ARVO 2014 Annual Meeting Abstracts
506 Corneal Epithelium and Development #2
Thursday, May 08, 2014 8:30 AM–10:15 AM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 5523–5546/A0097–A0120
Organizing Section: Cornea
Program Number: 5523 Poster Board Number: A0097
Presentation Time: 8:30 AM–10:15 AM
Assessment of corneal epithelial thickness in dry eye patients
evaluated by Fourier-domain optical coherence tomography
Xinhan Cui, Jiaxu Hong, Fei Wang, Yujing Yang, JianJiang Xu.
Ophthalmology, Eye and ENT Hospital of Fudan University,
Shanghai, China.
Purpose: To investigate the features of corneal epithelial thickness
topography with Fourier-domain optical coherence tomography
(OCT) in dry eye patients.
Methods: In this cross-sectional study, 100 symptomatic dry eye
patients and 35 normal subjects were enrolled. All participants
went through ocular surface disease index (OSDI) questionnaire,
corneal fluorescein staining, tear break-up time (TBUT), Schirmer I
test without anesthetic (S1t), and meibomian morphology. Corneal
epithelial thickness was mapped by Fourier-domain OCT with
Pachymetry+Cpwr scan pattern. Several epithelium statistics for
each eye, including central, superior, inferior, minmum, maximum,
minimum-maximum (Min-Max), and map standard deviation (MSD)
were averaged. Correlations of epithelial thickness with the signs and
symptoms of dry eye were calculated.
Results: The mean central, superior, and inferior corneal epithelial
thickness was 53.57±3.31μm, 52.00±3.39μm and 53.03±3.67μm in
normal eyes; and 52.71±2.83μm, 50.58±3.44μm and 52.53±3.36μm
in dry eyes. The superior corneal epithelium was thinner in dry eye
patients compared with normal subjects (P=0.037). In dry eye group,
patients with higher severity grades had thinner superior (P=0.017)
and minimum (P<0.001) epithelial thickness, more negative MinMax (P=0.032), and greater MSD (P=0.003). The average central
epithelial thickness had no correlation with TBUT, S1t or the severity
of meibomian glands; while average superior epithelial thickness
positively correlated with S1t (r=0.238, P=0.017).
Conclusions: Fourier-domain OCT demonstrated that the thickness
map of the dry eye corneal epithelium was thinner than normal eyes
in the superior region. In severer dry eye disease patients, the superior
and minimum epithelium was much thinner, with a greater range of
map standard deviation.
Commercial Relationships: Xinhan Cui, None; Jiaxu Hong, None;
Fei Wang, None; Yujing Yang, None; JianJiang Xu, None
Support: None in the Support field below
Program Number: 5524 Poster Board Number: A0098
Presentation Time: 8:30 AM–10:15 AM
Confocal microscopy: New tool for the follow-up of conjunctival
intraepithelial neoplasia
Vitor Maduro, luisa vieira, Rita Anjos, André Vicente, Nuno Alves.
Ophthalmology, CHL-ZC, Lisboa, Portugal.
Purpose: To analyze the contribution of in vivo confocal microscopy
for the diagnosis and follow-up of conjunctival intraepithelial
neoplasia
Methods: Retrospective case series of 5 patients with unilateral
conjunctival intraepithelial neoplasia (CIN) submitted to surgery plus
adjuvant therapy in a follow-up period of 12 months. All patients
were evaluated before and after surgery with slit-lamp photography
and confocal microscopy (Heidelberg Retina Tomograph II, Rostock
Cornea Module). The authors did a comparative analysis of slitlamp photographs, confocal microscopy images and histological
examination photographs of the same lesions.
Results: Five patients (4 males, 1 female) with a mean age of 79,6
years, were enrolled in this study. Three were identified as highgrade intraepithelial neoplasia and two as carcinoma in situ. The
histological findings correlated well with those seen by confocal
microscopy: both revealed epithelial disorganization with acanthosis,
parakeratosis, cellular pleomorphism, high cellular and nuclear
reflectivity, high nuclear to cytoplasmic ratio and sometimes
binucleation. The lesions were well demarcated and the subbasal
corneal nerves were not visualized in areas affected by CIN. Confocal
microscopy has identified 1 relapse and was useful to monitor the
treatment response.
Conclusions: In vivo confocal microscopy may be important
not only in the diagnosis of CIN, but also detecting relapses and
monitoring the treatment response, in a relatively non-invasive
manner.
Commercial Relationships: Vitor Maduro, None; luisa vieira,
None; Rita Anjos, None; André Vicente, None; Nuno Alves, None
Program Number: 5525 Poster Board Number: A0099
Presentation Time: 8:30 AM–10:15 AM
Normal development of central corneal thickness and intraocular pressure
Ian Cunningham, Arvind Chandna. Alder Hey Children’s NHS
Foundation Trust, Liverpool, United Kingdom.
Purpose: To measure central corneal thickness (CCT) and intraocular pressure (IOP) under general anaesthesia for infants under 8
years of age.
Methods: This study prospectively measured CCT and IOP in 195
Caucasian children attending Alder Hey Children’s Hospital, UK
for a surgical procedure unrelated to ophthalmology. Children had
no ocular anomalies and underwent general anaesthesia ensuring
stable fixation was maintained during IOP and CCT measurements.
Children with history of prematurity or other systemic complications
were not invited to participate. CCT was measured using a Palmscan
pachymeter with a 50Hz ultrasound probe (MicroMedical Devices,
USA). IOP was measured by electronic indentation tonometry using
Tonopen XL (Reichart, USA).
Results: Age ranged from 7 weeks to 7and ¼ years
(Mean±SD=138±95wks). Over half of children attended for ENT
procedures. There was a small trend for CCT to decrease within
the first year of life although overall there was no correlation with
increasing age (R=-0.0006). There was no statistical difference
between right and left CCT so right eye values only were analysed
(CCT: t=0.06, df=394, p=0.27; IOP: t=-0.65, df=340, p=0.26).
Average CCT was 539±39nm range 470-646nm. Average IOP
was 9.8±2.6mmHg range 4 to 18mmHg. There was no correlation
between CCT and IOP (R=-0.0025).
Conclusions: In healthy normally developing children CCT was
shown to slightly decrease in the first year of life although there
appears to be no consistent growth pattern such as that observed for
axial length or anterior chamber depth. It appears there is a large
range of corneal thickness in early childhood and it measurement
©2014, 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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
may assist in diagnosing of buphthalmos or infantile glaucoma yet
not used in isolation.
established Scheimpflug imaging–derived keratoconus classification
and anterior surface irregularity indices.
Results: Intra-individual repeatability of epithelial thickness was for
group-A ±1.13 μm, for group-B ±1.78 μm for center and average
±1.67 μm (center, superior, inferior, maximum and minimum).
In group-A, center epithelial was 52.54±3.23 μm, maximum
55.33±3.27 μm and minimum 48.50±3.98 μm. In group B, center
thickness was 51.75±7.02 μm, maximum and minimum were
63.54±8.85 μm and 40.73±8.51 μm. Topographic variability was
6.07±3.55 μm (range -22.81±12.55 μm) for the keratoconic group-B,
while for the control group-A 1.59±0.79 μm (-6.86±3.33 μm).
Epithelial thickness topographic variability and range correlated well
with keratoconus severity in the study group-B.
Conclusions: AS-OCT may offer high predictability of 3 dimensional
epithelial assessment in keratoconus. Overall epithelial thickness in
keratoconic eyes appears significantly different to normal even in
lower stages of keratoconus. Increased overall thickness correlated
remarkably with keratoconus severity, as defined by established
Scheimpflug imaging-derived anterior-surface irregularity indices.
