of purified HCECs at
different numbers of passages in vitro and compared it to the
transcriptome of freshly dissected corneal layers.
Methods: Human cadaveric corneas preserved in Optisol® were
procured by the Lions Eye Institute for Transplant and Research
(Tampa, FL). For some corneas, the HCECs were peeled off
Descemet’s membrane and cultured. For other corneas, epithelial,
stromal and endothelial layers were acutely dissected and minimally
processed for RNA extraction. RNA from at least 3 biological
replicates was independently collected, amplified and processed for
hybridization to Affymetrix GeneChip® arrays. We classified probes
by Gene Ontology and compared the gene expression profiles of
cultured HCECs and the three corneal layers.
Results: Cultured HCECs expressed ~82% of 28,869 total probes.
Further gene ontology analysis revealed the overrepresentation of
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
pathways related to cell survival, cytoskeletal remodeling, oxidative
phosphorylation and cell adhesion, among others. We also identified
a number of HCEC-specific markers that can be verified via
immunostaining and RT-PCR.
Conclusions: We have begun to describe the transcriptome of the
cornea, a tissue whose proper function is essential to maintaining
visual clarity, yet remains poorly characterized. In doing so we hope
to unveil the essential molecular players that regulate cell
morphology, survival, and proliferation and to better define HCECs’
identity for further use in cell therapy.
Commercial Relationships: Noelia J. Kunzevitzky, None; Karen
Alvarez-Delfin, None; Richard M. Merkhofer, None; Alejandra
D. Weisman, None; Jeffrey L. Goldberg, None
247 Surgery: Non-Refractive and Keratoprothesis
Monday, May 06, 2013 11:00 AM-12:45 PM
TCC 303 Paper Session
Program #/Board # Range: 1747-1753
Organizing Section: Cornea
Program Number: 1747
Presentation Time: 11:00 AM - 11:15 AM
Microstructural analysis of the cornea after Descemet membrane
endothelial keratoplasty using in vivo confocal microscopy
Akira Kobayashi, Hideaki Yokogawa, Natsuko Yamazaki, Toshinori
Masaki, Kazuhisa Sugiyama. Dept of Ophthalmology, Kanazawa
Univ Sch of Medicine, Kanazawa, Japan.
Purpose: To investigate the in vivo corneal changes in patients with
bullous keratopathy who underwent Descemet membrane endothelial
keratoplasty (DMEK) with the use of in vivo laser confocal
microscopy.
Methods: Five eyes of four patients (three men, one women; mean
age, 61.3±9.6 years [mean ± standard deviation]) with bullous
keratopathy who had undergone successful DMEK were enrolled in
this study. In vivo laser confocal microscopy was performed before
and one, three, and six months after DMEK. Selected confocal
images of corneal layers were evaluated qualitatively and
quantitatively for the degree of haze and the density of deposits.
Subepithelial haze, donor-recipient interface haze, donor-recipient
interface particles, and host stromal needle-shaped materials were
graded on a scale of 4 categories (grade 0; none, grade 1; mild, grade
2; moderate, grade 3; severe) at each time point. Time trends of the
outcomes were graphically displayed and evaluated with MantelHaenszel trend test.
Results: Preoperatively, the following were observed in all patients:
slight corneal epithelial edema, moderate subepithelial haze,
keratocytes in a honeycomb pattern, and tiny needle-shaped materials
in the stroma. After DMEK, moderate subepithelial haze persisted
during the follow-up period. Needle-shaped materials had a tendency
to decrease after DMEK. Most notably, donor-recipient interface
haze and donor-recipient interface particles were barely noticeable
after DMEK as early as one month postoperatively.
Conclusions: In vivo laser confocal microscopy can identify
subclinical corneal abnormalities after DMEK such as subepithelial
haze, host stromal needle-shaped materials, and minimum donorrecipient interface haze/particles. These abnormalities seemed subtle
compared to Descemet stripping automated endothelial keratoplasty;
this may explain the superior postoperative visual acuity after
DMEK. Further studies with this technology in a large number of
patients and long-term follow up are needed to fully understand the
long-term corneal changes after DMEK.
Commercial Relationships: Akira Kobayashi, None; Hideaki
Yokogawa, None; Natsuko Yamazaki, None; Toshinori Masaki,
None; Kazuhisa Sugiyama, None
Support: a Grant-in-Aid for Scientific Research (C) KAKENHI,
Japan (No. 22591934)
Program Number: 1748
Presentation Time: 11:15 AM - 11:30 AM
Impact of Donor Age on Endothelium-Descemet Membrane
Layer Harvesting and Roll Formation
Adam Bennett1, Shahira Rashad2, Donna Drury1, H D. Cavanagh1,
James P. McCulley1, Matthew Petroll1, Vinod V. Mootha1. 1Univ Tex
Southwestern Medical Center, Dallas, TX; 2Alexandria Faculty of
Medicine, Alexandria, Egypt.
Purpose: Descemet membrane endothelial keratoplasty (DMEK) is
an alternative to Descemet’s stripping automated endothelial
keratoplasty (DSAEK) to surgically replace diseased corneal
endothelium. Although graft rejection incidence has been reported to
be drastically lower in DMEK, wide adoption may be limited by two
factors. Harvesting the endothelium-Descemet membrane layer
(EDM) can be difficult, and tight EDM scrolling can hinder
unfolding once inserted into the patient’s eye. Anecdotally, surgeons
have noticed the use of younger donors has exacerbated these factors.
We sought to correlate donor age with EDM stripping difficulty and
scroll tightness.
Methods: EDM scrolls were harvested by a cornea-fellowship
trained ophthalmologist masked to donor age from 26 corneoscleral
buttons. An 11 mm partial trephination was used instead of blunt
dissection for a consistent and even outer cut. 7.0 to 8.25 mm EDM
scrolls were prepared using the SCUBA technique in Optisol GS.
VisionBlue® (.06% trypan blue) staining was used to harvest and
assess EDMs. The surgeon subjectively rated stripping difficulty on a
1 to 5 scale (easiest to unable to strip) based upon DM adherence to
underlying stroma and radial tear formation. Three different methods
were used to characterize scrolling severity: scroll width, normalized
scroll surface area (scroll width × scroll length/surface area of EDM),
and tendency for EDM scroll formation (referred to as scroll rating).
A scroll rating of 1 corresponded to opposite ends of the EDM not
touching, 2 when EDM ends touch, 3 when the EDM forms one
complete scroll and 4 when more than one scroll formed.
Results: Mean donor age was 59 ± 14 years (15-69). Mean diameter
of EDM scroll was 7.9 ± .23 mm (7.0-8.25). Stripping difficulty was
shown to be inversely correlated with donor age (p<.05). The three
methods to determine scrolling severity had different results. Inverse
relations between donor age was significant for scroll rating (p<.05),
nearly significant for normalized surface area (p=.0508), and not
significant for scroll width (p<.10).
Conclusions: Our data supports previous observations that
harvesting of EDM scrolls may be easier in older donor corneas.
There may be a decreased scrolling tendency based on an inverse
correlation between age and scroll rating (p<0.5) and normalized
surface area (p=0.508). Use of older donor corneas should reduce
surgical difficulty of DMEK procedures.
Commercial Relationships: Adam Bennett, None; Shahira
Rashad, None; Donna Drury, None; H D. Cavanagh, Menicon Ltd
(C); James P. McCulley, Alcon Laboratories, Inc. (C), PanOptica,
Inc. (C); Matthew Petroll, None; Vinod V. Mootha, None
Support: Research to Prevent Blindness; National Eye Institute
EY020799
Program Number: 1749
Presentation Time: 11:30 AM - 11:45 AM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Descemet’s Stripping Automated Endothelial Keratoplasty
(DSAEK) vs Ultra-Thin DSAEK (UT-DSAEK) vs Descemet’s
Membrane Endothelial Keratoplasty (DMEK)
Peng Yan, Salina Teja, Kashif Baig. Ophthalmology, University of
Ottawa - Eye Institute, Ottawa, ON, Canada.
Purpose: To compare the surgical and visual outcomes between
DSAEK, UT-DSAEK and DMEK as treatments for Fuch’s
Endothelial Dystrophy (FED) and Pseudophakic Bullous Keratopathy
(PBK).
Methods: The first ten consecutive DSAEK, UT-DSAEK, and
DMEK patients with either FED or PBK were reviewed
retrospectively. In DSAEK, a 350um head was used for all singlepass dissections. In UT-DSAEK, donor corneas were prepared by a
two-pass microkeratome dissection. In DMEK, a trephine-peel
technique was used to prepare the graft. Data was collected from
baseline up to 6-months follow-up, and outcomes including
intraoperative and postoperative complications, visual rehabilitation,
endothelial cell density and follow-up graft thickness were compared.
Results: The average age was 76, 69, and 67.5 years for DSAEK,
UT-DSAEK, and DMEK respectively. All patients had previous
cataract extraction and intraocular lens placement, with an equal
number of FED and PBK presentations. Mean donor endothelial cell
count was 2597 for DSAEK, 2590 for UT-DSAEK group and 2709
for DMEK. No donor tissues were lost during tissue preparation. The
DSAEK group had a mean preoperative best-corrected visual acuity
(BCVA) of 20/200 with mean intraocular pressure (IOP) of
17mmHg. The UT-DSAEK group had a mean preoperative BCVA of
20/80 with mean IOP of 13mmHg. The DMEK group had a mean
pre-operative BCVA of 20/120 with mean preoperative IOP of
14mmHg. One patient in the DMEK group had a large persistent
peripheral graft detachment despite 3 re-bubbling attempts and
required a second DMEK procedure. Six-month outcomes of visual
rehabilitation, endothelial cell loss, graft thickness and graft rejection
for all patients will be available by March 2013.
Conclusions: Endothelial Keratoplasty is constantly evolving, with
DSAEK currently being the standard of care. Available literature has
shown the benefits of UT-DSAEK and DMEK, including lower rates
of graft rejection, faster and greater visual recovery and comparable
endothelial cell loss. The difficulties with tissue preparation however,
have resulted in a slower transition to these two procedures. This
comparison of outcomes between our first 10 consecutive patients
having each procedure will shed light on the relative learning curve
and encourage corneal surgeons to consider the benefits of providing
these advanced treatments to their patients.
Commercial Relationships: Peng Yan, None; Salina Teja, None;
Kashif Baig, Bausch and Lomb (F), Allergan (C), Alcon (C)
Program Number: 1750
Presentation Time: 11:45 AM - 12:00 PM
Diamond knife assisted Deep Anterior Lamellar Keratoplasty
(Dia-DALK): A new surgical technique for management of
Keratoconus
Rasik B. Vajpayee, Prafulla K. Maharana, Namrata Sharma. R P
centre for Ophthalmic Sciences, All India Institute of Medical
Sciences, New Delhi, India.
Purpose: To evaluate outcomes of our new technique of DALK in
cases of Keratoconus
Methods: DALK was performed in 20 Keratoconic eyes using our
technique of Dia-DALK. The technique involved marking the host
cornea with a 8-8.5 mm trephine and performing an intraoperative
pachymetry along that mark at about 11 O'Clock. Subsequently,
diamond knife set at a depth 40µ less than that of pachymetry reading
was used to make an incision of 2mm at 11 O'Clock position. This
incision at that depth was then enlarged with the help of curved
Vannas scissors circumferentially and a blunt lamellar dissector was
used to dissect and remove the overlying stromal layers radially. A
0.25 mm oversized donor button whose descemet membrane had
been scrapped off, was sutured on the host bed. Main outcome
measures analysed were BCVA, keratometry (Km), spherical
equivalent (SEQ) and endothelial cell density.
Results: At 6 month the mean residual host thickness was 41.7±
13.8µ. The mean log MAR BCVA improved significantly from
preoperative value of 1.847±0.289 to 0.2117±0.061 (p= 0.005).The
average Km improved from 66.5±7.5D to 45.1±1.5D (p=0.03). The
mean SEQ decreased from -7.8 ±4.6D to -1.23±0.88 D (p=0.007). A
significant decrease was seen in refractive astigmatism from 5.93
±3.06D preoperatively to 3.23 ±1.14D (p=0.037). The mean
endothelial cell loss was 5.241+3.6 %. No intraoperative perforations
occurred in any of the cases.
Conclusions: Our technique of Dia-DALK is safe, predictable and
effective for the mangement of Keratoconus. It has the potential to
become an alternative to Big Bubble DALK
Commercial Relationships: Rasik B. Vajpayee, None; Prafulla K.
Maharana, None; Namrata Sharma, None
Program Number: 1751
Presentation Time: 12:00 PM - 12:15 PM
Femtosecond Laser-Assisted Deep Anterior Lamellar
Keratoplasty with Zig-zag Configuration: Initial Outcomes
Ijeoma Asota, Matthew Wade, John Xie, Sumit Garg, Roger F.
Steinert, Marjan Farid. Gavin Herbert Eye Institute, University of
California Irvine, Irvine, CA.
Purpose: To report the early refractive results and clinical outcomes
of deep anterior lamellar keratoplasty (DALK) performed using the
femtosecond laser with the zig-zag incision configuration.
Methods: 30 consecutive eyes underwent femtosecond laser assisted
DALK with zig-zag configuration. Clinical records were reviewed
retrospectively. Corrected distance visual acuity (CDVA), manifest
and topographic astigmatism, and complications were reviewed.
Results: In 26 eyes, a big-bubble was successfully achieved. The
remaining 4 eyes required dissection down to a very thin residual
stromal bed. Postoperative follow-up ranged from 3 months (n=28) to
2.5 years (n=3). At post-operative month 3, mean CDVA was 20/30
(range 20/20-20/60), mean manifest astigmatism was 3.5 D (range
1.25-6 D), and mean topographic astigmatism was 4.23 D (range 1.19.25 D). These outcomes remained stable throughout the follow up
period. Complications included suture revision at post-operative
month 3 for wound gape in one patient, and an episode of stromal
rejection in one patient.
Conclusions: Visual outcomes of femtosecond laser-assisted zig-zag
DALK are similar to our results with zig-zag full thickness
penetrating keratoplasty. In addition, this technique offers a
decreased risk of endothelial rejection in healthy eyes compared to
penetrating keratoplasty.
Commercial Relationships: Ijeoma Asota, None; Matthew Wade,
None; John Xie, None; Sumit Garg, None; Roger F. Steinert,
Abbott Medical Optics (C), OptiMedica (C), ReVision Optics (C),
WaveTec (C); Marjan Farid, None
Program Number: 1752
Presentation Time: 12:15 PM - 12:30 PM
Corneal Endothelial Cell Loss after Endothelial and Penetrating
Keratoplasty for Endothelial Disease
Sanjay V. Patel, Keith H. Baratz, Jay W. McLaren, Lori A. Bachman,
William M. Bourne. Ophthalmology, Mayo Clinic, Rochester, MN.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Purpose: To compare changes in the corneal endothelium after three
different keratoplasty techniques for the treatment of endothelial
disease.
Methods: Patients with corneal endothelial disease (predominantly
Fuchs dystrophy) were enrolled in two consecutive prospective
studies at Mayo Clinic, Rochester, MN. In a randomized controlled
trial, 28 eyes (26 patients) received penetrating keratoplasty (PK) or
deep lamellar endothelial keratoplasty (DLEK) with a 9 mm scleral
incision. In a consecutive prospective observational study, 52 eyes
(45 patients) received Descemet-stripping endothelial keratoplasty
(DSEK) with a 5 mm scleral incision. Endothelial images were
acquired through 3 years (or 5 years for some eyes after DSEK) by
using confocal microscopy (ConfoScan 3 or 4, Nidek Technologies).
Endothelial cell density (ECD) and morphology were determined by
the same masked observer by digitizing the apices of cells (HAI Cell
Analysis System) in images acquired at various intervals after
keratoplasty. Endothelial cell loss (ECL) was the percentage of cells
lost from preoperative ECD of the donor tissue, as measured by the
eye bank that provided the donor tissue. Endothelial variables were
compared between techniques by using generalized estimating
equation models to account for any correlation between fellow eyes
of the same subject.
Results: Donor diameter was 7.6 ± 0.1 mm (mean ± sd) for PK, 7.9 ±
0.1 mm for DLEK, and 8.2 ± 0.3 mm for DSEK. Preoperative donor
ECD did not differ between treatments (PK, 2,845 ± 306 cells/mm2;
DLEK, 2,749 ± 364 cells/mm2; DSEK, 2,925 ± 374 cells/mm2;
p≥0.10). Mean ECL is summarized in the Table. At one month, there
was a trend toward higher ECL after DSEK than after DLEK and PK
(p≥0.31, minimum detectable difference, 17% [α=0.05, β=0.20]). At
24 and 36 months, ECL after DSEK was lower than it was after
DLEK (p≤0.004) and PK (p≤0.03). At 60 months, ECL after DSEK
was similar to that at 36 months after DLEK and PK. By 3 years,
there were 0, 1, and 5 graft failures after PK, DLEK, and DSEK,
respectively.
Conclusions: Despite a trend toward higher early endothelial cell
loss after DSEK, cell loss at 3 years after DSEK is lower than that
after PK or DLEK. This difference might be related to graft diameter
or to postoperative anatomic differences of the posterior corneal
surface. Continued observation is required to determine the longerterm trend in cell loss after DSEK.
Commercial Relationships: Sanjay V. Patel, None; Keith H.
Baratz, Assessing the likelihood of developing Fuchs Corneal
Dystrophy (P); Jay W. McLaren, None; Lori A. Bachman, None;
William M. Bourne, None
Support: Research To Prevent Blindness, New York, NY; Mayo
Foundation, Rochester, MN
Clinical Trial: NCT00346138
Program Number: 1753
Presentation Time: 12:30 PM - 12:45 PM
Outcomes of new partial-thickness corneal grafts: a paradigm
shift in practice, but at what cost
Richard A. Mills, Miriam C. Keane, Keryn Williams. Dept of
Ophthalmology, Flinders University SA, Bedford Park, SA,
Australia.
Purpose: Lamellar corneal grafts are now being performed for
indications that previously were treated by full-thickness corneal
grafts. We examined the evidence for the success of these new
lamellar procedures, with a focus on graft survival and visual
outcome.
Methods: In a national register of >23,000 corneal grafts with up to
25 years of annual follow-up, 2983 lamellar grafts were identified, of
which 42% were endokeratoplasties (posterior corneal endothelial
cell grafts), 39% were traditional, peripheral lamellar keratoplasties,
and 19% were deep anterior lamellar keratoplasties (DALKs).
Kaplan-Meier plots were used to determine graft survival times, Cox
proportional hazards regression was used for multivariate graft
survival analysis, and visual outcomes were investigated using bestcorrected Snellen acuity, that is, with any prescribed spectacle lens or
contact lens.
Results: Kaplan-Meier graft survival at one year was 74% for
endokeratoplasties, 80% for traditional lamellar procedures, and 93%
for DALKs. The major indications for endokeratoplasty were Fuchs’
dystrophy (47%) and bullous keratopathy (33%). Over the time frame
2004-2012, penetrating corneal grafts performed for either of these
indications exhibited significantly better graft survival than did
endokeratoplasties for the same indications (p<0.001). The major
indication for DALK was keratoconus (76%). Over the time frame
1995-2012, penetrating corneal grafts performed for keratoconus
exhibited significantly better graft survival than did DALKs for
keratoconus (p<0.001). Approximately 80% of lamellar procedures
were performed to improve visual acuity, but at the time of most
recent follow-up, more patients with penetrating grafts achieved good
post-operative Snellen acuity than did those with lamellar grafts. A
best-corrected Snellen acuity of 6/12 or better (=20/40 or better) at
most recent follow-up was achieved in 18% of endokeratoplasties,
34% of traditional lamellar grafts, and in 37% of DALKs. Surgeons
who performed more than 15 lamellar procedures per year achieved
significantly better graft survival than those who performed fewer
grafts (p=0.02).
Conclusions: There appears to be a surgeon learning curve for new
lamellar corneal graft procedures. Currently, outcomes in terms of
graft survival and visual acuity are better for penetrating grafts than
for lamellar procedures, even when matched for era and indication
for graft.
Commercial Relationships: Richard A. Mills, None; Miriam C.
Keane, None; Keryn Williams, None
Support: Donate Life: Australian Organ & Tissue Donation &
Transplantation Authority
259 Corneal Immunology, Allergy, Neovascularization
Monday, May 06, 2013 11:00 AM-12:45 PM
Exhibit Hall Poster Session
Program #/Board # Range: 2053-2106/D0192-D0245
Organizing Section: Cornea
Contributing Section(s): Biochemistry/Molecular Biology
Program Number: 2053 Poster Board Number: D0192
Presentation Time: 11:00 AM - 12:45 PM
CICATRICIAL CHANGES IN OCULAR PEMPHIGUS
Patricia Chirinos-Saldaña, Isaac Zuñiga-Gonzalez, Julio C.
Hernandez-Camarena, Alejandro Navas, Tito Ramirez-Luquín, Atzin
Robles-Contreras, Maria C. Jimenez-Martinez, Arturo J. RamirezMiranda, Enrique O. Graue-Hernández. Cornea and Refractive
Surgery, Instituto de Oftalmología Conde de Valenciana, Mexico DF,
Mexico.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Purpose: To describe the ophthalmic clinical characteristics in
patients with pemphigus at an ophthalmological referral center.
Methods: A retrospective chart review of patients with the
immunopathological diagnosis of pemphigus examined between
January 1, 2000, to April 1, 2010 was performed. Uncorrected
distance visual acuity, best corrected distance visual acuity (BCVA),
and ocular symptoms as well as ocular surface inflammatory and
scarring changes were assessed.
Results: 15 patients were identified, with a mean age of 68.27 ±
14.35 years, and 80% (n = 12) were female. Extraocular involvement
was reported in 1 patient. All eyes showed cicatricial changes in the
conjunctiva. Six eyes (36.7%) were classified as stage I; 12 eyes
(40%), as stage II; 10 eyes (33%), as stage III; and 2 eyes (7%), as
stage IV. A statistically significant association was found between
BCVA and the severity of ocular involvement. Mean BCVA
logMAR was 1.66 (20/914), range from 0 (20/20) to 4 (NLP). Other
ocular diseases were found in 8 (53.3%), systemic diseases in 10
(66.7%) and the use of pemphigus inducing drugs in 10 patients
(66.7%).
