ARVO 2014 Annual Meeting Abstracts
242 Retina: physiology and pharmacology
Monday, May 05, 2014 11:00 AM–12:45 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 1881–1934/B0146–B0199
Organizing Section: Physiology/Pharmacology
Program Number: 1881 Poster Board Number: B0146
Presentation Time: 11:00 AM–12:45 PM
Prediction of Passive Permeability across the Retinal Pigment
Epithelium
Aapo Tervonen1, 2, Iina Vainio1, 2, Soile Nymark1, 2, Jari A. Hyttinen1, 2.
1
Electronics and Communications Engineering, Tampere University
of Technology, Tampere, Finland; 2BioMediTech, Tampere, Finland.
Purpose: Retinal pigment epithelium (RPE) is an important part
of the normal visual cycle. Located behind the retina, one of its
main functions as a part of the blood-retinal barrier is to regulate
the transport between the retina and systemic blood circulation.
The barrier properties, and changes in them, have a role in certain
retinal diseases, such as age-related macular degeneration (AMD).
Previously, mostly pharmacokinetic compartmental models have
been proposed to model the RPE barrier properties. In this study,
we introduce for the first time, an accurate physical structure-based
model of passive permeability across the RPE.
Methods: Our model relates the permeability coefficients of RPE
structures to the physicochemical properties of materials forming
the RPE. Our model structure bases on a corneal diffusion model by
Edwards, A. et al. (Pharm Res 18: 1497–508, 2001). Transcellular
and paracellular diffusion components are described by separate
permeability equations based on the material properties of each
pathway and the basic interactions between each of them and
the characteristics of the diffusing molecule, such as radius and
lipophilicity. Transcellular pathway is further divided into pathways
traversing the cell cytoplasm and diffusing laterally within the cell
membrane. The improved structure of our tight junction (TJ) model
takes into account both the pore pathway for small molecules and the
leak pathway for large molecules.
Results: Our RPE model is able to predict correct magnitude for
the molecular permeabilities and its behaviour corresponds to
experimental results. The results show that the paracellular pathway
is the dominant pathway, the transcellular pathway becoming
more permeable with small and lipophilic molecules. Further, the
permeability magnitude and behaviour of the TJ model appear similar
to the experimental data of molecules mainly traversing through the
TJs.
Conclusions: RPE barrier models would facilitate novel drug
development against retinal diseases. Our model forms, to our
knowledge, the mot advanced platform for development and
refinements of diffusional models of RPE and it can be used e.g. to
study the pathogenesis of AMD. Further, our model combines our
knowledge of the RPE structure and permeability. However, due to
the inconsistent experimental data of RPE permeability, rigorous
validation of this type of computational models cannot be made.
Commercial Relationships: Aapo Tervonen, None; Iina Vainio,
None; Soile Nymark, None; Jari A. Hyttinen, None
Support: TUT graduate school
Program Number: 1882 Poster Board Number: B0147
Presentation Time: 11:00 AM–12:45 PM
The role of purinergic P2X receptors and Ca++-dependent
chloride channels in ion transport of mouse retinal pigment
epithelium
Sighvatur S. Arnason, Sunna B. Skarphedinsdottir, Thor Eysteinsson.
Physiology, University of Iceland, Reykjavik, Iceland.
Purpose: The retinal pigment epithelium (RPE) is important for
normal function of the retina, one of which is to transport water and
Cl- from the subretinal space to the choroid, across the RPE cells.
It has been suggested that apical P2X purinergic receptors (P2XR)
transduce second messenger signals, such as Ca++, into stimulation
of Cl- transport across the RPE. The purpose of this study was to
assess the function of P2X receptors and Ca++-dependent chloride
channels (CaCC) in the mouse RPE.
Methods: Healthy mice (C57BL/6J) where euthanized and the RPE
together with the retina, choroid and sclera was mounted in special
miniature epithelial Ussing chambers with an aperture of 0.031 cm2
(EasyMount, Physiological Instruments) with normal Krebs on both
sides kept at 38°C and aired with 5%CO2-95%O2. The tissue was
voltage clamped to zero (WPI) to measure the short-circuit current
(Isc) as an indicator of net ion transport. Every 4th minute a 1 mV
pulse was passed to estimate the transepithelial resistance (TER).
The number of mice was 4-6 in each experiment series. The results
are presented as mean +/- SEM. Statistical significance was tested
by a paired t-test. The Ca++ dependent chloride channel blocker
CaCCA01 (Tocris) was added to both sides of the RPE at two
different doses, 0.58 mM and 1.15 mM, each tested over a 30 minute
period. The P2XR agonist β,γ-Met ATP (Sigma) followed by the
P2XR antagonist PPADS (Tocris) where added to the apical side of
the RPE and tested over a 30 minute period each.
Results: CaCCA01 applied apically to the mouse RPE caused a
significant decrease and reversal in the Isc, from -8.2+/- 2.4 to 4.7
+/- 0.9 mAmp/cm2 at the higher dose, 1.15 mM (p < 0.05), and an
increase in the TER (53 +/- 8 to 76 +/- 10 Ohm*cm2 (p < 0.05; n=4).
Neither β,γ-Met ATP (1 mM) nor PPADS (0.5 mM) induced any
change in the initial Isc of -10.5 +/- 2.1 mAmp/cm2, but both induced
a small but significant change in the TER, from 47 +/- 6 to 55 +/- 9
and 58 +/- 9 Ohm*cm2, respectively (p < 0.05; n=6).
Conclusions: The results indicate that the CaCC on the apical side
are open in the short-circuit state and drive a part of the ion transport
in the mouse RPE. Our experiments also suggest that CaCC are
situated on the apical side. On the other hand, P2XR on the apical
side do not mediate ion transport across the RPE.
Commercial Relationships: Sighvatur S. Arnason, None; Sunna
B. Skarphedinsdottir, None; Thor Eysteinsson, None
Support: Icelandic Research Council, Helga Jonsdottir and Sigurlidi
Kristjansson Memorial Fund, University of Iceland Research Fund
Program Number: 1883 Poster Board Number: B0148
Presentation Time: 11:00 AM–12:45 PM
Prolactin contributes to the regulation of retinal pigment
epithelial cell survival and monolayer resistance
Stephanie Thebault1, Edith Arnold1, Rodrigo D. Meléndez
García1, David Arredondo1, German D. Baeza-Cruz1, Juan D.
Riesgo-Escovar1, Vincent Goffin2, Carmen Clapp1. 1Instituto de
Neurobiologia, Univ Nacional Autonoma de Mexico, Queretaro,
Mexico; 2INSERM Unit 845, Research Center Growth and Signaling,
University Paris Descartes, Paris, France.
Purpose: We recently showed that the hormone prolactin (PRL)
acts as an endogenous retinal trophic factor able to limit retinal
degeneration. In addition to regulating glial-neuronal cell interactions
in the retina, we postulated thatPRL targets retinal pigment epithelial
(RPE) cells.
Methods: The presence of PRL receptor in the RPE of male albino
rats was determined by in situ hybridization and immunochemistry,
and in human ARPE-19 cell cultures by Western blot. The viability
of ARPE-19 cell cultures was examined by MTT assay, and ARPE19 cell monolayers were evaluated for transepithelial electrical
©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
resistance. A competitive PRL receptor antagonist was used to ensure
the specificity of recombinant human PRL actions.
Results: RPE in transverse sections of rat retinas was positive for
PRL receptor mRNA and protein. PRL receptor was also detected in
flat mounts of rat RPE and in ARPE-19 cell monolayers. A 48-hour
PRL treatment increased the viability of ARPE-19 cell cultures in a
dose-dependent manner, with a maximal effect dose of 100 pM (2.3
ng/ml). The PRL receptor antagonist increasingly reduced ARPE19 cell viability at 10 nM, 100 nM and 1 mM. The effect of 10 nM
PRL receptor antagonist was prevented by adding 100 pM rhPRL.
Moreover, PRL induced a transient increase in the resistance of
ARPE-19 monolayers that peaked at 30 minutes.
Conclusions: These findings show that the PRL receptor is present
in the RPE, and that the stimulation of PRL signaling may help to
regulate RPE permeability and survival.
Commercial Relationships: Stephanie Thebault, None; Edith
Arnold, None; Rodrigo D. Meléndez García, None; David
Arredondo, None; German D. Baeza-Cruz, None; Juan D. RiesgoEscovar, None; Vincent Goffin, None; Carmen Clapp, None
Support: CONACYT grant 176393 (S.T.)
Program Number: 1884 Poster Board Number: B0149
Presentation Time: 11:00 AM–12:45 PM
Iron Rescue of Deferoxamine Toxicity in human RPE cells
John B. Miller1, 2, Haijiang Lin1, 2, Peggy Bouzika1, 2, Alp Atik1, 2,
Yueran Yan1, 2, Yijun Hu1, 2, Joan W. Miller1, 2, Demetrios G. Vavvas1,
2 1
. Angiogenesis Laboratory, Harvard Department of Ophthalmology,
Boston, MA; 2Massachusetts Eye and Ear Infirmary, Boston, MA.
Purpose: Deferoxamine (DFX) is an iron chelator commonly used
to treat iron overload in patients requiring regular blood transfusions.
Several ocular complications have been reported, while in vitro
studies have confirmed its toxicity in hepatic, cortical brain cells and
bovine RPE cells. Our study is the first to examine DFX’s toxicity in
an established human RPE cell line (ARPE19) and whether apoptosis,
necrosis, or the chelation of minerals plays a role in toxicity.
Methods: ARPE19 cells were cultured on 96 well plates. MTT
assays and TUNEL staining were used to assess cell survival. To
investigate mechanisms of cell death, inhibitors of necroptosis (Nec1) and apoptosis (z-Vad) were added to DFX treated cells. Additional
plates were incubated with ZnCl2 or FeCl with and without DFX.
Results: DFX toxicity to ARPE19 cells was confirmed by an MTT
assay, and verified by TUNEL staining. No toxicity was observed at
24 hours, but decreased cell survival began at day 2 and progressed
through day 5. There was no significant difference in cell survival
between 0.2, 0.4, and 0.8 mg/mL of DFX. While investigating the
role of necroptosis and apoptosis in DFX-induced toxicity, we found
that neither Nec-1 nor Z-Vad improved cell survival at days 2 and 3.
We then focused on whether the chelation of minerals could reduce
DFX’s toxicity, but first checked for direct toxicity of iron and zinc.
Zinc alone showed a dose dependent response with high doses (0.04,
0.08, and 0.16M) demonstrating significant toxicity while lower
doses (10, 20, 40 uM) had no effect on cell survival. Iron alone
showed no toxicity at concentrations of 0.05, 0.1 and 0.2 M. Rescue
of DFX toxicity was then tested by adding zinc and iron to 0.4 mg/
mL DFX treated RPE cells. The addition of zinc did not improve cell
survival. However, iron was able to rescue DFX toxicity when used
at ratios of 1:1 and 2:1 (Fe:DFX). A favorable effect was also noted
when iron was premixed with DFX two hours before adding to cell
cultures. However, a two day delay in the addition of iron to DFX
treated cells was unable to prevent toxicity.
Conclusions: We are the first to confirm DFX toxicity in a human
RPE cell line. DFX toxicity appears to be time dependent. We found
no role for necroptosis or apoptosis in cell death. However, chelation
with iron at specific concentrations rescues RPE cells from DFX
toxicity.
Commercial Relationships: John B. Miller, None; Haijiang Lin,
None; Peggy Bouzika, None; Alp Atik, None; Yueran Yan, None;
Yijun Hu, None; Joan W. Miller, None; Demetrios G. Vavvas,
None
Support: Heed Ophthalmic Foundation
Program Number: 1885 Poster Board Number: B0150
Presentation Time: 11:00 AM–12:45 PM
PRESSURE TRIGGERS RETINAL SPREADING
DEPRESSION AND ITS BLOCKAGE BY BRIMONIDINE
TATRATE
Vinicius V. Oliveira, Adalmir M. Dantas, Marcio M. Rodrigues.
Federal Univ of Rio de Janeiro, Rio de Janeiro, Brazil.
Purpose: Spread depression (SD) was originally on retina by Gouras
(1958). SD is widely associated with neuronal damage. While it
spreads through retina, electrical and intrinsic optical signs can be
measured. The main purposes of this paper are: (1) demonstrate if
hydrostatic pressure can trigger SD and (2) to identify if brimonidine
tartarate (BT) is able to reduce, block, or even do not let start the
spreading phenomena.
Methods: We performed 25 experiments on fragments of retinal
preparations of White Leghorn chicks from 3 to 8 days after hatching.
Immediately after decapitation the eyeballs were removed and
sectioned along the equator. Fragments of retina were transferred to
a closed chamber and infused with modified Ringer solution (RS)
driven by a peristaltic pump in order to maintain the RS flowing at
a rate from 0.8 to 0.85 ml/min. The presence or absence of SD was
detected by recording its concomitant slow voltage variations.
The retina was infused with RS and the pressure inside the chamber
was kept at atmospheric pressure. After 15 minutes pressure was
raised to 20 mmHg. Then after 15 min another increase of 20 mmHg
was made, at a total of 40 mmHg. The same protocol was repeated
with fresh retinal tissue infusing RS + BT at 0.2%. Graphs and
statistical analysis were made with GraphPad Prism 5.03, using t-test
of Student.
Results: Our results demonstrate that pressure is capable of trigger
SD. There was no significant difference between SD voltage elicited
by 20 or 40 mmHg. We also observed that BT was able to prevent
SD.
Conclusions: SD has been associated to many central nervous
disorders as stroke, intracranial hypertension and trauma. On this
context, to evaluate if retina is susceptible to the same kind of injury
permits novel kind of understanding of the phenomenon. As it occurs
on glaucoma, high pressure applied to retinal tissue damage it as it
can trigger SD (a lesion wave).
Although the main BT pathway action is still not well known, it is
clear its powerful effect. The recent association of SD with many
traumatic disorders of the central nervous tissue leads us to wonder
how BT blocks SD, possibly acting as a neuroprotector drug. Its
possible acting pathway a Gi-protein mediated response, down
regulating AMPc is a strong possibility. Further studies are necessary
trying to explain the pathophysiology of the experiment.
Commercial Relationships: Vinicius V. Oliveira, None; Adalmir
M. Dantas, None; Marcio M. Rodrigues, 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: 1886 Poster Board Number: B0151
Presentation Time: 11:00 AM–12:45 PM
Comparative inhibition of excitatory neurotransmission by
N-Acetylcysteine and L-cysteine in bovine isolated retina
Catherine A. Opere1, Pratik S. Bankhele1, Ankita A. Salvi1, Jamal
Jamil1, Dan Munt1, Ya Fatou Njie-Mbye2, Madhura Chitnis2, Sunny
E. Ohia2. 1Pharmacy Sciences, Creighton University, Omaha, NE;
2
Department of Pharmaceutical Sciences, College of Pharmacy and
Health sciences, Texas Southern University, Houston, TX.
Purpose: We have evidence that L-cysteine, the substrate for
biosynthesis of hydrogen sulfide (H2S) can regulate potassium (K+)evoked glutamate release from bovine isolated retina. However, the
effect of the precursor for L-cysteine, N-acetyl cysteine (NAC) on
excitatory neurotransmission has not been fully elucidated. In the
present study, we compared the mechanisms by which L-cysteine
and NAC regulate K+-evoked [3H] D-aspartate release and glutamateinduced neurotoxicity in bovine retina.
Methods: Isolated neural retina were incubated in oxygenated Krebs
solution containing 200 nM of [3H] D-aspartate and then prepared
for studies of neurotransmitter release. The MTT assay was used to
assess retinal neuron survival.
Results: Both L-cysteine (0.1 mM to 10 mM) and NAC (10 mM
to 1 mM) attenuated K+-induced [3H] D-aspartate release in a
concentration-dependent manner. At an equimolar concentration
of 10 mM, L-cysteine and NAC inhibited evoked neurotransmitter
release by 54.3% (p < 0.001) and 8.3%, respectively. Whereas, the
CBS inhibitor, aminooxyacetic acid (AOA; 3mM) and the KATP
channel blocker,glibenclamide (300 mM) had no effect on K+-induced
[3H] D-aspartate release, they completely reversed the inhibitory
effect of L-cysteine (1 mM to 10 mM). Interestingly, glibenclamide
had no effect on NAC-induced inhibition of K+-induced [3H]
D-aspartate release while AOA partially reversed this response. Both
L-cysteine (1 mM) and NAC (1 mM) partially reversed glutamate (12
mM)-induced neuron degeneration by 31.1% (p<0.05) and 18.4%,
respectively.
Conclusions: Both L-cysteine and NAC regulate excitatory
neurotransmission in bovine retina by separate mechanisms.
Commercial Relationships: Catherine A. Opere, None; Pratik S.
Bankhele, None; Ankita A. Salvi, None; Jamal Jamil, None; Dan
Munt, None; Ya Fatou Njie-Mbye, None; Madhura Chitnis, None;
Sunny E. Ohia, None
Program Number: 1887 Poster Board Number: B0152
Presentation Time: 11:00 AM–12:45 PM
Effects of sildenafil on primate retinas
Thuy Doan1, Felice Dunn2, Fred Rieke3, 4. 1Department of
Ophthalmology, University of Washington, Seattle, WA; 2Department
of Biological Structure, University of Washington, Seattle,
WA; 3Department of Physiology and Biophysics, University of
Washington, Seattle, WA; 4Howard Hughes Medical Institute, Seattle,
WA.
Purpose: Phosphodiesterase type-5 inhibitors (PDE5), such as
sildenafil, are widely used for the treatments of erectile dysfunction
and arterial pulmonary hypertension. It has been documented
that ingestion of PDE5 inhibitors can lead to visual disturbances,
attributed to the nonspecific inhibition of PDE6 in rod and cone
photoreceptors. However, human retinas have been shown to
express PDE5 in the bipolar cell layer and inhibition at this site may
contribute to the visual disturbances with sildenafil intake. In this
study, we sought to quantify the direct effects of sildenafil on the
electroretinogram (ERG) responses in primate retinas.
Methods: Primate retinas (Macaca fascilaris, Macaca nemestrina,
and Macaca mulatta) were obtained through the Tissue Distribution
Program of the Regional Primate Research Center and in accordance
with the Institutional Animal Care and Use Committee at the
University of Washington. In-vitro ERGs (Azevedo and Rieke,
2011) were used to determine the changes in the transretinal
potential in response to light stimulation under application of various
concentrations of sildenafil and drug washout conditions.
Results: Application of sildenafil reduced the amplitudes of the
ERG a- and b-waves. The a- and b-wave implicit times also were
prolonged in the presence of sildenafil. Sildenafil washout led to the
partial recovery of both the a- and b-wave amplitudes and implicit
times.
Conclusions: We found that sildenafil reduced the amplitudes
and prolonged the kinetics of the a- and b-wave ERG responses.