Central corneal thickness (nm) against age (weeks)
Examples of epithelial mapping of a normal (left) and a keratoconic
patient (right) obtained by Optical Coherence Tomography
Commercial Relationships: Elana S. Rosenberg, None; Anastasios
J. Kanellopoulos, Alcon (C), Avedro (C); George Asimellis, None
Central corneal thickness (nm) correlation with intra-ocular pressure
(mmHg)
Commercial Relationships: Ian Cunningham, None; Arvind
Chandna, None
Support: Alder Hey Charity
Program Number: 5526 Poster Board Number: A0100
Presentation Time: 8:30 AM–10:15 AM
3-D epithelial mapping pattern assessment in normal and
keratoconus eyes
Elana S. Rosenberg1, Anastasios J. Kanellopoulos1, 2, George
Asimellis2. 1Ophthalmology, New York University School of
Medicine, New York, NY; 2LaserVision.gr Eye Institute, Athens,
Greece.
Purpose: To evaluate applicability of spectral domain optical
coherence tomography (AS-OCT) of epithelial thickness patterns
in normal and in the diagnosis of keratoconus, and to compare with
Scheimpflug imaging keratoconus severity.
Methods: 250 healthy (control group-A) and 155 untreated
keratoconic (study group-Β) eyes were subjected to anterior segment
OCT three-dimensional epithelial thickness imaging. Comparative
statistical analysis of patterns was performed, investigating central,
minimum, inferior, posterior, and topographic variability of epithelial
thickness. Epithelial thickness characteristics were correlated to
Program Number: 5527 Poster Board Number: A0101
Presentation Time: 8:30 AM–10:15 AM
Three-Dimensional Analysis of Corneal Epithelial Nerves in
Diabetes
Matthew S. Yorek1, Mark A. Yorek1, 3, Randy H. Kardon1, 2. 1Center for
the Prevention and Treatment of Visual Loss, Department of Veterans
Affairs, Iowa City, IA; 2Ophthalmology, University of Iowa, Iowa
City, IA; 3Internal Medicine, University of Iowa, Iowa City, IA.
Purpose: Diabetes mellitus is associated with corneal epithelial
manifestations including epithelial erosion, chronic epitheliitis,
superficial corneal ulcers and delayed epithelial healing. The
long-term complications of diabetic corneal neuropathy include
diminished corneal sensation and reduced tear secretion, both
symptoms contribute to epithelial manifestations and both symptoms
are related to the loss of the corneal innervation. Currently, in vivo
corneal confocal microscopy (CCM) is being employed to grade
the progression of diabetic neuropathy. This study combines both in
vivo corneal imaging and immunohistochemistry with laser scanning
confocal microscopy of the cornea in an effort to better understand
the neuropathic changes associated with diabetes.
Methods: At 12 weeks of age C57Bl/6 mice were treated with
streptozotocin (150 mg/kg i.p in saline) to induce diabetes. CCM was
performed, corneal nerves were imaged using the Rostock cornea
module on the Heidelberg Retina Tomograph confocal microscope.
Next, corneas were dissected from the eyes and trimmed around
the sclero-limbo region, then processed for immunohistochemistry
using various antibodies, and were finally visulized using a Zeiss
©2014, 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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
LSM 710 confocal microscope (Carl Zeiss; Germany). An analysis of
corneal nerves was completed and is presented with Imaris software
version 7.6.4 X64 (Bitplane; Zurich, Switzerland). Three-dimensional
representations of confocal stacks were reconstructued by surface
rendering, where a surface was created over the fluorescent staining
and this three-dimensional surface was used for quantitation of nerve
volume.
Results: Using a three-dimensional analysis of the epithelial nerves
we observed a significant decrease in the overall volume of nerve
immunofluoresence in diabetic mice when compared to control
animals. Control mice maintained a healthy 2.0% nerve volume,
while diabetic mice lost 0.5% (P = 0.035) nerve volume after 14
weeks of the disease. This result is in agreement with in vivo CCM
data showing a significant reduction (P = 0.016) in nerve density in
the same animals.
Conclusions: We observed a reduction of nerve density and nerve
volume in the mouse cornea during the progression of diabetes. Our
data suggest imaging of corneal nerves may be a useful marker of
diabetic neuropathy.
Cornea Subbasal Nerve Plexus (Vortex image). Control animal with a
healthy nerve structure. Nerves are labeled with β-III Tubulin.
Commercial Relationships: Matthew S. Yorek, None; Mark A.
Yorek, None; Randy H. Kardon, None
Support: VA Center for the Prevention and Treatment of Visual Loss
(C6810-C)
Program Number: 5528 Poster Board Number: A0102
Presentation Time: 8:30 AM–10:15 AM
Gamma-Glutamyl Transpeptidase in Human Diabetic and NonDiabetic Corneas
Margaret Young1, Marlyn P. Langford1, Thomas Redens1, Shubnum
Chaudhery2. 1Ophthalmology, Louisiana State University Health
Sciences Center, Shreveport, LA; 2Pathology, Louisiana State
University Health Sciences Center, Shreveport, LA.
Purpose: Diabetic keratopathy is common in longstanding diabetic
cases. Failure to protect against diabetes-associated increases in
oxidative stress and reactive oxygen species has been associated with
diabetic keratopathy. The current investigation assesses gammaglutamyl transpeptidase (GGT; key enzyme in the recapture of the
anti-oxidant glutathione) activity in corneal epithelium of diabetic
and non-diabetic human eyes.
Methods: The expression of GGT in the corneal epithelium
of 10 enucleated or eviscerated eyes from 5 diabetic (3 with
diabetic retinopathy) and 5 non-diabetic cases was assessed by
immunohistochemical analysis.
Results: Immunoreactive GGT was predominantly expressed by
the columnar epithelial cells and flat surface epithelial cells. GGT
was expressed weakly by the winged epithelial cells. GGT was
concentrated in the cell and nuclear membranes of the columnar
epithelial cells. Diabetic keratopathy was not detected, but thinning
of the central corneal epithelium was noted and immunoreactive GGT
was reduced from non-diabetic corneas in the columnar epithelial cell
membranes of the diabetic eyes.
Conclusions: The results of these basic studies suggest diabetesrelated loss of corneal GGT/glutathione recapture activity. The
results are consistent with the therapeutic importance of maintaining
anti-oxidant potential in cornea as well as other ocular tissues that are
susceptible to diabetes-induced oxidative stress.
Commercial Relationships: Margaret Young, None; Marlyn P.
Langford, None; Thomas Redens, None; Shubnum Chaudhery,
None
Program Number: 5529 Poster Board Number: A0103
Presentation Time: 8:30 AM–10:15 AM
Expression pattern of epithelial stem cell markers in the normal
human cornea
Michael J. Nicolas, Alexandre P. Moulin, Francois Majo. Department
of Ophthalmology, University of Lausanne, Jules-Gonin Eye hospital,
Lausanne, Switzerland.
Purpose: Many studies have tried to characterize corneal epithelial
stem cell markers, based on their restricted expression pattern in
the basal cell layer of the limbal epithelium. This study focused
on markers identified by a functional approach. ABCG2/BCRP, an
ATP-transporter protein, identifies a population of human limbal
epithelial cells with a high colony-forming efficiency in vitro. C/
EBPδ and Bmi-1 identify quiescent stem cells in the limbus and are
responsible for mitotic quiescence and self-renewal properties while
p63α sustain the stem cell proliferation potential. In order to validate
these experimental markers in the normal human cornea, we studied
their expression pattern in a large series of surgical specimens using
immunohistochemistry. These data were further compared with the
cytokeratin profile of the corneal and limbal epithelium.