Conclusions: This report represents the largest series of ocular
involvement in pemphigus confirmed by immunopathology. The
clinical manifestations varied from conjunctival hyperemia to corneal
scarring and perforation. There was a strong association between
scarring changes and low BCVA. Ocular and systemic diseases as
well as the use of pemphigus inducing drugs may predispose to
ocular cicatricial changes observed in this series.
Commercial Relationships: Patricia Chirinos-Saldaña, None;
Isaac Zuñiga-Gonzalez, None; Julio C. Hernandez-Camarena,
None; Alejandro Navas, None; Tito Ramirez-Luquín, None; Atzin
Robles-Contreras, None; Maria C. Jimenez-Martinez, None;
Arturo J. Ramirez-Miranda, Carl Zeiss Meditec (R); Enrique O.
Graue-Hernández, None
Program Number: 2054 Poster Board Number: D0193
Presentation Time: 11:00 AM - 12:45 PM
The Effect of Trigeminal Neurons on the Expression of
Maturation Markers by Bone Marrow-Derived Dendritic Cells
Sang-Mok Lee, William Stevenson, Kishore Reddy Katikireddy, Hyun
Soo Lee, Thomas H. Dohlman, Sunil K. Chauhan, Jing Hua, Zahra
Sadrai, Masahiro Omoto, Reza Dana. Schepens Eye Research
Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical
School, Boston, MA.
Purpose: The immune system interacts with the nervous system in a
variety of settings. For example, several neuropeptides have been
shown to exert immunomodulatory effects on antigen-presenting
cells including dendritic cells (DCs). The present study investigated
whether or not cultivated trigeminal neurons (TGNs), which provide
sensory innervation to the ocular surface, can affect the expression of
maturation markers by cultivated bone marrow-derived dendritic
cells (BMDCs).
Methods: Bone marrow and trigeminal ganglions were harvested
from C57BL/6 mice of 6-8 weeks age. After excluding red blood
cells, bone marrow cells were cultured in the presence of
granulocyte/macrophage colony-stimulating factor (GM-CSF,
20ng/ml) for 6 days to proliferate immature DCs. Loosely-adherent
immature DCs were collected and sub-cultured for 2 days in the
presence of IFN-γ (10ng/ml) without GM-CSF to induce maturation.
Primary cultured TGN or culture supernatants were added to IFN-γstimulated BMDCs to evaluate their effects on DC maturation. The
expression levels of MHC class II (mouse IA/IE) and CD86 on
CD11c+ DCs were analyzed using flow cytometry.
Results: The addition of either TGN or culture supernatant
significantly suppressed the expression of MHC class II by IFN-γ-
stimulated BMDC compared to IFN-γ-stimulated BMDCs without
TGN or their supernatants (control group) (relative frequencies:
79.64 ± 5.24% for control group, 45.55 ± 7.55% for TGN group (P ≤
0.05), and 63.68 ± 4.86% for supernatant group (P ≤ 0.05),
respectively; MFI: 31.72 ± 3.77 for control group, 19.38 ± 2.29 for
TGN group (P ≤ 0.05), and 24.48 ± 0.87 for supernatant group (P ≤
0.05), respectively, Mann-Whitney U test). The cell-surface
expression of CD86, a co-stimulatory molecule, demonstrated similar
changes to MHC class II expression as a result of TGN co-culture.
Conclusions: Cultivated TGN or their secreted factors are capable of
suppressing dendritic cell maturation.
Commercial Relationships: Sang-Mok Lee, None; William
Stevenson, None; Kishore Reddy Katikireddy, None; Hyun Soo
Lee, None; Thomas H. Dohlman, None; Sunil K. Chauhan, None;
Jing Hua, None; Zahra Sadrai, None; Masahiro Omoto, None;
Reza Dana, Allergan (C), Alcon (C), Bausch & Lomb (C), Eleven
Bio (I), GSK (F), Novabay (C), Revision Optics (C), Novaliq (C),
RIgel (F)
Support: NIH Grant R01 EY20889
Program Number: 2055 Poster Board Number: D0194
Presentation Time: 11:00 AM - 12:45 PM
Effect of Interleukin-17(IL-17) and IL-17 receptor on ocular
surface inflammation
Ai Yamada, Tohru Sakimoto, Akiko Ishimori, Takako Ohnishi,
Satoshi Sugaya, Mitsuru Sawa. Ophthalmology, Nihon University
School of Medicine, Tokyo, Japan.
Purpose: To investigate the effect of Interleukin-17(IL-17) and IL-17
receptor (IL-17R) on ocular surface inflammation, we studied
whether IL-17 stimulation can up-regulate inflammatory cytokines in
corneal epithelial cells and fibroblast cells. We also analyzed the IL17R expression in mouse alkali corneal burn model.
Methods: Expression of IL-17R, IL-8, and CCL20 after stimulation
by IL-17(100ng/ml) or tumor necrotizing factor(TNF)-α(50ng/ml)
was investigated using human corneal epithelial cell line(HCE) and
human corneal fibroblast cell line (HCF) by real-time reverse
transcription polymerase chain reaction (RT-PCR).Unilateral eye of
corneal alkali burn was made using filter paper presoaked in 1N
NaOH using A/J mice. After the treatment, both eyes were enucleated
on day 5. Two-μm-thick corneal section was made using laser
assisted microdissection method and the amount of RNA in the
samples was determined by real-time RT-PCR.
Results: TNF-α stimulation increased significantly IL-17R, IL-8, and
CCL20 expression in both HCE(1.7±0.2, 146.3±8.2, and
15.1±1.8times) and HCF(1.8±0.2 , 98.8±8.8, and 70.0±10.3 times )
compared to the non-treated control group(p<0.05, p<0.01,and
p<0.01, respectively). On the other hand, IL-17 did not stimulate IL17R expression significantly in both HCE and HCF, but increased IL8 expression(2.9 ±0.5 and 7.1±1.0 )and CCL20 expression(2.9 ±0.5
and 3.9±0.4) significantly(p<0.01). IL-17R expression in alkali
burned cornea increased by an average of 2.7 times compared with
that of non-treated cornea.
Conclusions: IL-17 and IL-17R play a role in pathophysiology of
ocular surface inflammation.
Commercial Relationships: Ai Yamada, None; Tohru Sakimoto,
None; Akiko Ishimori, None; Takako Ohnishi, None; Satoshi
Sugaya, None; Mitsuru Sawa, HOYA Co. (F), Santen
Pharmaceutical Co. (F)
Program Number: 2056 Poster Board Number: D0195
Presentation Time: 11:00 AM - 12:45 PM
Host Mesenchymal Stem Cells Home to Transplanted Cornea
and Promote Allograft Survival
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Masahiro Omoto, Yiping Jin, Sunil K. Chauhan. Schepens Eye
Research Institute, Harvard Medical School, Boston, MA.
Purpose: Along with their capacity for differentiating into cells of
multiple lineages, mesenchymal stem cells (MSC) have generated
great interest for their ability to display unique anti-inflammatory and
immunomodulatory properties. The purpose of this study was to
investigate whether systemically-injected host MSC can home to the
inflamed transplanted cornea, suppress induction of alloimmunity,
and promote allograft survival rate.
Methods: MSC (CD45-CD34-SCA1+CD29+) were generated from
the bone marrow of wild-type BALB/c or GFP+ C57BL/6 mice, and
1x106 cells were intravenously injected to allografted recipients 2h
after surgery. To track the homing of GFP+ MSC (C57BL/6), corneal
grafts from BALB/c (H-2d) mice were transplanted onto C57BL/6
(H-2b) recipient mice. MSC homing to the corneas was examined at
day 3 post-transplantation by immunohistochemistry. To investigate
the effect of MSC on alloimmunity and graft survival, corneal grafts
from C57BL/6 (H-2b) mice were transplanted onto BALB/c (H-2d)
recipient mice, and then wild-type BALB/c MSC were injected.
Frequencies of alloreactive IFNγ+ T cells were analyzed at day 14
post-transplantation using the ELISPOT assay. Frequencies of mature
CD11C+MHC-II+ antigen-presenting cells were analyzed by flow
cytometry. Graft survival was evaluated by slit-lamp biomicroscopy
weekly up to 8 weeks.
Results: Intravenously injected GFP+MSC were found in abundance
in the transplanted cornea, but not in the ungrafted (contralateral)
cornea. The frequencies of mature CD11C+MHC-II+ antigenpresenting cells were substantially decreased in the corneas (50.2%
vs. 76.7%) and draining lymph nodes (4.4% vs. 8.4%) of MSCinjected allograft recipients compared to control group. The draining
LN of MSC-injected allograft recipients showed significantly lower
frequencies allosensitized IFNγ-secreting T cells compared to the
control group (p=0.023). Allograft survival rate was significantly
(~2-fold) higher (p = 0.03) in the MSC-injected recipients (80%,
n=12) compared to the non-MSC injected group (40%, n =10).
Conclusions: Our data demonstrate that systemically-administered
MSC specifically home to transplanted corneas and promote allograft
survival by inhibiting APC maturation and induction of alloreactive T
cells. These data suggest that host MSC exert immunomodulatory
functions in corneal transplantation and may be used to prolong
transplant survival.
Commercial Relationships: Masahiro Omoto, None; Yiping Jin,
None; Sunil K. Chauhan, None
Program Number: 2057 Poster Board Number: D0196
Presentation Time: 11:00 AM - 12:45 PM
The Cross-reactivity of Subsequent Corneal Allografting After
Xenocorneal Transplantation Using Decellularized Porcine
Lamella Against Allo-antigens in Primates
Hyuk Jin Choi1, 2, Jong Joo Lee2, 3, Mee Kum Kim2, 3, Won Ryang
Wee2, 3, Hyun Ju Lee3, Ah Young Ko3, Jae-Il Lee4, Hee Jung Kang5.
1
Ophthalmology, Healthcare System Gangnam Center, Seoul
National University Hospital, Seoul, Republic of Korea;
2
Ophthalmology, Seoul National University College of Medicine,
Seoul, Republic of Korea; 3Laboratory of Corneal Regenerative
Medicine and Ocular Immunology, Seoul Artificial Eye Center,
Seoul National University Hospital Biomedical Research Institute,
Seoul, Republic of Korea; 4Xenotransplantation Research Center,
Seoul National University Hospital, Seoul, Republic of Korea;
5
Laboratory Medicine, Hallym University College of Medicine,
Anyang, Republic of Korea.
Purpose: To investigate the cross-reactivity of subsequent corneal
allografting after xenocorneal transplantation using decellularized
porcine lamella against allo-antigens in primates and to validate the
feasibility of decellularized porcine corneal lamella as a bridge to a
subsequent corneal allograft.
Methods: Five Chinese rhesus macaques, which had undergone
anterior partial thickness corneal transplantation using hypertonic
saline-treated decellularized porcine corneal lamellae in the
antecedent experiments, were used as recipients for subsequent fullthickness corneal allografts. To determine whether sensitization of
the recipients to xenoantigens led to cross-reactivity against
alloantigens, we compared; 1) allogeneic one way mixed lymphocyte
reaction (MLR) of the peripheral blood mononuclear cells (PBMCs)
from the recipients with that of PBMCs from randomly selected
rhesus macaques, 2) amount of IgG antibodies which bound to
PBMCs of a rhesus panel (5 monkeys) before sensitization with that
after sensitization. Graft survival and immunologic profiles including
memory T cell subset and donor rhesus-specific antibodies were
evaluated.
Results: There was neither hyperacute nor acute rejection within a
month post-transplantation in all recipients. Notable changes in all
memory T cell subsets were not seen during first one month after
transplantation even in two recipients which showed graft failure at
49 days and 35 days post-transplantation, respectively. Alloreactivity
on MLR was not different between rhesus recipients with
xenocorneal grafts and naïve rhesus monkeys. In either of the
recipients, IgG antibodies reactive against PBMCs from the panel
were not changed after sensitization by xenoantigens. Moreover,
there was no change in donor-rhesus specific antibodies in all
recipients.
Conclusions: Decellularized porcine corneal lamella seems not to be
cross-reactive against alloantigens and it is likely to be used as a
bridge before corneal allograft becomes available.
Commercial Relationships: Hyuk Jin Choi, None; Jong Joo Lee,
None; Mee Kum Kim, None; Won Ryang Wee, None; Hyun Ju
Lee, None; Ah Young Ko, None; Jae-Il Lee, None; Hee Jung
Kang, None
Support: Grant from the Korea Healthcare Technology R&D
Project, Ministry for Health, Welfare & Family Affairs, Republic of
Korea (Project No. A040004)
Program Number: 2058 Poster Board Number: D0197
Presentation Time: 11:00 AM - 12:45 PM
Tear expression profiling of cytokine, chemokine and soluble
receptor in keratoconus patients
Jeewon Mok, Choun-Ki Joo. Catholic Institutes of Visual Science,
Catholic Univ Korea, Seoul, Republic of Korea.
Purpose: To determine whether the expression levels of cytokines,
chemokines and soluble receptor in tear of keratoconus patients
contribute to the pathogenesis of keratoconus
Methods: The patients with keratoconus were diagnosed based on
the following criteria: (1) symptoms of keratoconus (Munson sign,
protrusion, Vogt’s striae, corneal thickness, scarring, Fleishcher ring,
photokeratoscopy signs, video keratography signs, and refractive
errors) and (2) medical histories (age, gender, contact lense wearing,
eye rubbing, systemic disease, atopy, and connective tissue disease).
Tears were collected from 28 keratoconus patients (56 eyes) and 30
healthy subjects (60 eyes) by a polyurethane minisponges. Control
subjects with no history of ocular disease were also enrolled. The
concentrations of cytokines/chemokines were analyzed by Luminex
200 using Human cytokine/chemokine (42 molecules), Human
cytokine/chemokine Panel II (23 molecules) and Human soluble
cytokine receptor (14 molecules). The Median Fluorescent Intensity
(MFI) was used to obtain the calculating cytokines, chemokines and
soluble receptor concentrations in tears
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Results: Of the 79 cytokines, chemokines and soluble receptors, we
detected 16 molecules that demonstrated a significant differences in
tears from Keratoconus patients. In cytokines, G-CSF (p<0.001),
IL1-ra (p<0.05), VEGF (p<0.05), IL-16 (p<0.05) and IL-20 (p<0.05)
were significantly increased in Keratoconus patients compared to
control subjects. MDC (p<0.05) and GRO (p<0.05) of chemokines
and sIL-1RI (p<0.05) and sIL4R (p<0.001) of soluble receptors were
increased in keratoconus patients. Whereas IL4 and IFN gamma (all
p<0.001) and FGF2 ( p<0.05) of cytokines, MIP1a and MIP1b (all
p<0.001) of chemokines, sCD30 and sIL6R (all p<0.05) were
significantly decreased in keratoconus patients
Conclusions: In tears of keratoconus patients, nine molecules were
elevated keratoconus patients, whereas 7 molecules were decreased.
It is suggested that different levels of inflammatory regulated
cytokines/chemokines/soluble receptors may all play an important
role in the pathogenesis of keratoconus
Commercial Relationships: Jeewon Mok, None; Choun-Ki Joo,
None
Program Number: 2059 Poster Board Number: D0198
Presentation Time: 11:00 AM - 12:45 PM
HC-HA Suppresses Inflammatory and Immune Responses and
Improves Murine Corneal Allograft Survival
Hua He1, Yaohong Tan3, Victor L. Perez3, 4, Scheffer C. Tseng1, 2.
1
TissueTech. Inc., Miami, FL; 2Ocular Surface Center, and Ocular
Surface Research Education Foundation, Miami, FL; 3Department of
Ophthalmology, Bascom Palmer Eye Institute, University of Miami
Miller School of Medicine, Miami, FL; 4Department of Microbiology
and Immunology, University of Miami Miller School of Medicine,
Miami, FL.
Purpose: Experimental and clinical studies have shown that amniotic
membrane (AM), AM extract, and HC-HA [a covalent complex
formed by heavy chain (HC) of inter-α-trypsin inhibitor (IαI) and
hyaluronan (HA)] suppress pro-inflammatory responses. Hence, we
decide to determine whether HC-HA can regulate T cell responses
and reduce murine corneal allograft rejection.
Methods: T cell activation was assessed by cell proliferation and
cytokine production in splenocytes from Ova T cell receptor
transgenic mice. Optimization of injection sites, volume, and
frequency with HC-HA before or during intracorneal injection of
LPS was determined by influx of EGFP + macrophages into corneas
of Mafia mice. At day 4 after HC-HA treatment, corneas were
digested with 820 units/ml of collagenase at 37 °C for 1 h. EGFP - and
EGFP+ cells were isolated by FACS. mRNA expression of Arg-1, IL10, and IL-12 was measured by qPCR. Allogeneic corneal
transplantation was performed using wild-type BALB/c mice as
recipients and C57BL/6 mice as donors, and its outcome scored by
graft clarity measured twice a week using slit lamp biomicroscopy.
Grafts that received two consecutive scores ≥ 3 without resolution
were considered rejected.
Results: HC-HA but not HA at 1 mg/ml significantly suppressed the
proliferation and production of IFN-γ and IL-2 in splenocytes with
OVA peptide (0 -10 μM) at day 2 and day 4 (all p < 0.05). The
injection regimen was optimized by giving 5 μl at each injection
between subconjunctiva and fornix to all four quadrants. Pretreatment
of HC-HA 3 days prior to LPS injection significantly suppressed the
influx of EGFP+ macrophages to LPS-insulted corneas (9.1±0.3
vs.12.3±0.4, HC-HA vs PBS, p =0.02). Importantly, even though
EGFP+ macrophages did migrate into corneas, some of them were
polarized into M2 phenotype as suggested by significant upregulation of Arg-1 and IL-10 but down-regulation of IL-12 (p <
0.05). Compared to PBS control, allograft rejection was significantly
suppressed by injection of 10 μl HC-HA at one quadrant twice a
week (p < 0.05), and further reduced by injection with 5 μl at 4
quadrants twice a week (p < 0.002).
Conclusions: HC-HA significantly suppresses murine corneal
allograft rejection. The mechanism of this action may be contributed
by HC-HA’s ability to down-regulate pro-inflammatory macrophages
and to suppress T cell immune response.
Commercial Relationships: Hua He, TissueTech, Inc. (E);
Yaohong Tan, None; Victor L. Perez, Alcon (C), Bausch & Lomb
(C), Genentech (C), Cleveland Clinic Foundation (P), Alcon (F),
Alcon (R); Scheffer C. Tseng, NIH, NEI (F), TissueTech, Inc. (F),
TissueTech, Inc. (E), TissueTech, Inc. (P)
Support: NIH, NEI, R44 EY017497 and R43 EY021045, R01
EY018624-01 (VLP), P30 EY014801, Research to Prevent Blindness
Program Number: 2060 Poster Board Number: D0199
Presentation Time: 11:00 AM - 12:45 PM
Polarization of Anti-inflammatory M2 Macrophages by HC-HA
Complex Purified from Amniotic Membrane or Reconstituted In
Vitro
Sean Tighe1, Hua He1, Suzhen Zhang1, Scheffer C. Tseng1, 2. 1Tissue
Tech Inc., Miami, FL; 2Ocular Surface Center and Ocular Surface
Research Education Foundation, Miami, FL.
Purpose: Native HC-HA [a covalent complex formed by heavy chain
(HC) of inter-α-trypsin inhibitor (IαI) with hyaluronan (HA)] purified
from human amniotic membrane (AM) possesses anti-inflammatory
actions by polarizing M2 macrophages and curtailing T cell
activation. Herein, we further delineate this anti-inflammatory role by
examining HA complex formed by in vitro reconstitution with
different components, i.e., HA, HC, and PTX3, with or without the
catalytic action of TSG-6.
Methods: High molecular weight HA was covalently immobilized on
CovaLink NH 96 wells using Sulfo-NHS and EDAC. Purified IαI and
recombinant PTX3 and TSG-6 were then added in different
sequences to form different HA complexes, of which the composition
was characterized by ELISAs and Western blotting. Their antiinflammatory actions were assessed in IFN-γ/LPS stimulated
RAW264.7 cells by qPCR and ELISAs to quantify mRNA and
protein expression of IL-10, IL-12, and IL-23.
Results: Only PTX3, but not TSG-6, could be released by
hyaluronidase digestion or a mild alkaline treatment with 50 mM
NaOH in native HC-HA purified from AM. HA complexes, formed
by either PTX3 or TSG-6, were resistant to extraction by 6M
guanidine HCl. TSG-6 dose-dependently inhibited PTX3 binding but
not vice versa. Transfer of HCs from IαI to HA only occurred in the
presence of TSG-6 but not PTX3. IFN-γ/LPS stimulated RAW264.7
cells on immobilized native HC-HA, but not on immobilized HA
alone, expressed significantly higher IL-10 but undetectable IL-12
and IL-23. HA complex with TSG-6 or HC/TSG-6 suppressed IL-12
expression but did not induce IL-10, while HA complex with PTX3
induced IL-10 expression and suppressed IL-12 expression. Neither
of the above conditions was able to suppress IL-23. Only HC-HA
complex formed by sequential addition of PTX3 followed by
IαI/TSG-6 inhibited IL-23.
Conclusions: PTX3 is an essential component in native HC-HA
purified from AM. The anti-inflammatory action of native HC-HA
can be recapitulated in vitro by step-wise addition of PTX3 followed
by IαI/TSG-6 to HA. Such information is useful for future scale-up
manufacturing and dissection of the molecular mechanism of AM’s
anti-inflammatory action.
Commercial Relationships: Sean Tighe, Tissue Tech Inc. (E); Hua
He, TissueTech, Inc. (E); Suzhen Zhang, TissueTech, Inc (E);
Scheffer C. Tseng, NIH, NEI (F), TissueTech, Inc. (F), TissueTech,
Inc. (E), TissueTech, Inc. (P)
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Support: NIH, NEI, R44 EY017497, R43 EY021045, and RO1
EY06819
Program Number: 2061 Poster Board Number: D0200
Presentation Time: 11:00 AM - 12:45 PM
In Vivo Administration of Interleukin-2 Increases Corneal
Allograft Survival through Expansion of CD4+CD25+ T
Regulatory Cells
Maryam Tahvildari, Parisa Emami-Naeini, Yihe Chen, Masahiro
Omoto, Jing Hua, Sunil K. Chauhan, Reza Dana. Schepens Eye
Research Institute, Massachusetts Eye and Ear, Department of
Ophthalmology, Harvard Medical School, Boston, MA.