Responses were partially reversible for the duration of our recordings.
Our experiments also demonstrate that in vitro ERG recordings
in primate retinas may be an effective and useful technique for
characterizing the effects of retinal drug toxicity in humans.
The first two authors contributed equally.
Commercial Relationships: Thuy Doan, None; Felice Dunn, None;
Fred Rieke, None
Program Number: 1888 Poster Board Number: B0153
Presentation Time: 11:00 AM–12:45 PM
The effects of stimulating and blocking the retinal A2A and A3
adenosine receptors on the components of the rat ERG
Gudmundur Jonsson, Thor Eysteinsson. Physiology, University of
Iceland, Reykjavik, Iceland.
Purpose: Adenosine is a neuromodulator that is present in the retina.
It has been suggested that adenosine may serve a neuroprotective role
in the retina, based on electroretinogram (ERG) recordings from the
rat retina. The purpose of this study was to assess the role of A2A
and A3 adenosine receptors, known to be present in the rat retina, in
generation and modulation of the rat ERG.
Methods: Sprague Dawley rats were anesthetized by an
intraperitoneal injection of S-ketamine (75mg/kg) and xylazine (6mg/
kg). The flash ERG was recorded between an electrode placed on the
cornea and a reference electrode on the lower canthus. Agonists and
antagonists for A2A receptors, and for A3 receptors, and adenosine
were each injected (5 mL) into the vitreous of six eyes with a NanoFil
IOKit system (WPI, Inc, USA). Their effects on the components of
the scotopic and photopic ERGs were examined, along with ERG
flicker responses.
Results: Adenosine [0.5 mM] caused an increase in the mean
amplitude of the scotopic ERG a-wave from 68.0 + 7.7 mV to 96.7
+ 13.7 mV (p=0.042). It also increased the mean amplitude of the
scotopic b-wave from 236.5 + 38.4 mV to 305.3 + 41.6 mV (p=0.035).
The A2A agonist CGS21680 [2mM] decreased the mean amplitude
of both the ERG b-wave of dark adapted (298.2 + 21.5 mV to 212.5
+ 19.3 mV; p=0.005) and light adapted eyes (124.3 + 17.7 mV to
87.8 + 11.2 mV; p=0.045). The mean scotopic oscillatory potentials
(OPs) were decreased by CGS21680 (99.9 + 9.4 mV to 47.2 + 11.4
mV; p=0.023). ZM241385 [4mM], an A2A antagonist did not have
any effect on any component of the ERG. The A3 agonist 2-CI-IBMECA [0.5mM] increased the mean amplitude of the a-wave (91.4
+ 15.4 mV to 152.9 + 21.2 mV; p=0.006), but decreased in the mean
amplitude of the b-waves of both dark adapted (290.9 + 40.3 mV to
210 + 21.4 mV; p=0.022) and light adapted (170 + 19 mV to 135.9
+ 11.4 mV; p=0.037) eyes. The scotopic OPs decreased in mean
amplitude (79.6 + 15.2 mV to 39.2 + 3.9 mV; p=0.038) after 2-Cl-IBMECA. The A3 antagonist VUF5574 increased the mean amplitude
of both the a-wave (65.8 + 8.0 mV to 139.5 + 29.3 mV; p=0.046) and
the b-wave of dark adapted eyes (223.7 + 20.3 mV to 312 mV + 38.7
©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
mV; p=0.037). None of the ligands tested had any effect on the ERG
flicker response.
Conclusions: Retinal neurons that contain A2A and/or A3 adenosine
receptors contribute to the generation of the ERG a- and b-waves and
OPs.
Commercial Relationships: Gudmundur Jonsson, None; Thor
Eysteinsson, None
Support: University of Iceland Research Fund
Program Number: 1889 Poster Board Number: B0154
Presentation Time: 11:00 AM–12:45 PM
Visual Responses in the Organotypically Cultured Mouse Retina
Daniel L. Rathbun1, 2, Ayse Sahaboglu1, Blanca Arango-Gonzalez1,
Eberhart Zrenner1, 2, Francois Paquet-Durand1. 1Institute for
Ophthalmic Research, University of Tuebingen, Tuebingen,
Germany; 2Centre for Integrative Neuroscience, University of
Tuebingen, Tuebingen, Germany.
Purpose: We investigated the appropriateness of the organotypic
retinal preparation for in vitro visual electrophysiology measurements
during early development of the mouse retina in comparison with
acutely dissected retinas.
Methods: Wild-type (C3H) mouse retinas with attached retinal
pigment epithelium were harvested at either postnatal day 5 (P5)
or P9 and maintained in culture up to P14. Retinal ganglion cell
(RGC) spiking responses to a full-field flashing visual stimulus were
recorded either from cultured or from age-matched, acutely-dissected
control retinas at P11, P12 and P14 using a multielectrode array.
Histological assessments of RGC viability were performed in parallel
using the TUNEL assay and BRN3A immunostaining.
Results: Retinas acutely prepared at P26 with the present methods
demonstrated ON and OFF response latencies, amplitudes and
durations similar to previous reports [Carcieri et al. J. Neurophysiol.
90:1704-13, 2003]. While latency and duration decreased from P11
to P26 in acute retina, amplitude increased over this time. At P11 and
P12 visual responses from cultured retina were more robust when
retinas were cultured at P9 vs. P5 – reflecting the observation that
RGC responsiveness degrades quickly in culture, possibly because
RGCs are axotomized in preparation. Responses from retinas cultured
at P9 and recorded at P11, P12 and P14 had longer latencies, shorter
durations, and - at most ages - lower amplitudes relative to acutely
prepared control retina. Response degradation correlated with the
histologically observed loss of RGCs, an effect that grew stronger
with time in culture. Neither exogenous brain-derived neurotrophic
factor (BDNF) nor retention of the optic nerve slowed degradation in
cultured responses.
Conclusions: For in vitro visual electrophysiology measurements, the
organotypic culture protocol provides only a limited methodological
basis for comparison of visual responses in healthy and degenerate
retinas. Although the number of RGCs numbers and their visual
responses decrease quickly in culture, baseline measurements have
been established for further investigation of RGC neuroprotection in
the organotypic retinal preparation.
Commercial Relationships: Daniel L. Rathbun, None; Ayse
Sahaboglu, None; Blanca Arango-Gonzalez, None; Eberhart
Zrenner, Retina Implant AG (F), Retina Implant AG (I), Retina
Implant AG (P), Retina Implant AG (R), Retina Implant AG (S);
Francois Paquet-Durand, None
Support: BMBF FKZ: 01GQ1002; DFG EXC307; BMBF HOPE2
FKZ: 01GM1108A; Kerstan Foundation
Program Number: 1890 Poster Board Number: B0155
Presentation Time: 11:00 AM–12:45 PM
HDAC Inhibition in Human Organotypic Retinal Cultures
(HORCs) protects against loss of THY-1 mRNA
Julie Sanderson1, Marina Hopes1, 2, David C. Broadway2, 1. 1School
of Pharmacy, University of East Anglia, Norwich, United Kingdom;
2
Department of Ophthalmology, Norfolk and Norwich University
Hospital, Norwich, United Kingdom.
Purpose: Histone deacetylase (HDAC) inhibitors have been
associated with potential neuroprotective properties in relation to
glaucoma. The purpose of these experiments was to investigate the
effect of the HDAC inhibitor trichostatin A (TSA) on gene expression
in human organotypic retinal cultures (HORCs).
Methods: Donor eyes were obtained from the East Anglian Eye Bank
within 24 hours post mortem. 4mm diameter paramacular explants
were dissected from the retina and cultured in serum free DMEM/
Ham F12 medium for 48 hours in the presence or absence of TSA
(0.1, 1 or 10mM). LDH release was used to assess cell death. Total
RNA levels were assessed by spectrophotometric analysis. Gene
expression was assessed by QRT-PCR.
Results: There was no significant change in LDH release from the
HORCs following 48 hours’ exposure to 0.1 - 10mM TSA indicating
that the HDAC inhibitor was not causing toxicity (n=4). During the
culture period, there was an approximate 60% decrease in total RNA
(n=4). TSA led to a significant amelioration of loss of total RNA
(approximately 25% at 10μM; n=4). Analysis of the expression of
two housekeeping genes (cytochrome c-1; CYC1 and topoisomerase
1; TOP1) showed no significant change in expression in TSA-treated
retina compared with control. The expression of three markers for
retinal neurons was also assessed: Thy-1 (THY-1) for retinal ganglion
cells (RGCs); recoverin (RCVRN) for photoreceptors and calbindin
(CALB) for horizontal cells. There was no significant change in
expression of CALB in HORCs treated with TSA compared with
control (n=4). RCVRN showed a significant increase in expression of
approximately 20% (n=4) and THY-1 an approximate 80% increase
(n=4) in expression in HORCs treated with 10mM TSA compared
with control at the 48 hour time point.
Conclusions: HDAC inhibition with TSA differentially inhibited
loss of gene expression in the cultured human retina. Of the genes
investigated, the RGC marker THY-1 showed the greatest protection.
Loss of normal gene expression has been shown to be an early
event in animal models of glaucoma. The current data may support
a potential neuroprotective role for HDAC inhibitors in relation to
glaucoma.
Commercial Relationships: Julie Sanderson, None; Marina
Hopes, None; David C. Broadway, None
Support: The Humane Research Trust and The Norwich Glaucoma
Research Fund
Program Number: 1891 Poster Board Number: B0156
Presentation Time: 11:00 AM–12:45 PM
Protected Retinal Function by Sulforaphane on Retinal Ischemic
Injury
Lindsay Ambrecht, James F. McDonnell, Jay I. Perlman, Ping Bu.
Loyola Hospital, Maywood, IL.
Purpose: Retinal ischemia is a major contributor to vision loss in
multiple diseases including acute angle-closure glaucoma, primary
open angle glaucoma, diabetic retinopathy, and retinal vascular
occlusions. The increase in oxidative stress is widely believed to
play an important role in retinal ischemic injury. Sulforaphane,
an isothiocyanate, and precursor of glucosinolate in cruciferous
vegetables such as broccoli, has demonstrated neuroprotective
effects in several experimental paradigms. It has been shown to
©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
protect neural cells in cell culture and animal models after stroke.
In this study, we propose to determine the neuroprotective effects
of sulforaphane on retinal ischemia in vivo, using a mouse model
of ischemic-reperfusion injury in mice as quantified functionally.
By quantifying relative changes in electrophysiology (ERG) (retinal
function), the neuroprotective effects of sulforaphane are determined.
Methods: Two groups of C56BL/6 wild type mice (6-8 weeks old)
(n= 8 per group) were used for this study. The retinal ischemicreperfusion injury was induced by elevation of intraocular
pressure for 45 minutes. Following ischemic insult, vehicle (1%
DMSO saline) or sulforaphane (25mg/kg/day) was administrated
intraperitoneally once per day for 5 days. Retinal function was
quantified by recording scotopic ERGs in dark-adapted mice prior to
and one-week following ischemic insult.
Results: Scotopic ERG a- and b-wave amplitudes prior to ischemic
injury were 408 ± 82 mV and 856 ± 146 mV, respectively. Following
ischemic-reperfusion injury, scotopic ERG a- and b-wave amplitudes
of vehicle-treated mice were 152 ± 50 mV and 332 ± 87 mV,
respectively. By comparison, ERG a- and b-wave responses from
sulforaphane-treated mice were 306 ± 73 mV and 664 ± 123 mV,
respectively.
Conclusions: Intraocular ischemic-reperfusion insult elicits
marked deficits in retinal function as quantified by scotopic ERG.
Administration of sulforaphane protects against ischemic-reperfusion
dependent deficits in retinal function. These preliminary findings
suggest that sulforaphane may have therapeutic value in the early
treatment of retinal ischemic diseases.
Commercial Relationships: Lindsay Ambrecht, None; James F.
McDonnell, None; Jay I. Perlman, None; Ping Bu, None
Support: The Richard A. Perritt Charitable Foundation, Illinois
Society for the Prevention of Blindness, and American Society of
Cataract and Refractive Surgery.
Program Number: 1892 Poster Board Number: B0157
Presentation Time: 11:00 AM–12:45 PM
Progesterone treatment shows greater protection in brain vs.
retina in a rat model of middle cerebral artery occlusion
Rachael S. Allen2, 3, Iqbal Sayeed1, Yuliya Oumarbaeva1, Katherine
Morrison1, Irina Lucaciu1, Heather Cale1, Paul H. Choi1, Jeffrey H.
Boatright2, Machelle T. Pardue2, 3, Donald G. Stein1. 1Emergency
Medicine, Emory University School of Med, Atlanta, GA;
2
Ophthalmology, Emory University School of Med, Atlanta, GA;
3
Rehab R&D Center of Excellence, Atlanta VA Medical Center,
Decatur, GA.
Purpose: To determine whether progesterone, which has been shown
to reduce inflammation and infarct size in the brain and improve
behavioral outcomes after middle cerebral artery occlusion (MCAO),
reduces inflammation and improves electroretinogram (ERG)
responses in the retina after MCAO.
Methods: MCAO surgery was performed on male Sprague-Dawley
rats. Progesterone or vehicle was given systemically at 1, 6, 24, and
48 h post-MCAO. Grip-strength and sticky tape task were performed
at 1-day post-MCAO and ERGs assessed at 2 days post-MCAO.
Brains and retinas were taken for histology at 3 days post-MCAO. In
another set of rats, protein levels of cytosolic NF-κB, nuclear NF-κB,
phosphorylated NF-κB, IL-6, TNF-α, CD11b, progesterone receptor
A and B, and pregnane X receptor were assessed in retinas and brains
at 24 and 48 h post-MCAO (n = 5/group).
Results: Following MCAO, vehicle-treated rats showed significant
deficits in behavioral tests, while progesterone-treated rats showed
significant improvements (71-84% recovery). Vehicle-treated rats
also showed significant reductions in ERG amplitude in ipsilateral
eyes post-MCAO, while progesterone-treated rats showed a trend
for increased ERG amplitudes (23% recovery). Contralateral eyes
from vehicle-treated rats also showed significant reductions in ERG
amplitude post-MCAO, while contralateral eyes from progesteronetreated rats showed significant increases (64% recovery). After
MCAO, vehicle-treated rats exhibited increased levels of pNF-κB,
nuclear NF-κB, IL-6, TNF-α, and CD11b and decreased levels of
cytosolic NF-κB in both brain and retina. Progesterone treatment in
MCAO rats significantly attenuated levels of nuclear NF-κB and IL-6
in both brain and retina, while levels of cytosolic NF-κB showed
significant increases. Progesterone treatment significantly attenuated
levels of pNF-κB, TNF-α, and CD11b after MCAO in brain, with
smaller reductions in retina. After MCAO, progesterone receptor A
and B were upregulated in brain and downregulated in retina.
Conclusions: While progesterone treatment reduced inflammation in
both brain and retina, protective effects were more dramatic in brain.
Progesterone treatment also resulted in greater improvements in
brain function-based behavioral tasks than in the retina-based ERG.
This differential effect may be due to differences in expression of
progesterone receptors in brain and retina after injury.
Commercial Relationships: Rachael S. Allen, None; Iqbal Sayeed,
None; Yuliya Oumarbaeva, None; Katherine Morrison, None;
Irina Lucaciu, None; Heather Cale, None; Paul H. Choi, None;
Jeffrey H. Boatright, None; Machelle T. Pardue, None; Donald G.
Stein, BHR Pharma (C), BHR Pharma (F), BHR Pharma (R), Emory
University (P)
Support: This material is based upon work supported by a generous
gift from H. Allen and Company, Atlanta VA Rehab R&D Service of
the Department of Veterans Affairs, The Abraham J. and Phyllis Katz
Foundation, Foundation Fighting Blindness, Research to Prevent
Blindness, NIH NEI R01EY014026, R01EY016470, R24EY017045,
P30EY006360, and T32EY007092-25.
Program Number: 1893 Poster Board Number: B0158
Presentation Time: 11:00 AM–12:45 PM
The Role Of Myd88 And Trif Signaling-Mediated Inflammation
In Ischemia-Reperfusion-Induced Retinal Damage
Galina Dvoriantchikova1, Andrea Rachelle Santos1, Xenia
Dvoriantchikova1, Dmitry V. Ivanov1, 2. 1Bascom Palmer Eye
Institute, Department of Ophthalmology, University of Miami Miller
School of Medicine, Miami, FL; 2Department of Microbiology
and Immunology, University of Miami Miller School of Medicine,
Miami, FL.
Purpose: Toll-like receptors have been demonstrated to play an
important role in ischemia-reperfusion (IR)-induced innate immune
response in the central nerve system (CNS). At the same time, the
role of two toll-like receptor signaling cascades, Myd88- and Trifdependent, in IR injury was poorly understood. We performed this
study to clarify the role of these pathways in IR induced inflammation
and neuronal damage.
Methods: Myd88- and Trif-deficient animals and C57BL/6J mice
as the wild type control were obtained from the Jackson Laboratory.
Retinal IR injury was induced by unilateral elevation of intraocular
pressure for 45 minutes by direct corneal cannulation. The changes
in expression of toll-like receptor family members, Myd88, Trif,
as well as pro-inflammatory genes 6 hours postreperfusion were
assessed by quantitative RT-PCR. Ganglion cell layer (GCL) neurons,
astrocytes and microglial cells were identified in flat-mounted retinas
by immunohistochemistry using cell type specific markers NeuN,
Gfap and Cd11b respectively. Cell death was evaluated by the direct
counting of neurons in the GCL of flat-mounted retinas seven days
postreperfusion.
Results: We found that while expression levels of Tlr1, Tlr2 and
Myd88 were increased in ischemic retinas, transcription levels of
©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
Tlr3, Tlr4 and Tlr9 were reduced in ischemic retinas after 6 hours. At
the same time, we found no significant differences in the level of Tlr7
and Trif expression between ischemic and sham-operated retinas.
The mice that lacked Trif showed significantly reduced expression of
pro-inflammatory genes 6 hours after reperfusion and significantly
increased survival of GCL neurons 7 days after IR. At the same
time, while Myd88 deficient animals had an even lower level of
inflammation in ischemic tissue compared to the mice that lacked
Trif, the levels of damage in ischemic tissue of Myd88 deficient
animals varied considerably.
Conclusions: Our findings suggest that while Trif signaling promotes
neurotoxic inflammation in ischemic tissue, Myd88 signaling cascade
may play a more complex role in IR injury. Thus, the design of
effective therapy for patients suffering from IR injury should be
based on a clear delineation of the beneficial and detrimental effects
of toll-like receptor signaling mediated inflammation in ischemic
tissue.