Methods: Histologically normal human corneas were obtained from
surgical specimens of 30 patients (age 7 m to 84 y) enucleated for
uveal melanoma (16/36) or retinoblastoma (14/36) at the Hôpital
Ophtalmique Jules-Gonin. Clinical charts were reviewed for corneal
disease. Paraffin sections were immunostained with antibodies
against C/EBPδ, Bmi-1, BCRP/ABCG2, p63α, CK3, CK5, CK15,
CK19 and Ki67.
Results: In all specimens, C/EBPδ and p63α were diffusely
expressed in the nucleus of basal epithelial cells in the limbus
but also in the central and peripheral cornea. By contrast, Bmi-1
expression was weak and restricted to the basal layer of the limbus.
ABCG2/BCRP was demonstrated in basal cells of limbus, but also in
central cornea,
Conclusions: We studied the expression of functional stem cell
markers ABCG2/BCRP, Bmi-1, p63α and C/EBPδ in the normal
human cornea and found that only Bmi-1 was always restricted
to limbus while ABCG2/BCRP, p63α and C/EBPδ were diffusely
expressed in the corneal epithelium. These data show that caution
should be exerted when extrapolating experimental data to normal
human cornea.
Commercial Relationships: Michael J. Nicolas, None; Alexandre
P. Moulin, None; Francois Majo, None
©2014, 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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
Program Number: 5530 Poster Board Number: A0104
Presentation Time: 8:30 AM–10:15 AM
ANALYSIS OF EXPRESSION KINETICS OF CELL CYCLE
MACHINERY DURING CORNEAL EPITHELIAL WOUND
HEALING AND EPITHELIAL GROWTH INFLUENCED BY
GROWTH FACTORS AND EXTRACELLULAR MATRIX
Gudiseva Chandrasekher, Swetha Pothula. Pharmaceutical Sciences,
South Dakota State University, Brookings, SD.
Purpose: Corneal injuries induce an increase in receptor tyrosine
kinase ligands and extracellular matrix proteins (ECMs) in lacrimal
and stromal secretions. These factors play very important roles in
promoting wound healing. This study compared the expression
pattern of various cell cycle proteins during corneal epithelial wound
healing and epithelial growth promoted by hepatocyte growth factor
(HGF), keratinocyte growth factor (KGF) and ECMs.
Methods: Wounds were created on live rabbit corneas by complete
epithelial debridement. Regenerated epithelium was collected from
1-, 2-, 4-, and 8-day post injury corneas. The protein expression was
analyzed by immunoblotting and immunofluorescence. The effect of
HGF, KGF and ECMs (laminin, collagens I and IV and fibronectin)
on cell cycle proteins expression was evaluated in rabbit corneal
epithelial primary cells cultured in DMEM-F12/FCS.
Results: While the expression of cell cycle progressing cyclin E,
cyclin A and cyclin-dependent kinases (CDK-2 and CDK4) increased
gradually between 1-4 days and declined by day 8, cell cycle
inhibitor p27kip exhibited biphasic expression. Its level was higher one
day after the injury but decreased by day 2. At later time points its
level increased and peaked at day 8. The kinetics of another inhibitor
p21cip expression was similar to cyclins and CDKs. DAPI-staining of
cornea cross-sections indicated restoration of multilayer epithelium
by day 8. Fluorescein staining revealed near complete wound closure
within 24 h following debridement indicating migration of epithelium
from the limbus. Treatment of cells with HGF and KGF (20 ng/ml)
for 24 h upregulated cyclins, CDKs and p21cip but suppressed p27kip
expression. Interestingly, all ECMs (10 μg/ml) increased p21cip and
p27kip levels in 24 h.
Conclusions: Increase in cyclins and CDKs levels between 2-4
days after the injury indicates robust proliferative activity which is
required to restore multilayer epithelial architecture. Elevated levels
of p27kip in 8-day post-wound cornea could lead to suppression of
cell division and prevention of epithelial hyperplasia. Further, the
different patterns of expression of various cell cycle proteins as
effected by HGF, KGF and ECMs could lead to the regulation of cell
adhesion, migration and proliferation phases during wound healing.
Commercial Relationships: Gudiseva Chandrasekher, None;
Swetha Pothula, None
Support: Department of Pharmaceutical Sciences Grduate research
program and State of South Dakota 2010 Translational Cancer
Research Center
Program Number: 5531 Poster Board Number: A0105
Presentation Time: 8:30 AM–10:15 AM
TRPV-1 expression in cornea of diabetic rats.
Lara C. Dias, Ana C. Dias, Luis Fernando Nominato, Eduardo M.
Rocha. Oftalmologia, Otorrinolaringologia e CCP, FMRP - USP,
Ribeirão Preto, Brazil.
Purpose: Diabetes mellitus (DM) is associated with dry eye disease.
TRPV-1 are receptors sensitive to hiperosmolarity. The aim of the
present work is to investigate the presence of hiperosmolarity in rat
models of DM and the expression of TRPV-1 in the lacrimal gland
and cornea of control and DM rats.
Methods: Diabetes mellitus was induced by streptozotocin (60mg/
kg)in male wistar rats (6 weeks old). Data were compared with
controls ( n = 5/group) after 5 weeks. Body weight, ocular findings,
glycemia, tear secretion, tears and systemic osmolarity was evaluated
in both groups. Tissues were harvested and TRPV-1 expression
measured by western blotting in the lacrimal gland and cornea.
Results: The body weight was significantly lower in DM group
(p=0.0099), Glycemia was 503.3 ± 21.07 mg/dl in DM and 261.5 ±
4.5 mg/dl in control group (p=0.0001), tear secretion was 5.5 ± 1.19
mm in control and 1.25 ± 1.50 mm in DM group, (p=0.0294). Tear
osmolarity and systemic osmolarity was significantly higher in DM
group (337 ± 17.35 mOsm and 305.5 ± 2.10 mOsm, respectively)
compared to control group (284 ± 3.29 mOsm and 328.8 ± 1.65
mOsm, respectively) (p=0.017 and p=0.0001). TRPV-1 expression in
cornea was similar in the DM control group (p=0.7904, repectively).
Conclusions: DM is associated with tear film and systemic
hyperosmolarity in rats. TRPV-1 is expressed in cornea of rats and
its expression in those tissues is not changed by DM. These findings
suggest a mechanism involving TRPV-1 signaling in the DM dry eye
disease.
Support: CAPES, CNPq, NAP-FTO USP
Commercial Relationships: Lara C. Dias, None; Ana C. Dias,
None; Luis Fernando Nominato, None; Eduardo M. Rocha, None
Program Number: 5532 Poster Board Number: A0106
Presentation Time: 8:30 AM–10:15 AM
Lack of tyrosine phosphatase Shp2 in corneal epithelium impacts
sensory innervation during development and homeostasis in
adult mice
Chia-Yang Liu1, Yujin Zhang1, Lung-Kun Yeh2, Winston W. Kao1.
1
Ophthalmology, University of Cincinnati, Cincinnati, OH;
2
Ophthalmology, Chang-Gung Memorial Hospital, Linko, Taiwan.
Purpose: Corneal sensory nerves contribute trophic factors to
maintain the integrity and function of the cornea and particularly
of the epithelium. Little is known whether corneal epithelium is
mutually important for the maintenance of corneal nerve function.