Purpose: T regulatory cells (Tregs) have a major role in inhibiting
growth and differentiation of activated T cells following alloantigen
stimulation, and it has been found that interleukin-2 (IL-2) is crucial
in the generation and maintenance of these cells. In this study of
murine corneal transplantation, we aimed to investigate the effect of
systemic administration of IL-2 on corneal allograft survival.
Methods: Corneal transplantation was performed on 8-10 week-old
male BALB/c (H-2d) mice using C57BL/6 (H-2b) mice as donors.
Prior to surgery, recipient mice received 3 intraperitoneal injections
(once a day) of either IL-2 (1µg/20 g body weight, in 100µl
phosphate buffer saline [PBS]) or PBS alone (as control). Injections
were continued once daily for one week after surgery followed by
twice a week for another 4 weeks. Allografts were evaluated up to 5
weeks post-transplantation and were scored using the standard
opacity grading system (from 0 to +5). Frequencies of
CD4+CD25+Foxp3+ Tregs in the draining lymph nodes were
evaluated by flow cytometry on the day of transplantation (day 0),
one week (day 7) and two weeks (day 14) afterwards. In addition, the
suppressive function of CD4+CD25+ Tregs derived from draining
lymph nodes of recipient mice was evaluated one week after
transplantation.
Results: Of the six corneas transplanted in each group, 66.6% in the
IL-2 treated group remained clear up to 5 weeks after surgery
compared to 33.3% in the control group; moreover, IL-2 treatment
delayed the incidence of graft rejection with mean rejection time of
32±1.4 days (mean±SD) for the IL-2 treated vs. 19.5±7.7 days for the
control group. Flow cytometric analysis showed significant increases
in the frequencies of CD4+CD25+Foxp3+ Tregs in the draining
lymph nodes of recipient mice in the IL-2 treated group compared to
the control group at day 0 (14.54% vs. 10.25%, p=0.014), day 7
(14.77% vs. 11.73%, p=0.009) and day 14 (16.15% vs. 12.41%,
p=0.0007). Treg suppression assay revealed 16.05% increased
suppression of effector T cells in the IL-2 treated group compared to
the control group one week after transplantation (p=0.01).
Conclusions: According to our results, systemic administration of
IL-2 is capable of enhancing the frequencies and suppressive function
of CD4+CD25+Foxp3+ Tregs, and increases corneal allograft
survival.
Commercial Relationships: Maryam Tahvildari, None; Parisa
Emami-Naeini, None; Yihe Chen, None; Masahiro Omoto, None;
Jing Hua, None; Sunil K. Chauhan, None; Reza Dana, Allergan
(C), Alcon (C), Bausch & Lomb (C), Eleven Bio (I), GSK (F),
Novabay (C), Revision Optics (C), Novaliq (C), RIgel (F)
Support: NIH Grant EY12963
Program Number: 2062 Poster Board Number: D0201
Presentation Time: 11:00 AM - 12:45 PM
Proteomic Analysis of Plasma and Mucosal Samples from
Patients with Stevens-Johnson Syndrome/Toxic Epidermal
Necrolysis
Julia Malalis1, Christine Mata1, Daniel Kahn3, Amy Lin1, Michael J.
Mosier2, Charles S. Bouchard1, Josephine Cunanan3, Omer Iqbal1, 3,
Debra Hoppensteadt3, Jawed Fareed3. 1Ophthalmology, Loyola
University Chicago Stritch School of Medicine, Maywood, IL;
2
Surgery, Loyola University Medical Center, Maywood, IL;
3
Pathology, Loyola University Chicago Stritch School of Medicine,
Maywood, IL.
Purpose: Stevens-Johnson syndrome and toxic epidermal necrolysis
(SJS/TEN) are life-threatening, immune-complex hypersensitivity
reactions that affect the skin and mucous membranes, often resulting
in significant ocular inflammation. The purpose of this study was to
determine and compare the proteomic and coagulation profile of
plasma and mucosal discharge samples in affected and unaffected
patients.
Methods: Following institutional review board approval, plasma and
swabs from ocular, oral, and skin lesions were obtained from patients
with clinically suspected SJS/TEN (n=5). Two patients had biopsyproven SJS/TEN. Three patients had alternative skin diagnoses not
consistent with SJS/TEN and were considered abnormal controls.
Samples from normal healthy controls were also obtained. Following
centrifugation, the plasma was frozen at -70°C and later thawed and
analyzed to determine thrombin-antithrombin complex (TAT,
Dade®, Marburg, Germany), fibrinopeptide (F1.2, Dade®),
plasminogen activator inhibitor-1 (PAI-1, Diagnostica Stago®,
Asnieres Sur Seine, France), ZYMUPHEN platelet microparticle
activity (Hyphen® BioMed, Neuville-Sur Oise, France),
HEMOCLOT protein C (Stago®), and STACHROME antithrombin
(Stago®), using ELISA kits as per manufacturer’s instructions. The
swabs were immediately frozen at -70°C and later thawed. The
discharges were isolated following addition of 0.25 ml of saline to
each swab and double centrifugation. The discharges and plasma
samples were analyzed using SELDI-TOF technique.
Results: Analyses of the SJS/TEN plasma samples revealed a
marked increase in the TAT complexes (6.3±5.9µg/ml), F1.2
(430.4±202.4 pmol/L), platelet microparticles (13.1±9.3nM) and
protein C levels (90.5±63.4%), with a corresponding decrease in PAI1 (53.3±18.8ng/ml) and antithrombin levels (80.7±42.4%) compared
to normal control human plasma, suggesting a procoagulant state.
Protein chip array of the SJS/TEN skin and oral mucosal samples
exhibited two major peaks at 14.2 kDa and 15.6 kDa, in the same
molecular weight range as recombinant human granulysin, a
molecule implicated in the pathophysiology of SJS/TEN. These
peaks were not present in the control group.
Conclusions: Procoagulant factors and unique peaks suggestive of
granulysin may lead to the development of targeted therapy aimed to
attenuate local and systemic inflammatory processes in patients with
SJS/TEN.
Commercial Relationships: Julia Malalis, None; Christine Mata,
None; Daniel Kahn, None; Amy Lin, None; Michael J. Mosier,
None; Charles S. Bouchard, None; Josephine Cunanan, None;
Omer Iqbal, None; Debra Hoppensteadt, None; Jawed Fareed,
None
Support: Illinois Society for the Prevention of Blindness (ISPB)
Grant
Program Number: 2063 Poster Board Number: D0202
Presentation Time: 11:00 AM - 12:45 PM
Morphologic Dendritic Immune Cells Parameters Reveal
Differential Characteristics between the Central and Peripheral
Cornea: an In Vivo Confocal Microscopy Normative Data
Clara M. Colon1, Bernardo M. Cavalcanti1, 2, Shruti Aggarwal1,
Andrea Cruzat1, 2, Candice Williams1, Douglas Critser4, Amy Watts3,
Christine W. Sindt4, Pedram Hamrah1, 2. 1Department of
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Ophthalmology, Ocular Surface Imaging Center, Massachusetts Eye
& Ear Infirmary, Harvard Medical School, Boston, MA; 2Department
of Ophthalmology, Cornea and Refractive Surgery Service,
Massachusetts Eye & Ear Infirmary, Harvard Medical School,
Boston, MA; 3Contact Lens Service, Massachusetts Eye & Ear
Infirmary, Harvard Medical School, Boston, MA; 4Department of
Ophthalmology & Visual Sciences, Contact Lens Service, University
of Iowa, Iowa City, IA.
Purpose: To determine the morphology of dendritic immune cells
(DC) in the central and four peripheral quadrants in corneas of
normal subjects.
Methods: Eighty-five normal non-contact lens wearers (85 eyes)
were prospectively enrolled and underwent laser in vivo confocal
microscopy (IVCM; HRT3/RCM) of the central cornea, as well as
inferior, nasal, superior and temporal quadrants. Slit-lamp
examination was performed to confirm lack of ocular disease.
Baseline images were assessed for DC morphology and density by
two masked observers. Morphology was assessed by number of
dendrites per cell, area of DC and DC field. Statistical analysis was
performed with ANOVA/Bonferroni to compare the differences
between corneal areas and intraclass coefficient was used to assess
interobserver reproducibility.
Results: The mean age of subjects was 31.4 years (range 20 to 69).
IVCM revealed the presence of central (35.6±4.3 cells/mm2) and
peripheral (74.1±4.5) corneal DC in all areas of all subjects. The
number of dendrites, area of DC, and DC field centrally were
2.4±0.04 n/cell, 71.6±4.3 µm2, and 304.2±19.2 µm2 respectively.
Number of dendrites (3.2±0.07 inferior, 2.9±0.03 nasal, 3.0±0.03
superior, 2.9±0.03 temporal), area of DC (134.6±3.5, 115.3±3.4,
117.0±3.5, 121.2±3.9), and DC field area (684.4±19.1, 586.2±19.7,
587.7±17.4, 601±18.6), were found to be significantly larger in all 4
peripheral quadrants (p<0.001). While the peripheral areas had larger
DC in general, the inferior quadrant demonstrated the largest area of
DC and DC field, as well as the highest dendrite numbers. Good
interobserver reproducibility coefficients were found for DC area
(0.83; 0.77-0.91 95%CI), number of dendrites (0.97; 0.95-0.99), and
DC coverage area (0.94; 0.85-0.98).
Conclusions: IVCM revealed significant difference in morphology
of peripheral versus central corneal DC. These changes suggest
increased DC activation in peripheral cornea. The morphological
parameters may be used to detect early changes in the corneal
inflammatory status. Further, they may provide additional data to
investigate cell activation state for corneal disease and monitor
response to anti-inflammatory therapy. With the increased use of
IVCM to detect subclinical inflammatory changes, the normative data
may serve as the basis for future clinical studies and trials.
Commercial Relationships: Clara M. Colon, None; Bernardo M.
Cavalcanti, None; Shruti Aggarwal, None; Andrea Cruzat, None;
Candice Williams, None; Douglas Critser, None; Amy Watts,
None; Christine W. Sindt, alcon (F); Pedram Hamrah, None
Support: NIH K08-EY020575, New England Corneal Transplant
Research Fund, Falk Medical Research Trust, Alcon Research LTD.
Clinical Trial: NCT01250925
Program Number: 2064 Poster Board Number: D0203
Presentation Time: 11:00 AM - 12:45 PM
Antimicrobial peptides protect the corneas from bacteria and
fungi infection
Chen Dong, Chen Dong, Nan Gao, Gi Sang Yoon, Fushin X. Yu.
Kresge eye institute, Detroit, MI.
Purpose: This study sought to determine bacteri- and fungi-cidal
activities of corneal epithelial secreted peptides, S100A8, S100A9,
CXCL10, and Chi3L1 and their expression in response to microbial
infection.
Methods: Three ocular pathogens, Staphyloccocus aureus, Canidia
albicans and Pseudomonas aeruginosa, were used to challenge B6
mouse cornea and the expression of antimicrobial peptides was
determined by Real-time PCR in corneal epithelial cells. In vitro
bacteri- and fungi-cidal activities were assessed using incubation of
pathogens with peptides in PBS, followed by plate colony counting.
In vivo role of S100A8 (100 ng in 5 μl PBS) and CXCL10 (500 ng in
5 μl PBS) were determine by subconjunctival injection of peptides
prior to inoculation and topical application 4 h post infection.
Results: The expressions of all 4 peptides, S100A8, S100A9,
CXCL10 and Chi3L1, were highly inducible in corneal epithelial
cells in response to microbial infection. In vitro, these peptides
exhibited different bactericidal and/or fungicidal activity under
physiological slat concentration. Subconjunctival injection S100A8
or CXCL10 prior to and 4h after ATCC or C. albicans inoculation
significantly attenuated the development of microbial keratitis using
pathogen burden as the readout of infection.
Conclusions: Corneal epithelial cells express and secrete
antimicrobial peptides in response to infection and synthetic AMPs,
alone or in combination can be used to control ocular infection.
Commercial Relationships: Chen Dong, None; Chen Dong, None;
Nan Gao, None; Gi Sang Yoon, None; Fushin X. Yu, None
Support: NIH grants R01 EY017960
Program Number: 2065 Poster Board Number: D0204
Presentation Time: 11:00 AM - 12:45 PM
Effect of hydroxychloroquine on the inflammation in antigen
presenting cells interacted with damaged corneal epithelial cells
Chang Ho Yoon1, 2, Jin Suk Ryu2, Mee Kum Kim1, 2, Won Ryang Wee1,
2 1
. Ophthalmology, Seoul National University Hostpital, Seoul,
Republic of Korea; 2Laboraory of Corneal Regenerative Medicine
and Ocular Immunology, Seoul Artificial Eye Center, Seoul National
University Hospital Clinical Research Institute, Seoul, Republic of
Korea.
Purpose: The favorable effect of hydroxychloroquine (HCQ) on the
dry eye in Sjögren syndrome is still matter of debates. We tried to
investigate effect of HCQ on the inflammation in antigen presenting
cells interacted with damaged corneal epithelial cells which simulated
damaged ocular surface in vitro.
Methods: The study protocol was approved by the Research Ethics
Committee at Seoul National University Hospital. Human primary B
cells which was provided from the healthy volunteers (n=6) was
interacted with damaged human corneal epithelial cells (DHE) which
were derived from primary cultivated human corneal epithelial cells
and were treated with 20% ethanol for 40 seconds with or without 5
μM of HCQ for 24 hours. B cells were used as negative control, and
B cells treated with CpG oligodeoxynucleotides (ODN) 3 μM as a
TLR9 stimulator with or without 5 μM of HCQ were used as positive
control. IFN- α, IL-6, TNF- α and IL-10 expressions were measured
by RT-PCR and ELISA.
Results: Increased IL-6 and TNF- α were shown in B cells interacted
with damaged corneal epithelial cells and it was significantly
decreased by the application of HCQ in ELISA (Wilcoxon signed
ranks test, p<.05) It was similar as elevation of IL-6 and TNF- α in B
cells treated with CpG (positive control) was significantly getting
down by application of HCQ. Other cytokines did not show
significant changes with or without HCQ treatment.
Conclusions: Our data suggest that hydroxychloroquine may have an
effect on decreasing inflammatory cytokines in TLR9-enriched B
cells which is stimulated by damaged corneal epithelial cells.
Commercial Relationships: Chang Ho Yoon, None; Jin Suk Ryu,
None; Mee Kum Kim, None; Won Ryang Wee, None
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Program Number: 2066 Poster Board Number: D0205
Presentation Time: 11:00 AM - 12:45 PM
Reduced expression of IL17 following rejection in baby rat
keratoplasty
Antonia Hildebrand, Thomas Reinhard, Johannes Schwartzkopff.
Ophtalmology, Universitätsklinik Freiburg, Freiburg, Germany.
Purpose: An unexplained increase of graft rejections following
keratoplasty is observed in very young patients (<1 year). Recently,
IL17 -that is secreted by certain T cell subsets and that usually
worsens the time course of an immune response- was described to be
beneficial following keratoplasty. We hypothesized that T cells are
crucial during this process and asked the question of an involvement
of Th17 cells. In order to analyze this the baby rat keratoplasty model
was used for T cell depletion experiments.
Methods: Orthotopic keratoplasty was performed between Fisher
donor and Lewis recipient rats. All experiments were divided in two
major groups: In group A, all recipients were 3 weeks-of-age and
were either treated with a T cell-depleting or an isotypic antibody for
one week. Group B contained untreated recipients only, that were
either 3 or 10 weeks old. Corneal grafts were monitored clinically
until rejection occurred but no longer than 35 days postoperatively. T
cell-depletion was monitored by FACS. Lymph nodes and cornea
were analyzed by qPCR for IL17-, IL6-, TGFβ-, and RORγt-mRNAlevels. Protein expression of IL17 was tested by intracellular FACS
staining.
Results: Group A: T cell depletion abrogated corneal graft rejection.
Even after the T cell-pool had recovered no delayed onset of rejection
episodes was observed. After complete T cell-recovery, mRNA of
IL-6, TGFβ, IL17 and RORγt were increased in draining lymph
nodes of initially T cell-depleted recipients compared to isotype
controls. At the same time increased mRNA-levels for IL17 and
RORγt were found in grafts of R73-treated animals.
Group B: Independently of recipient age, IL17 mRNA-levels were
decreased in draining lymph nodes of undepleted transplanted
compared to naïve rats. This was confirmed by FACS where
decreased numbers of CD4+IL17+ T cells were also detected.
Conclusions: Our results demonstrate that T cells are crucial during
corneal graft rejection in baby rats. Despite complete T cell-recovery,
no rejection occurs at a later time point in these animals. This is
accompanied by increased IL17-mRNA-levels, that are otherwise
reduced in untreated recipients of a corneal graft. We therefore
speculate, that IL17 influences the time course of rejection following
keratoplasty in baby rats.
Commercial Relationships: Antonia Hildebrand, None; Thomas
Reinhard, None; Johannes Schwartzkopff, None
Program Number: 2067 Poster Board Number: D0206
Presentation Time: 11:00 AM - 12:45 PM
Vitamin D3 attenuates Toll-like receptor 3 induced inflammation
in human corneal epithelial cells
Rose Y. Reins, Alison M. McDermott. College of Optometry, Univ of
Houston, Houston, TX.
Purpose: Vitamin D3 has been shown to have an important
immunomodulatory role during infection and inflammation in many
tissues. This study evaluated the in vitro anti-inflammatory actions of
vitamin D3 on human corneal epithelial cells in response to Toll-like
receptor (TLR) 3 activation.
Methods: Telomerase-immortalized human corneal epithelial cells
(hTCepi) were stimulated with 1,25D3 (10-7M) and/or TLR3 agonist
PolyI:C (1μg/ml) for 24 hours. RNA was collected from control and
treated cells and expression of IL-1β, IL-6, IL-8, IL-23, IFNβ, TNFα,
MIP3α, MMP-9, LL-37, and TLR3 were analyzed by real time RT-
PCR. Protein expression was examined using ELISA (IL-8, MMP9),
Luminex (IL-1β, IL-6, TNFα), western (LL-37), and flow cytometry
(TLR3). For evaluation of NFκΒ signaling, cells were incubated for 2
hours with PolyI:C and 1,25D3 and p65 nuclear translocation was
visualized through immunofluorescence. All experiments were
performed a minimum of three times.
Results: Stimulation with PolyI:C resulted in a significant increase
(range 38 to 1025 fold) in pro-inflammatory cytokine (IL-1β, IL-6,
IL-8, IL-23, IFNβ, TNFα, and MIP3α) expression in hTCepi.
Addition of 1,25D3 downregulated both the RNA and protein levels
of these cytokines following TLR3 activation (41-87%). MMP-9
expression was also attenuated by 1,25D3 during PolyI:C stimulation
by 51%. Additionally, 1,25D3 decreased TLR3 expression by 39%.
Interestingly, the coordinated response of both 1,25D3 and PolyI:C
resulted in increased production of the anti-microbial peptide, LL-37
(9.1±2 fold), above 1,25D3 stimulation alone (5.6±0.4 fold). Finally,
NFκΒ p65 nuclear translocation did not appear to be disrupted by
1,25D3 at the time points tested following PolyI:C stimulation.
Conclusions: Activation of hTCepi through TLR3 resulted in a
robust induction of inflammatory mediators. Vitamin D3 attenuated
this response by decreasing the production of these cytokines as well
as TLR3 expression during PolyI:C stimulation. At the same time,
LL-37 expression was augmented. While an effect on NFκΒ nuclear
localization was not seen at the times indicated, further studies are
ongoing to determine how vitamin D3 affects this signaling pathway.
These results suggest an important role for vitamin D3 in protecting
the ocular surface during inflammation and demonstrate its ability to
dampen the potentially harmful effects of an inflammatory response.
Commercial Relationships: Rose Y. Reins, None; Alison M.
McDermott, None
Support: NIH Grant EY13175, UHCO Core Grant EY07551,
NIH/NEI T32 EY07024
Program Number: 2068 Poster Board Number: D0207
Presentation Time: 11:00 AM - 12:45 PM
HC-HA Complex Purified from Amniotic Membrane Exerts
Anti-scarring Effect by Suppressing TGFβ1 but Activating
TGFβ3 Signaling in Human Corneal Fibroblasts
Fu Li2, Ying-Ting Zhu2, Hua He2, Su-Zhen Zhang2, Scheffer C.
Tseng1. 1Ocular Surface Research & Education Foundation, Miami,
FL; 2Tissue Tech Inc., Miami, FL.
Purpose: Experimental and clinical studies support an anti-scarring
therapeutic action by cryopreserved amniotic membrane (AM). Our
recent studies demonstrated that heavy chain-hyaluronan complex
(HC-HA) is uniquely produced by and can be purified from AM and
suppresses the TGF-β1 promoter activity in human corneal
fibroblasts. Herein, we further characterize this HC-HA’s antiscarring effect.
Methods: Human corneal fibroblasts were seeded on plastic with or
without immobilized HC-HA in DMEM or DMEM+10% FBS for 48
h, or DMEM for 24 h and then treated with 10 ng/ml TGFβ1 for 24 h.
Real-time qPCR was used to measure mRNA levels of TGFβ family
members. Immunostaining was performed to monitor α-smooth
muscle actin ( α-SMA) expression and SMAD2/3 signaling.
Results: On plastic as reported, TGFβ1 mRNA was increased 2-fold
by addition of FBS and 4-fold by addition of TGFβ1. In contrast, on
HC-HA, TGFβ1 mRNA was not increased by FBS but dramatically
decreased 8-fold by addition of TGFβ1. In parallel, on plastic, TGFβ3
mRNA was not increased by FBS, but increased 2-fold by TGFβ1.