Commercial Relationships: Galina Dvoriantchikova, None;
Andrea Rachelle Santos, None; Xenia Dvoriantchikova, None;
Dmitry V. Ivanov, None
Support: NIH grant R01EY022348, Bridge Grant 3KB01, NIH
center grant P30 EY014801
Program Number: 1894 Poster Board Number: B0159
Presentation Time: 11:00 AM–12:45 PM
CD4+T Cell Responses Contribute To Progressive
Neurodegeneration In Ischemic Neuropathy
T H Khanh Vu1, 2, Huihui Chen3, Djoeke Doesburg1, 2, Kin-Sang Cho1,
Martine Jager2, Dongfeng F. Chen1. 1Ophthalmology, Schepens Eye
Research Institute/Massachusetts Eye and Ear Infirmary, Boston,
MA; 2Ophthalmology, Leiden University Medical Center, Leiden,
Netherlands; 3Ophthalmology, the Second Xiangya Hospital of
Central South University and Institution of Ophthalmic Center,
Changsha, China.
Purpose: Retinal ischemia is a common cause of dysfunction
or death of retinal ganglion cells (RGCs) leading to progressive
visual loss and blindness. In this study we aimed to have a better
understanding of the pathophysiological processes and mechanisms
underlying ischemic retinopathy and the progressive damage of
RGCs. A mouse model of transient ischemic injury was thus used to
assess the role of the adaptive immune response.
Methods: Retinal ischemia was induced in C56BL/6J (B6), Rag1/- and TCR-/- mice, by raising the IOP to 110 mmHg for 60 minutes.
Sham-operated mice underwent the same procedure but without
elevated IOP. Mice were sacrificed at 3 days, and 1, 4, and 8 weeks
post-injury or at 28 days after sham operation. RGC loss was assessed
in retina whole-mounts that were immunostained with βIII-tubulin
antibody. Ocular immune responses were evaluated by assessing
CD11b+macrophage and CD4+T cell activation, and cytokine
expression in the retina. Systemic immune responses were measured
by detecting anti-Hsp responding T cells with IFN-γ ELISPOT assay.
Additionally, adoptive transfer of CD4+T cells isolated from B6 mice
after ischemia or sham operation was performed.
Results: Transient ischemic injury induces progressive RGC
degeneration, starting at as early as 3 days post-ischemia with
continued loss detectable up to 8 weeks after injury. Increased
numbers of macrophages and T cells and increased expression of
IFN- γ were observed in ischemic eyes compared to sham-operated
eyes. Concomitant with an increased induction of Hsp27 and Hsp60
expression in RGCs following ischemia, increased CD4+T cells
against Hsp27 and Hsp60 were also detected in the splenocytes of
ischemic mice compared to sham-operated mice. Rag1-/- and TCRβ/- mice showed significantly less RGC loss than B6 wild-type mice
after ischemic injury. Rag1-/- mice who received adoptive transfer of
T cells from B6 mice with retinal ischemia showed significantly more
RGC loss compared to Rag1-/- mice or Rag1-/- mice with T cells
from sham-operated mice.
Conclusions: T cell deficiency improved RGC survival, while
adoptive transfer of CD4+T cells isolated from ischemia-induced
mice into Rag1-/- mice, which carry both T and B cell deficiencies,
resumes the later phase RGC damage. Thus, the adaptive immune
response, especially CD4+TH1 type cell responses directed against
Hsp27 and Hsp60, has an essential role in retinal ischemia-induced
RGC loss.
Commercial Relationships: T H Khanh Vu, None; Huihui Chen,
None; Djoeke Doesburg, None; Kin-Sang Cho, None; Martine
Jager, None; Dongfeng F. Chen, None
Support: THKV: Stichting Glaucoomfonds, Stichting Nederlands
Oogheelkundig Onderzoek, Stichting Oogfonds Nederland, Prins
Bernhard Cultuurfonds. DFC: Department of Veterans Affairs
(1I01RX000110), Department of Defense (W81XWH-09-2-0091),
Lion’s Foundation Grants.
Program Number: 1895 Poster Board Number: B0160
Presentation Time: 11:00 AM–12:45 PM
Retinal Ganglion Cells Expressing Melanopsin Are InjuryResistant After Retinal Ischemia
Ruth E. Rosenstein, Maria F. Gonzalez Fleitas, Marcos L. Aranda,
Nuria de Zavalia, Pablo Sande, Damian Dorfman. Dept Human
Biochem-Sch Med, University of Buenos Aires, Buenos Aires,
Argentina.
Purpose: We investigated the effect of acute retinal ischemia on
the non-image forming visual system, particularly on melanopsin
expressing retinal ganglion cells (RGC).
Methods: Ischemia was induced in male Wistar rats by increasing
intraocular pressure (120 mm Hg for 40 min). Retinal function
(ERG), the number of Brn3a(+) and melanopsin(+) RGC
(immunohistochemistry), and melanopsin levels (Western Blot),
as well as the pupil light reflex (PRL) (after 30-s light flash) were
examined. Anterograde transport was examined after an intravitreal
injection of cholera toxin β-subunit, and circadian rhythms of general
locomotor activity were registered in cages equipped with infrared
detectors of motion.
Results: After 4 weeks of ischemia, clear alterations in the visual
function and retinal histology were observed. Concomitantly with a
significant decrease in the number of Brn3a(+) RGC, no differences
in the number of melanopsin(+) cells, and melanopsin levels were
observed between non-ischemic and ischemic retinas. Ischemia
decreased retinal projections to the superior colliculus, whereas the
anterograde transport to the suprachiasmatic nucleus and the olivary
pretectal nucleus remained unaffected. No differences in PRL were
observed between control and ischemic eyes, and the locomotor
activity pattern was conserved in animals submitted to bilateral
ischemia.
Conclusions: These results indicate melanopsin(+) RGC, and the
non-image forming visual system are resistant to ischemic injury.
Commercial Relationships: Ruth E. Rosenstein, None; Maria
F. Gonzalez Fleitas, None; Marcos L. Aranda, None; Nuria de
Zavalia, None; Pablo Sande, None; Damian Dorfman, None
Support: PICT 0610, PIP 1911, UBA 20020100100678
©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: 1896 Poster Board Number: B0161
Presentation Time: 11:00 AM–12:45 PM
Vulnerability of direction-selective ganglion cells and starburst
amacrine cells to ischemia-reperfusion in the adult mouse retina
Sandrine Joly, Vincent Pernet, Anna Guzik-Kornacka, Martin
E. Schwab. Brain Research Institute, ETH/Univ Zurich, Zurich,
Switzerland.
Purpose: Central vein and artery occlusion (CVAO) have devastating
effects on the inner retinal cells composed of retinal ganglion cells
(RGCs) and amacrine cells. In this study, we aimed at investigating
the degeneration time course of direction-selective ganglion cells
(DSGCs) and starburst amacrine cells (SACs) required for object
motion detection in a mouse model of CVAO.
Methods: To block the inner retinal circulation, the optic nerve
sheath containing the central vein and artery was ligated with a 9.0
suture in the orbit of C57BL/6 adult mice without damaging the
optic nerve. After 60 min, reperfusion was allowed by removing the
suture. Functional deficits were weekly monitored by measuring
the optokinetic response of each eye separately. At 10 and 21 days
after ischemia-reperfusion, the survival of the whole population of
RGCs, of ON-OFF direction-selective RGCs, of ON and OFF SACs
was assessed on retinal flat-mounts by immunohistochemistry using
β3Tubulin, cocaine and amphetamine regulated transcript (CART)
and Choline AcetylTransferase (ChAT) respectively as specific
markers..
Results: As early as 7 days after ischemia-reperfusion, the
optokinetic response was completely abolished while ~35 % and
~22% of RGCs stayed alive 10 and 21 days post injury respectively.
By immunohistochemistry, the density of CART-positive RGCs
decreased to 18% and 12% compared with intact retinae 10 and 21
days after reperfusion. The striking reduction in CART detection
could be due to CART protein down-regulation in DSGCs or to
cell death. In the ganglion cell layer (GCL), only 34% and 24% of
ON SACs remained in the retina at the two time points whereas
in the inner nuclear layer (INL) OFF SACs dropped to 78% and
54% of the control retina values. The percentage of surviving cells
suggest a faster and more massive loss of RGCs and ON SACs in
the GCL than of OFF SACs in the INL. In contrast, the elimination
of RGCs triggered by optic nerve crush did not affect the number of
ON and OFF SACS, indicating that SACs degeneration may not be
attributable to RGC cell death after ischemia-reperfusion.
Conclusions: RGCs and ON SACs are more vulnerable than
OFF SACs to ischemic insults caused by CVAO in mice. Future
experiments will be carried out to understand if remaining SACs and
DSGCs can be stimulated to restore the visual function measurable
with the optokinetic test.
Commercial Relationships: Sandrine Joly, None; Vincent Pernet,
None; Anna Guzik-Kornacka, None; Martin E. Schwab, None
Support: Forschungskredit UZH#54150602; Swiss National
Foundation (SNF#31003A-149315-1), Velux Stiftung #817
Program Number: 1897 Poster Board Number: B0162
Presentation Time: 11:00 AM–12:45 PM
Suppression of Interferon-gamma protects retinal ischemiainduced neuron death
Kin-Sang Cho1, T H Khanh Vu1, 2, Djoeke Doesburg1, 2, Dongfeng
F. Chen1, 3. 1Schepens Eye Research Institute, Massachusetts Eye
and Ear, Harvard Medical School, Boston, MA; 2Ophthalmology,
Leiden University Medical Center, Leiden, Netherlands; 3Boston VA
Healthcare System, Boston, MA.
Purpose: Transient retinal ischemic-reperfusion injury is a popular
mouse model to be used for the study of mechanisms of ischemiainduced neuron death. Our recent study showed that retinal ischemia
induced interferon gamma (IFN-γ), suggesting that IFN-γ may
contribute to the death of retinal neurons. We thus propose to test if
suppression of IFN-γ protects retinal ischemia-induced neuron death.
Methods: Acute retinal ischemia was induced in C57BL6/J mice.
Briefly, the anterior chamber of right eye was cannulated with a
glass needle connected to a sterile saline bag via plastic tubing.
The intraocular pressure was elevated to 110 mmHg for 60 min by
raising the saline bag to 120 cm high. In sham-operated group, same
procedure of cannulation was performed without infusing saline into
the anterior chamber. Two μg of anti-IFN-γ IgG or vehicle control
solution (saline or IgG isotype) was administrated to the vitreous
cavity at day 3, 7, 10 and 14 post-induction of retinal ischemia. At 3
weeks post injury, electroretinographic (ERG) response was recorded
in mice. The mice were sacrificed at 4 weeks post injury. Wholemount retinas were collected and incubated with Tuj1 antibody
which recognizes a marker for retinal ganglion cells (RGC). Tuj1+
RGC were visualized by incubating with Cy3 conjugated secondary
antibody, and Tuj1+ RGC were quantified. The percentage of
RGC loss was calculated by comparing with that in normal retina.
To determine the T cell-mediated responses potentially involved
in the ischemia induced pathological process, cells from cervical
lymph nodes of the mice were stimulated with phorbol 12-myristate
13-acetate and ionomycin for 4 hr in culture and analyzed by FACS.
Results: Significantly less number of Tuj1+ RGC loss was observed
in the ischemic retina that received anti-IFN-γ treatments than that
were treated with saline or IgG isotype (p<0.01). However, we did
not observe significant improvement of ERG response in the ischemic
retina that were treated with anti-IFN-γ if compared with saline or
IgG isotype-treated groups, of which all have shown significant
reduction of ERG a- and b-wave amplitudes comparing to the normal
retina. Nor did administration of anti-IFN-γ altered the population
of CD4+IFN-γ+ cells in the cervical lymph node as compared to the
saline treated group
Conclusions: Suppression of IFN-γ by administration of neutralizing
IFN-γ antibody improves RGC survival following retinal ischemia.
Commercial Relationships: Kin-Sang Cho, None; T H Khanh Vu,
None; Djoeke Doesburg, None; Dongfeng F. Chen, None
Support: Department of Veterans Affairs (1I01RX000110) and
Lion’s Foundation Grants to D.F.C. and K.S.C.
Program Number: 1898 Poster Board Number: B0163
Presentation Time: 11:00 AM–12:45 PM
Arginase blockade preserves retinal neurons during ischemia/
reperfusion injury
Zhimin Xu1, Esraa Shosha1, S. Priya Narayanan1, Harumasa Yokota4,
Robert William Caldwell2, Ruth B. Caldwell1, 3. 1Vascular Biology
Center, Georgia Regents University, Augusta, GA; 2Department of
Pharmacology and Toxicology, Georgia Regents University, Augusta,
GA; 3VA Medical Center, Augusta, GA; 4Ophthalmology, Asahikawa
Medical University, Asahikawa, Japan.
Purpose: Our previous studies have shown that deletion of the urea/
ornithine generating enzyme arginase 2 (A2) significantly reduces
neuronal injury in a model of retinopathy of prematurity (Narayanan
et al., 2011). To evaluate whether A2 could be a therapeutic target in
other forms of ischemic retinopathy, we examined the role of A2 in
neuronal death following retinal ischemia/reperfusion injury (I/R).
Methods: I/R injury was induced in the right eye of wild type
(C57BL/6, WT) or A2-/- mice by raising the intraocular pressure
to 110 mmHg for 40 minutes followed by reperfusion for different
times. The left eye was used as sham control. Other WT mice
were treated with the arginase inhibitor ABH (Amino-2-Borono6-Hexanoic Acid, 10mg/kg/day, ip, 8 days). Whole mounted
©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
retinas were stained with NeuN antibody and neuronal cell loss in
the ganglion cell layer (GCL) was quantified at 7 days after I/R.
Thickness of the retina was evaluated by morphometric analysis
of H&E stained retinal sections collected at 7 days after I/R injury.
Western blotting was performed to examine the activation of stress
pathways.
Results: There was a 40% reduction in the number of NeuN positive
GCL neurons in the WT retinas exposed to I/R as compared to the
WT sham controls (p<0.01). This neuronal cell loss was markedly
attenuated in the A2-/- mice (p<0.05) and ABH-treated WT mice
(p<0.01). The neuronal loss in the WT I/R retinas was accompanied
by a significant thinning of the retina (10%, p<0.01). Retinal
thinning was significantly inhibited in the A2-/- mice (4%, p<0.01)
as compared to the WT. The neuronal cell protection in the A2-/- I/R
retinas was associated with blockade of stress pathways as shown by
~50% decreases in phosphorylated JNK and p38MAPK as compared
to the WT mice at 3 hours after I/R injury (p<0.05). Expression of
receptor-interacting protein (RIP kinase 1), a regulator of necroptosis,
was also reduced by 50% in A2-/- I/R retinas as compared to the WT
retinas (p< 0.05).
Conclusions: Deletion of A2 or inhibition of arginase prevents the
loss of GCL neurons and limits retinal thinning following I/R injury.
Stress activated MAPK/JNK pathway and RIP-mediated necroptosis
may be involved in the neuronal cell injury. These data demonstrate
that A2 plays an important role in neuronal degeneration during I/R.
Inactivation of A2 may offer a therapeutic strategy for preventing
neuronal cell death in ischemic retinopathy.
Commercial Relationships: Zhimin Xu, None; Esraa Shosha,
None; S. Priya Narayanan, None; Harumasa Yokota, None;
Robert William Caldwell, None; Ruth B. Caldwell, None
Support: NIH Grants EY11766 (R.B.C. and R.W.C.); EY04618
(R.B.C.); HL70215 (R.W.C.); VA Merit Award (R.B.C.); AHA
11SDG7440088(S.P.N.)
Program Number: 1899 Poster Board Number: B0164
Presentation Time: 11:00 AM–12:45 PM
Delta Opioid-Receptors activation mitigates detrimental effects of
TNF-α during Retinal Ganglion Cells Degeneration.
Yasir Abdul, Matthew J. Nutaitis, Shahid Husain. Ophthalmology,
Medical Univ of South Carolina, Charleston, SC.
Purpose: To determine if TNF-α causes a direct injury to the retinal
ganglion cells (RGCs) and TNF-α-induced RGCs injury is alleviated
by the activation of delta opioid-receptors.
Methods: TNF-α was injected intravitreally (10 ng TNF-α in 3ml) in
deeply anesthetized Brown Norway rats. Contralateral eye was used
as sham or control and injected with vehicle (3 ml PBS). Animals
were treated with a selective delta opioid-receptor agonist (SNC121; 1mg/kg; i.p) right after the intravitreal injection of TNF-α
and subsequently daily for 7 days. Intraocular Pressure (IOP) was
measured as the average of 6-8 consecutive measurements prior to
surgery (baseline IOP) and weekly after TNF-α injection for 28 days.
Functional response of RGCs and retina was measured by pattern
electroretinograms (Pattern-ERGs) and scotopic-ERGs, respectively.
Retinal ganglion cells (RGCs) were visualized by retrograde-labeling
using fluorogold. Optic nerve morphology was determined by
toluidine blue staining in optic nerve head sections.
Results: The intraocular pressure was neither changed in vehicle
nor in TNF-α treated animal. The pattern-ERGs were reduced by
44% in TNF-α treated eyes on day 28th when compared with vehicle
treated eyes (Sham eyes 100 ± 00% vs TNF-α treated eyes 66 ±
7%; p<0.05). The loss in pattern-ERGs was significantly reduced in
SNC-121 treated groups (TNF-α treated eyes 66 ± 7% vs SNC-121
+ TNF-α treated eyes 98 ± 11%; p<0.05). In addition to pattern-
ERGs, scotopic-ERGs were also improved in SNC-121 treated group
when measured on day 28th, post injury. The numbers of RGCs
were reduced significant (p<0.05) in TNF-α treated eyes, which was
improved in the presence of SNC-121. Additionally, TNF-α treatment
causes morphological changes and axonal loss, which was preserved
by SNC-121 treatment.
Conclusions: We have shown earlier that TNF-α is produced from
glial cells very early during the progression of glaucoma, which
subsequently lead to the RGC death. Current data supported our
previous findings and provide new evidences that TNF-α caused
direct injury to the axons and RGCs. Additionally, we have shown
that activation of delta opioid-receptors by exogenous ligand rescue
axons and RGCs against TNF-α-induced retinal injury.
Commercial Relationships: Yasir Abdul, None; Matthew J.
Nutaitis, None; Shahid Husain, None
Support: NIH Grant EY-019081
Program Number: 1900 Poster Board Number: B0165
Presentation Time: 11:00 AM–12:45 PM
Endothelin B (ETB) Receptors Contribute to Neurodegeneration
in a Rodent Model of Glaucoma via Upregulation of c-Jun and
Bax
Alena Z. Minton1, 2, Shaoqing He1, 2, Hai-Ying Ma3, Raghu R.
Krishnamoorthy1, 2. 1Cell Biology and Immunology, Univ of North
Texas Hlth Sci Ctr, Fort Worth, TX; 2North Texas Eye Research
Institute, Fort Worth, TX; 3Pharmacology & Neuroscience, University
of North Texas Health Science Center, Fort Worth, TX.