Herein, we test the possibility that impaired stratified corneal
epithelium due to ablation of Shp2 may cause failure of corneal
innervation during development and nerve degeneration in adult.
Methods: Krt14-rtTA/tet-O-Cre/Shp2f/f/Thy-1-YFP quadruple
transgenic mice administrated without (referring to Shp2 wild-type)
or with (referring to Shp2 null) doxycycline (Dox) from embryonic
day 7 (E7) to E12.5 and E15.5, from postnatal day 1 (P1) to P21, and
from P21 to P33, respectively. Mouse excised corneas were subjected
to whole-mount histology examination under confocal microscopy.
Thy-1-YFP was served as a fluorescent marker for comparing
the pattern of peripheral corneal innervation with or without
Shp2 expression in the corneal epithelium. Transmission electron
microscopy (TEM) was also used to examine detail morphology of
the corneal nerves.
Results: YFP signal was first detected from four quadrants of the
eye at E12.5 and they covered the entire corneal surface at E16.5
of the control mice, whereas Shp2 conditional knockout in K14+
cells rendered the failure of corneal innervation during embryonic
development. Interestingly, Shp2 ablation from fully stratified corneal
epithelium at P21 resulted in the degeneration of corneal nerve
bundle at P33. TEM examination revealed that individual nerve fibers
and nerve bundles wrapped around the basal epithelial cells and the
wing cells and the large single fibers contain many mitochondria,
clusters of glycogen particles, and vesicles of different types and
sizes of the wild-type cornea. In contrast, however, little of such was
found in the Shp2 null cornea.
Conclusions: These data suggest that formation of stratified
corneal epithelium is indispensable for corneal innervation during
development and maintenance of nerve integrity in adults, and Shp2-
©2014, 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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
mediated receptor tyrosine kinase signaling may play a pivotal role in
this corneal epithelium-nerve interaction which is paramount for the
corneal morphogenesis and homeostasis.
Commercial Relationships: Chia-Yang Liu, None; Yujin Zhang,
None; Lung-Kun Yeh, None; Winston W. Kao, None
Support: NIH Grant EY21501, EY013755, Research to Prevent
Blindness, Ohio Lions Eye Research Foundation
Program Number: 5533 Poster Board Number: A0107
Presentation Time: 8:30 AM–10:15 AM
Cultured limbal stem cell versus limbal tissue transplantation for
treating severe limbal deficiency
Vincent M. Borderie1, 2, Djida Ghoubay-Benallaoua1, 2, Nacim
Bouheraoua1, 2, Otman Sandali1, Elena Basli1, Pablo L. Goldschmidt1,
2
, Laurent Laroche1, 2. 1Ophthalmology, CHNO des Quinze-Vingts,
Paris, France; 2Institut de la Vision, Paris, France.
Purpose: To compare cultured limbal stem cell transplantation with
limbal tissue transplantation for treating severe limbal deficiency.
Methods: Thirteen patients with total limbal deficiency
were included in a phase II prospective clinical trial (TC181,
ClinicalTrials.gov Identifier: NCT01619189). Treatment consisted
of transplantation of limbal stem cells cultured from superficial
limbal explants, either autologous (n=6) or allogeneic (n=7), on
human amniotic membrane. This study group was compared with
a retrospective control group including 20 limbal tissue autografts
(n=12) or allografts (n=8) performed for total limbal deficiency.
Before transplantation all eyes featured irregular corneal epithelium
with abnormal fluorescein permeability on the whole corneal surface,
superficial corneal vascularization, and presence of goblet cells
in the corneal epithelium in vivo (confocal microscopy or corneal
impression cytology) and ex vivo (histology).
Results: The mean follow-up time was 41 months. The postoperative visual acuity at M6 (1.6 LogMAR + 7 lines), M12
(1.5 LogMAR + 8 lines), and M24 (1.6 LogMAR + 8 lines) was
better than the preoperative visual acuity (2.0 LogMAR + 5 lines)
(p<0,005). The mean improvement in visual acuity was 8.0 lines
in the study group and 3.0 lines in the control group (p=0.02).
This figure was 5.2 lines for autografts and 4.8 lines for allografts
(p=0.85).
Conclusions: Cultured limbal stem cell transplantation was
associated with better visual recovery compared with limbal tissue
transplantation. Visual recovery was not significantly better in
autologous cases compared with allogeneic ones.
Commercial Relationships: Vincent M. Borderie, None; Djida
Ghoubay-Benallaoua, None; Nacim Bouheraoua, None; Otman
Sandali, None; Elena Basli, None; Pablo L. Goldschmidt, None;
Laurent Laroche, None
Support: PHRC 2001, PHRC 2009
Clinical Trial: NCT01619189
Program Number: 5534 Poster Board Number: A0108
Presentation Time: 8:30 AM–10:15 AM
Prognostic factors in chronic complications of Stevens-Johnson
syndrome and toxic epidermal necrolysis
DongHyun Kim1, 2, Mee Kum Kim1, 2, Kyung Chul Yoon3, Hyo
Seok Lee3, Kyoung Yul Seo4, Sang Chul Yoon4. 1Department of
Ophthalmology, Seoul National University College of Medicine,
Seoul, Republic of Korea; 2Laboratory of Ocular Regenerative
Medicine and Immunology, Seoul Artificial Eye Center, Seoul
National University Hospital Clinical Research Institute, Seoul,
Republic of Korea; 3Department of Ophthalmology, Chonnam
National University Medical School, Gwangju, Republic of Korea;
4
Department of Ophthalmology, Institute of Vision Research, Yonsei
University College of Medicine, Seoul, Republic of Korea.
Purpose: To investigate prognostic factors in chronic ocular
complications of Stevens-Johnson syndrome (SJS) or Toxic
epidermal necrolysis (TEN) patients, we analyzed systemic and
ocular factors in acute stages and effect of early treatment modalities
on chronic complications.
Methods: The medical records of the 86 eyes in 43 patients who
had been diagnosed as SJS/TEN were retrospectively reviewed.
Main outcome measures were final VA and grading of chronic ocular
surface complications(GCOC, 0-15). Age, sex, causative drugs,
initial visual acuity(VA), grading of the ocular complication at onset
(0-3), grading of the systemic involvement at onset (0-16), systemic
steroid dosage, intravenous immunoglobulin (IVIG) dosage, amniotic
membrane transplantation(AMT) were analyzed as prognostic factors
to be related with poor final VA (less than 20/200) and high GCOC
(more than 8 points).
Results: Grading of the systemic involvement at onset in IVIG
therapy alone, combination therapy of steroid and IVIG, steroid
therapy group were significantly higher than that of conservative
treatment group (p<0.001, Kruskal Wallis test). Final VA and GCOC
were not significantly different depending on the treatment modalities
despite of correcting the patients’ demographics (p=0.169, 0.117,
Nonparametric ANCOVA). Poor final VA was related with initial
AMT and high GCOC were related with women or AMT. (p=0.048/
p=0.012, 0.040, Multiple logistic regression).
Conclusions: The facts that female or initial AMT due to severe
ocular surface complication at onset suggest worse chronic ocular
complications in SJS/TEN as prognostic factors.
Commercial Relationships: DongHyun Kim, None; Mee Kum
Kim, None; Kyung Chul Yoon, None; Hyo Seok Lee, None;
Kyoung Yul Seo, None; Sang Chul Yoon, None
Program Number: 5535 Poster Board Number: A0109
Presentation Time: 8:30 AM–10:15 AM
Lateral Diffusion Coefficient in CXL with Corneal Channels
Rebecca M. McQuaid1, 2, JiaJun Li1, Michael Mrochen2, Brian
Vohnsen1. 1Physics, University College Dublin, Dublin 4, Ireland;
2
Ophthalmology, IROC Innocross, Zurich, Switzerland.