On HC-HA, TGFβ3 mRNA was increased 3-fold than plastic, and 2fold and 5-fold after addition of FBS and TGFβ1, respectively.
TGFβ2 mRNA was not affected by any treatments when cells were
seeded on plastic or HC-HA. In addition, HC-HA inhibited
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
SMAD2/3 nuclear translocation and substantially reduced
cytoplasmic α-SMA normally upregulated by TGFβ1.
Conclusions: Collectively, these results indicate that HC-HA
purified from AM is responsible for AM’s anti-scarring action by
marked downregulation of TGFβ1 but upregulation of TGFβ3, which
is known to counteract TGF-β1 signaling. Furthermore, such an antiscarring effect is more apparent under the challenge of TGF-β1 or
serum, during which time HC-HA also suppresses Smad2/3
signaling, and expression of α-SMA.
Commercial Relationships: Fu Li, Ocular Surface Research &
Education Foundation (F); Ying-Ting Zhu, Tissue Tech (F), Tissue
Tech (E), Tissue Tech (P); Hua He, TissueTech, Inc. (E); Su-Zhen
Zhang, Tissue Tech, Inc (E); Scheffer C. Tseng, NIH, NEI (F),
TissueTech, Inc. (F), TissueTech, Inc. (E), TissueTech, Inc. (P)
Support: NIH, NEI, R44 EY017497, R43 EY021045, and RO1
EY06819
Program Number: 2069 Poster Board Number: D0208
Presentation Time: 11:00 AM - 12:45 PM
The response of tear film neutrophils to occasional overnight lens
wear
Maud Gorbet1, 2, Doerte Luensmann2, Lyndon W. Jones2. 1Systems
Design Engineering, University of Waterloo, Waterloo, ON, Canada;
2
Centre for Contact Lens Research - School of Optometry, University
of Waterloo, Waterloo, ON, Canada.
Purpose: During sleep, in the closed-environment, an influx of
polymorphonuclear neutrophils (PMNs) occurs, which typically
returns to normal levels a few hours after waking. This clinical study
was conducted to investigate the response of tear film PMNs to
occasional overnight lens wear.
Methods: Adapted lotrafilcon A lens wearers and non-lens wearers
were included (n=20 per group). Lenses were worn on a daily basis
for 30 days and two nights, one at the beginning and one at the end of
the lens wear cycle (Day1 & Day30). After each of these two nights,
ocular surface cells were collected immediately upon waking using
an eye-wash technique. Cells were also collected from the contact
lenses. Cells were incubated with fluorescently labeled antibodies
against ICAM-1, Mac-1, CD66b and CD33 (degranulation membrane
markers) and CD45 (pan leukocyte-marker) with and without
Lipopolysaccharide. Flow cytometry was used to determine the level
of expression and the ratio between unstimulated and stimulated
PMNs was calculated.
Results: After 8hrs of sleep, leukocyte cell count collected from the
ocular surface ranged from 60,000 to 2 million. There was no
significant difference in the total number and leukocyte type
collected from lens wearers and non-lens wearers (p>0.05). Cells
collected from the lenses represented 9.4 ± 8.2 % of the total PMN
count. Cell samples collected from the contact lenses were only
included for flow cytometry analysis if > 500 PMNs events were
observed (n=7).
Eye-wash collected PMNs showed a similar expression and ratio
between the two groups for Mac-1 and ICAM-1 (p>0.05). Significant
lower levels of expression of CD66b and CD33 were found in the
lens wearers (p<0.05 for both), while the ratio LPS
stimulated/unstimulated was similar to the non-lens wearers
suggesting that cells were able to appropriately respond to stimuli.
The tear film PMNs collected from the contact lenses had
significantly lower CD66b expression and higher ICAM-1 expression
when compared to the PMNs collected from the ocular surface of the
lens wearers (p=0.03).
Conclusions: Occasional overnight lens wear may have an effect on
the degranulation profile of tear film PMNs, particularly for PMNs
that remain in direct contact with the contact lens. Changes in PMN
phenotype could play a role in the development of ocular
inflammatory events; however, further research is needed to better
understand this mechanism.
Commercial Relationships: Maud Gorbet, CIBA Vision/Alcon
(F); Doerte Luensmann, Alcon (R); Lyndon W. Jones, Alcon (F),
Alcon (R), Allergan (F), Abbott Medical Optics (R), Bausch & Lomb
(R), Ciba Vision (F), Ciba Vision (R), CooperVision (F), Johnson &
Johnson (F), Johnson & Johnson (R)
Clinical Trial: NCT01379768
Program Number: 2070 Poster Board Number: D0209
Presentation Time: 11:00 AM - 12:45 PM
TLR3, RIG-1 and MDA5 are constitutively expressed on human
corneal limbal fibroblasts and induce proinflammatory response
Alfredo Domínguez1, 2, Rodrigo Bolanos1, Jeanet Serafín2, Jessica
Nieves-Hernández1, Yonathan Garfias1, 3. 1Instituto de Oftalmología
Conde de Valenciana, Mexico City, Mexico; 2Department of
Immunology, National School of Biological Sciences, Instituto
Politécnico Nacional, Mexico City, Mexico; 3Department of
Biochemistry, Faculty of Medicine, Universidad Nacional Autónoma
de México, Mexico City, Mexico.
Purpose: The ocular surface is exposed to a wide array of
microorganisms some of which could corneal opacification and loss
of vision. The principle cause of corneal damage is an exacerbated
immune response. Innate immunity is the first line of defense against
corneal infection. In response to virus infections, viral components
are recognized by pattern recognition receptors (PRRs). Upon ligand
binding, PRRs activate cytokine genes that set into motion a complex
inflammatory cascade. We previously reported that human corneal
limbal fibroblast (HCLF) stimulated with polyI:C elicited the
elevated production and mRNA expression of TLR3 and great variety
of pro-inflammatory cytokines. It was recently reported that RIG-I
and MDA-5 also recognize polyI:C. In this study, we investigated
whether RIG-I and/or MDA-5 contribute to polyI:C inducible
responses in HCLF.
Methods: HCLF were obtained from cadaveric sclero-corneal rims.
All the assays were carried out using 1x104 HCLF incubated or not
with poly I:C at a concentration of 10µg/ml during 12 h. After
stimulation period, the cell culture supernatant was used to detect 36
different cytokines. The total RNA was obtained retro-transcribed to
cDNA, were performed PCR assays for pro-inflammatory cytokines,
TLR3, RIG-1 and MDA-5. On the other hand, the total protein were
isolated and separated by SDS-PAGE and transferred to a
nitrocellulose membrane for immunoblot analysis with rabbit antiRIG-I or rabbit anti-MDA5 or rabbit anti-TLR3 antibodies.
Results: The mRNA levels of both the pro-inflammatory cytokines
(IL-6, IL-8, CCL5, CCL2, CXCL1, CXCL10 and IFN-β), and
receptors TLR3, and RIG were augmented when the cells were poly
I:C stimulated; similarly, the levels the pro-inflammatory cytokines
in the culture supernatants of HCLF were also augmented.
Interestingly, TLR3 protein was constitutively presented and
increased its expression when HLCF were poly I:C stimulated.
Conclusions: Our results showed that HLCF express transcripts and
proteins for PRR such as TLR3, MID-5 and RIG-1. After poly I:C
stimulation, these cells are able to respond increasing the synthesis,
production and secretion of proinflammatory cytokines.
Commercial Relationships: Alfredo Domínguez, None; Rodrigo
Bolanos, Institute Ophthalmology (P); Jeanet Serafín, None; Jessica
Nieves-Hernández, Institute of Ophthalmology (P); Yonathan
Garfias, Institute of Ophthalmology (P)
Support: CONACYT-SALUD-2011-C01-160286; CB 167438;
Fundación Conde de Valenciana IAP; CVU: 406706.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Program Number: 2071 Poster Board Number: D0210
Presentation Time: 11:00 AM - 12:45 PM
Bilateral Up-regulation of Limbal Intravascular Adhesion
Molecules Mediates Cell Infiltration into the Cornea after
Unilateral Trigeminal Nerve Axotomy
Deshea L. Harris1, 2, Takefumi Yamaguchi2, 1, Aslihan Turhan2,
Ulrich von Andrian3, Pedram Hamrah2, 1. 1Ophthal-Harvard Med
School, Schepens Eye Research Inst, Boston, MA; 2Ophthal-Harvard
Med School, Massachusetts Eye and Ear Infirmary, Boston, MA;
3
Program in Cellular and Molecular Medicine-Children's Hospital
Boston, Immune Disease Institute, Boston, MA.
Purpose: We have recently demonstrated that P-selectin and
MAdCAM-1 mediate the recruitment of corneal dendritic cells after
inflammation. Further, our group has demonstrated increased density
of leukocytes in unaffected contralateral eyes in unilateral corneal
diseases. The purpose of this study was to determine the neural
involvement of the trigeminal nerve in up-regulation of these
adhesion molecules.
Methods: Six- to 8-week-old adult BALB/c mice underwent
trigeminal axotomy to transect the ophthalmic branch of the
trigeminal nerve. Normal, axotomized, contralateral and suturedinduced inflamed corneas (n=3/group) were excised on postoperative days 3 and 7. RNA was isolated, DNAse treated, and
converted to cDNA. Real-time PCR was performed using primers
against the integrin MAdCAM-1 and P-selectin. Beta-actin and
GAPDH were used as housekeeping genes and results were
compared to normal and sutured controls. Homing of DC to the
cornea was studied by immunostaining and confocal microscopy.
Results: Following trigeminal axotomy, MAdCAM-1 was upregulated both on post-operative day 3 (1.8-fold) and day 7 (1.6fold). P-selectin was also up-regulated at day y (1.6-fold) and day 7
(1.9-fold) after axotomy. Interestingly, both MAdCAM-1 and Pselectin expression were even higher in contralateral unaffected
corneas at day 3 (2.5-fold) and day 7 (2.8-fold) for MadCAM-1, and
1.8-fold for day 3) and 2.3-fold for day 7 for P-selectin, as compared
to normal controls. The density of CD45+ cells in the center of the
cornea significantly increased from 189+/-21 /mm2 to 550 +/-134 at
day 3 and 979+/-45 at day 7 in the axotomized eye (P = 0.0015) and
slightly increase to 247+/-25 at day 3 and 245+/-35 at day 7 in the
contralateral eye (P = 0.595).
Conclusions: Our work demonstrates changes in the contralateral
unaffected eyes as a response to ipsilateral trigeminal axotomy in
mice. Trigeminal axotomy results in up regulation of the integrin
MAdCAM-1 and P-selectin in both axotomized and in contralateral
corneas, and suggest a neural regulation of limbal intravascular
adhesion molecules.
Commercial Relationships: Deshea L. Harris, None; Takefumi
Yamaguchi, None; Aslihan Turhan, None; Ulrich von Andrian,
None; Pedram Hamrah, None
Support: NIH K08-EY020575 (PH), Research to Prevent Blindness
Career Development Award (PH), Falk Medical Research Trust (PH),
Japanese Eye Bank (TY)
Program Number: 2072 Poster Board Number: D0211
Presentation Time: 11:00 AM - 12:45 PM
Tear Molecule Levels and Conjunctival Inflammatory Gene
Expression in Atopic Keratoconjunctivitis (AKC)
Amalia Enriquez-De-Salamanca1, 2, Erma Trias1, Verónica MartinezTottil1, Lidia Cocho1, Carmen García-Vázquez1, Maria J. GonzalezGarcia1, 2, Margarita Calonge1, 2. 1Ocular Surface Group, IOBAUniversity of Valladolid, Valladolid, Spain; 2Biomedical Research
Networking center in Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Valladolid, Spain.
Purpose: To measure a panel of tear cytolkines/chemokines and
mRNA expression of a panel of inflammatory molecules and their
receptors in conjunctival cells in atopic keratoconjunctivitis (AKC)
and to correlate their levels with AKC-related features and
extraocular atopic disorders.
Methods: 22 AKC patients and 18 age and sex-matched controls
were evaluated after 20 min in a controlled environment chamber
(45% relative humidity, 22°C, 930 mb). Patients were medically
controlled and had discontinued medications for one week before
examination. Symptom questionnaires were answered by patients and
controls. Unstimulated tears were collected from most symptomatic
eye. Levels of 19 molecules (EGF, IL-1RA, IL-1β, IL-2, IL-4, IL-5,
IL-6, IL-8, IL-10, IL-12, IL-13, IL-17A, IP-10, eotaxin 1, fractalkine,
IFN-γ, VEGF, TNF-α and RANTES) were measured by multiplex
bead assay and correlated with clinical parameters. Conjunctival
epithelial cells were collected by impression cytology in upper bulbar
conjunctiva and gene expression of 84 inflammatory molecules was
determined by RT2-PCR in pooled cDNA samples.
Results: IL-5 tear levels were decreased (p=0.0121) in AKC
compared to controls. IL-6 and IL-1β tear levels were increased in
AKC tear samples near to statistical significance (p=0.0509;
p=0.0713). AKC patients with active atopic dermatitis (AD) had
increased IFN-γ and TNF-α tear levels (p=0.0104, p=0.0176)
compared to non-active AD. AKC patients with concomitant asthma
had decreased tear IL-2 (p=0.0282). Several molecule tear levels
correlated with clinical signs. All molecules were detected, except
IL-17A and eotaxin 1. Expression of 45 genes was detected in AKC
conjunctival epithelial cells: 10 genes (IFNγ, CXCL2, CXCL3,
CCL11, CCL24, CCL26, CCR2, CCR3, IL-17C, and NAMPT) were
≥ 2 fold upregulated and 5 genes (CCL5, CXCR1, IL27, LTA, and
LTB) were ≥ 2 fold dowregulated.
Conclusions: IL-5 tear levels were decreased in AKC patients
compared to healthy individuals, probably related to the absence of
proliferative lesions, as described. Interestingly, the addition of active
AD correlated with 2 highly inflammatory molecules, IFN-γ and
TNF-α, in tears. In spite of the fact that these patients had mild
inflammation, the expression of several inflammatory genes was
altered in their conjunctival cells. These results add further
knowledge to the search for potential biomarkers in AKC.
Commercial Relationships: Amalia Enriquez-De-Salamanca,
Allergan Inc. (F); Erma Trias, None; Verónica Martinez-Tottil,
None; Lidia Cocho, None; Carmen García-Vázquez, None; Maria
J. Gonzalez-Garcia, None; Margarita Calonge, Allergan (C)
Support: SAF2010-15631, Ministry of Science and Innovation,
Spain
Program Number: 2073 Poster Board Number: D0212
Presentation Time: 11:00 AM - 12:45 PM
Measurement of wheat-specific tear IgE in patients with allergic
conjunctivitis
Tatsuya Mimura. Department of Ophthalmology, Tokyo Women's
Medical University Med. Cent. East, Arakawa-ku, Japan.
Purpose: The allergy to the hydrolyzed wheat in facial soap is a
major social issue in Japan. It has been reported that the most
frequent early symptoms of allergy to hydrolyzed wheat protein in
soap are allergic conjunctivitis and rhinitis, and the development of
wheat-dependent exercise-induced anaphylaxis (WDEIA) could be
induced by its long term use. We evaluated the relation between tear
fluid levels of wheat-specific tear IgE and features of allergic
conjunctivitis.
Methods: A prospective, nonrandomized, cross-sectional study was
conducted in 103 moderate to severe cases of allergic conjunctivitis
(allergic group) and 10 age- and sex-matched healthy control subjects
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
(control group). Specific IgE level for wheat was measured in tear
fluid with an immunochromatographic assay (1-4) and a skin prick
test (SPT) was also performed. A severity score (0, 1, 2, or 3) was
assigned for various changes of the palpebral and bulbar conjunctiva,
as well as for limbal and corneal lesions.
Results: The positive rate and specific IgE score were higher in the
allergic group than in the control group (71.8 % vs. 40.0% and1.9 ±
0.7 vs. 1.4 ± 0.5). The prevalence of a positive SPT to wheat was
higher in the allergic group than in the control group (6.8% vs. 0.0%).
In the allergic group, the specific IgE score was higher in patient with
positive SPT results than in patients with negative SPT results (3.3 ±
0.5 vs. 1.8 ± 0.6, p<0.001). In the allergic group, the wheat-specific
tear IgE score was correlated with five features of allergic
conjunctivitis (p<0.05).
Conclusions: These results suggest that wheat may be involved in
the development of allergic conjunctivitis.
Commercial Relationships: Tatsuya Mimura, None
Clinical Trial: 2612
Program Number: 2074 Poster Board Number: D0213
Presentation Time: 11:00 AM - 12:45 PM
In vitro toxicity of omalizumab in conjuctival epithelial cells
Anne-Sophie Benischke1, Albert Neutzner1, 3, Christoph Tappeiner2,
David Goldblum3. 1Department for Biomedicine, University Basel,
Basel, Switzerland; 2Department of Ophthalmology, University
Hospital of Bern, Bern, Switzerland; 3Department of
Ophthalomology, University Basel, Basel, Switzerland.
Purpose: The anti-IgE antibody omalizumab is used for the
treatment of severe allergic reactions and might be also applied
topically for the prevention of allergic reactions in the eye. Thus, the
short as well as long-term toxicity of omalizumab formulated as
Xolair® or in pure form on human conjunctival epithelial cells was
tested.
Methods: Chang cells were treated with increasing doses ranging
between 125 μg/ml and 125 mg/ml of Xolair® or purified
omalizumab from 125 μg/ml to 40mg/ml for 20 minutes or seven
days. Cell death was measured using ethidium bromide exclusion
while cell survival was assessed using calcein AM staining.
Results: Neither treatment with omalizumab from 125 μg/ml to 125
mg/ml formulated as Xolair® or treatment with purified omalizumab
from 125 μg/ml to 40mg/ml proved harmful to human conjunctival
epithelial cells.
No morphological cell alterations were observed in any
concentrations.
Conclusions: Our data did not reveal an acute or long-term toxicity
to conjunctival epithelial cells. Thus, for severe allergic reactions in
the eye or in case of side effects towards established anti-allergic eye
medication, omalizumab in the formulation of Xolair® or in form of
eye drops might offer additional treatment options employing
alternative mechanisms of action.
Commercial Relationships: Anne-Sophie Benischke, None; Albert
Neutzner, None; Christoph Tappeiner, None; David Goldblum,
None
Support: Swiss National Foundation
Program Number: 2075 Poster Board Number: D0214
Presentation Time: 11:00 AM - 12:45 PM
Effects of IL-13 Stimulation on Primary Mouse Conjunctival
Epithelial Cultures
Johanna Tukler Henriksson, Xiaobo Zhang, De-Quan Li, Cintia S.
De Paiva, Stephen C. Pflugfelder. Ophthalmology, Baylor College of
Medicine, Houston, TX.
Purpose: To investigate the impact of IL-13 stimulation on goblet
cell differentiation in primary mouse conjunctival cultures.
Methods: Young adult, (6-8 week) female C57BL/6 mice were
utilized. Explants were obtained from the forniceal conjunctiva and
plated, one explant per well in 48 well plates. First, growth kinetics
were evaluated with different media and EGF concentrations.
Second, the explants received 200 µl per week of either Keratinocyte
media with 3% fetal bovine serum defined (KSFM) or KSFM with
added 1ng/ml or 10ng/ml of interleukin-13 (IL-13). Cultures were
incubated for 1 or 2 weeks. Subsequently, either cell proliferation
was assessed by WST-1 assay or cultures were fixed in methanol and
immunostained for MUC5AC and cytokeratin7 (K7), IL- 4 receptor
alpha (Rα) or IL-13 receptor alpha1 (Rα1).
Results: On average 50% of explants showed outgrowth using
KSFM with 80ng/ml EGF. Both the IL-13 treated and the non-treated
cultures showed a statistically significant increase in growth at 2
weeks compared to 1 week (P < 0.05), and the IL-13 1 ng/ml treated
cultures had significantly greater outgrowth than control (P < 0.05).
100% of the cultures in all conditions were K7 positive. After 1 week
the MUC5AC/K7 ratio was 0.11 in control media versus 0.89 and
0.99 in IL-13 1 ng/ml and IL-13 10 ng/ml treated cultures,
respectively (P < 0.05). There was no difference in MUC5AC/K7
ratio at 2 weeks [(0.99 controls, 0.95 IL-13 1 ng/ml and 0.98 IL-13
10 ng/ml (P > 0.05)]. Positive expression for the signal transducing
IL- 4 (Rα) and IL-13 (Rα1) receptors was also noted.
Conclusions: This study illustrated successful growth of primary
mouse conjunctival cultures with increased proliferation over time.
IL-13 cytokine stimulation appears to increase goblet cell
differentiation in a dose dependent manner at the early stages of
outgrowth. The results from these experiments indicate that
conjunctival goblet cell growth and differentiation is regulated by the
T helper (Th) 2 cytokine IL-13.
Commercial Relationships: Johanna Tukler Henriksson, None;
Xiaobo Zhang, None; De-Quan Li, None; Cintia S. De Paiva,
Glaxo Smith Kline (C), Baylor College of Medicine (P); Stephen C.
Pflugfelder, Allergan (C), Glaxo Smith Kline (C), Bausch and Lomb
(C), Baylor College of Medicine (P)
Support: NIH Grant EY11915 (SCP), Research to Prevent
Blindness, Oshman Foundation, William Stamps Farish Fund, Hamill
Foundation
Program Number: 2076 Poster Board Number: D0215
Presentation Time: 11:00 AM - 12:45 PM
Increased proliferation of cultured conjunctival epithelium in
IFN-γ deficient strains
Meng Chen, Terry G. Coursey, Johanna Tukler Henriksson, Cintia S.
De Paiva, De-Quan Li, Stephen C. Pflugfelder. Ophthalmology,
Baylor College of Medicine, Houston, TX.
Purpose: Previous experiments have demonstrated that IFN-γ
promotes conjunctival epithelial apoptosis and goblet cell loss. The
objective of this study was to compare epithelial growth and
differentiation in conjunctival epithelial cultures initiated from IFN-γ
deficient mice and wild type (WT) C57BL/6 mice.