Purpose: Previously, our lab has demonstrated that increased levels
of ETB receptors contribute to the death of retinal ganglion cells
(RGCs) and degeneration of optic nerve axons in the Morrison’s
elevated intraocular pressure (IOP) model of glaucoma in rats.
Moreover, these pathological changes were greatly attenuated in
ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, an
increase in ETB receptor levels in RGCs, following 2 weeks of IOP
elevation in Brown Norway rats, was shown to be associated with
increased expression of c-Jun, a member of the activator protein-1
(AP-1) family. The current study was aimed at investigating whether
the increased expression of c-Jun observed in wild type rats is
reduced in ETB receptor-deficient Wistar Kyoto rats subjected to
the Morrison’s model of glaucoma. The status of another apoptotic
protein, Bax, was also assessed in these rats.
Methods: IOP was elevated in one eye of adult wild type and ETB
receptor-deficient transgenic Wistar Kyoto rats using the Morrison’s
method (injection of hypertonic saline through episcleral veins),
while the contralateral eye served as control. After IOP was elevated,
rats were maintained for 2 weeks and sacrificed. Retinal sections
were obtained and stained with specific antibodies to detect the
expression of c-Jun and Bax by immunohistochemistry. In addition,
retinal sections were immunostained using an antibody to βIIItubulin, which is selectively expressed by RGCs in the retina. Images
were taken using Zeiss LSM-510 confocal microscope with Z-scan.
Results: Immunohistochemical analysis showed that IOP elevation
for 2 weeks caused increased expression of c-Jun and Bax mainly in
the ganglion cell layer (GCL) of wild type transgenic Wistar Kyoto
rats as compared to ETB receptor-deficient transgenic Wistar Kyoto
rats. Interestingly, using the Promo 3 software, we found 15 binding
sites for members of the AP-1 family of proteins on the rat 1.95 kb
upstream promoter region of Bax. Therefore, the transcription factor
c-Jun may be an upstream regulator of Bax (pro-apoptotic factor).
Conclusions: Transcription factor AP-1 could be involved in the
elevation of the ETB receptor levels in the Morrison’s model of
glaucoma. Conversely, deletion of the ETB receptor results in the
©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
downregulation of c-Jun. Taken together, there may be a reciprocal
feedback loop between the AP-1 and ETB receptors.
Commercial Relationships: Alena Z. Minton, None; Shaoqing He,
None; Hai-Ying Ma, None; Raghu R. Krishnamoorthy, None
Support: NEI: 1RO1 EY0199952-01
Program Number: 1901 Poster Board Number: B0166
Presentation Time: 11:00 AM–12:45 PM
INVOLVEMENT OF RETINAL RENIN ANGIOTENSIN
PATHWAY IN EXPERIMENTAL MODEL OF RETINOPATHY
OF PREMATURITY
Madhu Nath1, Rajvardhan Azad1, Ashok kumar Deorari3, Baskar
Singh4, Neelima Aron1, Thirumurthy Velpandian2. 1Ophthalmology,
All India Institute of Medical Sciences, New Delhi, India; 2Ocular
Pharmacology & Pharmacy, All India Institute of Medical Sciences,
New Delhi, India; 3Peadiatrics, All India Institute of Medical
Sciences, New Delhi, India; 4Biophysics, All India Institute of
Medical Sciences, New Delhi, India.
Purpose: Renin angiotensin system is well established in ocular
tissues, alteration in this system during developing stage of
retinal vessel in prematurely born neonate can lead to ROP, which
is the leading cause of blindness and visual morbidity in the
surviving premature infants.Modulation of RAS pathway through
pharmacological intervention can be proved as effective therapy for
this neonatal blinding disease in near future.
Methods: Wistar rat pups were exposed to high oxygen saturation
(75%) in a controlled chamber, from 7th -12th postnatal day. On day
12th pups were randomised in to receive ACE inhibitor (lisinopril
0.07mg/kg), AT1 receptor blocker (telmisartan 7mg/kg)) and both
in combination. Bevacizumab was used as a positive control and
saline treated pups served as untreated control. Drugs and saline were
injected through subcutaneous route in two divided doses in 48 hours
interval. Structural and functional health of retina were assessed by
tortuosity index of blood vessels and electroretinography respectively
on PD 17 and 25 using MICRON III rodent imaging system. ROP
rats were compared with normoxia rat pups. Rat pups were sacrificed
and retina were extracted to study the gene expression of RAS
components in various test groups.
Results: Retinopathy was assessed in the terms of tortuosity of
vessels from optic nerve head to posterior pole of retina. It is been
observed that arterioles were more effected than venules. There was a
significant increase in arteriole tortuosity in sham with comparison to
normoxia group (p=0.002). When compared with sham, bevacizumab
(p=0.040), lisinopril (p=0.003), telmisartan (p=0.002) and Lisinopril
plus Telmisartan combination (p=0.002) group has shown significant
decrease in tortuosity and were comparable to normoxia group. In
ERG responses, on Day 17 there was a 66% reduction in the b wave
amplitude in Sham as compared to Room air (p=0.002). The b wave
amplitude of the drug treatment groups were comparable to room air.
Expression of RAS components of various groups were also altered.
Conclusions: Retinopathy was hindered in various groups viz.
Lisinopril, Telmisartan and their combination, respectively as
evidenced by improved ‘b’ wave and lesser tortuosity index. Further
studies are in process to evaluate its the clinical utility with the help
of intravitreal delivery system.
Commercial Relationships: Madhu Nath, None; Rajvardhan
Azad, None; Ashok kumar Deorari, None; Baskar Singh, None;
Neelima Aron, None; Thirumurthy Velpandian, None
Program Number: 1902 Poster Board Number: B0167
Presentation Time: 11:00 AM–12:45 PM
Evaluation of the Neuroprotective Potential of the 5HT2A
Receptor Antagonist Sarpogrelate Hydrochloride in a Light
Damage Model of Retinal Degeneration
Brandon E. Tullis1, 2, Michael J. Gale1, Alexander J. Nicholson1, 2,
Michelle Sorensen1, Shreya Datta1, Mark E. Pennesi1. 1Casey Eye
Institute, Oregon Health & Science University, Portland, OR; 2School
of Medicine, Oregon Health & Science University, Portland, OR.
Purpose: To assess the potential of the 5HT2A receptor antagonist
sarpogrelate hydrochloride (MCI-9042; Anplag) for protecting rodent
retinal structure and function from light-induced phototoxicity
Methods: Albino BALB/c mice received intraperitoneal injections of
5 mg/kg sarpogrelate hydrochloride (5 mice), 50 mg/kg sarpogrelate
hydrochloride (5 mice), or 0.9% saline solution (4 mice). The mice
received injections 48, 24, and 0 hours prior to light damage, as well
as 24 and 48 hours afterward. Light damage was induced by exposing
the mice to 9.0-9.4×103 lux from four compact fluorescent light bulbs
inside a custom-built light box. The light box was well-ventilated
and temperature-controlled, and the mice were checked on each hour
to ensure comfort. One week after light damage, retinal structure
was viewed in cross-section using spectral domain optical coherence
tomography (SD-OCT). Retinal function was analyzed through
electroretinography (ERG).
Results: SD-OCT imaging of the high dose sarpogrelate mice
showed a marked preservation of retinal structure in comparison
to the low dose and saline control mice. The outer nuclear layer
was severely thinned in the saline control mice and the low dose
sarpogrelate mice, but was only slightly thinned in most retinal
quadrants imaged in the high dose sarpogrelate mice. Total retinal
thickness was also visibly thicker in the high dose sarpogrelate mice;
numeric comparisons of retinal thicknesses will be made available
following further analysis. The five low dose sarpogrelate mice did
not exhibit a grossly appreciable preservation of retinal structure
when compared to the control group. The ERG data followed a
similar pattern as the OCT data. The b wave amplitudes of the saline
control and low dose sarpogrelate mice were 50% and 37% as large
as those seen in normal BALB/c mice, respectively, but the b wave
amplitudes of the high dose sarpogrelate mice were 83% of normal
on average.
Conclusions: Preliminary evidence indicates that high dose, but
not low dose, sarpogrelate hydrochloride appears to be effective in
protecting albino BALB/c mouse retinal structure (as measured by
OCT) and preserving retinal function (as measured by ERG) from
light-induced phototoxicity compared to saline-injected animals.
Commercial Relationships: Brandon E. Tullis, None; Michael J.
Gale, None; Alexander J. Nicholson, None; Michelle Sorensen,
None; Shreya Datta, None; Mark E. Pennesi, Imagine Eyes (F)
Support: This research was made possible with support from the
Oregon Clinical and Translational Research Institute (OCTRI), grant
number TL1 RR024159 from the National Center for Advancing
Translational Sciences (NCATS), a component of the National
Institutes of Health (NIH), and NIH Roadmap for Medical Research.
Additional support was provided by the following grants: Research to
Prevent Blindness (unrestricted, CEI); Foundation Fighting Blindness
(Pennesi CDA, CD-CL-0808-0469-OHSU).
©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: 1903 Poster Board Number: B0168
Presentation Time: 11:00 AM–12:45 PM
Prolonged NMDA Stimulation Induces Neuroprotective Pathways
and Enhances Survivability of Primary Retinal Ganglion Cells
Brett H. Mueller1, 2, Yong H. Park1, 2, Hai-Ying Ma1, 2, Thomas Yorio1,
2 1
. Pharmacology & Neuroscience, Univ of North Texas Hlth Sci Ctr,
Fort Worth, TX; 2North Texas Eye Research Institute, Univ of North
Texas Hlth Sci Ctr, Fort Worth, TX.
Purpose: Calcium influx through postsynaptic NMDA receptors
has been shown to stimulate a number of key pro-survival genes;
however, prolonged stimulation has been shown to have excitotoxic
effects leading to apoptosis in neurons. Previous studies have
shown a rapid dephosphorylation of CREB in primary hippocampal
neurons treated for 1-2 hours with100mM NMDA. It is hypothesized
that the activation of CREB-specific phosphatases is one of the
main pathways that cause apoptosis during NMDA excitotoxicity.
The current study investigated the role of NMDA stimulation on
the phosphorylation of CREB in primary RGCs, and assessed if
NMDA overstimulation caused excitotoxic changes similar to
those seen in primary hippocampal neurons. The occurrence of
NMDA excitotoxicity in bipolar and photoreceptor cells was also
investigated.
Methods: Purification and culture of RGCs were performed by
sequential immunopanning using Thy 1.1 antibody from P3-P7
Sprague-Dawley rats. Mixed retinal cultures that remained following
isolation of RGCs from the retina were plated once the RGCs were
separated and purified. Western blots were performed to determine
signaling pathways linked to NMDA induced cell survival or
excitotoxicity. Calcein AM and ethidium homodimer were used to
quantify cell survival and cell death. Cells were also subjected to a
trophic factor deprivation insult for 6 hours and 24 hours.
Results: Treatment of primary RGCs with NMDA (100 mM) for 6
hours caused a greater than 2-3 fold induction of the transcription
factor pCREB. MK801 (NMDA antagonist) completely abolished
endogenous levels of pCREB and blocked NMDA induction of
pCREB. NMDA (100 mM) treatment for 6 and 24 hours under trophic
factor deprivation, protected RGCs from trophic factor deprivation
induced cellular death. The mixed retinal cultures (retinal cells
without RGCs) had an opposite effect, where the levels of pCREB
were diminished and the neurons died when treated with 100 mM of
NMDA.
Conclusions: The data suggests that NMDA signaling is essential
for RGC survivability and blocking calcium ion influx through this
receptor by the NMDA blocker, MK801, can be detrimental to RGC
function and survival. These results also demonstrate that primary
RGCs behave differently than other neurons in the retina, and are not
susceptible to NMDA excitotoxicity.
Commercial Relationships: Brett H. Mueller, None; Yong H.
Park, None; Hai-Ying Ma, None; Thomas Yorio, None
Support: Department of Defense (W81XH-10-2-0003)
Program Number: 1904 Poster Board Number: B0169
Presentation Time: 11:00 AM–12:45 PM
Retinal Ganglion Cells are Resistant to AMPA Receptor
Mediated Excitotoxicity
Yong H. Park1, 2, Brett H. Mueller1, 2, Nolan McGrady2, 3, Adnan
Dibas1, 2, Thomas Yorio1, 2. 1Pharmacology & Neuroscience, UNT
Health Science Center, Fort Worth, TX; 2North Texas Eye Research
Institute, Fort Worth, TX; 3Cell Biology & Immunology, UNT Health
Science Center, Fort Worth, TX.
Purpose: The ionotropic glutamate receptors (iGluR) have been
hypothesized to play a role in glaucoma pathogenesis by mediating
excitotoxic death of retinal ganglion cells (RGC). Previous studies
on iGluR in the retina have been focused on two broad classes of
receptors: NMDA and non-NMDA receptors including the α-amino3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and
Kainate receptor. In this study, we examined the specific excitotoxic
effects of activation of the AMPAR in RGCs in-vitro.
Methods: Purified rat RGCs were isolated from P3-P5 SpragueDawley rats by a double immunopanning technique using an antibody
to Thy 1.1. RGCs were cultured for 7 days before s-AMPA (100μM)
treatments. s-AMPA excitotoxicity was determined by Caspase3/7
luciferase activity assay, immunoblot analysis for α-fodrin and
Live (calcein AM)/Dead (ethidium homodimer-1) assay. Gap-43
expression was assessed by immunocytochemistry.
Results: Treatment of cultured RGCs with s-AMPA (100μM)
for 24, 48 and 72 hr, both in the presence and absence of trophic
factors (BDNF and CNTF), did not alter caspase 3/7 activity and
cleavage of α-fodrin (neuronal apoptosis marker), compared to
untreated controls. A significantly higher (p<0.05) cell survival of
RGCs (85.3±1.5% alive cells) was observed after a 72h treatment
with 100μM s-AMPA compared to control untreated RGCs
(74.8±3.1% alive cells). Quantification of s-AMPA (100μM) –
mediated excitotoxicity in purified RGCs incubated for 24h in an
oxygen/glucose deprived (0.5% oxygen) medium demonstrated
no statistically significant differences in cell survival compared
to control RGCs maintained under either normoxia or hypoxia.
Additionally, immunocytochemical analysis showed increased GAP43 staining in RGCs after 24h of treatment with s-AMPA (100μM).
Conclusions: These results indicate that purified RGCs in-vitro are
not susceptible to AMPA excitotoxicity as previously hypothesized.
Activation of AMPAR increased GAP-43 expression, suggesting
AMPAR could possibly increase neurite outgrowth. The ability of
AMPA receptors to promote neuroprotection of RGCs remains to be
confirmed.
Commercial Relationships: Yong H. Park, None; Brett H.
Mueller, None; Nolan McGrady, None; Adnan Dibas, None;
Thomas Yorio, None
Support: NIA Training Grant: T32AG020494; Department of
Defense: W81XH-10-2-0003; Sigma Xi GRIAR
Program Number: 1905 Poster Board Number: B0170
Presentation Time: 11:00 AM–12:45 PM
Role of Ca2+-permeable AMPA receptor in retinal ischemia
models
Ai Ling Wang1, Scott A. Nawy2. 1Ophthal & Visual Sci, Albert
Einstein School of Medicine, Bronx, NY; 2Ophthalmology, Albert
Einstein College of Medicine, Bronx, NY.
Purpose: Purpose: Recent work has found that expression of the
Ca2+-permeable AMPA receptor (CP-AMPAR) is elevated following
transient global forebrain ischemia, resulting in increased neuronal
death. Retinal ischemia is also associated with neuronal death,
particularly the death of retinal ganglion cells, and can lead to
blindness. At present, there is no effective treatment against retinal
ischemic damage. The purpose of the present study was two fold: 1)
We tested the hypothesis that CP-AMPARs are upregulated following
ischemic insult, and that Ca2+ influx through these channels
contributes to RGC death in retinal ischemia. 2) We investigated the
idea that global forebrain ischemia can also damage RGCs through
the same mechanisms as retinal ischemia.
Methods: Two in vivo animal models were used. One was a
transient, global cerebral ischemia (4-VO) model, and the other was
a retinal ischemia/reperfusion model. In addition, the oxygen/glucose
deprivation (OGD) model was established in cultured primary RGCs.
To test for changes in AMPAR expression, we measured levels of
GluA2, a subunit that, following Q/R editing by ADAR2, renders
©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
AMPARs impermeable to Ca2+. In separate experiments, siRNA
was used to knockdown levels of ADAR2 in primary culture to
directly test the hypothesis that increased expression of CP-AMPARs
contributes to RGCs death.
Results: Using real-time RT-PCR, we found a 75% decrease in
GluA2 expression in the retina in the 4-VO model, and a 65%
decrease in GluA2 expression in response to increased IOP in the
ischemia/reperfusion model at 1 week, suggesting that both forms
of ischemia can induce changes in AMPAR composition. We also
observed a decrease in GluA2 immunoreactivity in retinal neural cell
cultures exposed to OGD. This decrease was confirmed by real-time
RT-PCR and western blots. In a second series of experiments, we
used Co2+ staining and electrophysiology to confirm that ADAR2
siRNA increased Ca2+ permeability through AMPARs. Importantly,
the susceptibility of RGCs to AMPA induced excitotoxicity was
dramatically increased in siRNA-treated RGCs compared with
control or sham siRNA treated RGCs.
Conclusions: We investigated the function of increased CP-AMPARs
levels on RGCs function and viability. Our work suggests that global
ischemia can increase expression of CP-AMPARs in RGCs, and that
Ca2+ influx into RGCs as a result of increased CP-AMPARs may
play a role in the pathogenesis of retinal ischemia.
Commercial Relationships: Ai Ling Wang, None; Scott A. Nawy,
None
Support: R01EY010254, Unrestricted RPB grant
Program Number: 1906 Poster Board Number: B0171
Presentation Time: 11:00 AM–12:45 PM
The neuroprotective role of Nrf2 in ischemia-reperfusion mouse
model
Zhenhua Xu, Matthew Hartsock, Junsong Gong, Yanhong Wei,
Shuang Wang, Elia J. Duh. Smith Building, Room 3001B, Johns
Hopkins University, Baltimore, MD.
Purpose: Nrf2 is known to play a protective role in regulating
oxidative stress and inflammation. Our group previously found that
Nrf2 plays a cytoprotective role in murine models of retinal ischemiareperfusion and diabetic retinopathy. The objective of this study was
to explore the potential neuroprotective role of Nrf2 in the mouse
ischemia-reperfusion model.