Purpose: Recent studies involving corneal cross-linking (CXL)
have investigated other methods of riboflavin delivery to the cornea
such as Iontophoresis, femtosecond laser-created pockets and transepithelium CXL. Standard CXL sees riboflavin diffusion to the
cornea over a 30 minutes interval. In this study, we investigate a new
method of riboflavin delivery to the cornea without removal of the
corneal epithelium. The purpose is to gain insight into the temporal
dynamics of corneal riboflavin diffusion via surgically-created intrastromal channels.
Methods: Porcine globes were obtained from the local
slaughterhouse 4 hours post mortem and kept at a temperature of
4 degrees Celsius. An incision was made using a cannula needle,
©2014, 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 2014 Annual Meeting Abstracts
followed by insertion of keraring (Mediphacos Ltd) to create
a channel in the cornea for riboflavin diffusion. Monitoring of
the cornea with a CCD camera facilitates the study of diffusion
characteristics in real time.
Results: The results show that riboflavin can be effectively injected
into the cornea via the created diffusion channels without removal of
the epithelium layer. The recorded videos show characteristic timescales on the order of seconds to minutes from which a characteristic
diffusion coefficient can be derived on the basis of Fick’s 2nd law of
diffusion.
Conclusions: This study investigated the distribution of riboflavin
diffusion in the cornea through creation of surgically-created
channels in post-mortem porcine eyes. This could be an effective
method for riboflavin application for CXL without the need to
remove the epithelial layer and thus with reduced patient discomfort..
Commercial Relationships: Rebecca M. McQuaid, None; JiaJun
Li, None; Michael Mrochen, None; Brian Vohnsen, None
Program Number: 5536 Poster Board Number: A0110
Presentation Time: 8:30 AM–10:15 AM
Fibrin glue in cornea epithelial cell migration
Aaron M. Yeung, Lana A. Faraj, Owen D. McIntosh, Harminder
S. Dua. Ophthal & Visual Sciences, University of Nottingham,
Nottingham, United Kingdom.
Purpose: Fibrin glue has been used successfully in numerous
ophthalmic surgical procedures. Recently fibrin glue has been used
in limbal stem cell transplantation to reduce both operative time and
negate the need for sutures. The aim of this study was to determine
the effects of fibrin glue on epithelial cell migration in vitro.
Methods: Cornea-scleral rims were split to retain the superficial
layer. Rims were cut into 8 equal sized pieces and were either placed
on culture plates with or without fibrin glue. Rims were cultured
over a 16 day period to allow epithelial cells to growth. Epithelial
cells were photographed and immunofluorescence for anti-fibrin was
performed.
Results: Explants that were glued demonstrated delayed epithelial
cell growth and migration, compared to explants without glue. By
Day 16, all fibrin glue had dissolved permitting cells to freely migrate
and expand.
Conclusions: Fibrin glue delays epithelial cell growth and
migration and thus can be used to our advantage in limbal stem cell
transplantation.
Commercial Relationships: Aaron M. Yeung, None; Lana A.
Faraj, None; Owen D. McIntosh, None; Harminder S. Dua, None
Program Number: 5537 Poster Board Number: A0111
Presentation Time: 8:30 AM–10:15 AM
Conjunctival cell viability and apoptosis after one month of
treatment with Thealoz® in dry eye subjects
Hayette Rebika1, Alberto López-Miguel3, 4, Roberto Reinoso2, 3,
Carmin Martín2, 3, Alfredo Corell2, 3, Itziar Fernández3, Margarita
Calonge2, 3, Maria-Jesus J. Gonzalez2, 3. 1R&D, Laboratoires
Thea, Clermont-Ferrand, France; 2, IOBA, Universidad de
Valladolid, Spain, Valladolid, Spain; 3CIBER-BBN (Biomedical
Research Networking Center in Bioengineering, Biomaterials
and Nanomedicine), Spain, vallalolid, Spain; 4VISIÓN I+D, SL,
Valladolid, Spain.
Purpose: to analyze the viability and apoptosis of conjunctival cells
after 1 month of treatment with Thealoz® (threalose 3%) compared
with control (saline solution 0.9%) in dry eye subjects.
Methods: 29 dry eye subjects were analyzed from a total of 50
patients foreseen for the study. After a wash-out period of 1 week
(minimum 3, maximum 8 instillations per day of saline solution
0.9%), they were randomly assigned to use Thealoz® or saline
solution 0.9% for 1 month, 6 times per day. Brush cytology was
performed in the conjunctival inferior fornix before (V1) and after
(V2) four weeks of treatment. Cell viability and apoptosis were
analyzed by flow cytometry. Lissamine green conjunctival staining
was performed and was graded in a 0-4 scale (Oxford scale). Change
of variables was defined as the rate of change at V2 respect to the
initial value (V1).
Results: In the group treated with Thealoz® it was found an
increase in the percentage of viable cells after the treatment (V1:
37.41±27.57%; V2: 49.85±28.01), and a decrease in the control
group (V1: 47.14±25.67%; V2: 46.61±21.57), these changes were
not significant. Percentage of cells in early apoptosis decreased in
Thealoz® (V1: 25.75±12.72, V2: 22.83±15.73) and in control group
(V1: 22.06±9.84, V2: 19.78±7.52), but changes were not significant.
Percentage of cells in late apoptosis (significant change) decreased
in the Thealoz® group (V1: 33.09±20.76, V2: 24.62±14.12), and
increased (non significant change) for the control group (V1:
28.73±15.65, V2: 32.19±15.81). A significant change in both groups
was found in the percentage of dead cells (Thealoz®, V1: 3.75±2.24,
V2: 2.72±2.71; control, V1: 2.05±2.03, V2: 1.42±0.95). A significant
change was found in conjunctival staining, in the Thealoz® group
(V1: median: 1, 95% confidence interval -CI-:1,2; V2: median: 0,
95% CI:0,1 ), and this change was not significant for the control
group (V1: median: 1, 95% confidence interval -CI-:1,2; V2: median:
1, 95% CI:1,2 ).
Conclusions: This intermediate analysis results show positive
tendencies and significant changes on the way that Thealoz® seems
to protect the conjunctival epithelial cells in dry eye subjects,
probably by rescuing them from apoptosis. This finding seems to
have a clinical expression in the decrease of conjunctival staining.
New analysis to confirm positive tendencies will be done at the end
of the study.
Commercial Relationships: Hayette Rebika, Laboratoires Thea
(E); Alberto López-Miguel, None; Roberto Reinoso, None;
Carmin Martín, None; Alfredo Corell, None; Itziar Fernández,
None; Margarita Calonge, None; Maria-Jesus J. Gonzalez, None
Clinical Trial: NCT01742884
Program Number: 5538 Poster Board Number: A0112
Presentation Time: 8:30 AM–10:15 AM
Blue Light-induced Oxidative Stress in Human Corneal
Epithelial Cells: Protective Effect of Ethanol Extracts from
Mixture of Various Medicinal Plants
Kyung Chul Yoon1, Zhengri Li1, Je Moon Woo2, Soo Hyun Kim3,
Jee Bum Lee3. 1Department of Ophthalmology, Chonnam National
University Medical School and Hospital, Gwangju, Republic of
Korea; 2Department of Ophthalmology, Ulsan University Hosital,
Ulsan, Republic of Korea; 3Department of Dermatology, Chonnam
National University Medical School and Hospital, Gwangju,
Republic of Korea.