Methods: Primary conjunctival epithelial cultures were established
in parallel from fresh tissue explants of forniceal conjunctiva
dissected from IFN-γ gene knockout (IFN-γKO) and WT C57BL/6
age- and gender-matched mice. Cultures were incubated with
Keratinocyte SFM (KSFM) medium supplemented with 80 ng/mL
murine EGF and 3% fetal bovine serum. Cell proliferation was
measured by WST-1 assay. Explant outgrowth areas were quantified
using Celltrace Calcein Green AM staining and Nikon Elements
software analysis. Keratin 7 (K7) and MUC5AC were
immunodetected by immunofluorescent staining. K7 and MUC5AC
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
mRNA expression were measured by quantitative real time PCR.
Results: Seventy-seven percent of IFN-γKO cultures reached 7080% confluence after 2 weeks, compared to 61% of WT C57BL/6
cultures. Positive K7 and MUC5AC immunofluorescent staining
confirmed the growth of conjunctival epithelium and goblet cells in
culture. WST-1 cell viability assay showed IFN-γKO cultures had
2.24 fold (p<0.05, n=9) and 2.01 fold (p<0.05, n=6) greater number
of viable cells at 7 and 14 days, respectively, compared to WT
C57BL/6 cultures. Calcein Green AM staining also demonstrated
statistically significant increased epithelial outgrowth from IFN-γKO
explants compared to C57BL/6 . At 4 days, mean growth area of WT
C57BL/6 and IFN-γ cultures were 1.52 x 107 µm and 2.80 x 106 µm
(p<0.05, n=5), respectively, and at 7 days, they were 2.33 x 10 7 µm
and 6.14 x 106 µm (p<0.05, n=4), respectively. A significant 2.58
fold elevation in MUC5AC mRNA transcripts was observed in 2
week old IFN-γKO cultures compared to WT C57BL/6 cultures, but
K7 mRNA transcripts were not significantly different between the
two strains.
Conclusions: Our findings show that cultured conjunctival epithelial
and goblet cells from IFN-γ deficient mice have increased growth and
differentiation compared to wild-type mice. This suggests IFN-γ
suppresses conjunctival epithelial growth and goblet cell
differentiation.
Commercial Relationships: Meng Chen, None; Terry G. Coursey,
None; Johanna Tukler Henriksson, None; Cintia S. De Paiva,
Glaxo Smith Kline (C), Baylor College of Medicine (P); De-Quan
Li, None; Stephen C. Pflugfelder, Allergan (C), Glaxo Smith Kline
(C), Bausch and Lomb (C), Baylor College of Medicine (P)
Support: NIH Grant EY11915 (SCP), Research to Prevent
Blindness, Oshman Foundation, William Stamps Farish Fund, Hamill
Foundation
Program Number: 2077 Poster Board Number: D0216
Presentation Time: 11:00 AM - 12:45 PM
Detection of natural helper cells in innate immune systemdependent mouse conjunctivitis model
Yosuke Asada1, 2, Akira Matsuda1, Kanji Hori1, Satoshi Iwamoto1,
Nobuyuki Ebihara1, Susumu Nakae2, Akira Murakami1.
1
Ophthalmology, Juntendo University School of Medicine, Tokyo,
Japan; 2Frontier Research Initiative, Institute of Medical Science,
University of Tokyo, Tokyo, Japan.
Purpose: Natural helper (NH) cells are identified innate lymphocyte
population, which respond to a combination of interleukin (IL)-2 and
IL-25 or IL-33 to produce large amounts of TH2 cytokines and
induce eosinophil infiltration in the lung- and gut-associated
lymphoid tissues. We identified NH cells in mouse lacrimal- and
conjunctival- tissues obtained from papain-induced conjunctivitis
model, in which innate immune system dependent conjunctival
eosinophilic infiltration was observed.
Methods: To made papain-induced conjunctivitis model mice,
papain-soaked soft contact lens (2mm diameter) was inserted into the
conjunctival sacs of C57/BL6(B6) mice or B6-Rag2 knockout (KO)
mice, which lack acquired immune systems, and the eye lids were
sutured. 4days after contact lens installation, the eyes were subjected
to histological analysis. Flow cytometric analyses were performed
using anti-Siglec-F and anti-CCR3 antibodies to quantify infiltrated
eosinophils. Spleen, thymus, cervical lymph node, lacrimal gland,
and conjunctival tissues obtained from the papain-induced
conjunctivitis model mice were subjected to cytometric analysis. At
the same time, serum IgE concentration of the mice was quantified.
Results: We identified the existence of NH cells (Lineage- ST2+
CD25+CD127+Sca1+Thy1+) in the conjunctival- and lacrimal gland
-tissues obtained from papain-induced conjunctival model. Papain-
induced conjunctivitis model showed comparable degree of
eosinophil infiltration in the conjunctivae of Rag2 KO mice
compared to those of wild-type mice. (53 ± 34 versus 73 ± 44, mean
number of eosinophil ± SD per slides, n = 6 , P> 0.05 by MannWhitney’s U test ) Siglec-F+CCR3+ eosinophils were increased in
the conjunctivae of papain-induced conjunctivitis model compared to
naïve conjunctivae. (0.126 versus 0.0679, the ratio of SiglecF+CCR3+/CD45+ cells). Total IgE concentrations were unchanged
during conjunctivitis induction using papain-soaked contact lens.
Conclusions: We made innate immune system-dependent
conjunctivitis model with papain-soaked soft contact lenses. NH cells
were identified in the conjunctival tissue and in the lacrimal tissue of
the papain-induced conjunctivitis model.
Commercial Relationships: Yosuke Asada, None; Akira Matsuda,
None; Kanji Hori, None; Satoshi Iwamoto, None; Nobuyuki
Ebihara, None; Susumu Nakae, None; Akira Murakami,
SEED(Japan) JP4855782 (P), SEED(Japan) JP5132958 (P)
Program Number: 2078 Poster Board Number: D0217
Presentation Time: 11:00 AM - 12:45 PM
The Synergistic Effects between Bevacizumab and Cyclosporin A
(Cys A) reduced the MMP-3 and MMP-13 Expression and the
Migration on Cultured Pterygial Fibroblast Cells and Tissues
Yeoun-Hee Kim1, 2, Young Jeung Park1, Sun-Hee Kang2, Jae-Chang
Jung2, Kyoo Won Lee1. 1ophthalmology, Cheil eye hospital, Daegu,
Republic of Korea; 2Biology, Kyungpook National University,
Daegu, Republic of Korea.
Purpose: To investigate the effects of Bevacizumab or/and
Cyclosporin A (Cys A) on the matrix metalloproteinase (MMP) 3 and
MMP-13 expression in cultured human pterygial fibroblast cells and
tissues.
Methods: Pterygial and normal conjunctival tissue of Korean
patients (10 males and 10 females) were analyzed. Immunostaining,
Western blotting and RT-PCR were performed to detect the
localization and expression level of MMPs. After being scratched,
human pterygial fibroblast cells were exposed to Cys A
concentrations of 1 µg/ml (0.0001%) and 100 µg/ml (0.01%) for 3 or
10 minutes and/or bevacizumab 1 µg/ml with serum depletion
DMEM-F-12 (1:1) media for 48 hours. Changes in expression of
MMP-3 and MMP-13 of fibroblast after the stimulation were studied.
Results: MMP-3 and MMP-13 expression in pterygium tissues was
greater than those of normal conjunctiva and was closely associated
to the progression of pterygium. Bevacizumab-injected pterygium
tissues were significantly reduced MMP-3 and MMP-13 expression.
In vitro, bevacizumab and/or Cys A-treated pterygial fibroblast was
also markedly inhibited cell migration and MMPs expression.
Interestingly, pre-exposed pterygial fibroblast with Cys A and
followed by bevacizumab treatment blocked the cell migration and
MMP-3 and MMP-13 expression (P<0.01), significantly higher.
Conclusions: Other previously reported studies demonstrated that
subconjunctival bevacizumab injection was useful in management of
patients with primary and recurrent pterygium without significant
local or systemic adverse effects, however, the exact mechanism still
remains unclear. Our results suggested high expression of MMPs
induced proliferation and migration of pterygial fibroblast. Secondly,
expressionof MMPs were down-regulated by bevacizumab or Cys A
treatment. Here, we show that combined treatment of Cys A and
bevacizumab was more effective in inhibition of MMPs. These
finding also provide therapeutic potential for pterygium progression.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Commercial Relationships: Natasha V. Nayak, None; Anton M.
Kolomeyer, None; Jason S. Kim, None; Eliott S. Kim, None;
Christina Fang, None; David S. Chu, Abbott (F), Novartis (F),
Santen (F), Eyegate (F), Lux Biosciences (F), Bausch & Lomb (R)
Associate Principal Researcher Department of Ophthalmology Cheil
Eye Research Institute Cheil Eye Hospital
Commercial Relationships: Yeoun-Hee Kim, None; Young Jeung
Park, None; Sun-Hee Kang, None; Jae-Chang Jung, None; Kyoo
Won Lee, None
Program Number: 2079 Poster Board Number: D0218
Presentation Time: 11:00 AM - 12:45 PM
Topical Cyclosporine A for the Management of Chronic
Follicular Conjunctivitis
Natasha V. Nayak1, Anton M. Kolomeyer1, Jason S. Kim2, Eliott S.
Kim2, Christina Fang1, David S. Chu1, 2. 1Institute of Ophthalmology
and Visual Science, University of Medicine and Dentistry of NJ,
Newark, NJ; 2Metropolitan Eye Research & Surgery Institute,
Palisades Park, NJ.
Purpose: To describe the use of Cyclosporine A (CsA) in patients
with chronic follicular conjunctivitis (CFC).
Methods: This study was a retrospective chart review at a tertiary
care center. Patients treated with topical CsA (1% emulsion) for
idiopathic CFC between August 2001 and November 2012 were
included. Biopsies were performed. Main outcome measures included
time to reduced inflammation, final grade of inflammation (0-4;
grading performed by one physician [DSC]), ability to taper steroids,
final visual acuity (VA), and reported side effects. Unless otherwise
noted, mean ± standard deviation (SD) were reported.
Results: Twenty-two eyes of 13 patients (nine [69%] females; 12
[92%] Caucasians; age of 49.5 ± 14.7 years) met study criteria. Two
(15%) patients had an associated autoimmune disease (Sarcoidosis
and Hashimoto’s thryroiditis/Sjogren’s syndrome). Mean follow-up
time was 233 days (range, 33-887 days). CsA was initiated 50.6 days
± 46.9 days after diagnosis. Six (46%) patients ended CsA use after
182.2 ± 95.0 days, in all cases after inflammation was controlled. The
remaining seven (54%) patients have ongoing management with CsA
(currently for 278.9 ± 206.1 days). Final grade of inflammation (0.2 ±
0.4) was improved compared to initial grade of inflammation (2.1 ±
1.0) [two-tailed t-test, p-value < 0.0001]. Inflammation was
controlled on average 40.1 ± 23.1 days (range, 5-84 days) after
initiation of CsA. Eleven (85%) patients tapered and eventually
discontinued topical steroids 31.5 ± 27.9 days after initiation of CsA
treatment. One patient required re-initiation of topical steroids three
months after initial discontinuation secondary to a flare up. Mean
initial log MAR VA for right and left eyes was 0.090 and 0.067,
respectively, whereas mean final VA was 0.089 and 0.028. Three
(23%) patients reported mild ocular irritation and/or burning;
however, none discontinued the eye drops.
Conclusions: Management of CFC with topical CsA resulted in
inflammation control and allowed for steroid taper in the majority of
patients without severe complications. Most patients require longterm CsA treatment.
Program Number: 2080 Poster Board Number: D0219
Presentation Time: 11:00 AM - 12:45 PM
Suppression of TLR3-Inducible Gene Expression by EP3 in
Conjunctival Epithelium
Mayumi Ueta1, 2, Shuh Narumiya3, Shigeru Kinoshita1.
1
Ophthalmology, Kyoto Prefectural Univ of Medicine, kyoto, Japan;
2
Research Center for Inflammation and Regenerative Medicine,
Faculty of Life and Medical Sciences, Doshisha University,
kyotanabe, Japan; 3Department of Pharmacology and Faculty of
Medicine, Kyoto University, kyoto, Japan.
Purpose: We previously reported that prostaglandin E receptor 3
(EP3) negatively regulates the eosinophilic infiltration of murine
experimental allergic conjunctivitis induced by toll-like receptor 3
(TLR3), which causes reduced eosinophilic conjunctival
inflammation in TLR3/EP3 double knockout (DKO) mice, despite
the pronounced eosinophilic conjunctival inflammation in EP3 KO
mice. It was also reported that EP3 was dominantly expressed in
epithelial cells such as conjunctival epithelial cells, airway epithelial
cells, and keratinocytes. In this study, to examine the effects of EP3
against TLR3-inducible gene expression in conjunctival epithelium,
we performed gene expression analysis of polyinosinic:polycytidylic
acid (polyI:C)-stimulated murine conjunctival epithelium in wildtype (WT)-, EP3-, and EP3/TLR3 DKO mice.
Methods: First, we examined the comprehensive effects of gene
expression in polyI:C-stimulated murine conjunctival epithelium of
WT mice using GeneChip® oligonucleotide microarrays. Then, to
find the transcript regulated by EP3, we compared the gene
expression of the transcripts, which were significantly and more than
3-fold upregulated, in polyI:C-stimulated conjunctival epithelium
between WT and EP3 KO mice by quantitative reverse transcription
polymerase chain reaction (RT-PCR).
Results: GeneChip® analysis showed that 131 transcripts were
upregulated more than 3-fold and that 31 transcripts were upregulated
more than 10-fold upon polyI:C stimulation of murine for 6 hours.
Quantitative RT-PCR findings confirmed that 21 of 31 transcripts
were significantly and more than 3 fold upregulated upon polyI:C
stimulation in murine conjunctival epithelium. All of those 21
transcripts were found to be expressed in the polyI:C-stimulated
conjunctival epithelium of EP3 KO mice significantly stronger than
in that of WT mice. Moreover, mRNA expression of those 21
transcripts were confirmed to be significantly reduced in the polyI:Cstimulated conjunctival epithelium of EP3/TLR3 DKO mice than in
that of EP3 KO mice.
Conclusions: EP3 suppressed the TLR3-inducible genes in polyI:Cstimulated conjunctival epithelium, which causes reduced gene
expression in the conjunctival epithelium of TLR3/EP3 DKO mice,
despite the pronounced gene expression in the conjunctival
epithelium of EP3 KO mice.
Commercial Relationships: Mayumi Ueta, None; Shuh Narumiya,
ONO Pharmaceuticals (F); Shigeru Kinoshita, Senju Pharmaceutical
Co (P), Santen Pharmaceutical Co (P), Otsuka Pharmaceutical Co
(C), Alcon (R), AMO (R), HOYA (R)
Support: Japanese Ministry of Education, Culture, Sports, Science
and Technology
Program Number: 2081 Poster Board Number: D0220
Presentation Time: 11:00 AM - 12:45 PM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Model of allergic keratoconjunctivitis - Stat6 signaling in chronic
inflammation
Michael Conwell1, Sonia DaSilva-Arnold2, Na Luo1, Wei Li3, Jeffery
Travers2, Yang Sun1, 2. 1Ophthalmology, Glick Eye Institute,
Indianapolis, IN; 2Dermatology, Indiana University, Indianapolis, IN;
3
Xiamen Eye Institute, Xiamen University, Xiamen, China.
Purpose: The purpose of this study is to characterize the corneal
inflammatory lesions found in the Stat6VT mouse as a model for
atopic dermatitis. We hypothesize that the constitutive activation of
Stat6 results in corneal neovascularization and pannus formation.
Methods: SKH1-HrHr wild-type mice and mice which express an
integrally active Stat6 (Stat6VT), V547A/T548A, (N=20, 24
respectively) where examined for the development of
keratoconjunctivitis. Corneas were removed and examined by H&E
and PAS staining. Immunoblot analysis was performed with
Interleukin-4, Interferon gamma, and CD3. Steroid treatment with
clobetasol was performed on affected lesions.
Results: Compared to wild-type, Stat6VT transgenic mice exhibited
increased frequency of corneal neovascularization (N=24, P<0.05).
The Stat6VT transgenic mice demonstrated corneal infiltrate as well
as neovascularization (N=5, P<0.05). Real-time PCR analyses
performed on the mRNA from both the wild type and Stat6VT mice
yielded elevated levels of Th2 cytokines isolated from the Stat6VT
mice (N=5,5 respectively P<0.05). We showed that clobetasol
treatment slowed the development of periocular and corneal lesions.
Conclusions: Stat6 signaling may play a role in the pathogenesis of
chronic ocular inflammation. IL-4 may mediate corneal inflammation
in allergic keratoconjunctivitis.
Figure 1. Corneal infiltrates in STAT6VT mice. (A) WT and (B)
STAT6VT mice cornea were stained by H&E. Scale bar represents
10 microns.
Commercial Relationships: Michael Conwell, None; Sonia
DaSilva-Arnold, None; Na Luo, None; Wei Li, None; Jeffery
Travers, None; Yang Sun, NIH (F), American Glaucoma Society
(R), Knights Templar Eye Foundation (R), Reeves Foundation (R)
Support: NIH-EY-K08-022058; American Glaucoma society;
Knights Templar Eye foundation
Program Number: 2082 Poster Board Number: D0221
Presentation Time: 11:00 AM - 12:45 PM
Molecular mechanism of corneal neovascularization inhibition by
decorin therapy
Ashish Tandon, Ajay Sharma, Jason T. Rodier, Rajiv R. Mohan.
Ophthalmology, Mason Eye Institute, Columbia, MO.
Purpose: Recently we showed that targeted AAV-decorin gene
therapy significantly inhibits corneal angiogenesis in vivo in rabbits.
This study tested the hypothesis that decorin’s anti-angiogenic effects
in the cornea are mediated via the restoration of normal physiologic
balance between the pro- and anti-angiogenic cytokines, VEGFR
modulation, and VEGFR-mediated ERK signaling.
Methods: New Zealand white rabbits and human corneal fibroblast
(HCF) cultures were used. The HCF cellular lysates treated with
decorin (250nM) +/- VEGF (50ng/ml) were used for time dependent
analysis of vascular endothelial growth factor receptor (VEGFR),
VEGFR1 and VEGFR2 phosphorylation using proteome human
receptor tyrosine kinases (RTKs) array analysis. Corneal
neovascularization in rabbits was produced with VEGF-implant
micro-pocket assay. Quantitative real-time qPCR,
immunohistochemistry and fluorescence confocal microscopy
analyzed pro-/anti-angiogenic cytokines (VEGF, MCP1,
angiopoietin, PEDF etc.), VEGFR mRNA/protein expression, and
VEGFR-mediated ERK1/2 signaling in AAV-decorin-transduced,
AAV-gfp-transduced neovascularized, and naive rabbit corneas.
Results: VEGF treatment caused phosphorylation of VEGFR in HCF
as detected by RTK array assay. Detection of no VEGFR
phosphorylation in HCF incubated with VEGF+decorin suggests that
decorin abrogated VEGF-induced phosphorylation of VEGFR in
corneal fibroblasts. Decorin also inhibited VEGFR phosphorylation
in vivo as revealed by a marked decrease in ERK1/2 phosphorylation
in AAV-decorin-delivered neovascularized rabbit corneal sections.
Additionally, AAV-decorin gene delivery rescue normal physiologic
balance between pro- and anti-angiogenic factors by down-regulating
VEGF, MCP1 (mRNA 3-5 fold p< 0.05 and protein 11-27%, p<0.01)
and up-regulating PEDF (mRNA 1.8-2.5 fold p<0.05 and protein 913%, p<0.01) in neovascularized rabbit corneas. The results of other
signaling studies are pending.
Conclusions: Decorin inhibits corneal angiogenesis largely by
interrupting VEGF signaling, and restoring the pro- and antiangiogenic factors balance required for corneal transparency.
Commercial Relationships: Ashish Tandon, None; Ajay Sharma,
None; Jason T. Rodier, None; Rajiv R. Mohan, None
Support: 1I01BX000357-01Veteran Health Affairs Merit (RRM),
RO1EY17294 National Eye Institute, National Institutes of Health
(RRM)
Program Number: 2083 Poster Board Number: D0222
Presentation Time: 11:00 AM - 12:45 PM
Recurrent corneal inflammation stimulates lymphatic vessel
memory
Richard M. Tempero, Alicia L. Conner, Philip M. Kelley. Boys Town
National Rsch Hosp, Omaha, NE.
Purpose: To determine how recurrent corneal inflammation, a
common clinical condition, regulates corneal lymphatic vessel
remodeling.
Methods: A mouse corneal model was developed in which recurrent
inflammation was stimulated by suture placement following wound
recovery. Immunofluorescent microscopy was used to visualize and
quantify the lymphatic vessel response. VEGFR-2 and VEGFR-3
decoy receptors were used to block VEGF-A and -C pathways during
initial inflammation and recurrent inflammation.
Results: Recurrent inflammation accelerated the development a
functional lymphatic vessel network. This observation revealed a
novel program of lymphangiogenesis and identified a property of
lymphatic vessel memory in response to recurrent inflammation. A
brief episode of initial corneal inflammation and regressed lymphatic
vessels were associated with the development of lymphatic vessel
memory. The spatial distribution of specialized lymphatic vessel
features was distinct in these vessels. Results using the VEGFR-2 and
R-3 decoy receptors demonstrated that the lymphatic vessel memory
response was not dependent upon the VEGF-C or VEGF-A
pathways, indicating that different molecular pathways regulate
inflammatory lymphangiogenesis and lymphatic vessel memory.
Conclusions: These findings uncover a priming mechanism to
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
facilitate a rapid lymphatic vessel memory response: a potential
important component of corneal defense.
Commercial Relationships: Richard M. Tempero, None; Alicia L.
Conner, None; Philip M. Kelley, None
Support: NH grants EY021571 and P20 RR018788
Program Number: 2084 Poster Board Number: D0223
Presentation Time: 11:00 AM - 12:45 PM
The Effects and Underlying Mechanism of Bevacizumab
(Avastin) in Inhibiting Corneal Neovascularization in a Rabbit
Closed Eye Contact Lens Wear Model
Wei-Li Chen1, Yan-Ming Chen1, 2, Fung-Rong Hu1. 1Ophthalmology,
National Taiwan University Hospital, Taipei, Taiwan;
2
Ophthalmology, E-Da University Hospital, Kaohsiung, Taiwan.