Methods: Retinal ischemia-reperfusion model was performed
by elevating intraocular pressure to 90mmHg for 90 min. NeuN
immunostaining of retina flat-mount and H/E staining of paraffinembedded sections were used to determine cell loss in the ganglion
cell layer. RGC-5 cells, a retinal neuronal cell line, were treated with
different doses of Tert-Butyl hydroperoxide (TBH), and the level of
reactive oxygen species (ROS) was measured by DCF assay. Keap1
siRNA was used to up-regulate Nrf2 activity in RGC-5 cells. The
synthetic triterpenoid CDDO-Im (2-Cyano-3,12-dioxooleana-1,9dien-28-imidazolide) was used to activate Nrf2 expression in RGC-5
cells.
Results: In the retinal ischemia-reperfusion mouse model, cell
loss in ganglion cell layer was much more severe in Nrf2 knockout
mice than in wild-type mice at 2 and 7 days after I/R. There was
significantly more apoptosis in Nrf2 KO retinas compared to wildtype. Treatment with TBH increased ROS level in RGC-5 cells.
siRNA-mediated knockdown of the Nrf2 inhibitor Keap1 exacerbated
this TBH-induced ROS increase. Pharmacologic activation of
Nrf2 using the triterpenoid CDDO-Im inhibited TBH-induced ROS
increase in a dose-dependent fashion.
Conclusions: These results indicate that Nrf2 exhibits a neuronal
protective function in the retinal ischemia-reperfusion model and
suggest that pharmacologic activation of Nrf2 could be a therapeutic
strategy.
Commercial Relationships: Zhenhua Xu, None; Matthew
Hartsock, None; Junsong Gong, None; Yanhong Wei, None;
Shuang Wang, None; Elia J. Duh, None
Support: NIH EY022383 (EJD) and Bright Focus Foundation (ZX).
Program Number: 1907 Poster Board Number: B0172
Presentation Time: 11:00 AM–12:45 PM
Loss of cholinergic amacrine cells in an ischemia-reperfusion
animal model
Heiko Schmid, Marina Renner, H. Burkhard Dick,
Stephanie C. Joachim. Experimental Eye Research Institute,
Knappschaftskrankenhaus, Bochum, Germany.
Purpose: Ocular ischemic injuries like ocular vein occlusion are
huge risk factors for damage of retinal neurons and often lead to
loss of vision. In the last couple of years, extensive research has
been performed to avoid retinal ganglion cell (RGC) loss after an
ischemic event. However, very little is known about the global effect
of ischemia on different retinal neurons. The goal of this study was
to investigate functional and morphological changes of different cell
types of the retina after ischemia-reperfusion (I/R).
Methods: I/R was induced by raising the intraocular pressure (IOP)
in one eye of rats to 140 mmHg for 1h (N=5). The other eye served
as control (Co). The IOP was measured regularly. 21 days after
ischemia, scotopic flash electroretinogram (ERG) measurements were
performed on both eyes using intensities ranging from 0.1-25 cd*s/
m2. H&E staining was used to measure the retinal layer thickness.
Changes of RGC, amacrine-, rod bipolar-, and glia cells were
analyzed using immunohistochemistry (IHC).
Results: No changes in IOP were noted between I/R and Co
(p>0.05). The ERG measurement revealed a decrease of a-wave
(p<0.01) and b-wave amplitude (p<0.001) in I/R animals at different
light intensities. Histology showed a reduction of the RGC (Co:
7.1±0.4 mm, I/R: 5.8±0.2 mm, p=0.0022) and the inner plexiform
layer (Co: 25.6±1.2 mm, I/R: 15.4±1.8 mm, p<0.001) thickness in
I/R. No changes of the other layers were observed. IHC revealed a
RGC decrease (Co: 34±2 cells/mm, I/R: 24±2 cells/mm, p=0.0016)
caused by apoptosis (Co: 68.5±3.7%, I/R: 88.5±2.3%, p<0.001). A
loss of cholinergic amacrine cells (Co: 11±1 cells/mm, I/R: 4±1 cells/
mm, p<0.001) as well as an increase of GFAP+ area was detected
in I/R (Co: 3.1±0.3, I/R: 9.4±1.4, p<0.001). Counting of rod bipolar
cells stained with PKC± revealed no differences between both groups
(p>0.05) and no changes in the morphology of these cells were
observed.
Conclusions: We hypothesize that, although I/R is a global event,
the inner retina is the primary site of damage after I/R. RGCs and
amacrine cells seem to be affected first. We also assume that damage
later spreads to the outer retina. We could show by a reduced b-wave
amplitude that cells like rod bipolar cells, while morphologically
intact, are functionally impaired. This leads us to the conclusion
that only using both histology and electrophysiology gives accurate
insight on the pathomechanisms of I/R.
Commercial Relationships: Heiko Schmid, None; Marina Renner,
None; H. Burkhard Dick, None; Stephanie C. Joachim, None
Support: Novartis
©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: 1908 Poster Board Number: B0173
Presentation Time: 11:00 AM–12:45 PM
Suppression of T cell function with anti-CD4 antibody leads to
increased RGC survival after retinal ischemia-reperfusion injury
Djoeke Doesburg1, 2, T H Khanh Vu1, 2, Kin-Sang Cho2, Martine
Jager1, 2, Dongfeng F. Chen2. 1Ophthalmology, Leids Universitair
Medisch Centrum, Leiden, Netherlands; 2Ophthalmology, Schepens
Eye Research Institute, Massachusetts Eye and Ear Infirmary,
Harvard Medical School, Boston, MA.
Purpose: Ischemia contributes to the pathophysiology of a variety
of neurodegenerative disorders, including stroke and glaucoma.
Insufficient blood flow to the retina can lead to the development of
ischemic retinopathy which results in the destruction of neural tissue
in the eye and subsequent vision loss. CD4+ T cell-mediated immune
responses have been proposed as critical elements in triggering
ischemia-induced inflammation and neural damage. Therefore, we
studied whether blocking CD4+ T cell function had a protective
effect on retinal ganglion cell (RGC) survival in a transient retinal
ischemic-reperfusion mouse model.
Methods: Transient retinal ischemia was induced in C57BL/6J
mice by acute elevation of intraocular pressure to 110 mmHg for 60
min, followed by intravitreal injections of saline, isotype IgG (as an
isotype control), or anti-CD4 antibody on day 3, 7, 10, and 14. To
assess the retinal function, electroretinography (ERG) was performed
on day 21 post injury. At 28 days after the ischemic injury, all mice
were sacrificed and RGC quantification was performed by βIIItubulin staining in retinal whole-mounts. In addition, FACS analysis
of the draining cervical lymph nodes was performed to identify
different T cell-mediated responses.
Results: The level of RGC loss was comparable between mice
who received intravitreal injections of saline and isotype IgG after
ischemic injury. In contrast, mice injected with anti-CD4 antibody
showed significantly less RGC loss compared to mice treated with
sterile saline. T cell responses measured in the draining lymph nodes
did not differ between mice who received injections with either antiCD4, isotype control or sterile saline. Retinal function as measured
by ERG showed that anti-CD4 treated mice had better responses to
the stimulations at 21 days after transient ischemia than mice who
received saline injections.
Conclusions: Our data demonstrate that suppression of local CD4+
T cell-mediated responses in the eye protects against RGC loss and
retinal function impairment after transient ischemic injury. This
local suppression of T cell responses has no significant effect on the
systemic immune response. The findings suggest possibilities for a
new therapeutic approach in the treatment of ischemic neuropathy.
Commercial Relationships: Djoeke Doesburg, None; T H Khanh
Vu, None; Kin-Sang Cho, None; Martine Jager, None; Dongfeng
F. Chen, None
Program Number: 1909 Poster Board Number: B0174
Presentation Time: 11:00 AM–12:45 PM
Placental Growth Factor Levels Correlated with Retinal Ischemia
Progression
Kyle V. Marra1, Kyle Kovacs2, Sushant Wagley3, Sudheer Akella1,
Gianna C. Teague4, Walter Johnson5, Kameran Lashkari4, Jorge G.
Arroyo1. 1Ophthalmology, Beth Israel Deaconess Medical Center,
Brighton, MA; 2Albert Einstein College of Medicine, Bronx, NJ;
3
College of Human Medicine, Michigan State University, East
Lansing, NJ; 4Ophthalmology, Schepens Eye Research Institute,
Massachusetts Eye & Ear, Boston, MA; 5Physics, Suffolk University,
Boston, MA.
Purpose: To characterize the vitreous cytokine, chemokine, and
growth factor profiles in patients with increasing retinal ischemia.
Methods: This IRB-approved study retrospectively analyzed 81
undiluted vitreous samples obtained from patients undergoing pars
plana vitrectomy by a single surgeon at Beth Israel Deaconess
Medical Center from November 2010 to September 2012. The
specimens underwent a Bio-Plex Pro Human Cytokine Assay to
determine the levels of 34 proteins including chemokines, cytokines,
non-inflammatory proteins, and growth factors. Specimens were
divided into the following four groups based on whether the patient
underwent: 1.) vitrectomy for epiretinal membrane peeling and/
or macular hole with no history of diabetes (control group), 2)
vitrectomy for epiretinal membrane peeling and/or macular hole with
a history of diabetes (DM group), 3) vitrectomy for proliferative
diabetic retinopathy (PDR group), and 4) vitrectomy for neovascular
glaucoma (NVG group). Parametric and non-parametric analyses
were performed using SPSS software comparing demographics, as
well as protein levels between each group.
Results: There was no significant difference in age and gender
between groups. Numerous proteins were noted to be significantly
elevated comparing the control and DM group (G-CSF, sCD40L,
Endoglin, IL-6, PlGF, VEGF-D), the DM and PDR group (leptin, IL8, PlGF, VEGF-A), as well as the DM to NVG group (G-CSF, leptin,
TIE-2, sCD40L, EGF, HB-EGF, IL-6, IL-8, PlGF, TNF-alpha). Of
note, placental growth factor (PlGF) exhibited a significant increase
in all the aforementioned comparisons. Most proteins elevated in the
PDR and NVG groups were significantly elevated compared to the
control group as well.
Conclusions: Both angioproliferative growth factors as well as
inflammatory proteins are elevated in eyes with severe retinal
ischemia. We found that vitreous levels of PlGF increase significantly
in patients with worsening retinal ischemia.
Commercial Relationships: Kyle V. Marra, None; Kyle Kovacs,
None; Sushant Wagley, None; Sudheer Akella, None; Gianna C.
Teague, None; Walter Johnson, None; Kameran Lashkari, None;
Jorge G. Arroyo, None
Support: This research is supported by the Grimshaw-Gudewicz
Charitable Foundation.
Program Number: 1910 Poster Board Number: B0175
Presentation Time: 11:00 AM–12:45 PM
NIR Photobiomodulation Does Not Alter Retinal Function or
Morphology in Non-dystrophic Sprague Dawley Rats.
Janis T. Eells1, Betsy Abroe1, Heather M. Schmitt3, Alina GonzalezQuevedo4, Phyllis Summerfelt5, Adam M. Dubis6, Joseph Carroll5,
Sandeep Gopalakrishnan2. 1Health Sciences, Univ of Wisconsin
- Milwaukee, Milwaukee, WI; 2College of Nursing, University of
Wisconsin-Milwaukee, Milwaukee, WI; 3Ophthalmology, University
of Wisconsin-Madison, Madison, WI; 4Biochemistry, Medical
University of Havana, Havana, Cuba; 5Ophthalmology, Medical
College of Wisconsin, Milwaukee, WI; 6Ophthalmology, University
College London, London, United Kingdom.
Purpose: Previous studies in our laboratory have shown that 670nm
and 830nm photobiomodulaton (PBM) protects against retinal
dysfunction and photoreceptor cell death in rodent models of retinal
injury and retinal degeneration. The purpose of this study was to
test the hypothesis that NIR PBM would not alter retinal function or
morphology in a non-dystrophic Sprague-Dawley rat.
Methods: All studies were conducted in compliance with the
ARVO Statement for the Use of Animals in Ophthalmic and Visual
Research. Sprague Dawley (Harlan Sprague-Dawley, Madison, WI)
rats were treated once per day with either 670nm or 830nm light
(180 s; 25 mW/cm2; 4.5 J/cm2) using a light-emitting diode array
(QDI, Barneveld WI) from postnatal day p10 to p25. Sham-treated
rats were restrained for 180 seconds, but not exposed to 670nm or
©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
830nm light. Retinal function and structure were assessed at p30 by
measuring photoreceptor function with electroretinography (ERG)
and retinal morphology using spectral domain optical coherence
tomography (SD-OCT).
Results: Photon irradiation with 670nm or 830 nm light did not
alter ERG parameters in SD rats compared to sham-treated control
animals. ERG a-wave amplitude and latency and ERG b-wave
amplitude and latency were not altered by NIR PBM treatment.
Retinal imaging studies using SD-OCT imaging revealed no
differences in the structural integrity of the retina in NIR PBM treated
rats compared to sham-treated control animals.
Conclusions: Our findings demonstrate the safety of 670nm and
830nm photobiomodulation applied to the retina in Sprague-Dawley
albino rats. They confirm other experimental and clinical studies
demonstrating an absence of adverse effects of photobiomodulation.
Further, they provide essential safety data for the continued
development and clinical application of PBM for the treatment of
retinal degenerative disease.
Commercial Relationships: Janis T. Eells, QBMI Photomedicine
(C); Betsy Abroe, None; Heather M. Schmitt, None; Alina
Gonzalez-Quevedo, None; Phyllis Summerfelt, None; Adam M.
Dubis, None; Joseph Carroll, None; Sandeep Gopalakrishnan,
None
Support: JE: FFB (TA-NP-0709-0465-UWI), IRRF, NIH
(EY001931). JC: NIH ( EY001931, EY014537), MCW Research
Affairs Committee, RPB
Program Number: 1911 Poster Board Number: B0176
Presentation Time: 11:00 AM–12:45 PM
Quantitative High Throughput Screening for Small Molecules
That Promote Photoreceptor Differentiation and Survival
John A. Fuller1, Karl Wahlin1, Cynthia Berlinicke1, Douglas
Yasumura2, Michael T. Matthes2, Ryan MacArthur3, Patricia
Dranchak3, Matthew M. LaVail2, James Inglese3, Donald J. Zack1.
1
Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD;
2
Beckman Vision Center, UCSF School of Medicine, San Francisco,
CA; 3Department of Preclinical Innovation, National Center for
Advancing Translational Sciences, Rockville, MD.
Purpose: Retinal degenerations are a heterogeneous group of
diseases in which there is slow but progressive loss of photoreceptors.
There are currently no approved therapies for treating retinal
degenerations. In an effort to identify novel small molecules that are
1) neuroprotective and 2) promote photoreceptor differentiation, we
have developed microscale (1536-well) high throughput assays using
primary retinal neurons.
Methods: Primary cells from rhodopsin-GFP knock-in, QRXEGFP transgenic, and C57BL/6 wild-type mice were dissociated,
seeded into 1536-well plates, and treated with small molecule
libraries in a concentration-dependent fashion. GFP positive cell
generation is assessed by fixing cells after 7-14 days in culture and
imaging with a microplate-based laser cytometer. Positive wells
are verified with an automated microscopy platform. Cell viability
of CD73 immunopanned photoreceptors is assayed following 96h
in culture. Concentration-response curves are then generated to
pharmacologically profile each small molecule. Putative actives from
the primary screen are then verified by immunostaining and qRT-PCR
analysis of an array of photoreceptor-associated genes.
Results: We have developed an assay with a signal-to-background
ratio of 3.2 and a coefficient of variation <20%. As an example
of the potential of this screening paradigm, we have identified a
small molecule that conferred a 4-fold increase in the number of
differentiated rhodopsin-EGFP cells, and a 100% increase in the
expression of QRX-GFP. Gene expression analysis of photoreceptor-
associated genes showed a significant increase in expression of
rhodopsin, NR2E3, NRL, Gnat1, and Pdc.
Conclusions: We have developed a primary cell-based high
throughput screen to identify small molecules that influence
photoreceptor development and survival. Our preliminary screening
results demonstrate that our approach can successfully identify
photoreceptor differentiation and survival promoting molecules, and
ongoing screening will likely identify additional and more potent
such molecules. It is hoped that this work will identify potential
preclinical candidates, as well as molecular probes that will be useful
for analysis of the molecular mechanisms that mediate photoreceptor
differentiation and survival.
Commercial Relationships: John A. Fuller, Johns Hopkins
University (P); Karl Wahlin, None; Cynthia Berlinicke, None;
Douglas Yasumura, None; Michael T. Matthes, None; Ryan
MacArthur, None; Patricia Dranchak, None; Matthew M. LaVail,
None; James Inglese, None; Donald J. Zack, Johns Hopkins
University (P)
Support: NIH, Foundation for Fighting Blindness: Wynn-Gund
TRAP, Research to Prevent Blindness
Program Number: 1912 Poster Board Number: B0177
Presentation Time: 11:00 AM–12:45 PM
An oxo-indolinone that targets mitochondria reduces
photoreceptor cell loss in models of retinitis pigmentosa (RP)
Mausumi Bandyopadhyay1, Cecile Nasarre1, Nathan Perron1,
Christopher Lindsey2, 3, Richard Comer2, Craig Beeson2, 3, Baerbel
Rohrer1, 4. 1Ophthalmology, MUSC, Charleston, SC; 2Mitochem,
Charleston, SC; 3Pharmaceutical Sciences, MUSC, Charleston, SC;
4
Ralph H. Johnson VA Medical Center, Charleston, SC.
Purpose: Mitochondria generate the majority of a cell’s energy and
integrate life and death decisions. The process of fission/fusion,
biogenesis and mitophagy is critical to maintain mitochondrial
integrity and thereby cellular health. We have hypothesized that loss
of mitochondrial homeostasis underlies degenerative pathologies.
Previously, we screened for molecules that prevent loss of
mitochondrial homeostasis during metabolic stress to protect against
cell loss. Here we examined the effects of one of the compounds,
1-butyl-3-hydroxy-3-(2-oxo-2-(pyridin-2-yl) ethyl) indolin-2-one
(termed CB11), in models of RP.
Methods: Effects of CB11 were tested for efficacy in reducing cell
loss in vitro (661w cells treated with calcium ionophore A23187), in
situ (organ cultures of genetic RP models) and in vivo (constant light
damage in Balb/c mice). Cell loss was quantified using MTT assays
or cell counts. Cytotoxicity was determined by dose-escalation. The
effects on mitochondrial capacity were assessed using Seahorse
respirometry. Quantitative RT-PCR was performed to assess potential
mechanisms of action for CB11. Finally, biodistribution of CB11 was
determined in pig eyes after topical administration, using HPLC.
Results: CB11 increased survival and mitochondrial function in
661w cells challenged with A23187 or IBMX, respectively, but had
no cytotoxic effects in two cell lines. Photoreceptor loss in RPEretina explants derived from rd1 mouse or S334ter rat pups was
significantly attenuated when treated with CB11 in the culture media.
Similarly, daily eyedrops with CB11 reduced photoreceptor cell loss
in Balb/c mice exposed to constant light as shown in cell counts
and protein content for rhodopsin. In these retinas, mRNA levels for
genes involved in mitochondrial dynamics were found to be elevated.