Purpose: To investigate the effects of visible light on human corneal
epithelial cells and the impact of natural antioxidants on oxidative
stress produced by light overexposure.
Methods: Light emitting diode with various wavelengths
(410–830nm) was used to irradiate on human corneal epithelial
cells, and then cell viability was assessed. Production of
reactive oxygen species (ROS) was analyzed using the
2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA). Ethyl
alcohol (EtOH) extracts were prepared from the mixture of various
medicinal plants. After application of the EtOH extracts, freeradical-scavenging activity was measured using 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging assay. The induction
©2014, 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 2014 Annual Meeting Abstracts
of antioxidant enzymes including heme oxygenase-1 (HO-1),
peroxireoxin-1 (Prx-1), catalase (CAT), and superoxide dismutase-2
(SOD-2) and inhibitory capability of ROS by the extracts were
evaluated by real time-PCR and DCF-DA in human corneal epithelial
cells.
Results: The viability of human corneal epithelial cells was
diminished after irradiation of blue light (above 10J at 410nm and 50J
at 480nm). ROS production by blue light irradiation increased in a
dose dependant manner. EtOH extracts had potent radical scavenging
activity. The application of the extracts increased the expression of
HO-1, Prx-1, CAT, and SOD-2 and attenuated ROS production by
blue light.
Conclusions: Overexposure of blue light (410-480nm) may have a
harmful impact on human corneal epithelial cells compared to other
visual light wavelengths. Medicinal plant extracts can have potent
protective effects on blue light-induced oxidative stress.
Commercial Relationships: Kyung Chul Yoon, None; Zhengri Li,
None; Je Moon Woo, None; Soo Hyun Kim, None; Jee Bum Lee,
None
Program Number: 5539 Poster Board Number: A0113
Presentation Time: 8:30 AM–10:15 AM
Novel primary epithelial cell toxicity assay using porcine corneal
explant
Hiroki Takahashi, Kazuki Tajima, Takaaki Hattori, Hiroshi Goto.
Ophthalmology, Tokyo Medical University, Shinjyuku-ku, Japan.
Purpose: The purpose of this study is to develop novel primary
epithelial cell toxicity assay using porcine corneal explant and
evaluate this assay using benzalkonium chloride (BAK), which has
been reported to be especially toxic to the ocular surface.
Methods: The circular corneal explant, which is approximately 1/4
depth of the cornea, including epithelium and stroma was trephined
from the peripheral cornea of porcine eye using 2.0 mm biopsy
punches under microscope. Explants were placed on collagen gel
coated 6-well culture dishes and cultured in DMEM/F12 medium
with fetal bovine serum in a 37 °C incubator supplied with 5% CO2
for 12 hours. After 12 hours incubation, 50 ml BAK at 0.00001%,
0.0001%, 0.001%, or 0.01% was topically applied to each well for
2-minutes. After washing in PBS, explants were cultured again for
24 hours. The image of migrating epithelial cell areas at 36 hours
were taken and measured by Image J software, Cell expansion rate
(%) was calculated as follow; area of each BAK concentrations / area
of PBS control. Explants and migrated epithelial cells were stained
with cytokeratin (CK) 3, CK12 differentiated corneal epithelial cells
and ki67. The number of ki67 positive cells in corneal explant and
migrated epithelial cells (close tissue and edge) was calculated by
using Image J software. The viable cell number was measured by
wst-8 assay.
Results: The epithelial cells migrated roundly from corneal explant
as the time elapses and these corneal explant and migrated cells were
positive for CK3 and CK12. Cell expansion rate was significantly
decreased with 0.0001%, 0.001%, and 0.01% BAK group compared
to PBS-treated control (p < 0.01). Viable cell number was also
significantly decreased with 0.001% and 0.01% BAK group
compared to PBS-treated control (p < 0.05). Ki67 positive cells were
present in corneal explant and migrated epithelial cells, especially in
explant and close to the corneal tissues. Moreover, ki67 positive cells
in corneal explant, close tissue, and edge were significantly decreased
with 0.01% BAK group compared to PBS-treated control (p < 0.05).
Conclusions: The primary epithelial cell toxicity assay using porcine
corneal explant is a new technique that detects corneal toxicity
depends on BAK concentrations. This toxicity assay could be useful
in estimating epithelial proliferation of the cornea and toxicity of
BAK.
Commercial Relationships: Hiroki Takahashi, None; Kazuki
Tajima, None; Takaaki Hattori, None; Hiroshi Goto, None
Program Number: 5540 Poster Board Number: A0114
Presentation Time: 8:30 AM–10:15 AM
DNA damage in Human Limbal Epithelial Cells expanded ex vivo
Yolanda Lorenzo Corrales1, 2, Kristiane H. Berg1, Liv K. Drolsum1, 3,
Morten Carstens Moe1, 3, Andrew R. Collins2, Bjorn Nicolaissen1, 3.
1
Ophthalmology, Oslo University Hospital.Center for Eye Research
and The Norwegian Eye Bank., Oslo, Norway; 2Nutrition., Medical
Faculty, University of Oslo., Oslo, Norway; 3Ophthalmology,
University of Oslo., Oslo., Norway.
Purpose: Limbal stem cell deficiency, secondary to insults and
diseases, may be treated by transplantation of ex vivo engineered
epithelial grafts. We here present preliminary data on levels of
cellular DNA damage in grafts produced in two different types of
culture medium.
Methods: Cultures were initiated using corneo-limbal donor tissue
after removal of the central area for transplant purposes. Explants
(approx. 2x2 mm) were positioned epithelial side down on tissue
culture treated polyester membranes and expanded for four weeks
in DMEM/F12(1:1) with either 10% human serum (H medium) or
with 5% FBS, EGF, ITS, cholera toxin-A, DMSO and hydrocortisone
(COM medium). Cells were dissociated using Trypsin- EDTA
(0.05%) for 30 min., the enzyme activity was inhibited by medium
and serum, and the cell suspension was transferred to tubes on ice
and processed using the Comet Assay. Duplicate samples from
each culture were analyzed in each assay by visual scoring. Using
a fluorescence microscope, 100 comets (50 from each gel) were
classified into five categories, 0- 4, representing increasing relative
tail intensities. Summing the scores (0- 4) of 100 comets therefore
gives an overall score of between 0 and 400 arbitrary units.
Results: Preliminary data show low levels of DNA damage in cells
dissociated from the grafts regardless of the type of culture medium
used. Visual scoring of DNA damage in cells obtained from central
areas of the grafts was 63.5 in COM medium and 50.25 in H medium.
Scoring of DNA damage in cells obtained from peripheral areas of
the grafts was 55.5 in COM medium and 40 in H medium.
Conclusions: Recent studies have shown that medium with human
serum equally support production of grafts containing differentiated
as well as undifferentiated cells suitable for clinical transplantation.
Preliminary data from our experiments indicate that levels of
molecular damage to the DNA do not increase in cells cultured in H
medium despite its lack of complexity.
Commercial Relationships: Yolanda Lorenzo Corrales, None;
Kristiane H. Berg, None; Liv K. Drolsum, None; Morten Carstens
Moe, None; Andrew R. Collins, None; Bjorn Nicolaissen, None
Program Number: 5541 Poster Board Number: A0115
Presentation Time: 8:30 AM–10:15 AM
Cytotoxicity Effect of Boric Acid-based Multipurpose Contact
Lens Care Solutions (MPSs) on Human Corneal Epithelial Cells
HCET
Kissaou T. Tchedre1, Masaki Imayasu2, Yuichi Hori3, Cavanagh H.