Purpose: To evaluate the therapeutic effects of early and late
subconjunctival injection of bevacizumab on the inhibition of corneal
neovascularization (NV) in a rabbit closed eye contact lens (CL) wear
model. The underlyng mechanism focusing on VEGF and VEGF
receptors were also evaluated.
Methods: Corneal NV was induced by closed eye CL wear in
rabbits. Group 0 included rabbits eyes without bevacizumab
treatment. Weekly subconjunctival injections of bevacizumab (5.0
mg) for 1 month were started immediately (Group I) and 1 month
after induction of corneal NV with continuous induction (Group II.
Digital photographs were taken weekly. The percentage of involved
corneal surface (PICS)/centricity/extent of corneal NV were
evaluated. Immunohistochemical staining was used to determine the
intracorneal diffusion of bevacizumab in the different groups.
Immunostaining with anti-CD31, α- smooth muscle actin (αSMA)
and high-molecular-weight melanoma-associated antigen (HMWMAA) was used to evaluate the formation of pericytes and smooth
muscle around the corneal NV. The expression of vascular
endothelial growth factor (VEGF) and its receptors (VEGFR1,
VEGFR2) was also evaluated. TUNEL staining was performed to
evaluate cellular apoptosis after bevacizumab injection. In each
group, 6 rabbtit eyes were examined. There were 6 eyes in each
experimental conditions
Results: Early treatment with bevacizumab (Group I) more
significantly inhibited corneal NV than late treatment administered
(Group II) after 4 weeks (p<0.01 in NV length, surface area and
extent). Immunostaining showed pericyte and smooth muscle
coverage on the vessel wall as early as 2 weeks after induction.
Intracorneal diffusion of bevacizumab was not different among the 3
groups. VEGF/VEGFR1/VEGFR2 expressed on corneal epithelial
cells was found in group 0/1/2, and on corneal vessels in group 0/2
but not group 1. Corneal vessels with evidence of apoptosis was not
found in group 1 and group 2 at 4 weeks after bevacizmbab injection.
Conclusions: Early but not late injection of bevacizumab inhibited
corneal NV induced by closed eye CL wear in rabbits. The difference
in therapeutic effect was not related to intracorneal diffusion of
bevacizumab. Bevacizumab did not influence the expression of
VEGF/VEGFR1/VEGFR2 on corneal vessels if treated late.
Commercial Relationships: Wei-Li Chen, None; Yan-Ming Chen,
None; Fung-Rong Hu, None
Support: None in the Support field
Program Number: 2085 Poster Board Number: D0224
Presentation Time: 11:00 AM - 12:45 PM
SAMHD1 is a Novel Regulator of Soluble Flt-1
leah owen, Derick G. Holt, Hironori Uehara, Balamurali K. Ambati.
Ophthalmology, University of Utah John Moran Eye Center, Salt
Lake City, UT.
Purpose: Aberrant angiogenesis is fundamental to numerous ocular
pathologies and represents a significant clinical problem. Current
therapeutics primarily target vascular endothelial growth factor
(VEGF) though with incomplete efficacy. This may be due to the fact
that the full complement of disease mechanisms is not addressed, or
alternatively that there is a more central “molecular switch”
responsible for this pathology. Soluble vascular endothelial growth
factor receptor-1 (sFLT1) has emerged as an important endogenous
regulator of both physiologic and pathologic angiogenesis in multiple
tissues including the eye. sFlt1 is a physiologic splice variant of the
full length FLT1 receptor which contains only the ligand binding
domain and therefore functions as an endogenous VEGF inhibitor.
While sFLT1 plays an important role in physiologic corneal
avascularity, molecular pathways important for its production remain
incompletely understood.
Methods: Genome-wide microarray was performed using murine
models of corneal neovascularization expressing a gradient of sFlt1.
Corneal samples were assessed from MRL mice which are known to
expression supra-physiologic levels of sFlt1 and have avascular
corneas, Pax6+/- mice which express low levels of sFlt1 and have
spontaneously vascularized corneas, as well as wild-type controls.
Candidate genes were identified for which expression correlated
significantly with sFlt1 gradient expression. These data were
confirmed using real-time PCR. Functional relevance was determined
using si-RNA mediated knock-down of candidate regulators in
human umbilical vein endothelial cells followed by real-time PCR
assessment of sFlt1 expression.
Results: SAMHD1 expression was found to correlate significantly
with decreased sFlt1 expression and the presence of corneal
neovascularization. These data were confirmed using siRNA
mediated knock-down of SAMHD1 which demonstrated increased
sFlt1 expression in the presence of SAMHD1 knock-down.
Conclusions: SAMHD1 represents a novel regulator of sFlt1 which
is necessary for suppression of sFlt1 expression. The physiologic
function of SAMHD1 is unknown, though its SAM domain
classically mediates protein interactions and its HD domain is
thought to have phosphohydrolase activity. Further work will clarify
whether SAMHD1 function is sufficient to mediate sFlt1 suppression
and investigate the mechanism of action allowing for SAMHD1 to
evolve as a possible therapeutic target in the future.
Commercial Relationships: leah owen, None; Derick G. Holt,
None; Hironori Uehara, None; Balamurali K. Ambati, None
Support: National Eye Institute 5R01EY017950
Program Number: 2086 Poster Board Number: D0225
Presentation Time: 11:00 AM - 12:45 PM
Serum eye drops promote vascular endothelial cell proliferation
in vitro and antagonize (antiangiogenic) VEGF blockade in vivo
Deniz Hos, Konrad R. Koch, Felix Bock, Claus Cursiefen, Ludwig M.
Heindl. Department of Ophthalmology, University of Cologne,
Cologne, Germany.
Purpose: Autologous serum eye drops lead to faster wound closures
and help treating non-healing and relapsing corneal ulcerations.
Although having a well defined wound-healing profile, the influence
of serum eye drops on corneal neovascularization is so far unclear.
We therefore analyzed the corneal vascular effects of serum eye
drops in comparison as well as in combination with topical VEGF
blockade.
Methods: For in vitro studies, human blood or lymphatic endothelial
cells (BEC or LEC, respectively) were treated with 10% fresh serum,
50 ng/ml Bevacizumab or a combination of both. Endothelial cell
proliferation was assessed after 48 hours using a BrdU-based ELISA.
For in vivo studies, inflammatory corneal neovascularization was
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
induced by suture placement in BALB/c mice (n=8 per group). Mice
then received serum or Bevacizumab eye drops (3x daily,
respectively) or a combination of both (3x daily for each treatment).
After one week, corneal wholemounts were stained for blood (CD31)
and lymphatic (LYVE-1) vessels.
Results: In vitro, serum addition significantly increased whereas
Bevacizumab significantly reduced endothelial cell proliferation
(BEC: serum mean m= +55.3%, p<0.01; Bevacizumab m= -13.6%,
p<0.05; LEC: serum m= +9.5%, p<0.01; Bevacizumab m= -33.0%,
p<0.01). The combined treatment with serum and Bevacizumab led
to cell proliferation levels that presented between the monotreated
groups (BEC: m= +42.2%, p<0.05; LEC: m= -5.9%, p>0.05). In
vivo, inflammatory corneal hem- and lymphangiogenesis was not
influenced by treatment with serum eye drops, whereas treatment
with Bevacizumab significantly reduced blood and lymphatic vessel
growth (hemangiogenesis: serum m= +9.5%, p>0.05; Bevacizumab
m= -19.8%, p<0.05; lymphangiogenesis: serum m= -7.8%, p>0.05;
Bevacizumab m= -33.3%, p<0.05). The combination of serum and
Bevacizumab eye drops attenuated the anti(lymph)angiogenic effect
of Bevacizumab (hemangiogenesis: m= -8.4%, p>0.05;
lymphangiogenesis: m= -15.2%, p>0.05).
Conclusions: Although promoting blood and lymphatic endothelial
cell proliferation in vitro, serum eye drops show no significant impact
on inflammatory corneal hem- and lymphangiogenesis in vivo.
Interestingly, serum eye drops seem to antagonize the
anti(lymph)angiogenic effect of topical VEGF blockade.
Commercial Relationships: Deniz Hos, None; Konrad R. Koch,
None; Felix Bock, None; Claus Cursiefen, Gene Signal (C), Alcon
(R), Allergan (R), Bayer (R); Ludwig M. Heindl, None
Support: German Research Foundation Sonderforschungsbereich
SFB 643 (B10), DFG Cu 47/4-1; GEROK-Programme, University
Hospital of Cologne
Program Number: 2087 Poster Board Number: D0226
Presentation Time: 11:00 AM - 12:45 PM
Sterculic Acid Antagonizes 7-ketocholesterol-mediated Corneal
Angiogenesis
Joshua Chou, Juan Amaral, Jung W. Lee, Ignacio R. Rodriguez. Lab
of Retinal Cell & Molecular, NEI, Bethesda, MD.
Purpose: The purpose of this study is to determine if sterculic acid
(SA) and sterculia oil (SO) will inhibit 7-ketocholesterol (7KCh)mediated inflammation and angiogenesis in vivo.
Methods: An angiogenesis model was developed in rats by placing
wafers containing 7KCh in the anterior chamber of the eye. The
wafers were prepared using a mixture of poly (2hydroxyethymethacrylate) and polyethylene glycol 20K (50:50 w/w).
The appropriate amounts of 7KCh, SA and/or SO were then added to
the mixture. India ink was added to the wafers to ensure proper
mixing of all the components. Corneal vessel growth was imaged at
7, 10, 14, and 21 days after implantation by fluorescein angiography
using a dissecting microscope equipped with a fluorescent lamp.
Neovessels were quantified by area measurement using the Nikon
NIS elements software.
Results: Four different 7KCh concentrations were used in the wafers,
5%, 7%, 10% and 15% (w/w). All concentrations of 7KCh
consistently induced corneal angiogenesis. Peak neovessel growth
was observed for all concentrations between 7 and 10 days after
implantation. Neovessel area regressed thereafter. Various
combinations of 7KCh and SA were tested (0.1, 0.5, 1, 2.5, 5% SA +
5% 7KCh; 10% SA + 10% 7KCh). All concentrations of SA reduced
7KCh-mediated corneal neovascularization. SA significantly
inhibited 7KCh-mediated angiogenesis at a concentration as low as
0.1% SA with 5% 7KCh (w/w). The reduction in neovessel growth in
SA-containing wafers ranged from 30% to 60%. Several
combinations of 7KCh and SO were also tested (1% SO + 7% 7KCh,
10% SO + 10% 7KCh). All concentrations of SO reduced 7KChmediated corneal neovascularization in all 7KCh concentrations. SO
significantly reduced 7KCh-mediated angiogenesis at a concentration
as low as 1% SO with 7% 7KCh wafers. The reduction in neovessel
growth in SO-containing wafers ranged from 42% to 55%.
Conclusions: 7KCh promotes angiogenesis and inflammation in
vivo, while SA and SO effectively antagonizes these effects. Our
results further support our hypothesis that chronic 7KCh
accumulation could be involved in the development of chronic
inflammation and angiogenesis. SA and/or SO may be potential
therapeutic agents for the treatment of age-related diseases in which
chronic inflammation caused by oxidized lipids play a major role in
their pathogenesis (e.g. atherosclerosis and age-related macular
degeneration).
Commercial Relationships: Joshua Chou, None; Juan Amaral,
None; Jung W. Lee, None; Ignacio R. Rodriguez, None
Program Number: 2088 Poster Board Number: D0227
Presentation Time: 11:00 AM - 12:45 PM
Role of IL-7 in Corneal Lymphangiogenesis
Tatiana Ecoiffier1, Maria Iolyeva2, Cornelia Halin2, Lu Chen1.
1
Vision Science, University of California, Berkeley, School of
Optometry, Center for Eye Disease and Development, Berkeley, CA,
94720, USA, CA; 2Institute of Pharmaceutical Sciences, Swiss
Federal Institute of Technology, ETH Zurich, Zurich, Switzerland.
Purpose: Interleukin-7 (IL-7) is an immunoregulatory cytokine that
have been recently identified on lymphatic endothelial cells. The
purpose of this study was to investigate the in vivo effect of IL-7 on
corneal lymphangiogenesis (LG, the growth of new lymphatic
vessels).
Methods: Uniform slow-release IL-7 pellets were prepared by
mixing IL-7 (Peprotech, Rocky Hill, NJ) or appropriate control with
sucralfate and hydron (Sigma Aldrich, St. Louis, MO). A
micropocket was surgically created in BALB/c mouse cornea and a
pellet was implanted 1.0 mm apart from the limbal vascular arcade.
Corneas were harvested after 2 weeks and stained with LYVE-1,
lymphatic specific marker. Percentage LG covered area under both
control and IL-7 treated conditions were evaluated using NIH Image
J software.
Results: Pellets containing IL-7 induced a 6.4 fold increase in the
corneal area covered by lymphatic vessels compared to the control
condition (p < 0.05).
Conclusions: Our data demonstrate that IL-7 is a strong stimulator of
lymphangiogenesis in vivo. This study will shed some new light on
our understanding of corneal lymphangiogenesis and potentially
promotes the development of novel therapeutic strategies for
lymphatic related diseases occurring inside and outside the eye.
Commercial Relationships: Tatiana Ecoiffier, None; Maria
Iolyeva, None; Cornelia Halin, None; Lu Chen, None
Support: : This work is supported in part by research grants from
National Institutes of Health and the University of California at
Berkeley (to L.C.). Tatiana Ecoiffier is an Ezell Fellow from the
American Optometric Foundation
Program Number: 2089 Poster Board Number: D0228
Presentation Time: 11:00 AM - 12:45 PM
Suppression of Corneal Angiogenesis by Toll-like Receptors
Lei Liu, Jiahui Wu, Andrew D. Dick. Dept of Ophthalmology, School
of Clinical Sciences, University of Bristol, Bristol, United Kingdom.
Purpose: To investigate whether TLR activation regulates corneal
hem- and lymph-angiogenesis.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Methods: B6LY5 mice aortic ring explants were cultured for 5 d
with the presence of 10ng/mL VEGF before TLR ligands 1-9 were
added into culture media. Sprouting outgrowth was measured and
compared between TLR ligands treated groups and water treated
control group after 5 further days of culture. Corneal angiogenesis
was induced by 3 Nylon corneal sutures on BALB/c mice before
water, TLR3, TLR7 or TLR9 ligand was applied topically on
debrided cornea at day 0, day 2, and day 4. The corneal vessel
ingrowth was scored under surgical microscope after 7 days. The
volumes of blood and lymphatic corneal vessels were quantified after
whole mount immunofluorescence staining with CD31 and LYVE-1
antibodies.
Results: TLR7 and TLR9 activation suppressed vascular outgrowth
from aortic ring explants compared to control. TLR9 engagement
inhibited corneal blood vessel ingrowth after 7 d post corneal
suturing, while there was no difference between control and TLR3
and TLR7 treatment groups. TLR9 activation also decreased the
volumes of blood and lymphatic corneal vessels compared to control.
Conclusions: TLR9 activation could suppress aortic vessel sprouting
in vitro as well as corneal hem- and lymph-angiogenesis in vivo.
Commercial Relationships: Lei Liu, None; Jiahui Wu, None;
Andrew D. Dick, Novartis (C), Novartis (F), GSK (F), Abbott (F)
Support: Rosetrees Trust
Program Number: 2090 Poster Board Number: D0229
Presentation Time: 11:00 AM - 12:45 PM
Subconjunctival Cyclosporine A implants do not affect corneal
neovascularisation after transplantation: results of a randomized
clinical trial
Claus Cursiefen1, Thomas Reinhard2, Felix Bock1, Hans-Ulrich
Prokosch3. 1Dept of Ophthalmology, University of Cologne, Koln,
Germany; 2Dept. of Ophthalmology, University of Freiburg,
Freiburg, Germany; 3Medical Informatics, University of Erlangen,
Erlangen, Germany.
Purpose: To test whether subconjunctivally implanted Cyclosporine
A affects the incidence and degree of postkeratoplasty corneal
neovascularisation.
Methods: Prospective, randomized, controlled phase II/III clinical
trial comprising trial sites in Germany, India and the USA. Overall,
97 enrolled patients were randomized to receive either two dosages of
subconjunctival Cyclosporine A implants (A: 36 and B: 40) or
placebo (n=21) at time of penetrating keratoplasty. The incidence and
degree of postkeratoplasty corneal neovascularisation were evaluated
(LX201-01 study). A web-based image upload system was
developed. Quantitative and objetive evaluation of standardized
digital slit lamp pictures was performed using AnlysisB morphometry
sofware.
Results: No significant difference in the incidence and the degree of
corneal neovascularisation was found between treatment groups and
placebo group. Mean neovascularisation was 3.2% ± 0.3 in treatment
A versus placebo (3.5% ± 0.27; p= 0.5) and 3.0% ± 0.4 and in
treatment B versus placebo (3.5% ± 0.27; p= 0.3).
Conclusions: Subconjunctival cyclosporine A does not affect
postkeratoplasty corneal neovascularisation.
Commercial Relationships: Claus Cursiefen, Gene Signal (C),
Alcon (R), Allergan (R), Bayer (R); Thomas Reinhard, None; Felix
Bock, None; Hans-Ulrich Prokosch, None
Clinical Trial: NCT00447187
Program Number: 2091 Poster Board Number: D0230
Presentation Time: 11:00 AM - 12:45 PM
Role of Non-Endothelial Cells in Corneal Angiogenesis
Jin Zhao, Takayuki Nagasaki. Ophthalmology, Columbia University,
New York, NY.
Purpose: It has been suggested that growth of blood and lymphatic
vessels in the cornea is assisted by non-endothelial cells in the
stroma, in particular, during later stages when a vascular network is
established by anastomosis. Supporting roles of stromal myeloid cells
and an existing nerve network have been suggested, but molecular
and cellular mechanisms remain unresolved. The aim of this study is
to examine evidence for the participation of stromal, non-endothelial
cells during the vascular network formation.
Methods: For tests of myeloid cells, four corneal angiogenesis
models in the mouse eye were used. 1) suture placement, 2) topical
application of ML9 (MLCK inhibitor), 3) Dstncorn1 mouse
(spontaneous corneal vascularization model), 4) corneal
conjunctivalization following total epithelial removal. Blood vessel
growth was monitored in a live eye with fluorescence angiography.
Cell distribution was determined by whole-mount
immunofluorescence with CD11b, CD45, CD90.2, F4/80, MHC-class
II, CD31 (blood vessels) and LYVE1 (lymphatic vessels). Cell
distribution was quantified in an area of vascular growth front where
a vascular network is formed by anastomosis. For a nerve guidance
test, suture was placed in the cornea of Thy1-CFP mice, and both
blood vessels (fluorescence angiography) and nerves (CFP) were
tracked by time-lapse imaging in live animals.
Results: Untouched, avascular corneas contained a small number of
F4/80, CD11b and MHC-class II positive cells, primarily in the
limbal area and sparsely throughout the entire cornea. CD90.2
positive cell distribution had no limbal preference but was closely
associated with sub-basal nerve plexus. After initiation of
experimental angiogenesis, the number of cells with one or more of
myeloid markers greatly increased within an area of vascular growth.
There were frequent instances of a F4/80 positive cell being present
between two sprouting tips of growing vessels, appearing to bridge
them. Time-lapse imaging revealed no clear correlation between
vessel growth and a CFP-positive nerve fiber network.
Conclusions: Myeloid cells may play a supporting role in network
formation of pathological corneal vasculature either in a paracrine
manner or by means of fusion with endothelial tip cells, as reported
in developing brain. A guiding role of an existing nerve network, on
the other hand, is not supported by the data.
Commercial Relationships: Jin Zhao, None; Takayuki Nagasaki,
None
Support: Eye Surgery Fund, Research to Prevent Blindness
Program Number: 2092 Poster Board Number: D0231
Presentation Time: 11:00 AM - 12:45 PM
Impaired angiogenic response in cornea by lacking TRPV1 in
mice
Katsuo Tomoyose, Yuka Okada, Takayoshi Sumioka, Kumi Shirai,
Shizuya Saika. Wakayama Medical University, Wakayama, Japan.
Purpose: To investigate the effects of loss of Transient receptor
potential
(TRP)V1 in the development of neovascularization (NV) in a corneal
stroma in
mice. TRP channels are polymodal receptors that are activated by a
host of
stimuli to mediate sensory transduction.
Methods: Corneal NV from the limbal vessels were induced by
cauterization of
the central cornea of an eye by using disposable tool of Optemp. WT
and
TRPV1-null (KO) mice (n = 16 in each genotype) were used and
killed at day 3
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
and 7. The eye was enucleated and processed or cryosectioning for
histology
and immunohistochemistry. To examine the expression of angiogenic
growth
factors in vivo, centrally cauterized cornea (n = 40 in each of WT or
KO
group) was excised at day 3. Total RNA was extracted from samples
and
processed for real-time RT-PCR for VEGF, TGFb1.
Results: The development of CD31-labeled new vessels from the
limbus toward
the center of the cornea was impaired in KO mice as compared with
WT mice at
day 3 days, but not at day 7. Expression of mRNAs of VEGF and
TGFb1 was
significantly less in a KO cornea as compared with a WT cornea at
day 3.
Conclusions: TRPV1 signal is involved in the development of
cauterization-induced NV in corneal strom via modulation of
angiogenic gene
expression in the affected tissue.
Commercial Relationships: Katsuo Tomoyose, None; Yuka
Okada, None; Takayoshi Sumioka, None; Kumi Shirai, None;
Shizuya Saika, None
Program Number: 2093 Poster Board Number: D0232
Presentation Time: 11:00 AM - 12:45 PM
Photodynamic Ablation of Lymphatic Vessels in the Cornea
Franziska Bucher1, Yanlong Bi2, Claus Cursiefen1, Felix Bock1.
1
Department of Ophthalmology, University of Cologne, Cologne,
Germany; 2Department of Ophthalmology, Tongji University School
of Medicine, Shanghai, China.