Finally, CB11 accumulated in the pig retina at physiologically
relevant concentrations 2 hours after eye drop application.
Conclusions: Hence, irrespective of the trigger for degeneration
(environmental or genetic mutations), CB11 reduced photoreceptor
cell loss. Since CB11 increased mitochondrial respiratory capacity
©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
and expression for genes involved in mitochondrial dynamics,
we suggest that CB11 targets mitochondria. Improvement of
mitochondrial integrity and capacity allows for maintenance of
cellular homeostasis and thereby extends the life span of cells.
Commercial Relationships: Mausumi Bandyopadhyay, None;
Cecile Nasarre, None; Nathan Perron, US119869 (P); Christopher
Lindsey, US119869 (P); Richard Comer, None; Craig Beeson,
US119869 (P); Baerbel Rohrer, Mitochem Therapeutics (F),
US119869 (P)
Support: WG-TRAP award from Foundation Fighting Blindness and
SRA from Mitochem Therapeutics
Program Number: 1913 Poster Board Number: B0178
Presentation Time: 11:00 AM–12:45 PM
670nm LED Photobiomodulation is Retinoprotective in a
Transgenic Mouse Model of Parkinson’s Disease
Michele Salzman1, Kristina DeSmet2, Harry T. Whelan3, Ellen V.
Buchmann3, Janis T. Eells4. 1Marshfield Clinic Research Foundation,
Marshfield, WI; 2Stiefel, a GSK Company, Raleigh-Durham, NC;
3
Neurology, Medical College of Wisconsin, Milwaukee, WI; 4College
of Health Sciences, University of Wisconsin-Milwaukee, Milwaukee,
WI.
Purpose: Parkinson’s disease (PD) is a progressive
neurodegenerative disease characterized by specific altered motor
symptoms. These movement alterations are due to the loss of
dopaminergic neurons in the substantia nigra and the accumulation
of Lewy bodies consisting primarily of alpha-synuclein. Non-motor
symptoms, including altered visual functions, are also associated
with PD. Our laboratory previously documented the neuroprotective
effects of 670nm LED photobiomodulation (PBM) in a transgenic
mouse model of Parkinson’s disease. The purpose of the present
study was to examine the potential of 670nm LED PBM to protect
dopaminergic amacrine cells in the retina of these same mice.
Methods: Studies were conducted in a transgenic mouse model
of PD that expresses the A53T mutation of alpha-synuclein. These
transgenic mice develop a Parkinson’s-like syndrome characterized
by neurodegenerative changes in the basal ganglia and severe motor
dysfunction. Two therapeutic protocols were tested, a prevention
protocol and a treatment protocol. For the prevention protocol, mice
received 670nm LED PBM (300 sec at 25 mW/cm2; 7.5 J/cm2) or
sham treatment 3 times weekly beginning at 2 months of age and
extending to 20 months of age. For the treatment protocol, the 670nm
LED PBM treatment was initiated at 8 months of age and extended to
20 months of age.
Results: The onset of Parkinsonian motor symptoms was
significantly delayed in both therapeutic protocols. Moreover, striatal
dopamine concentrations were significantly greater in the PBMtreated mice compared to sham-treated mice indicative of protection
of the nigrostriatal pathway. Retinal dopamine concentrations were
also significantly greater in PBM-treated mice than in sham-treated
mice indicative of protection of dopaminergic amacrine cells in the
retina.
Conclusions: These data document the neuroprotective and
retinoprotective actions of 670nm LED PBM in an experimental
model of PD and support the potential of PBM in the treatment of
Parkinson’s disease.
Commercial Relationships: Michele Salzman, None; Kristina
DeSmet, None; Harry T. Whelan, None; Ellen V. Buchmann,
None; Janis T. Eells, QBMI Photomedicine (C)
Support: NIH Grant 5R21AT3002-2, NIH Grant EY001931, and the
Bleser & Baumann Endowments
Program Number: 1914 Poster Board Number: B0179
Presentation Time: 11:00 AM–12:45 PM
Neurodegenerative Role of HIF-1α in Glaucomatous Injury.
Margaret Brown, Yasir Abdul, Sudha Singh, Melissa Nix, Shahid
Husain. Medical University of South Carolina, Charleston, SC.
Purpose: This study was designed to determine the
neurodegenerative role of hypoxia-inducible transcription factor1α (HIF-1α) during glaucomatous injury in a chronic glaucoma rat
model.
Methods: Brown Norway rats were used to elevate intraocular
pressure (IOP) by injecting 50 mL of 2 M hypertonic saline into the
circumferential limbal veins. IOP was recorded as the average of 6-8
consecutive measurements using a calibrated Tonolab tonometer.
Animals were treated with HIF-1α inhibitor (17-DMAG; 2 mg/
kg; i.p.) right after injury and subsequently daily for 7 days. The
levels of HIF-1α expression was measured by Western blotting and
immunohistochemistry. The functional deficiencies were measured by
Pattern electroretinograms (PERG). Primary cultures of human optic
nerve head (ONH) astrocytes were treated with hypoxia (oxygen,
glucose deprivation [OGD]) 24 hours and changes in HIF-1α
expression and cytokines were measured by Western blotting.
Results: The mechanisms of retinal ganglion cell (RGC) death
in response to glaucomatous injury are not clearly defined, some
evidence suggests that tissue hypoxia may adversely affect RGC
survival via pro-apoptotic pathways. Immunohistochemistry data
show an up-regulation of HIF-1α in rat glaucomatous retina on day 7,
post injury, which remained elevated up to 28 days. HIF-1α staining
was mainly localized to the nerve fiber layer (NFL) and RGC layer.
Additionally, Western blotting data show an up-regulation of HIF-1α
by 51±15 and 54 ±14 % at days 7 and 28, respectively, post injury.
Pattern-ERGs were not changed at day 7 post injury, while HIF1α was increased at 7th day. In contrast, pattern-ERG amplitudes
were reduced by 25% by day 28 post injury. We measured HIF-1α
expression in response to hypoxia-related stress (oxygen, glucose
deprivation [OGD]) for 24 hours. OGD increased the expression of
HIF-1α (742 ±104%), TNF-α (103 ±19%), and IL-1β (141 ±15%)
over the control levels, respectively.
Conclusions: We conclude that hypoxia develops in an early stage
of the glaucoma pathology. In addition, data supports an idea that
under glaucomatous injury hypoxic microenvironments develop not
only in RGCs but also in ONH astrocytes. Such changes during the
progression of glaucoma will lead to the production of neurotoxic
proteins (e.g., HIF-1α and cytokines) and that will subsequently lead
to the RGC death.
Commercial Relationships: Margaret Brown, None; Yasir Abdul,
None; Sudha Singh, None; Melissa Nix, None; Shahid Husain,
None
Support: NIH/NEI EY019081 and Research to Prevent Blindness
Program Number: 1915 Poster Board Number: B0180
Presentation Time: 11:00 AM–12:45 PM
Overexpression of the POU Domain Transcription Factor Brn3b
Causes Neurite Outgrowth in Cultured PC 12 Cells Under
Condition of Oxygen Glucose Deprivation
Nitasha R. Phatak1, 2, Dorota L. Stankowska1, 2, Raghu R.
Krishnamoorthy1, 2. 1Cell Biology and Immunology, Univ of North
Texas Hlth Sci Ctr, Fort Worth, TX; 2North Texas Eye Research
Instutute, Fort Worth, TX.
Purpose: Brn3b is a POU domain transcription factor shown to
play a key role in regulating retinal ganglion cell axon outgrowth
during development. Hypoxia is a contributing factor in many
neurodegenerative diseases including glaucoma. The purpose of this
study was to determine if overexpression of Brn3b could promote
©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
neurite outgrowth in cultured PC 12 cells during conditions of
oxygen glucose deprivation (OGD).
Methods: Rat Pheochromocytoma cells (PC12) were grown
on poly-D-lysine coated 100 mm dishes and transfected either
with pCMV6-Brn3b (an expression vector encoding Brn3b) or
pCMV6-Empty (empty vector). After 6 h of transfection, cells were
maintained overnight in a differentiating medium containing NGF
(100ng/ml). Then, the cells were transferred to glucose free DMEM
and maintained for 2 h in 0.5% O2 and 5% CO2 (for hypoxia) in
an Invivo2 200 hypoxia chamber. For the normoxia controls, PC12
cells overexpressing Brn3b or Empty vector were maintained in
differentiating medium for 2 h in 5% CO2 and 95% air in a standard
incubator. Protein extracts were isolated from these cells and
analyzed for Brn3b and GAP43, TUBA-1 protein expression by
immunoblot analysis. In another set of experiments, PC 12 cells were
seeded on Poly-D-Lysine coated 25mm cover slips and transfected
with either pCMV6-Brn3b or pCMV6 -Empty and maintained in
differentiating medium for 4 days. The cells were subjected to either
hypoxia (2h) or normoxia. Brn3b, GAP43 and TUBA-1 expression
were analyzed using immunocytochemistry. Morphological changes
in PC 12 cells transfected with Brn3b were studied by using confocal
microscopy.
Results: Immunoblot analysis confirmed overexpression of Brn3b
in PC12 cells transfected with Brn3b cDNA in normoxic as well as
in OGD conditions. Interestingly, a marked upregulation of GAP-43
and ac-TUBA expression was observed in Brn3b overexpressing cells
under conditions of normoxia and OGD. Overexpression of Brn3b in
PC12 cells produced a statistically significant increase in maximum
neurite length and number of neurites per cell under both conditions.
A marked increase in immunostaining for Brn3b and neurite-specific
GAP43, TUBA-1 were also observed in PC12 cells overexpressing
Brn3b in condition of normoxia and OGD.
Conclusions: The transcription factor Brn3b could promote neurite
outgrowth in PC12 cells under conditions of normoxia and OGD.
Commercial Relationships: Nitasha R. Phatak, None; Dorota L.
Stankowska, None; Raghu R. Krishnamoorthy, None
Program Number: 1916 Poster Board Number: B0181
Presentation Time: 11:00 AM–12:45 PM
Evaluation of a Novel, Reversible, Fluorescent Probe for the
Assessment of Retinal Oxidative Status
Nigel L. Barnett1, 2, Cassie Rayner1, Glen A. Gole2, Steven E.
Bottle3. 1Queensland Eye Institute, South Brisbane, QLD, Australia;
2
University of Queensland, Brisbane, QLD, Australia; 3Queensland
University of Technology, Brisbane, QLD, Australia.
Purpose: To evaluate the utility of a novel, reversible, profluorescent
nitroxide (PFN) probe that selectively detects superoxide radicals in
live cells, as a reporter of in vivo retinal oxidative status in a model of
retinal metabolic challenge.
Methods: Following an intravitreal injection of PFN (2 mM),
unilateral acute retinal ischaemia was induced in anaesthetized
Sprague Dawley rats by elevation of intraocular pressure (IOP) to
120 mmHg for 60 mins. After restoration of normal IOP, retinal
fluorescence (556 nm / 590 nm) was assessed at various time-points
(5, 10, 15, 30, 45, 60 mins) during reperfusion using a Micron III
rodent fundus camera. Changes in fluorescence were quantified with
Image J and compared with the fluorescence intensity measured
before the ischaemic insult. Control fluorescence time-course data
were obtained from the non-ischaemic contralateral eyes. The effects
of known antioxidants, lutein (0.2 mg/kg i.p.) and edaravone (3 mg/
kg i.p.), either alone or upon the ischaemia/reperfusion-induced
fluorescence response, were quantified. The effect of intraocular PFN
on retinal function was assessed by electroretinography (ERG).
Results: Restoration of blood flow after retinal ischaemia, which
stimulates free radical production, induced a marked decrease in
retinal PFN probe fluorescence intensity (59.8 ± 4.3 SEM % of the
pre-ischaemic value, n=5, at 15 mins reperfusion). Administration of
lutein or edaravone ameliorated the ischaemia/reperfusion-induced
decrease in retinal PFN fluorescence: lutein (increased to 85.5 ± 6
%pre-ischaemic, n=7), edaravone (increased to 88.5 ± 3.1 %preischaemic, n=4). The antioxidants did not significantly alter the
fluorescence intensity in non-ischaemia/reperfusion retinas. The
intraocular injection of the PFN probe did not adversely affect the
ERG a- or b-waves.
Conclusions: PFN probes can detect changes in retinal oxidative
status in real-time in vivo, under pro-oxidant and anti-oxidant
conditions. Because the probes are reversible and react to both
reducing and oxidizing conditions, we can look for the first time at
novel anti-oxidant treatment effects in real-time for the myriad retinal
diseases that involve oxidative stress.
Commercial Relationships: Nigel L. Barnett, None; Cassie
Rayner, None; Glen A. Gole, None; Steven E. Bottle, None
Support: Ophthalmic Research Institute of Australia
Program Number: 1917 Poster Board Number: B0182
Presentation Time: 11:00 AM–12:45 PM
Changes in Endothelin A Receptor (ETA) Expression in a Rat
Model of Ocular Hypertension
Nolan McGrady1, 2, Alena Z. Minton1, 2, Raghu R. Krishnamoorthy1.
1
Cell Biology and Immunology, UNT Health Science Center, Fort
Worth, TX; 2Visual Sciences, UNT Health Science Center, Fort
Worth, TX.
Purpose: The endothelin system of peptides and their receptors have
been implicated for their neurodegenerative role in glaucoma. The
purpose of this study was to determine changes in the ETA receptor
expression in the retina in the Morrison’s elevated IOP model of
glaucoma in rats.
Methods: IOP was elevated in the left eye of adult male retired
breeder Brown Norway rats using the Morrison’s model of glaucoma
(by injection of hypertonic saline through episcleral veins) while
the contralateral eye served as the control. The rats were maintained
for two to four weeks following IOP elevation and sacrificed.
Retinal sections were obtained from both control and IOP-elevated
eyes, and analyzed for changes in ETA receptor expression using
immunohistochemistry. ETA receptor immunostaining was colocalized with β-III-Tubulin, which is selectively expressed in retinal
ganglion cells.
Results: After two weeks of IOP elevation, rat eyes with IOP
elevation showed an increase in immunostaining for ETA receptors in
several retinal layers including the inner and outer plexiform layers,
with a modest increase in the retinal ganglion cell layer. Following
four weeks of IOP elevation, ETA receptor expression was slightly
increased in the inner and outer plexiform layers of the retina,
compared to that in the corresponding contralateral eyes.
Conclusions: Elevated intraocular pressure results in a timedependent change in ETA receptor expression. Increased ETA
receptor expression is associated with neurodegenerative changes in
glaucoma.
Commercial Relationships: Nolan McGrady, None; Alena Z.
Minton, None; Raghu R. Krishnamoorthy, None
Support: NIH 1R01EY019952
©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: 1918 Poster Board Number: B0183
Presentation Time: 11:00 AM–12:45 PM
Endothelins’ effects on gene expression in rat retinal ganglion
cells
Shaoqing He1, Yong H. Park2, Thomas Yorio2, Raghu R.
Krishnamoorthy1. 1Cell Biology and Immunology, University of
North Texas Hlth Sci Ctr, Fort Worth, TX; 2Pharmacology and
Neuroscience, University of North Texas Hlth Sci Ctr, Fort Worth,
TX.
Purpose: A growing body of evidence suggests that endothelin (ET),
a family of 21-amino-acid vasoactive peptides, and their receptors
(ETA and ETB receptors) are contributors to the neuronal damage in
glaucoma. However, ET’s actions in retinal ganglion cells (RGCs)
are not fully understood. The aim of this study is to reveal the roles of
ETs and their receptors in primary rat retinal ganglion cells.
Methods: Primary RGCs were isolated from postnatal day 4-6
rats by panning with Thy-1 antibody. After 7 days culture, isolated
RGCs were treated with 100nM of ET-1, ET-2 or ET-3 for 24 hours.
Total RNA was extracted using Qiagen RNeasy Mini kit followed
by cDNA synthesis and a gene microarray analyses. Affymetrix Rat
Genome 230 2.0 Microarray was used to analyze the gene expression
in RGCs in response to 100nM ET-1, ET-2 and ET-3 treatments.
Realtime PCR to detect gene expression was used to validate the
result of Microarray, and immunocytochemical staining was used
to confirm the protein expression of regulated genes in RGCs in the
same treatment conditions.
Results: 31100 gene transcripts and variants from over 28000 rat
genes were detected using the probe chip. There was a more than 2.5
fold up-regulation of 1897, 2459 or 2259 genes and down-regulation
of 561, 409 or 343 genes with ET-1, ET-2 or ET-3 treatment
respectively, compared with control. In real-time PCR validation,
there was no appreciable change for Bax, Caspase-2, Caspase-8
and c-Jun. Gene expression of ETA and Bcl-2 in ET-1 treatment was
decreased to 70% of control, whereas ETB increased to 13.6 fold
compared to sham control. Immunostaining showed a significant
increase in ETA, ETB, GAP-43, phosphorylated c-Jun, c-Jun and C/
EBPβ with ET-1 treatment, and a slight increase in Bax, Bim and
Bcl-XL.
Conclusions: Endothelins induced profound expression alteration in
varieties of genes including cytokines, structural proteins, signaling
pathways, transcription factors and matrix molecules in RGCs,
and also triggered significant changes in neuronal gene expression.
The exploration of endothelins’ roles will help understanding on
molecular mechanisms underlying glaucomatous changes with ocular
hypertension.
Commercial Relationships: Shaoqing He, None; Yong H. Park,
None; Thomas Yorio, None; Raghu R. Krishnamoorthy, None
Support: NIH Grant EY019952 to RRK
Program Number: 1919 Poster Board Number: B0184
Presentation Time: 11:00 AM–12:45 PM
Development of Biomarker Equations based on MultiDimensional Modeling of Cytokine Shifts in Retinal Diseases
Gianna C. Teague1, Namrata Nandakumar1, Jie Ma1, Kyle V.
Marra2, Jorge G. Arroyo2, Megan E. Baldwin3, Walter Johnson4,
Kameran Lashkari1. 1Schepens Eye Research Institute, Massachusetts
Eye & Ear Infirmary, Boston, MA; 2Ophthalmology, Beth Israel
Deaconess Medical Center, Boston, MA; 3Opthea Pty Ltd, Circadian
Technologies Ltd, South Yarra, VIC, Australia; 4Physics, Suffolk
University, Boston, MA.
Purpose: We have hypothesized that levels of individual cytokines
reflect underlying disease processes. Cumulative (multi-dimensional)
shifts among cytokine families may be used to define specific disease
processes and lead to novel disease-specific biomarkers.
Methods: Human vitreous samples collected from a variety of retinal
diseases were subjected to multiplex analysis of cytokine markers.