Dwight4. 1R&D and Innovation Center, Menicon LTD, Le Mans,
France; 22R&D and Innovation Center, Menicon LTD, Kasugai,
Japan; 3Ophthalmology, 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
©2014, 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 2014 Annual Meeting Abstracts
(MPSs) have any cytotoxicity effect on human corneal epithelial
cells. MPSs effect on membrane-associated mucins (Muc1 &
16) expressions in the Rat cornea was also assessed. Membraneassociated 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: Human corneal epithelial 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.
Results: The viability/cytotoxicity assay result showed that MPSs
containing boric acid induce apoptosis 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.
Commercial Relationships: Kissaou T. Tchedre, Menicon (E);
Masaki Imayasu, Menicon (E); Yuichi Hori, None; Cavanagh H.
Dwight, None
Program Number: 5542 Poster Board Number: A0116
Presentation Time: 8:30 AM–10:15 AM
Improvement in Visual Function and Acuity with Prosthetic
Replacement of the Ocular Surface Ecosystem (PROSE)
Treatment in Patients with Chronic Graft Versus Host Disease
Christos Theophanous1, Gloria Chiu2, 3, John A. Irvine4. 1USC Keck
School of Medicine, Los Angeles, CA; 2Keck Medical Center of
USC, Los Angeles, CA; 3USC Ophthalmology, Los Angeles, CA;
4
Ophthalmology, USC Keck School of Medicine, Los Angeles, CA.
Purpose: Chronic Graft versus Host Disease (cGvHD) is a significant
complication of hematopoietic stem cell transplantation that can
result in ocular discomfort and impaired visual functioning. This
study explores the impact of Prosthetic Replacement of the Ocular
Surface Ecosystem (PROSE) treatment, utilizing customized scleral
devices, on visual acuity and ocular-related function in cGvHD
patients whose symptoms are refractory to conventional therapy.
Methods: This is a prospective study of 40 patients with ocular
cGvHD who had previously failed conventional therapy and
were referred to the USC Department of Ophthalmology between
November 2009 and July 2013 for PROSE treatment. Ocular
discomfort and visual function were assessed before and after
treatment using the standardized Ocular Surface Disease Index
(OSDI) survey. Visual acuity changes were also assessed through
a retrospective chart review. OSDI and visual acuity changes were
analyzed to assess the impact of PROSE wear on ocular symptoms
and function.
Results: Of the 40 treated patients (79 eyes), 38 (76 eyes) showed
bilateral improved or unchanged visual acuity. On average, logarithm
of the minimal angle of resolution (logMAR) visual acuities
improved 67.6% from 0.49 ± 0.52 to 0.16 ± 0.44, an equivalent
Snellen change of ~20/60 to ~20/30 (Fig. 2). At survey follow-up,
8 patients had expired and 3 stopped wearing their device. Of the
remaining 29 “active wear” patients using the device “all,” “most,” or
“half” of the time, OSDI scores improved an average of 70.9%, from
72.6 ± 20.1 and 21.1 ± 14.9 (Fig. 1).
Conclusions: PROSE therapy can both reduce ocular symptoms
and improve visual acuity in patients with cGvHD and should be
considered for patients refractory to conventional therapy. Even
patients who did not report better visual acuity benefited from
improved ocular comfort.
Figure 1. OSDI Scores pre and post PROSE treatment for “active
wear” patients at follow-up (n=29). Patients showed an average preOSDI Score of 72.6 ± 20.1 (SD) and post- OSDI of 21.1 ± 14.9 (SD)
(p < 0.0001).
Figure 2. Visual Acuity (VA) pre and post treatment represented as
logarithm of the minimal angle of resolution for all patients (n=40).
Patients showed an average pre- VA of 0.49 ± 0.52 (SD) and post- VA
of 0.16 ± 0.44 (SD) (p < 0.0001).
Commercial Relationships: Christos Theophanous, None; Gloria
Chiu, None; John A. Irvine, None
Program Number: 5543 Poster Board Number: A0117
Presentation Time: 8:30 AM–10:15 AM
CULTURED AUTOLOGOUS ORAL MUCOSAL
EPITHELIAL CELL SHEET GRAFTS FOR TREATMENT
OF EXPERIMENTALLY-INDUCED LIMBAL STEM CELL
DEFICIENCY
Fawzia Bardag-Gorce1, Richard H. Hoft3, Joan Oliva Vilana1,
Andrew Wood1, Derek Pan1, Jacquelyn Thropay1, Andrew Makalinao1,
Hiroyuki Sota1, Samuel W. French2, Yutaka Niihara1. 1Hematology,
LA BioMed at Harbor UCLA Med Ctr, Torrance, CA; 2Pathology,
LA BioMed at Harbor UCLA Med Ctr, Torrance, CA; 3SurgeryOphtalmogloy, LA BioMed at Harbor UCLA Med Ctr, Torrance, CA.
Purpose: This pilot study investigates the effects of autologous oral
mucosal cell sheet grafts for experimentally-induced limbal stem cell
deficiency (LSCD) in rabbits
Methods: Severe LSCD was created by superficial surgical excision
of all limbal tissue. Three months later, when rabbits showed stable
corneal vascularization and opacification, oral mucosal biopsies were
©2014, 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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
performed. Autologous oral mucosal cell sheets were engineered
and transplanted onto the surface of the corneas. Follow-up exams
were performed on transplanted cornea (n=5) and sham cornea (n=4),
every 4-6 weeks for 6 months after transplantation, using slit lamp
observation and photography
Results: Three out of five grafted corneas showed a decrease in
superficial vascularization, while almost all the sham corneas did
not show similar decrease. H&E staining showed similar squamous
epithelium in the grafted corneas compared to a normal eye. The
sham corneas showed more attenuated and abnormal epithelium
with numerous areas of thinning, and other areas with erosions and
complete absence of normal squamous epithelium. The sham cornea
also showed very significant infiltration of mononuclear inflammatory
cells. Progenitor stem cell marker deltaNp63 stained markedly in the
grafted cornea and to a lesser extent in the sham cornea. Proliferating
cell nuclear antigen (PCNA) and Ki-67 staining were much greater
in the sham corneas compared to the grafted and normal corneas. K3
and K13 staining results show that oral mucosa epithelial cell sheet
transplantation resulted in a regenerated epithelium in the grafted
corneas compared to the sham corneas. Mu5ac was decreased in the
central region of grafted corneas, compared to sham corneas. Very
little alpha smooth muscle actin staining (a-SMA, which indicates the
presence of myofibroblast cells) was present in grafted corneas, while
there was more a-SMA staining in sham corneas. Staining for antiangiogenic factor TIMP-3 was increased, and pro-angiogenic factor
MMP-3 was decreased in grafted corneas compared to sham corneas.
Conclusions: In conclusion, our results indicate that autologous
oral mucosa epithelial cell sheet grafts resulted in improved
epithelialization of the corneal surface and decreased vascular fibrosis
of the cornea
Commercial Relationships: Fawzia Bardag-Gorce, CellSeed Inc.