Purpose: Corneal lymphangiogenesis is one of the main risk-factors
for immune mediated allograft rejection after corneal transplantation.
Whereas topical VEGF inhibitors can decrease pathological
neovascularization, regression of existing pathological blood and
lymph vessels in the cornea remains an unsolved problem.
Verteporfin photodynamic therapy (PDT) is an established treatment
for subfoveal choroidal neovascularization (CNV) in patients with
age-related macular degeneration (AMD). This experiment shows for
the first time the effect of photodynamic ablation of lymphatic
vessels in the cornea.
Methods: Ten female BALB/c mice (aged 6-8 weeks) were used in
the mouse model for suture induced inflammatory corneal
neovascularization. After two weeks the treatment group (n=5)
received an injection of 2.5µl Verteporfin (2mg/ml) into the corneal
stroma. Control mice (n=5) received an intrastromal injection of
2.5µl PBS. After a two-hour dispersion, we performed PDT in all ten
mice (t = 40s, P = 46mW). Corneas were excised 20 hours later and
corneal wholemounts were stained with CD31 and LYVE-1 to detect
blood and lymphatic vessels. Hemangiogenesis and
lymphangiogenesis were analyzed morphometrically by using a
semiautomatic method based on the image analyzing program Cell^F.
Results: Blood vessels were reduced to 78% and lymphatic vessels
dropped to 38% after PDT in the group treated with Verteporfin
compared to control mice.
Conclusions: Corneal PDT can be used to relatively selectively and
specifically reduce lymphangiogenesis in the cornea. Future studies
using this new technique can investigate the role of lymph vessels in
graft rejection more in detail. In the clinic, this is a promising new
preoperative technique to “precondition” high-risk corneas prior to
transplantation to reduce allograft rejection in high-risk patients.
Commercial Relationships: Franziska Bucher, None; Yanlong Bi,
None; Claus Cursiefen, Gene Signal (C), Alcon (R), Allergan (R),
Bayer (R); Felix Bock, None
Program Number: 2094 Poster Board Number: D0233
Presentation Time: 11:00 AM - 12:45 PM
Formation and Regulation of Lymphatic Valves during
Inflammation
Tan N. Truong1, Eda I. Altiok1, Eric Huang1, Tatiana Ecoiffier1, Don
Yuen1, Toshimitsu Uede2, Lu Chen1. 1School of Optometry & Vision
Science, University of California, Berkeley, Berkeley, CA; 2Division
of Molecular Immunology, Institute for Genetic Medicine, Hokkaido
University, Sapporo, Japan.
Purpose: Lymphatic dysfunction is associated with a wide array of
disorders from transplant rejection to cancer metastasis. To date,
there is little effective treatment for lymphatic diseases. The cornea
provides an ideal tissue for lymphatic research due to its unique
features. We have recently provided the first evidence showing that
lymphatic valves are formed during corneal inflammatory
lymphangiogenesis (LG) via the up-regulation of integrin α9 (Truong
et al. 2011). These structures play a critical role in directing proper
lymph flow within the vessels. The purpose of this study is to further
characterize the time course of valve formation in relation to vessel
formation during corneal LG. In addition, integrin α9 antibody
therapy was evaluated to determine whether integrin α9 blockade
would interfere with the process of lymphatic valve formation during
inflammatory corneal LG.
Methods: Our standardized suture-induced corneal inflammatory LG
murine model was used. Whole-mount corneas were harvested every
2 weeks for up to 8 weeks post-suture for immunofluorescent
microscopic analysis. Additionally, mice were randomized to receive
subconjunctival injections of either integrin α9 antibody or isotype
control twice a week for 2 weeks. Whole-mount corneas were
harvested for immunofluorescent microscopic analysis. Digital
images were analyzed by NIH Image J software to quantify
lymphatic vessels as well as valve distribution.
Results: Corneal lymphatic vessel invasion area at 4, 6, and 8 weeks
were significantly less than the peak at 2 weeks post-suture. In
contrast, the ratio of corneal lymphatic valves to vessel invasion area
was at its lowest at 2 weeks with a peak around 6 weeks post-suture.
Moreover, therapeutic intervention with integrin α9 antibody
significantly reduced the number of lymphatic valves after sutureinduced corneal LG (p<0.05).
Conclusions: This study presents new insight into the time course of
corneal lymphatic valve morphogenesis during inflammatory
processes. Additionally, we have shown that it is possible to inhibit
corneal valve formation via integrin α9 interference. Since lymphatic
valves are closely associated with vessel function, they may serve as
an important indicator of vessel maturation for future research and
provide a novel therapeutic approach for lymphatic related diseases,
such as inflammation and transplant rejection.
Commercial Relationships: Tan N. Truong, None; Eda I. Altiok,
None; Eric Huang, None; Tatiana Ecoiffier, None; Don Yuen,
None; Toshimitsu Uede, Gene Techno Science (C); Lu Chen, None
Support: This work is supported in part by research grants from the
National Institutes of Health, University of California at Berkeley
(L.C.), and National Institutes of Health K-12 training grant (T.T.)
Program Number: 2095 Poster Board Number: D0234
Presentation Time: 11:00 AM - 12:45 PM
MicroRNA-184 Regulates Corneal Lymphangiogenesis
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Lu Chen, Sammy Grimaldo, Tatiana Ecoiffier, Don Yuen. Center for
Eye Disease & Development, Program in Vision Science and School
of Optometry, University of California, Berkeley, CA.
Purpose: MicroRNAs are a class of small non-coding RNAs that
negatively regulate gene expression by binding to complimentary
sequences of target messenger RNAs. Their roles in the cornea still
remain largely unknown. Here we present our new findings on the
anti-lymphangiogenesis function of microRNA-184 in the cornea
during an inflammatory response in vivo.
Methods: The murine in vivo suture placement model was used to
induce corneal inflammatory lymphangiogenesis (LG). Mice were
randomly selected to receive subconjunctival injections of either
synthetic microRNA-184 mimics or control twice a week after the
procedure. Whole-mount corneas were sampled and stained with
LYVE-1, the lymphatic marker, for immunofluorescent microscopic
assays. Results were analyzed by NIH-ImageJ software.
Results: Compared to the control condition, the lymphatic invasion
area was significantly reduced after the treatment of MicroRNA-184
mimics in the inflamed cornea.
Conclusions: MicroRNA-184 suppresses corneal
lymphangiogenesis. Its further investigation may provide novel
therapies for lymphatic related disorders in the cornea, such as
inflammation and transplant rejection.
Commercial Relationships: Lu Chen, None; Sammy Grimaldo,
None; Tatiana Ecoiffier, None; Don Yuen, None
Support: This work is supported in part by research grants from NIH
and University of California at Berkeley (LC).
Program Number: 2096 Poster Board Number: D0235
Presentation Time: 11:00 AM - 12:45 PM
Live Imaging of Lymphatic Valve Formation after Corneal
Transplantation
Gyeong Jin Kang1, Tatiana Ecoiffier1, Young-Kwon Hong2, Lu
Chen1. 1Center for Eye Disease and Development, Program in Vision
Science and School of Optometry, University of California, Berkeley,
CA; 2Department of Surgery, Norris Comprehensive Cancer Center,
University of Southern California, Los Angeles, CA.
Purpose: The lymphatic pathway is a major mediator for transplant
rejection. Most recently, we have provided the first evidence on
lymphatic valve formation during corneal inflammatory
lymphangiogenesis (Truong et al. 2011). The purpose of this realtime in vivo study is to investigate the time course and pattern of
lymphangiogenesis as well as lymphatic valve formation induced by
corneal transplantation with our newly developed live imaging
system and Prox-1-GFP mice.
Methods: Standard orthotopic corneal transplantation was performed
with Prox1-GFP mice as recipients. Corneal grafts of the same mice
were continuously observed by the live imaging system for
longitudinal analysis. Prox-1 positive lymphatic vessels and valves
were evaluated at both limbal and corneal areas.
Results: Prox-1 positive lymphatic vessels and valves were formed
after corneal transplantation. As corneal lymphangiogenesis
proceeded, more valves were observed inside vessel cavities.
Furthermore, lymphatic valvulogenesis was initiated inside limbal
vessels before spreading into the central cornea.
Conclusions: We have shown, for the first time, corneal
transplantation induces both lymphangiogenesis and valvulogenesis
in vivo and in real time. Further investigation on this new
phenomenon may reveal new mechanisms underlying transplant
rejection. Since the lymphatic system plays an important role in many
other functions, this study may also offer new insights to our
understanding and treatment of other lymphatic related disorders
outside the eye.
Commercial Relationships: Gyeong Jin Kang, None; Tatiana
Ecoiffier, None; Young-Kwon Hong, None; Lu Chen, None
Support: This work is supported in part by research grants from NIH
and University of California at Berkeley (LC).
Program Number: 2097 Poster Board Number: D0236
Presentation Time: 11:00 AM - 12:45 PM
Novel Characterization of MHC-Class II Positive Cells in
Embryonic Cornea during Spontaneous Lymphatic Events
Don Yuen, Guangyu Li, Lu Chen. Center for Eye Disease and
Development, Program in Vision Science and School of Optometry,
University of California, Berkeley, CA.
Purpose: We recently reported that unlike the adult cornea which is
devoid of lymphatic vessels, the immature cornea is supplied by
lymphatics which undergo spontaneous regression during
development. Since lymphatic vessels mediate antigen presenting cell
trafficking during an immune response, this study is to investigate
whether the distribution of dendritic cells in immature cornea differs
from the adult stage and how it correlates with the lymphatic events.
Methods: Corneal samples of different developmental stages were
isolated from C57B6 mice and stained with a panel of specific
markers for MHC-Class II, CD45, CD11c, and LYVE-1 for
immunofluorescent microscopic assays.
Results: In contrast to the adult cornea where MHC Class II positive
cells were found in the peripheral area, embryonic cornea at E16.5
also exhibited these cells in the central area. The cells expressed
CD45 and CD11c as well. As the cornea became mature and
underwent spontaneous lymphatic formation and regression, the
MHC Class II positive cells were more restricted to the peripheral
area.
Conclusions: Our findings have shown, for the first time, MHC
Class II positive cells are present in the central cornea during
development. Further investigation on this new phenomenon may
provide novel insights into corneal embryogenesis as well as the
creation and regulation of immune privilege in the cornea.
Commercial Relationships: Don Yuen, None; Guangyu Li, None;
Lu Chen, None
Support: This work is supported in part by research grants from NIH
and University of California at Berkeley (LC)
Program Number: 2098 Poster Board Number: D0237
Presentation Time: 11:00 AM - 12:45 PM
Deletion of Foxc1 and/or Foxc2 from neural crest cells leads to
corneal vascularization and anterior segment dysgenesis
Tsutomu Kume, Kathryn M. Schultz, Amy Sasman, Seungwoon Seo.
Medicine, Northwestern Univ Sch of Med, Chicago, IL.
Purpose: Foxc1 and Foxc2, members of the forkhead/Fox
transcription factor family, are critical regulators of vascular
development. Foxc1 and Foxc2 are both expressed in ocular
mesenchymes of neural crest origin. However, little is known about
the role of Foxc1 and Foxc2 in ocular development, mainly due to the
mid-gestation lethality of global knockout mice. Previously, we have
demonstrated that deletion of Foxc1 in neural crest-derived cells
(NCC) leads to aberrant vessel growth in the normally avascular
corneas of mice, and that the effect is cell-type specific, because the
corneas of mice lacking Foxc1 expression in vascular endothelial
cells remained avascular. Therefore, we investigate the role of Foxc1
and Foxc2 in NCC during ocular development.
Methods: To specifically delete Foxc1 and/or Foxc2 from NCC,
conditional knockout mice for Foxc2 (NC-Foxc2-/-) and for Foxc1
and Foxc2 (NC-Foxc1-/-; Foxc2-/-) were generated by crossing
floxed-Foxc1 and floxed-Foxc2 mice with Wnt1-cre mice.
Results: Adult NCC-specific Foxc2 mutant mice exhibit corneal
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
neovascularization and lymphangiogenesis. Mutant eyes exhibit
abnormal thickness in the peripheral-to-central corneal stroma and
limbus (Figure 1). Interestingly, some mutant corneas have
hyperplastic stroma with increased cell density. Mutant pupils are
displaced from the normal central position. The anterior chamber is
normally formed in NC-Foxc2-/- mice, but not in NC-Foxc1-/-;
Foxc2-/- mice. The severity of the ocular phenotype is dependent on
the cumulative “dose” of functional Foxc1 and Foxc2 genes and
abnormalities are more extensive in NC-Foxc1-/-; Foxc2-/-. In fact,
in NC-Foxc1-/-; Foxc2-/- mice the cornea is not present at all (Figure
1). In the alkali-burn model, corneas of adult NC-Foxc2+/- mice and
NC-Foxc1+/-; Foxc2+/- mice have increased corneal
neovascularization and lymphangiogenesis compared with those of
control mice (Figure 2).
Conclusions: Taken together, these data suggest that the overlapping
function of Foxc1 and Foxc2 in the neural crest plays an important
role in maintaining corneal avascularity and development of the
anterior eye segment.
Commercial Relationships: Tsutomu Kume, None; Kathryn M.
Schultz, None; Amy Sasman, None; Seungwoon Seo, None
Support: NIH Grant EY019484
Program Number: 2099 Poster Board Number: D0238
Presentation Time: 11:00 AM - 12:45 PM
Topical Infliximab as an inhibitor of corneal hemangiogenesis
and lymphangiogenesis
Giulio Ferrari, Fabio Bignami, Chiara Giacomini, Paolo Rama.
Ophthalmology -Cornea Unit-Eye Repair, San Raffaele Scientific
Institute, Milan, Italy.
Purpose: To test whether topical application of the anti-TNF alpha
antibody Infliximab can reduce lymphangiogenesis and/or
hemangiogenesis in an alkali burn mouse model of corneal
neovascularization.
Methods: Eighteen C57BL/6 mice were induced alkali burn model
of the right cornea. Animals were then divided in two groups: group
1 received topical Infliximab 10mg/ml six times a day (10ul), group 2
received regular saline. After two weeks, animals were sacrificed,
corneas stained for CD31 and LYVE1 to identify hemangiogenesis
and lymphangiogenesis. Finally, the corneas were whole mounted
and imaged. Neovascularization was calculated as neovascular area,
i.e. the area of the cornea covered by vessels normalized for the total
area of the cornea.
Results: Following topical application of Infliximab
hemangiogenesis decreased from 0.81±0.12 to 0.6±0.24 (26%
reduction, P<0.05). Lymphangiogenesis was also reduced from 0.11±
0.04 to 0.05± 0.02 (55% reduction, P<0.001), after topical treatment
with Infliximab.
Conclusions: Topical application of Infliximab 10mg/ml for two
weeks is effective in reducing corneal hemangiogenesis and
lymphangiogenesis in a mouse model of corneal alkali burn.
Interestingly, Infliximab appeared more effective inhibting
lymphangiogenesis than hemangiogenesis. Given the high prevalence
of corneal neovascularization in a wide range of corneal diseases, we
suggest these findings may have implications in the treatment of
corneal neovascularization in human subjects.
Commercial Relationships: Giulio Ferrari, None; Fabio Bignami,
None; Chiara Giacomini, None; Paolo Rama, None
Program Number: 2100 Poster Board Number: D0239
Presentation Time: 11:00 AM - 12:45 PM
Effect of LYVE-1 on FGF2-induced lymphangiogenesis in vivo
Birgit Regenfuss1, Natalia Platonova2, 3, Géraldine Miquel2, 3, Said
Taouji4, Eric Chevet4, Andreas Bikfalvi2, 3, Claus Cursiefen1.
1
Ophthalmology, University of Cologne, Cologne, Germany;
2
INSERM U1029, Talence, France; 3Université Bordeaux I, Talence,
France; 4INSERM U1053, Bordeaux, France.
Purpose: Hyaluronan receptor LYVE-1 (lymphatic vessel
endothelial hyaluronan receptor-1) is expressed mainly on the surface
of lymphatic endothelium and is involved in binding and
internalization of the extracellular matrix glycosaminoglycan
hyaluronan. However it was recently shown that LYVE-1-deleted
mice have no obvious structural or functional lymphatic defects and
normal hyaluronan levels. As the interplay between hyaluronan
receptor CD44, a close homologue of LYVE-1, and the growth-factor
FGF2 is already known, a possible interaction of LYVE-1 with FGF2
and its effect on lymphatic vasculature was analyzed in this study.
Methods: The mouse corneal micropocket assay was performed with
sucralfate pellets containing a combination of 80 ng recombinant
human FGF2 (rhFGF2) or 200 ng rhVEGF-C respectively and 1 µg
recombinant human LYVE-1. Control pellets contained 80 ng
rhFGF-2 or 200 ng rhVEGF-C alone. Pellets were coated with
hydron and implanted into a corneal micropocket. On postoperative
day seven the mice were euthanized, the cornea with limbus was
excised and the lymphangiogenic response was analyzed
semiautomatically.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Results: Treatment of corneas with pellets containing the
combination of FGF2/LYVE-1 resulted in the outgrowth of
lymphatic vessels with a mean neovascularization area of 0.12 ± 0.07
mm2 (n=20) whereas corneas with pellets containing FGF2 alone
showed a mean neovascularization area of 0.23 ± 0.11 mm2 (n=21) (p
= 0.0008). Contrary no significant difference was found between
VEGF-C induced lymphangiogenesis and the combination of VEGFC/LYVE-1 induced lymphangiogenesis in the micropocket assay.
Conclusions: LYVE-1 inhibited the FGF2-induced outgrowth of
lymphatic vessels in a corneal micropocket assay suggesting that
LYVE-1 may bind FGF2 and interact with bFGF influencing
lymphangiogenesis in vivo.
Commercial Relationships: Birgit Regenfuss, None; Natalia
Platonova, None; Géraldine Miquel, None; Said Taouji, None;
Eric Chevet, None; Andreas Bikfalvi, None; Claus Cursiefen,
Gene Signal (C), Alcon (R), Allergan (R), Bayer (R)
Support: German Research Foundation (DFG) SFB 643/B-10 and
Cu 47/4-1) to C.C. Agence National de la Recherche (ANR), Institut
National du Cancer (INCA), Conseil Régional d’Aquitaine to A.B.
and INCA, Association de Recherche contre le Cancer, Inserm
(Avenir) to E.C.
Program Number: 2101 Poster Board Number: D0240
Presentation Time: 11:00 AM - 12:45 PM
Analysis of VEGFR-1 Isoforms in Pax6-deficient Mice Corneas
Phillip A. Moore1, Peter J. Accola1, Barbara Artelt1, Megan Aarnio1,
James D. Lauderdale2. 1Dept Small Animal Med/Surg, College Vet
Med Univ Georgia, Athens, GA; 2Department of Cellular Biology,
Franklin College of Arts and Sciences, The University of Georgia,
Athens, GA.
Purpose: To identify VEGFR-1 isoforms in the cornea of Pax6deficient mice.
Methods: The limbal region, central cornea and conjunctiva of Pax6
+/- mice were compared to wild type mice for the presence of
VEGFR-1 and sVEGFR-1. Immunostaining was performed to wild
type (n=5) and Pax6 +/- mice (n= 5) with three antibodies: one
antibody to the N-terminus that detected both membrane and soluble
VEGFR-1; one antibody to the C-terminus that detected the
membrane bound form of VEGFR-1; and one antibody that
specifically detected the soluble form of VEGFR-1. Immunoblotting
was performed on protein extracts prepared separately from the
cornea and conjunctiva of wild type and Pax6 +/- mice.
Results: Immunostaining revealed that both VEGFR-1 and sVEGFR1 are present in the cornea of both wild type and Pax6 +/- mice.
Immunoblotting revealed that both VEGFR-1 and sVEGFR-1 are
present in the conjunctiva and central cornea of wild type and Pax6
+/- mice. Interestingly, this analysis also revealed VEGFR-1 isoforms
that appeared to be specific to Pax6 +/- corneas.
Conclusions: Our results are in contradiction to previous reports that
sVEGFR-1 is deficient in Pax6 +/- corneas. The presence of
sVEGFR-1 and VEGFR-1 isoforms specific to Pax6 +/- corneas
suggests that a deficiency in sVEGFR-1 is not the cause for corneal
neovascularization in Pax6 +/- mice.
Commercial Relationships: Phillip A. Moore, None; Peter J.
Accola, None; Barbara Artelt, None; Megan Aarnio, None; James
D. Lauderdale, None
Support: UGA Veterinary Ophthalmology Research Fund, Sharon
Stewart Aniridia Research Trust, Vision for Tomorrow Foundation
Program Number: 2102 Poster Board Number: D0241
Presentation Time: 11:00 AM - 12:45 PM
Use of Amaranthus leucocarpus lectin to determine corneal neovascularization
Valeria L. Coria1, 2, Gibran A. Estua1, Alfredo Domínguez1, Edgar
Zenteno2, Jessica Nieves-Hernández1, Yonathan Garfias1, 2.
1
Research Unit, Institute of Ophthalmology, Mexico city, Mexico;
2
Biochemistry Department, Faculty of Medicine, Universidad
Nacional Autónoma de México, Mexico city, Mexico.
Purpose: The cornea is the interphase between the eye and the
external environment, and is also the most powerful refractive surface
in the eye. In non-pathological conditions, the cornea is one of the
few avascular tissues from the body. The mechanism responsible for
establishing the avascular nature of the cornea is unknown; but, in
several pathologycal situations like trauma, infection or surgical
procedures, the stroma can be invaded by abnormal vessels
(neovascularization) leading it to opacification. The
neovascularization is the creation of new vessels from preexisting
ones; in these abnormal vessels, the glycosylation could be aberrant.
The glycosylation process, is the post translational modification in
which saccharide residues get added to amino acid chains. Some of
the tools used for studying neo-vessels in eye are lectins, such as
Griffonia simplicifolia. Amaranthus leucocarpus (ALL) is a lectin
that recognizes specifically Galbeta1,3GalNAc carbohydrates
structures.