The five disease groups examined were proliferative diabetic
retinopathy (PDR), proliferative vitreoretinopathy (PVR), retinal
vascular occlusions (RVO) and neovascular glaucoma (NVG). The
control samples included epiretinal membranes, floaters and macular
holes. Mean, chi-squares and t-scores of log-transformed data were
calculated and used to model multi-dimensional equations.
Results: From the 35 cytokines examined, 29 were selected for
which a Gaussian distribution of log-transformed data was observed
in the control group. Single cytokine analysis for individuals in
particular disease groups showed that certain diseases were associated
with large t-score shifts from the controls for specific cytokines. The
three cytokines showing the greatest shifts for the diseases studied are
as follows: For PDR, PLGF, VEGF-A, and TNF-α; for PVR, TIE-2,
Prolactin, and FGF-basic; for RVO: PLGF, VEGF-A, and EGF; for
NVG: PLGF, VEGF-A, and EGF.
Conclusions: Elevated t-scores correlate with the impact of a
particular cytokine on a specific disease process. Biomarker equations
could be derived from multi-dimensional t-scores to adequately
predict ocular disease processes and distinguish one disease from
another.
Commercial Relationships: Gianna C. Teague, None; Namrata
Nandakumar, None; Jie Ma, None; Kyle V. Marra, None; Jorge
G. Arroyo, None; Megan E. Baldwin, Circadian Technologies Ltd
(E); Walter Johnson, None; Kameran Lashkari, None
Support: Grant supported by Opthea Pty Ltd, Circadian
Technologies Limited
Program Number: 1920 Poster Board Number: B0185
Presentation Time: 11:00 AM–12:45 PM
Adverse Retinal Effect of Fenretinide in Association with
Reduced Pigmentation
Leif E. Johnson1, 2, Michael Larsen2, Maria-Thereza Perez1. 1Division
of Ophthalmology, Lund University, Lund, Sweden; 2Dept. of
Ophthalmology, Glostrup Hospital, Glostrup, Denmark.
Purpose: Fenretinide is a synthetic retinoid derivative used e.g.,
to treat some forms of cancer. Fenretinide reduces serum levels
of retinol and can lead to night blindness, which is reversible.
This compound has also been tested in patients with dry macular
degeneration and geographic atrophy (GA) with positive results. The
purpose of this study was to analyze the effect of fenretinide in other
models of photoreceptor degeneration.
Methods: Pink-eyed Royal College of Surgeon (RCS) rats, a model
of Retinitis Pigmentosa (RP), pigmented Brown Norway (BN) rats
and albino Sprague Dawley (SD) rats received 20 mg/kg fenretinide
intraperitoneally every second day from postnatal (PN) day 10 to
PN22 and were sacrificed at PN24. Two fully pigmented mouse
models of RP, the rd1 and rd2 strains, and congenic C3H mice
received 20 mg/kg fenretinide daily from PN7 through PN13 (rd1) or
PN22 (rd2 and C3H control). All mice were sacrificed the day after
the final injection. Retinas were analyzed using hematoxylin-eosin
staining and the TUNEL in situ cell-death assay.
Results: Fenretinide did not delay photoreceptor cell death in the
models of RP. On the contrary, degeneration was accelerated in
treated RCS rats, with the outer nuclear layer being reduced to about
half the thickness of age-matched controls. A similar deleterious
effect was not observed in the pigmented rd1 and rd2 mouse models.
On the other hand, fenretinide also induced an almost sevenfold
increase in the number of TUNEL positive photoreceptors in albino
SD rats, but did not appear to have any adverse effect on BN rats.
©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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Conclusions: Fenretinide accelerated retinal degeneration in selected
strains of rats with preexisting degenerative disease related to
pigmentation defects. This negative effect was further compounded in
the RCS rat, in which there was an exceptionally rapid thinning of the
outer nuclear layer, probably because photoreceptors are particularly
vulnerable in this strain due to disease-induced stress.
Commercial Relationships: Leif E. Johnson, None; Michael
Larsen, None; Maria-Thereza Perez, None
Support: Velux Stiftung, Crown Princess Margareta’s Committee
for the Blind, Stf för Synskadade fd Malmöhus Län, Greta och Johan
Kocks Stf, Edwin Jordans Stf för Oftalmologisk Forskning, Gun och
Bertil Stohnes Stf, Magnus Bergvalls Stf, Beckett Foundation, Spar
Nord Foundation.
Program Number: 1921 Poster Board Number: B0186
Presentation Time: 11:00 AM–12:45 PM
Retinal function and morphology in the rabbit eye after
intravitreal injection of the TNF alpha inhibitor adalimumab
Anna Cardiakidis Myers, Fredrik K. Ghosh, Sten Andreasson, Vesna
Ponjavic. Ophthalmology, Lund University, Lund, Sweden.
Purpose: To study the effects of the tumor necrosis factor alpha
inhibitor adalimumab on rabbit retina after injection into the vitreous
body.
Methods: 48 rabbits of mixed strain (9-12 months old, weighing ≈
3,5 kg) were randomized into four groups. Adalimumab was injected
at one of two concentrations (1.25 mg or 2.5 mg) into the eyes of two
groups, and balanced salt solution into the eyes of the third group.
The fourth group acted as controls. Full-field electroretinography
(ffERG) was performed before injection and 1 and 6 weeks postinjection. At 6 weeks post-injection the rabbits were euthanized and
the sectioned retinas were studied. Retinal histology was studied with
hematoxylin - eosin staining. Immunohistochemical analysis was
performed on rods, cones, rod bipolar cells, horizontal cells, amacrine
cells and Müller cells.
Results: No significant difference in ffERG amplitudes or implicit
times was observed between the four groups at any time point.
Histological and immunohistochemical findings were similar in all
groups.
Conclusions: Injection of adalimumab into the vitreous body of
healthy rabbits, at doses up to 2.5 mg, does not appear to be toxic to
the rabbit retina.
Commercial Relationships: Anna Cardiakidis Myers, None;
Fredrik K. Ghosh, None; Sten Andreasson, None; Vesna Ponjavic,
None
Support: Synskadade i fd Malmöhus län, Stiftelsen Olle Engkvist,
Stiftelsen Synfrämjandets Forsknings-fond, Kronprinsessans
Margaretas Arbetsnämnd, Torsten and Ragnar Söderbergs Stiftelser,
the Swedish Medical Research Council (project no. 2007-3385) and
the Faculty of Medicine at Lund University
Program Number: 1922 Poster Board Number: B0187
Presentation Time: 11:00 AM–12:45 PM
Changes in the ERG D-wave with Vigabatrin Treatment in a
Pediatric Cohort
Rachel Dragas1, Carol A. Westall1, 2, Thomas Wright1.
1
Ophthalmology and Vision Sciences, The Hospital for Sick Children,
Toronto, ON, Canada; 2Ophthalmology and Vision Sciences; Institute
of Medical Science, The University of Toronto, Toronto, ON, Canada.
Purpose: Vigabatrin (VGB), a common treatment for the childhood
epilepsy, infantile spasms (IS), is implicated in visual field reduction.
Electroretinograms (ERGs) are a substitute to visual field testing in
infants and VGB-associated ERG reduction (VAER) is a reduction
in age-corrected light adapted 3.0 flicker response amplitude from a
pre-treatment measurement. The d-wave ERG response is the result
of OFF bipolar cell depolarization to light offset. The purpose of
this study is to evaluate if the ERG d-wave response is a marker for
VAER toxicity in a pediatric population.
Methods: Eighty-seven children with IS treated with VGB were
assessed prospectively and tested for the cone-OFF response elicited
to a 250 cd.s.m2 flash with 200 ms duration. Twenty with IS had
been tested before VGB and 9 of the 87 developed VAER during the
time frame of the study. Thirteen retinally normal controls were also
tested. Amplitude and implicit timing of the d-wave response were
measured manually.
Results: There was no difference in d-wave amplitude between the
IS group tested before VGB treatment (IS-baseline) and controls.
Combining data from the IS-baseline and control group, there was no
effect on d-wave amplitude whilst there was a reduction in implicit
time with increasing age. The d-wave amplitude was reduced in the
IS group with VAER compared to those without VAER (p<0.01).
Longer duration of treatment was associated with reduced d-wave
amplitude (anova p=0.054) in those with and without VAER.
Conclusions: Reduced amplitude of the cone OFF response may be
good indicator for VGB-induced adverse retinal changes.
Commercial Relationships: Rachel Dragas, None; Carol A.
Westall, Lundbeck Pharmaceuticals (F); Thomas Wright, None
Support: This research is supported by funding from Lundbeck
Pharmaceuticals
Program Number: 1923 Poster Board Number: B0188
Presentation Time: 11:00 AM–12:45 PM
Oral Mineralocorticoid Antagonists for the Treatment of Central
Serous Chorioretinopathy
Eric K. Chin, David Almeida, Stephen R. Russell, James C. Folk.
Ophthalmology and Visual Sciences, University of Iowa Hospitals &
Clinics, Iowa City, IA.
Purpose: To evaluate the effect and tolerance of oral
mineralocorticoid antagonists, eplerenone and/or spironolactone, in
central serous chorioretinopathy (CSCR).
Methods: We performed a retrospective observational case series.
The medical records of one-hundred twenty patients diagnosed
with CSCR between January 1, 2012 and September 1, 2013 were
reviewed. Twenty-nine patients were treated with one or more
mineralocorticoid antagonists. Six patients were excluded from final
analysis. Primary outcome measures included best corrected visual
acuity (BCVA, Snellen), central macular thickness (CMT, μm), and
macular volume (MV, mm^3) via Spectralis® Heidelberg OCT.
Secondary outcomes included duration of treatment, treatment dose,
tolerable and intolerable side effects, and prior treatment failures.
Results: The average age was 58.4 ± 10.5 years (range, 36.9-72.6
years) and 15 patients (65.2%) were male. Nine patients (39.1%)
had a history of prior steroid use. Sixteen patients (69.6%) had
been previously treated with other interventions (e.g. ketoconazole,
rifampin, anti-vascular endothelial growth factor agents, or
photodynamic therapy) before trying oral mineralocorticoid
antagonists. Fifteen patients were treated with eplerenone only,
three patients were treated with spironolactone only, and five
patients were treated with spironolactone following a trial of
eplerenone. Medication dose varied from 25 mg to 50 mg twice
daily. The average entire duration of treatment (eplerenone and/or
spironolactone), or time to nearest follow-up while on treatment, was
3.9 ± 2.3 months (range, 1-8.5 months). Twelve patients (52.2%)
showed decreased CMT and MV, six patients (26.1%) had increased
in both, and five patients (21.7%) had negligible changes. For all
patients, the mean decrease in CMT from start of therapy to final
follow-up was 42.4 μm (range, -136 to 255 μm), and the mean
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ARVO 2014 Annual Meeting Abstracts
decrease in MV was 0.20 mm^3 (range -2.33 to 2.90 mm^3). Median
BCVA at start of therapy was 20/30 (range, 20/20-20/250), and at
final follow-up 20/40 (range, 20/20-20/125). Nine patients (39.1%)
experienced systemic side effects, of which three patients (13.0%)
were unable to tolerate continuation of therapy.
Conclusions: Mineralocorticoid-antagonist treatment had a positive
treatment effect in half of our patients who may have not responded
to other therapies. Systemic side effects, even at low doses, may limit
its usage in some patients.
Commercial Relationships: Eric K. Chin, None; David Almeida,
None; Stephen R. Russell, None; James C. Folk, None
Program Number: 1924 Poster Board Number: B0189
Presentation Time: 11:00 AM–12:45 PM
Spironolactone reduces sub retinal fluid and choroidal thickness
in CSR
Francine F. Behar-Cohen1, 3, Elodie Bousquet2, 3, Talal Beydoun2,
Min Zhao2, 3, Pierre Raphael Rothschild3, Alain Gaudric4, Emmanuel
Curis5, François Chast6. 1Jules Gonin Hospital, University of
Lausanne, Paris, France; 2University Paris Descartes, Hotel-Dieu of
Paris Department Ophthalmology, Paris, France; 3Physiopathology
of ocular diseases: Therapeutic Innovations, Inserm UMRS 872,
Paris, France; 4Ophthalmology, Lariboisière Hospital, Paris, France;
5
Statistic, Université Paris Descartes, Paris, France; 6Pharmacy,
Cochin-Hôtel-Dieu Hospital, Paris, France.
Purpose: We have previously shown that mineralocorticoid receptors
(MR) activation by high doses glucocorticoids induced choroid
vessels dilatation and leakage in rats through endothelial SK3 channel
upregulation. Specific MR antagonists prevented glucocorticoidsinduced choroidal enlargement. The aim this study is test whether
spironolcatone exerts significant effect of sub retinal fluid and
choroidal enlargment in patients with chronic Central Serous
Chorioretinopathy (CSR).
Methods: Sixteen patients with chronic CSR (> 4 months) were
randomized to either oral spironolactone (50mg/d) or placebo for
30 days. After 8 days wash-out period, patients were switched to
the other treatment for 30 days and followed up to 90 days. The
primary endpoint was the reduction of sub-macular fluid, measured
by thickness and volume from ELM to bruch, or ILM to bruch using
segmentation methods. The secondary endpoint was the reduction of
sub foveal macular choroidal thickness measured by EDI-OCT.
Statistical analysis Crossover data were analyzed using the linear
mixed effects model framework ; Since a strong and asymetric carryover effect was present, analysis was made on the first period results
only. This analysis was made on the absolute and relative difference
between inclusion and day30. These differences were compared using
Student’s T-test or Mann-Whitney test, according to the results of the
normality tests for the crossover analysis. Difference between the two
groups was considered significant for p < 0.05
Results: The differences of geometrical parameters (width, volume
of whole, intern or extern retina) were very strongly correlated (>
0.98). Reduction of subretinal fluid was significantly reduced in the
spironolactone treated group as compared to the placebo treated
group, either when measuring the ELM / bruch thickness or the
CMT; and also when measuring the sub retinal volume or central
macular volume. Moreover, the macular choroidal thickness was
significantly reduced after spironolactone treatment but not after
placebo treatment.
Conclusions: In this prospective randomized controlled study, even
if the number of patients is low, the results clearly show a significant
effect of spironolactone after 30 days of treatment on macular and
choroidal thickness validating the hypothesis that MR is involved in
CSR physiopathogenesis.
Commercial Relationships: Francine F. Behar-Cohen, inserm
(P); Elodie Bousquet, None; Talal Beydoun, None; Min Zhao,
None; Pierre Raphael Rothschild, None; Alain Gaudric, None;
Emmanuel Curis, None; François Chast, None
Support: Inserm
Clinical Trial: NCT01552044
Program Number: 1925 Poster Board Number: B0190
Presentation Time: 11:00 AM–12:45 PM
Treatment of Central Serous Retinopathy with the
Mineralocorticoid Receptor Antagonist Eplerenone
Robert Beardsley, Sandra Montezuma, Richard Johnston.
Ophthalmology and Visual Neurosciences, University of Minnesota,
Minneapolis, MN.
Purpose: To evaluate the efficacy of treatment of chronic central
serous retinopathy (CSR) with the mineralocorticoid receptor
antagonist eplerenone.
Methods: IRB approval was obtained through the University of
Minnesota. Subjects with CSR based on symptoms, biomicroscopy,
fluorescein angiography (FA) and optical coherence tomography
(OCT) were enrolled. Subjects must have had symptoms and
subretinal fluid on OCT for at least three months prior to study
enrollment. Previous treatment was not exclusionary however
concomitant therapy was. Exclusion criteria included co-existing
macular disease, pre-existing electrolyte anomalies, or previous
sensitivity to eplerenone. Baseline best corrected visual acuity
(BCVA), OCT, and FA was obtained on each subject prior to study
drug initiation. Each subject was given 25mg eplerenone twice daily
for 1 week, subsequently increased to 50mg twice daily. Subjects
were monitored for response to therapy over 3 months. Baseline
electrolyte studies were obtained as well as at 1 month and 3 months.
The primary outcome was change in central macular thickness
(CMT) based on OCT. Secondary outcomes included change in best
corrected visual acuity and subjective visual response.
Results: A total of at least ten subjects were enrolled. At least eight
were enrolled for the study duration of three months. Six patients
showed statistically significant OCT improvement of CMT. In terms
of secondary endpoints, four subjects showed improvement of BCVA
by at least one line and eight said that their vision was subjectively
improved. There were no complications to the therapy and no
electrolyte anomalies.
Conclusions: Eplerenone shows efficacy in the treatment of chronic
CSR with a low side effect profile. Larger, randomized trials are
needed to further refine its utility in clinical settings.
Commercial Relationships: Robert Beardsley, None; Sandra
Montezuma, None; Richard Johnston, None
Program Number: 1926 Poster Board Number: B0191
Presentation Time: 11:00 AM–12:45 PM
Eplerenone use to treat Central Serous Chorioretinopathy
Javier Moreno. Grupo VISTA Gutiérrez Amorós, La Coruña, Spain.
Purpose: Based on experimental data showing that Central Serous
Chorioretinopathy (CSC) could result from overactivation of
mineralocorticoid receptor pathway in choroid vessels, eplerenone,
a mineralocorticoid receptor antagonist, was studied as a potential
treatment for CSC.
Methods: Three patients diagnosed of unilateral CSC were treated
with eplerenone 25 mgrs/day. All three patients did not received
any previous treatment, all of them had an onset of symptoms for
more than three months. Prior to treatment, Visual Acuity (VA) was
taken (Snellen chart), Optical Coherence Tomography (OCT, Cirrus
Humphrey-Zeiss) of the macular area, was performed also, evaluating
the height of the subretinal fluid at the fovea, and the total volumen
©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
at the macular cube. The evaluation was repeated at one month, two
months and three months after treatment.
Results: There was an improve in VA in all three patients since the
first month after initiating treatment and it was related with a decrease
in the subretinal fluid, measured with OCT, with less height at the
subfoveal space and a decrease in total volume measured through
macular cube.
Conclusions: Eplerenone seems to be an alternative to treat CSC
with good results. It could be interesting to prove this treatment in a
ramdomized controlled trial with more cases.
Commercial Relationships: Javier Moreno, None
Program Number: 1927 Poster Board Number: B0192
Presentation Time: 11:00 AM–12:45 PM
Oral Eplerenone For The Management Of Chronic Central
Serous Chorioretinopathy
Rishi P. Singh, Jonathan Sears, Rumneek Bedi, Andrew Schachat,
Peter K. Kaiser, Justis Ehlers. Cole Eye Institute, Cleveland Clinic,
Cleveland, OH.
Purpose: The proposed pathogenesis for central serous
chorioretinopathy (CSCR) is excessive glucocorticoid-dependent
mineralocorticoid receptor (MR) activation in the choroidal
vasculature. In this study, we examine eplerenone (Inspra, Pfizer), an
MR antagonist, as a treatment option for patients with CSCR.