Japan (F), Emmaus Medical, Inc (F), Emmaus Medical, Inc (R);
Richard H. Hoft, Emmaus Medical Inc., (R); Joan Oliva Vilana,
CellSeed Inc. (F), Emmaus Medical Inc., (R); Andrew Wood,
Emmaus Medical Inc., (E); Derek Pan, None; Jacquelyn Thropay,
None; Andrew Makalinao, None; Hiroyuki Sota, Emmaus Medical
Inc., (E); Samuel W. French, None; Yutaka Niihara, Emmaus
Medical In., (F)
Program Number: 5544 Poster Board Number: A0118
Presentation Time: 8:30 AM–10:15 AM
Repeatability of Measurements from Fourier-Domain OCT for
Detecting Ectatic Diseases of Corneal Epithelial Thickness
Ana Laura C. Canedo, Bruno F. Valbon, Rosane Correa, Renato
Ambrosio. Cornea & Refrative Surgery, Rio de Janeiro Corneal
Tomography & Biomechanics Study Group, Rio de Janeiro, Brazil.
Purpose: To evaluate the repeatability of corneal epithelial thickness
measurements from Fourier domain optical coherent tomography
(FD-OCT; RTVue, Optovue, Fremont, CA) on normal, forme fruste
keratoconus (FFKC) and keratoconus (KC).
Methods: One eye randomly selected from 10 normal and from
10 keratoconus patients, and from 10 eyes with forme fruste
keratoconus were included. FFKC criteria was the fellow eye with
normal topography from patients with very asymmetric keratoconus.
The minimum, maximum, central, superior and inferior epithelial
thickness values were computed for the central 6mm zone. For each
eye, 5 consecutive measurements were taken by the same observer
using the RTVue. The standard deviation (repeatability [SD]) and
coefficient of variation (CV [SD divided by average]) of the 5
consecutive measurements taken for each parameter.
Results: The repeatability (SD) for the normal, keratocons and FFKC
groups were respectively: 1.19, 2.22 and 0.752 for minimal; 0.742,
1.175 and 1.595 for maximal; 0.639, 1.6 and 1.166 for central; 0.71,
0.879 and 0.846 for superior; 0.627, 1.161 and 0.907 for inferior;
and 0.322, 0.306 and 0.415 for the variation of epithelial values. The
CV for the normal, keratocons and FFKC groups were respectively
(%): 2.838, 5.893 and 1.626 for minimal; 1.295, 1.894 and 2.761 for
maximal; 1.224, 3.199 and 2.245 for central; 1.412, 1.6 and 1.631 for
superior; 1.206, 2.285 and 1.743 for inferior.
Conclusions: OCT RTVue was found to be very precise with less
than 2 microns of repeatability for minimum, maximum, central,
superior and inferior corneal epithelial thickness values on the central
6mm zone. Corneal epithelial measurements should be tested for their
accuracy for detecting ectatic diseases.
Commercial Relationships: Ana Laura C. Canedo, None; Bruno
F. Valbon, None; Rosane Correa, None; Renato Ambrosio, Oculus
(C)
Program Number: 5545 Poster Board Number: A0119
Presentation Time: 8:30 AM–10:15 AM
Vitamin D Activation and TLR2 Activity in Human Corneal
Epithelial Cells
Rose Y. Reins, Hasna Badouri, Alison M. McDermott. College of
Optometry, Univ of Houston, Houston, TX.
Purpose: Vitamin D has been shown to play a role in the wound
healing response, increasing effectors of the innate immune system
important for host protection. Toll-like receptors (TLR), sensors
of the innate immune system, are also activated during wounding,
and can interact with vitamin D signaling. In particular, TLR2
engagement has been demonstrated to enhance vitamin D activation
and antimicrobial peptide production (LL-37) in other tissues. LL-37
itself is able to aid in corneal epithelial wound healing, increasing cell
migration. Therefore, we examined the interaction between vitamin
D and TLR2 signaling, as well as the involvement of the vitamin D
receptor (VDR) in antimicrobial peptide expression.
Methods: Telomerase-immortalized human corneal epithelial cells
(hTCEpi) were stimulated with 1,25D3 (10-7M) or TLR2 agonists
FSL1, Pam3CSK4 (1μg/ml), and zymosan (10μg/ml) alone or
concurrently for 24 hours. RNA was collected from control and
treated cells and expression of CYP27B1, TLR2, and LL-37 were
analyzed by real time PCR. For VDR silencing, 10nM control
or VDR-specific siRNA were transfected into hTCEpi using
Lipofectamine. VDR knock-down was confirmed by RT-PCR and
Western analysis at 24, 48, and 72 hours post-transfection. Cells were
stimulated with 1,25D3 24 hours after transfection.
Results: Stimulation of hTCEpi with TLR2 agonists resulted in a
significant increase (~2.5 fold) of CYP27B1, the vitamin D activating
hydroxylase. In addition, the coordinated response of both 1,25D3 and
TLR2 agonists resulted in increased production of the antimicrobial
peptide, LL-37 (8-12 fold increase), above 1,25D3 stimulation alone
(2.5 fold). 1,25D3 did not modulate TLR2 expression, however. VDR
expression was effectively knocked down with siRNA (77% at 24
hours) and 1,25D3 did not increase LL-37 in these samples, compared
to cells with normal VDR expression.
Conclusions: Activation of hTCEpi through TLR2 ligation resulted
in an increase in CYP27B1, the enzyme responsible for producing the
active form of vitamin D, 1,25D3. Also, vitamin D and TLR2 acted
synergistically to produce LL-37. The vitamin D-mediated expression
of LL-37 was shown to be dependent on VDR, demonstrating
functional activity of the receptor in hTCEpi. These results suggest
that upon TLR2 activation, such as during wound healing, vitamin D
activity can be augmented and can cooperate with TLR2 to enhance
innate immunity and antimicrobial peptide production.
Commercial Relationships: Rose Y. Reins, None; Hasna Badouri,
None; Alison M. McDermott, None
Support: NIH Grant EY13175, NIH Training Grant T32 07024
©2014, 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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
Program Number: 5546 Poster Board Number: A0120
Presentation Time: 8:30 AM–10:15 AM
RNA processing of gamma secretase complex members, Pen2 and
Nicastrin, corneal Epithelia
Stephen P. Sugrue, Daniel Ryu, Qian Peng, Jeong-Hoon Joo.
Anatomy & Cell Biology, University of Florida, Gainesville, FL.
Purpose: It is now evident that RNA processing has a significant
and broad influence over cellular phenotype. The gamma secretase
complex, which contains both Pen2 and Nicastrin, functions through
the proteolytic cleavage of Notch after ligand binding releasing
an intracellular fragment of Notch, which then translocates to the
nucleus and activates transcription factors that impact both cell
differentiation and morphogenesis. Our previous studies showed
that knockdown of PNN promotes inclusion of introns 3 and 15
in presenilin enhancer 2 homolog (Pen-2) and nicastrin (Ncstn)
transcripts respectively and hence premature stops in both transcripts.
Methods: Here we ask, through RT-PCR and fluorescence in situ
hybridization, whether these alternatively spliced mRNA isoforms are
expressed in human in vivo corneal epithelia and cultured epithelial
explants.
Results: We have observed low levels of intron 3-containing PEN-2
and intron 15-containing Ncstn isoforms in human in vivo epithelia
and epithelial explants, suggestive that these isoforms are stabile
in vivo. In addition, in situ hybridizations suggest that Ncstn-(i15)
message may be exported from the nucleus and accumulates in the
cytoplasm in a similar pattern to the WT Ncstn.
Conclusions: Our work demonstrates that normal human corneal
epithelial cells express low levels of splicing isoforms of both PEN-2
and Nicastrin.
Commercial Relationships: Stephen P. Sugrue, None; Daniel Ryu,
None; Qian Peng, None; Jeong-Hoon Joo, None
Support: EY07883
©2014, 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 pubs@arvo.org.