The aim of this study was at determining Galbeta1,3GalNAc
structures in a corneal neovascularization murine model
Methods: This study was carried out in accordance with the
Institutional Animal Care and Use Committe and Vision Research
with the ARVO statement for the Use of Animals in Ophthalmic and
Vision Research. All experiments were performed on 6-8 week-old
female BALB/c mice. Corneas were chemically burned with NaOH.
Burned and healthy mice corneas were obtained, and were processed
for immunohistochemistry assays. ALL coupled to FITC was used to
perform immunolabeling on corneal tissue; CD31 was used to colocate the neovessel labeling of ALL-recognized structures
Results: The presence of ALL was exclusively observed in the
chemically-burned mice corneal tissues, in contrast to the burned
tissues, there was not ALL-immunostaining in the healthy mice
corneal tissues. Interestingly, ALL colocalized with CD31
immunostaining in the burned and neovascularized corneal tissue.
Conclusions: Amaranthus leucocarpus lectin is able to identify
corneal neo-vessels, suggesting that o-glycosylation process could be
an important process in developing this aberrant corneal condition.
Commercial Relationships: Valeria L. Coria, None; Gibran A.
Estua, None; Alfredo Domínguez, None; Edgar Zenteno, None;
Jessica Nieves-Hernández, Institute of Ophthalmology (P);
Yonathan Garfias, Institute of Ophthalmology (P)
Support: CONACYT 160286 and 167438
Program Number: 2103 Poster Board Number: D0242
Presentation Time: 11:00 AM - 12:45 PM
Netrin-4 mediates corneal neovascularization
Anna-Karina Maier, Sabrina V. Klein, Norbert Kociok, Aline
Riechardt, Enken Gundlach, Claudia Steger, Olaf Strauss, Antonia
M. Joussen. Charité, University Medicine Berlin, Berlin, Germany.
Purpose: Netrin-4, a secreted protein, regulates neuronal and
vascular growth. It is found in the basement membranes of blood
vessels. Previous results are contradictory supporting Netrin-4 as
either a pro- or anti-angiogenic factor. In the presented study we
investigated the role of Netrin-4 in the mouse-model of sutureinduced, inflammatory corneal neovascularization.
Methods: Corneal neovascularization was induced in Netrin-4deficient (Ntn4-/-) and wild-type mice by suturing three 11-0 nylon
intrastromally into the cornea. Vascularized area of corneal
flatmounts were analyzed 14 days after suturing by
immunocytochemistry in conjunction withimage and statistical
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
evaluation. The mRNA levels for VEGF-A and Netrin-4 in mouse
corneas were analyzed by qPCR.
Results: In the wild-type mouse the netrin-4 expression was
increased when corneal vascularization was increased by nylon
saturation. Accordingly, Ntn4 -/- showed an increased corneal
vascularized area compared to that in wild-type mice after suturing.
Additionally, mRNA level of VEGF-A was increasedin Ntn4-/compared to that in wild-type mice.
Conclusions: Netrin-4 probably acts as an anti-angiogenic factor
through down-regulation of VEGF-A expression in the suture
induced corneal neovascularization.
Commercial Relationships: Anna-Karina Maier, None; Sabrina
V. Klein, None; Norbert Kociok, None; Aline Riechardt, None;
Enken Gundlach, None; Claudia Steger, None; Olaf Strauss,
None; Antonia M. Joussen, None
Support: "Friedrich C. Luft" Clinical Scientist Pilot Program funded
by Volkswagen Foundation and Charité Foundation
Program Number: 2104 Poster Board Number: D0243
Presentation Time: 11:00 AM - 12:45 PM
Subconjunctival and intrastromal Bevacizumab to control of
corneal neovascularization and opacification
Silvia Mendez, Maria Santiago, Elena Raposo, Rosario Touriño,
Maria Teresa Rodriguez-Ares. Department of Ophthalmology,
University of Santiago de Compostela (USC) Hospital Complex,
Santiago de Compostela, Spain.
Purpose: Bevacizumab is a recombinant humanized monoclonal
antibody directed against vascular endothelial growth factor (VEGF).
The purpose of this study was to evaluate the therapeutic effect of
subconjunctival and intrastromal injection of Bevacizumab on
corneal neovascularization (CNV) and the secondary opacification.
Methods: We have performed a prospective nonrandomized
interventional study, including 15 eyes with CNV secondary to:
keratoplasty due to corneal ectasia (n = 2), keratoplasty due to
herpetic keratitis (n = 3), keratoplasty due to bullous keratopathy (n =
2), herpetic keratopathy previously treated with amniotic membrane
transplantation (n = 2), chemical burns (n = 3) and CNV after
pterygium surgery (n = 3). We have injected 2.5 mg / 0.1 ml of
Bevacizumab, subconjunctival and intrastromal, in the area with
CNV. We have analyzed the CNV regression by anterior segment
phothography in relation to the total corneal area. Furthermore, we
have evaluated the decrease of corneal opacity regarding lipid
deposits, corneal edema and local inflammation.
Results: We have observed regression of CNV in all patients. The
mean area of CNV regression was 37% with a range between 6 and
86%. We have noticed improvement especially in younger vessels
and smaller caliber vessels (p<0.001). In 6 cases there was a decrease
of corneal opacification (p=0.46). There were no significant adverse
effects.
Conclusions: Intrastromal and subconjunctival injections of
Bevacizumab are effective for the reduction of CNV and seem to be
useful in terms of decreasing lipid deposits, corneal edema and local
inflammation.
Commercial Relationships: Silvia Mendez, None; Maria Santiago,
None; Elena Raposo, None; Rosario Touriño, None; Maria Teresa
Rodriguez-Ares, None
Program Number: 2105 Poster Board Number: D0244
Presentation Time: 11:00 AM - 12:45 PM
Corneal Organ Culture Angiogenesis Model
Sally S. Twining, Hanzhu Zhang, Debra J. Warejcka. Biochemistry
and Ophthalmology, Medical College of Wisconsin, Milwaukee, WI.
Purpose: Corneal angiogenesis occurs under numerous conditions
including corneal injury, infection and hypoxia. These vessels can
block light entering the eye and alter vision. Corneal angiogenesis is
usually studied in vivo, however, use of animals is expensive and
time consuming. An in vitro model that uses limbal endothelial cells
and corneal components would be useful for initial screening of
angiogenesis stimulators and inhibitors. In this study, an organ
culture model of angiogenesis was developed using rat corneas.
Methods: Whole rat eyes were obtained from PelFreez, washed,
treated with Ciprofloxin and the cornea with a 2mm scleral rim was
dissected. The corneas were placed over sterile agarose coated glass
beads tethered to wells of a 24 well plate. Medium alone or medium
containing 100 ng/ml FGF-2 with and without the angiogenesis
inhibitor maspin (1µM) and the related non-inhibitor protein
ovalbumin (1µM) was added to the level of the limbal region of the
cornea. This medium was changed daily and 125 µl dropped on the
corneal surface. After seven days the corneas were removed from
culture and radial cuts were made in the corneas. The corneas were
fixed in acetone, blocked with BSA and stained with phycoerythrinPECAM (CD31) antibodies. The corneas were flat mounted and the
vessels visualized. Vessel lengths were determined using ImageJ. The
data was analyzed using an ANOVA and post hoc individual
comparisons (Sigma Plot). n=7-12 corneas per group.
Results: FGF-2 induced the ingrowth of vessels into the cornea
relative to medium alone (Fig 1). Seven days in culture was optimal
for vessel ingrowth and corneal clarity. The angiogenesis inhibitor,
maspin inhibited vessel ingrowth with an average vessel length
similar to that of the control. Ovalbumin, a related non-inhibitory
protein, did not inhibit vessel ingrowth.
Conclusions: Corneal angiogenesis can be initially studied using the
developed organ culture model. It will be useful for the screening of
pro and antiangiogenic molecules.
Fig 1: Rat organ culture angiogenesis model. Air lifted rat corneas
were treated with medium alone (Control, A), FGF-2 (B) and FGF-2
+ Maspin (C). The tips of the vessels are outlined by the white line.
D. Enlargement of FGF-2 induced vessels. E. The lengths of corneal
vessels were averaged for each treatment. n= 7-12 corneas per group.
* p < 0.001 relative to control, maspin, FGF-2 + maspin and
ovalbumin.
Commercial Relationships: Sally S. Twining, None; Hanzhu
Zhang, None; Debra J. Warejcka, None
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Support: NIH Grants RO1 EY12731 and EY021152 and P30
EY001931
Program Number: 2106 Poster Board Number: D0245
Presentation Time: 11:00 AM - 12:45 PM
UV-Crosslinking for Fixation of Biosynthetic Corneal Collagen
Implants
Kerstin M. Wand1, Karin Kobuch1, Michael Baumann3, May
Griffith2, Mohammad M. Islam2, Johannes Junger3, Raphael T.
Neuhann1, Chris Lohmann1. 1Ophthalmology, Klinikum rechts der
Isar, Technische Universität München, Munich, Germany;
2
Regenerative Medicine, Linkoping University, Linkoping, Sweden;
3
MLase AG, Germering München, Germany.
Purpose: In times of donor organ shortage, also in corneal
transplantation, biosynthetic corneal implants as an alternative to
human donor tissue have been presented. Previously a Phase I
clinical study demonstrated good results regarding stability and
integration. However suture related delay in epithelial closure lead to
thinning of the implant, irreversible formation of haze and
irregularity of the surface in these areas. Therefore an alternative
suture-free fixation method as UV-crosslinking is expected to
improve the visual outcome.
Methods: We implanted different cell-free corneal implants
consisting of recombinant human collagen type III: RHC III and
RHC/MPC (methacrylphosphorylcholine).
Ex vivo the biosynthetic corneal implants (350µm in depth, 6mm in
diameter) were placed on the anterior cornea of porcine and rabbit
eyes after performing deep anterior lamellar keratoplasty with either
femtosecond or excimer laser. After application of Riboflavin 0.1%
solution for 5 minutes UV-crosslinking was performed at either
standard (3mW/cm2 for 30 minutes) or rapid procedure (18mW/cm2
for 5 minutes). Thereafter the corneas were excised, fixed in PFA4%
and embedded in paraffin.
Crosslinking effects (thickness and diameter of implant, adhesion
between implant and cornea) were evaluated by slitlamp
biomicroscopy, OCT images and finally histologically (HE-stained/
picrosirius stained sections, electronmicroscopy).
Results: Before and after crosslinking the precise fitting of the
implant could be demonstrated in OCT images. This was more
accurate in porcine eyes than in rabbit eyes, maybe due to the
difference in corneal thickness. Histologically we could prove
crosslinks between implant and corneal stroma. After crosslinking the
different types of implants showed different degrees of shrinkage.
There was no difference in the outcome between standard and rapid
crosslinking procedure.
Conclusions: UV-crosslinking as a fixation method for biosynthetic
corneal implants was demonstrated to be promising. It can reduce
suture-related complications as neovascularization, haze formation
and surface irregularity. Biostability, integration and long term
outcome is further evaluated in in vivo animal experiments.
Commercial Relationships: Kerstin M. Wand, None; Karin
Kobuch, None; Michael Baumann, None; May Griffith, Univ. of
Ottawa - OHRI (P); Mohammad M. Islam, None; Johannes
Junger, MLase AG (E); Raphael T. Neuhann, None; Chris
Lohmann, None
Support: Euro Nanomed I-CARE, Transnational collaborative
project
260 Conjunctiva Cell Biology
Monday, May 06, 2013 11:00 AM-12:45 PM
Exhibit Hall Poster Session
Program #/Board # Range: 2107-2130/D0334-D0357
Organizing Section: Cornea
Program Number: 2107 Poster Board Number: D0334
Presentation Time: 11:00 AM - 12:45 PM
Development of a conjunctival tissue substitute on the basis of
plastic compressed collagen
Cornelia Corinne Drechsler1, Alvena Kureshi2, Stephan Reichl3,
Gerd Geerling1, Julie T. Daniels2, Stefan Schrader1. 1Ophthalmology,
University Duesseldorf, Duesseldorf, Germany; 2Institute of
Ophthalmology, UCL - University College London, London, United
Kingdom; 3Institut für Pharmazeutische Technologie, TU
Braunschweig, Braunschweig, Germany.
Purpose: Ocular surface disorders can cause severe ocular surface
inflammation, conjunctival scarring, fornix shortening and
ankyloblepharon. There is the need for the development of new
matrices for conjunctival reconstruction for this group of patients.
Therefore, aim of this study was, to evaluate the suitability of
compressed collagen as a substrate for the expansion of human
conjunctival epithelial cells in order to develop an epithelialized
conjunctival substitute for fornix reconstruction.
Methods: Human conjunctiva epithelial cells (HCEC) were cultured
on acellular compressed collagen constructs or those containing
human conjunctival fibroblasts (HCF). The epithelial cells were
evaluated for cytokeratin expression (CK19 /CK4) and the
appearance of goblet cells. The presence of putative progenitor
epithelial cell markers such as p63α, ABCG2 and CK15 was assessed
by immunostaining. The proliferative capacity of the epithelial cells
after expansion on the plastic compressed collagen gels was
evaluated by their colony forming efficiency and compared to cells
cultured on amniotic membrane.
Results: The results showed successful expansion of the conjunctival
epithelial cells on both the acellular compressed collagen constructs
and those containing human conjunctival fibroblasts. The
immunofluorescence staining revealed the expression of the
cytokeratins such as CK19 as well as the putative progenitor cell
markers such as CK15 and ABCG2. After expansion on the collagen
constructs, the conjunctival epithelial cell population was still able to
form epithelial cell colonies, as tested by colony forming efficiency
assay.
Conclusions: Plastic compressed collagen seems to be a suitable
substrate for the expansion of human conjunctival epithelial cells.
The presence of epithelial cells, positive for putative progenitor cell
markers and the sustained proliferative capacity of the epithelial cell
population indicates the maintenance of conjunctival cells with
progenitor cell characteristics on the collagen gels. Therefore, an
epithelialized conjunctival tissue construct on the basis of
compressed collagen, might be a promising alternative bioartificial
tissue substitute for conjunctival reconstruction.
Commercial Relationships: Cornelia Corinne Drechsler, None;
Alvena Kureshi, None; Stephan Reichl, None; Gerd Geerling,
Alcon (C), Allergan (C), Thea Pharma (C), Novagali (C), Bausch &
Lomb (C), Tearlab Inc. (C); Julie T. Daniels, TAP Biosystems (P);
Stefan Schrader, None
Support: DFG SCHR 1210/3-1
Program Number: 2108 Poster Board Number: D0335
Presentation Time: 11:00 AM - 12:45 PM
Lymphatic Microvessel Density as a Predictive Marker for the
Recurrence Time of Pterygium: A 3-year Follow-up Study
Haotian Lin1, Lixia Luo1, Shiqi Ling2, Wan Chen1, Zhaochuan Liu1,
Xiaojian Zhong1, Changrui Wu1, Weirong Chen1, Yizhi Liu1. 1State
Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center,
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Guangzhou, China; 2Dept of Ophthalmology, Third Affiliated
Hospital of Sun Yat-sen University, Guangzhou, China.
Purpose: To investigate whether lymphatic microvessel density
(LMVD) could be used as a predictive marker for the recurrence time
of pterygia
Methods: This was a prospective case series study. A total of 96
patients with unilateral eye primary nasal pterygia were included. The
included patients were clinically evaluated to grade the severity of
their pterygia (32 Grade 1, 29 Grade 2, and 35 Grade 3) before they
underwent bare sclera resection with the use of mitomycin C. Excised
tissues from the 96 patients and the 10 normal nasal conjunctiva
obtained from age-matched donor eyeballs (controls) were
immunostained with LYVE-1 and CD31 monoclonal antibodies to
evaluate lymphatic microvessel density (LMVD) and blood
microvessel density (BMVD). The included patients were followed
up for 3 years or until the identification of pterygium recurrence,
which was defined as fibrovascular regrowth past the limbus in a
previously compromised area. The recurrence time (RT) for a
pterygium was calculated, and its relationship with LMVD and/or
BMVD was statistically analyzed.
Results: In total, there were 24 cases of pterygium recurrence. The
recurrence rate (RR) of Grade 1 was 28.1% (9/32), Grade 2 was
24.1% (7/29), and Grade 3 was 22.9% (8/35), as classified in the
primary pterygium (p>0.05); the overall RR was 25% (24/96) for all
patients during the 3-year follow-up. In the tissue analysis, there were
a small number of CD31 (+), LYVE-1(-) BMVD and only a few
CD31 (weak), LYVE-1(+) LMVD in the 10 normal nasal conjunctiva
tissues. BMVD and LMVD increased significantly in the pterygium
tissue compared to the control tissue and were significantly correlated
with both the width and area of pterygium in Grades 1-3 (all p values
< 0.05). However, RT was not correlated with BMVD or pterygium
grade, but LMVD was significantly and negatively correlated with
RT within each group and in the total patient cohort. Furthermore, we
determined that an LMVD greater than 20 in the surgical specimens
was predictive of pterygium recurrence.
Conclusions: The LMVD of surgical specimens is an independent
risk factor and a valuable predictive factor for the recurrence time of
pterygia.
Commercial Relationships: Haotian Lin, None; Lixia Luo, None;
Shiqi Ling, None; Wan Chen, None; Zhaochuan Liu, None;
Xiaojian Zhong, None; Changrui Wu, None; Weirong Chen,
None; Yizhi Liu, None
Program Number: 2109 Poster Board Number: D0336
Presentation Time: 11:00 AM - 12:45 PM
NHE8 Participates in Electrolyte and Water Secretion in
Conjunctival and Lacrimal Gland Epithelia
Mingwu Wang1, Jing Li2, Fayez K. Ghishan2, Hua Xu2.
1
Ophthalmology, University of Arizona College of Medicine,
Tucson, AZ; 2Pediatrics, University of Arizona College of Medicine,
Tucson, AZ.
Purpose: NHE8 is a membrane protein in the sodium/hydrogen
exchanger (NHE) family. It involves mucin and bicarbonate secretion
in the intestine. But it is not known if NHE8 is expressed in
conjunctival and lacrimal gland tissues, and what role it may play in
maintaining ocular surface homeostasis.
Methods: The study was conducted in compliance with the Tenets of
the Declaration of Helsinki and ARVO statement for the use of
animals in Ophthalmic and Visual Research. Mouse conjunctival and
lacrimal gland tissues were harvested for immunohistochemistry,
total RNA extraction, and protein isolation. Age- and gender-matched
wild type (WT) and NHE8 knockout (KO) mice (eight in each group)
were subjected to examiner-masked ocular surface evaluations
including tear pH measurement with narrow range pH paper, and
under anesthesia, phenol cotton thread test, tear break up time
(TBUT), and corneal fluorescein staining.
Results: The expression of NHE8 was confirmed at both mRNA and
protein levels in conjunctival and lacrimal gland tissues.
Immunohistochemistry staining showed that NHE8 was located on
the membrane of both epithelial and goblet cells of conjunctiva. In
lacrimal gland, NHE8 is differentially expressed on cell membrane,
with only certain gland acinar and ductal epithelial cells being
positive. Tear pH value of KO mice was lower than that of WT mice
(p < 0.001). KO mice also had significantly lower phenol cotton
thread test, TBUT, and significantly higher corneal staining scores (p
< 0.05, respectively).
Conclusions: For the first time, we have demonstrated that NHE8 is
involved in ocular surface pH regulation. Mice lacking NHE8
function showed signs of dry eye. Our results shed new insights into
the understanding of dry eye syndrome, which could lead to new
therapeutic strategies.
Commercial Relationships: Mingwu Wang, None; Jing Li, None;
Fayez K. Ghishan, None; Hua Xu, None
Support: NIH R01DK073638, Unrestricted RPB grant to
Department of Ophthalmology University of Arizona
Program Number: 2110 Poster Board Number: D0337
Presentation Time: 11:00 AM - 12:45 PM
Carboxypeptidase A3 expression in intraepithelial mast cells of
chronic allergic conjunctivitis
Kanji Hori, Nobuyuki Ebihara, Toshinari Funaki, Kaori Ohtomo,
Yosuke Asada, Akira Murakami, Akira Matsuda. Ophthalmology,
Juntendo Univ School of Med, Bunkyo-ku, Japan.
Purpose: Carboxypeptidase A3 (CPA3) is a protease known to be
expressed in mast cells. Preferential CPA3 expression in
intraepithelial mast cells in atopic asthma was reported previously.
We investigated the expression of CPA3 in the giant papillae
obtained from vernal keratoconjunctivitis (VKC) and atopic
keratoconjunctivitis (AKC) patients, and compared the expression
with known mast cell immunohistochemical markers
(tryptase/chymase), and a mast cell activation marker; high affinity
IgE receptor β chain (FcεRIβ).
Methods: 10 giant papillae were resected for therapeutic purpose and
sections were prepared as previously described. (IOVS.50:28712877) Anti-CPA3/anti-tryptase or anti-CPA3/anti-chymase double
immnunohistochemical staining was carried out by indirect
immunofluorescent method. Anti-CPA3/anti-FcεRIβ double
immunohistochemical staining was carried out by direct
immunofluorescent method using Zenon labeling system
(Invitorogen, CA).
Results: CPA3-tryptase double immunostaining showed that CPA3
positive staining was observed in the intraepithelial and subepithelial
tryptase positive mast cells. CPA3-chymase double immunostaining
showed both CPA3+ chymase - mast cells and CPA3+ chymase+
mast cells in the conjunctival epithelium of the giant papillae
samples. CPA3+ chymase+ mast cells preferentially located at near
the epithelial-stromal borders. CPA3/ FcεRIβ double positive cells
were also observed the giant papillae samples.
Conclusions: Preferential CPA3 expression in the intraepithelial
mast cells of giant papillae was observed. Tryptase positive/Chymase
negative (MCT) mast cells are predominant mast cell type for CPA3
expression. CPA3 expression may relate to mast cell activation or
maturation.
Commercial Relationships: Kanji Hori, None; Nobuyuki
Ebihara, None; Toshinari Funaki, None; Kaori Ohtomo, None;
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea
Yosuke Asada, None; Akira Murakami, SEED(Japan) JP4855782
(P), SEED(Japan) JP5132958 (P); Akira Matsuda, None