Methods: A retrospective consecutive case series was conducted
for patients who received oral eplerenone for the treatment of
chronic CSCR with a minimum follow-up of 90 days. At baseline
and each follow-up visit, spectral domain OCT (SDOCT) imaging
was performed, including macular cube and 5-line raster scans (both
horizontal and vertical) were performed with a Zeiss Cirrus HD-OCT
(Cirrus version 6.1 software). SDOCT analysis included manual
measurements of the height and diameter size of subretinal fluid. The
primary outcome measure was reduction in subretinal fluid following
initiation of therapy. Secondary outcome measures included logMar
visual acuity and central subfield retinal thickness.
Results: A total of 17 eyes of 13 patients with a history of chronic
CSCR treated with 25 and 50 mg of oral eplerenone per day were
identified. Subretinal fluid decreased over time following eplerenone
therapy (p = 0.007 and p = 0.002, diameter and height respectively).
Maximum subretinal fluid diameter decreased from a mean of 131.5
μm at baseline to 15.3 μm at day 181+. Subretinal fluid height
decreased from an average of 2174.4 μm at baseline to 46.9 μm
at day 181+. LogMar visual acuity improved from 0.42 (Snellen
equivalent: 20/53) at baseline to 0.29 (Snellen equivalent: 20/39) at
day 181+ (p = 0.013). CST decreased from 339.5 μm at baseline to
270.3 μm at day 181+ (p = 0.029).
Conclusions: Following eplerenone oral therapy, there was a
significant reduction in subretinal fluid in eyes with chronic CSCR.
Additionally, a reduction in CST and improved visual acuity were
noted. Additional research, including larger prospective randomized
trials, is needed to validate these findings.
Commercial Relationships: Rishi P. Singh, Alcon (C), Bausch and
Lomb (C), Genentech (C), Regeneron (C); Jonathan Sears, None;
Rumneek Bedi, None; Andrew Schachat, None; Peter K. Kaiser,
Alcon (C), Bausch and Lomb (C), Carl Zeiss (C), Topcon (C); Justis
Ehlers, Bioptigen (I), Regeneron (C), Thrombogenics (C)
Program Number: 1928 Poster Board Number: B0193
Presentation Time: 11:00 AM–12:45 PM
Antagonists of mineralocorticoid receptors in the treatment of
chronic central serous chorioretinopathy: a case series
Sabrina FALAH, Thomas Pugliese, Jonathan Benesty, Marie-Helene
Errera, José-Alain Sahel, Michel Paques. CHNO des 15-20, Paris,
France.
Purpose: The optimal management of CSC remains controversial.
Recent clinical and experimental studies suggest that chronic central
serous chorioretinopathy (CSR) may result from an overactivation of
mineralocorticoid receptor pathway and therefore may provide the
rationale for the use of eplerenone (Zhao and al. Mineralocorticoid
receptor is involved in rat and human ocular chorioretinopathy; J Clin
Invest. 2012 Jul 2;122(7):2672-9). Herein, we report our experience
about the use of eplerenone in these patients.
Methods: Thirteen patients (14 eyes) with a minimum of 6 months
duration of chronic CSR were enrolled in the study. They had no
previous treatment for CSR. They all received eplerenone (50mg/d
orally) during 1 to 3 months. The median duration of follow-up was
6 months. Outcome measures were: best corrected visual acuity
(BCVA), central macular thickness, subretinal fluid amount, and
choroidal thickness measured by enhanced depth imaging (EDI)spectral domain optical coherence tomography (EDI-OCT). For
each subject, OCT image sets were obtained using a Spectralis OCT
(Heidelberg Engineering, Germany).
Results: The mean age of patients included was 47 y. Ten were
male (77%) and mean duration of chronic CSR was 36 months. No
statistical significant difference for BCVA [p=0.40], central macular
thickness [p=0.15] and retrofoveal choroidal thickness [p=0.11] was
found at 1 and 3 months following the start of eplerenon treatment.
However, a statistically significant reduction of subretinal fluid was
found under treatment [p<0.05] with complete resolution in 4 eyes
during the first month of treatment.
Conclusions: Eplerenone may represent an effective approach to
decrease the amount of subretinal fluid in eyes with chronic CSR.
However, our study failed to show a significant benefit on visual
outcome, maybe due to preexistent damage to photoreceptors and
RPE. Randomized controlled trials are needed to confirm these
results before formulating recommendations on the use of antagonists
of mineralocorticoid receptors in the treatment of chronic CSR.
©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
supplementation would aid in establishing guidelines for their use
and potential side effects.
Commercial Relationships: Sabrina FALAH, None; Thomas
Pugliese, None; Jonathan Benesty, None; Marie-Helene Errera,
None; José-Alain Sahel, None; Michel Paques, None
Program Number: 1929 Poster Board Number: B0194
Presentation Time: 11:00 AM–12:45 PM
Central serous chorioretinopathy associated with homeopathic
adrenal medication
Derek Huang1, Robert Millay2, Brian Y. Kim2. 1Department of
Ophthalmology, California Pacific Medical Center, San Francisco,
CA; 2Department of Ophthalmology, University of Vermont,
Burlington, VT.
Purpose: Central serous chorioretinopathy (CSC) has been
well described with its association with stress, steroid use and
hypercotisolism. However, we present a case of its association with a
homeopathic adrenal medication, ADHS. When this medication was
discontinued, the patient’s CSC also resolved.
Methods: Central serous chorioretinopathy is characterized by
leakage of fluid into the subretinal space from the choroid that can
lead to serous retinal detachments. CSC is most prevalent in males
aged 20-50 years old. Its association with type A personality, stress,
steroid use and hypercortisolism has been reported. The prognosis
is generally good with spontaneous resolution in weeks to months;
however about 5% of patients fail to regain greater than 20/30 acuity.
Results: This is the first case report describing central serous
chorioretinopathy in association with the use of homeopathic
adrenal medication. A 42 year old man presented with acute
visual complaints characterized as “blurry, dark spot in his central
vision” in his left eye of two days duration. He acknowledged
using 2 tablets of ADHS supplement for adrenal health. ADHS is
a homeopathic adrenal support supplement, marketed to support
desired dehydroepiandrosterone (DHEA), Secretory IgA and normal
cortisol levels. Examination revealed best-corrected visual acuity
was 20/20 in the right eye (OD) and 20/30-2 in the left eye (OS). Slit
lamp examination showed left retinal pigment epithelial mottling
and subretinal fluid. Angiographic examination revealed two areas
of leakage in the left eye. OCT was consistent with a diagnosis of
central serous chorioretinopathy. During a follow up examination (1
week later), he denied further use of his homeopathic medication and
experienced improvement in the dark central spot. His visual acuity
was 20/20 OD and 20/30+2 OS. OCT demonstrated improvement in
his subretinal fluid.
Conclusions: CSC has been linked to endogenous Cushing’s
syndrome, sympathomimetic use,as well as elevated catecholamine
levels. ADHS supplies raw materials necessary for the synthesis
of steroid hormones and epinephrine, the conversion of tyrosine
to catecholamines, and hormonal secretion. The ingredients and
amino acids contained in ADHS could play a role in its pathogenesis
in CSC. Additional investigation into the mechanism of adrenal
Commercial Relationships: Derek Huang, None; Robert Millay,
None; Brian Y. Kim, None
Program Number: 1930 Poster Board Number: B0195
Presentation Time: 11:00 AM–12:45 PM
Retinal Macular Volumes May Correlate with Serum VEGF
Levels in Women with Severe Preeclampsia
Victoria North1, Huy V. Nguyen1, Aakriti Garg1, Serge Cremers2,
Cande Ananth3, 4, Ronald J. Wapner3, Rando Allikmets1, 5, Srilaxmi
Bearelly1. 1Department of Ophthalmology, College of Physicians
and Surgeons, Columbia University, New York, NY; 2Irving Institute
for Clinical and Translational Research, Columbia University, New
York, NY; 3Department of Obstetrics and Gynecology, College of
Physicians and Surgeons, Columbia University, New York, NY;
4
Department of Epidemiology, Joseph L. Mailman School of Public
Health, Columbia University, New York, NY; 5Department of
Pathology and Cell Biology, College of Physicians and Surgeons,
Columbia University, New York, NY.
©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
Purpose: To compare serum levels of vascular endothelial growth
factor (VEGF) between patients with severe preeclampsia (sPE)
and normotensive postpartum controls using enzyme-linked
immunosorbent assay (ELISA), and to determine whether VEGF
levels correlate with retinal macular volumes in patients with sPE
using enhanced depth imaging spectral domain optical coherence
tomography (EDI SD-OCT).
Methods: This incident case-control study examined 12 women
with sPE and 5 women with normotensive pregnancies who were
age-matched (mean age 33.6 years ± 7.5 SD and mean age 32.6 years
± 8.0 SD, respectively). Nineteen eyes of women with sPE were
included in the imaging portion of the analysis. Exclusion criteria
included chronic or gestational hypertension without proteinuria, and
diabetes. All subjects were recruited in the immediate postpartum
period and underwent EDI SD-OCT imaging as well as blood draw.
Heidelberg Eye Explorer software was used to automate retinal
macular volume measurements. ELISA was used to measure serum
VEGF in picograms (pg/ml).
Results: Mean serum VEGF in sPE was 319.5 pg/ml and in
normotensive postpartum controls was 160.5 pg/ml (P = 0.2401).
Mean retinal macular volume in sPE was 8.82 mm3 and in controls
was 8.42 mm3 (P = 0.0004). Amongst cases, retinal macular volumes
correlated with serum VEGF levels with a Pearson’s correlation
coefficient of R = 0.44 (P = 0.0594).
Conclusions: Our results support the hypotheses that VEGF levels
are increased in sPE as compared with normotensive postpartum
controls, and that systemic VEGF levels may correlate with retinal
thickening in the setting of sPE. This study is limited by the small
number of subjects.
Commercial Relationships: Victoria North, None; Huy V. Nguyen,
None; Aakriti Garg, None; Serge Cremers, None; Cande Ananth,
None; Ronald J. Wapner, None; Rando Allikmets, None; Srilaxmi
Bearelly, None
Support: This work was supported by a grant from the Doris Duke
Charitable Foundation to Columbia University (AG), grants from the
National Eye Institute/NIH EY013435 and EY019007 (Core Support
for Vision Research), unrestricted funds from Research to Prevent
Blindness (New York, NY) to the Department of Ophthalmology,
Columbia University (RA), and the Robert L. Burch III Fund, NY,
NY (SB).
Program Number: 1931 Poster Board Number: B0196
Presentation Time: 11:00 AM–12:45 PM
Effects of long duration dive (8 and 10 hours) with hyperbaric
hyperoxia on Navy divers’ eye and visual function: preliminary
results
Bernard Valero1, Rodolphe Vignal1, Olivier Castagna2, Aurélie
Brugier1, Alexandra de Faria1, Marie Bourniquel1, Rim Sekfali1,
Jean-Eric Blatteau2, Jean-Marie Giraud1. 1Sainte Anne Military
Hospital, Toulon, France; 2Underwater research team (ERSSO).
Military biomedical research (IRBA), Toulon, France.
Purpose: To assess anatomical and functional effects of long duration
dive (8 and 10 hours) on the eye, by reproducing all the constraints
undergone by professional divers and to make sure that this kind
of diving doesn’t present any danger to the diver’s eye and visual
function.
Methods: 12 professional male Navy divers (31,5 +/- 3,2 y/o)
executed a prolonged immersion (8 and 10 hours), breathing 50%
Nitrox or 100% 02, depending on the depth of immersion (from 7 to
20 msw), corresponding to 1.54 to 1.7 ATA partial oxygen pressure
(PaO2). They were regularly fed and hydrated.
For each diver, we studied several parameters 24 hours before and 15
hours after immersion: visual acuity, low spatial contrast sensitivity,
color vision (desaturated 15 Hue® test), eye refraction, ocular
examination, visual field (Metrovision®, MIXTE and STAT 57C
program, studying 24° and 10° central visual field), full-field clinical
electroretinography (Metrovision®) using a short protocol (pupillary
dilation, photopic response with 16 white standard flash and 16 red
flash, 4 minutes to dark adaptation, scotopic response with 8 blue
flash and 8 attenuated white flash), and multifocal electroretinography
(Metrovision®).
Wilcoxon test was used to assess differences between results for each
diver, before and after immersion.
Results: None of the 12 divers had any loss of visual acuity, nor any
loss of spatial contrast sensitivity. We didn’t notice any difference
on color vision. Moreover, we didn’t observe any modification of
eye refraction nor any alteration of visual field parameters (corrected
mean deviation, temporal and spatial deviation, foveolar threshold).
Ocular examination was normal for all the divers, before and after
immersion. Regarding full-field clinical electroretinography, b-wave
was significantly decreased after immersion (p = 0,002) on scotopic
response with white attenuated stimulation. Regarding to multifocal
electroretinography, amplitude of P1-wave beyond central 15°was
increased (p = 0,035) and N2-wave beyond 15° was deeper (p =
0,032).
Conclusions: These findings suggest long duration dive with
hyperbaric hyperoxia doesn’t induce any immediate danger for the
eye. However, subclinical functional effects on vision seem to exist,
affecting rod-cell function, as it’s suggested by electroretinography
results. Further studies are needed to try and confirm these results.
Commercial Relationships: Bernard Valero, None; Rodolphe
Vignal, None; Olivier Castagna, None; Aurélie Brugier, None;
Alexandra de Faria, None; Marie Bourniquel, None; Rim Sekfali,
None; Jean-Eric Blatteau, None; Jean-Marie Giraud, None
Clinical Trial: Ref Afssaps : B120310-20 / Ref Promoter 2012_
RBM_CASTAGNA / N°ID RCB : 2012-A00008-35 / Ref CPP :
12.012
Program Number: 1932 Poster Board Number: B0197
Presentation Time: 11:00 AM–12:45 PM
Retinal and intravitreal temperature during vitreous surgery
Benjamin Buck, Gabriela Lopezcarasa, Veronica A. Kon Jara, JeanClaude Mwanza, Maurice Landers. Ophthalmology, The University
of North Carolina at Chapel Hill, Chapel Hill, NC.
Purpose: The purpose of this study is to demonstrate how retinal
and vitreous temperatures fluctuate during vitrectomy with room
temperature infusion fluid and to demonstrate the extent to which the
retina is cooled during routine vitreous surgery.
Methods: Prospective study of 16 patients already schedule for
vitrectomy; a 23-gauge flexible wire thermoprobe was used to
measure intraocular temperatures before, at the end of active
vitrectomy, and 5 minutes after closing infusion line. The ocular
temperature measurements were taken by contact in middle of
vitreous, retina nasal to optic disk, retina just outside of inferior
arcade, 2 disc diameter temporal to the fovea and immediately above
the fovea.
Results: Total of 16 patients, 6 males and 10 females, room t° and
infusion t° were 68.9°F, Patient t° average was 97.5°F, positive
correlation 0.04 was found. The patients’ diagnostics were VH, DME,
MH, TRD, RRD and ERM. We found that basal retinal temperature
was physiologically mild hypothermia and that temperature went
down 13 to 14 degrees in average, which was deep hypothermia,
described under 86°F; after 5 minutes with infusion line closed it
recovered an average of 6.5°F.
1) No statistical differences between patient t, Room t, or infusion t
at any time point.
©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
2) Significant differences were observed in t between the 3 time
points at all vitrous locations. Pre-vitrectomy t were significantly
higher than both intra-operative and post-vitrectomy t. The perioperative temps were all significantly lower than post-vitrectomy
t. The temperature of the vitreous adjacent to the fovea positively
correlated with the patients temperature (r = 0.625, P = 0.0096)
Conclusions: During surgery the vitreous and retina are cooled to
deep hypothermia. Rapid re-warming begins once infusion is closed.
These t fluctuation are extreme and rapid when compared with
therapeutic hypothermia.
The retina is physiologically in mild hypothermia.
The effects of these t on ocular tissues is unclear and studies that
change the infusion t during vitrectomy and long term follow are
required.
Commercial Relationships: Benjamin Buck, None; Gabriela
Lopezcarasa, None; Veronica A. Kon Jara, None; Jean-Claude
Mwanza, None; Maurice Landers, None
Support: Research to Prevent Blindness
Program Number: 1933 Poster Board Number: B0198
Presentation Time: 11:00 AM–12:45 PM
Direct Visualization of Silicone Oil Removed From Vitrectomized
Eyes with Transmission Electron Microscopy Reveals
Microemulsions and Nanoemulsions
Jesse T. McCann1, 2, Yale Fisher3, 2. 1Ophthalmology, NYU Medical
Center, New York, NY; 2Ophthalmology, Manhattan Eye, Ear,
and Throat Hospital, New York, NY; 3Vitreous Retina Macula
Consultants, New York, NY.
Purpose: Poly(dimethylsiloxane) polymers (commonly known
as silicone oil) have been frequently used in retinal surgery as a
tamponade. Emulsification of these oils has been a difficult and
persistent problem. Persistence of the oil following vitrectomy
with fluid exchange has been confirmed by contact B-scan
ultrasonography. In addition, infiltration of the silicone oil into
the retina and optic nerve has been demonstrated by SD-OCT and
adaptive optics imaging. This study intends to directly visualize
the emulsions formed by silicone oil used in retinal surgery by
multimodal imaging.
Methods: Silicone oil that was removed from eyes by fluid exchange
and vitrectomy having been used as a surgical tamponade was
subjected to chemical and electron micrographic analysis. 0.1 mL of
silicone oil was washed with 2 mL of sterile irrigating solution (BSS,
Alcon, TX). The mixture was then vortexed for 60 s and allowed to
sit at 25 °C for 24 hours. The supernatant was decanted and placed
on carbon-coated copper grids for transmission electron microscopy
(TEM) and cryo-TEM analysis.
Results: BSS was sufficient to emulsify the removed silicone oil.
Transmission electron microscopy revealed nano-size (<100 nm) and
micron-sized (<1 mm) oil-in-water emulsions. These emulsions are
several orders of magnitude smaller than previously reported. These
droplet sizes are similar to the size of silicone oil droplets that have
been found in the tissues of the retina and optic nerve using optical
coherence tomography and adaptive optics imaging.
Conclusions: This demonstrates that silicone-soluble components of
the eye partition into the silicone oil after its instillation as a surgical
tamponade. These silicone-soluble components are alone sufficient to
cause emulsification. The equilibration of the silicone oil with lipidsoluble components in the eye is consistent with phase-equilibrium
behavior. Further analysis of the silicone oil to determine the identity
of the emulsifying agents is ongoing.
Commercial Relationships: Jesse T. McCann, None; Yale Fisher,
None
Program Number: 1934 Poster Board Number: B0199
Presentation Time: 11:00 AM–12:45 PM
Pupillography under Hypobaric Hypoxia- The THAO trial
Tobias Peters, Andreas Schatz, Max Schultheiss, Gabriel Willmann,
Helmut Wilhelm, Barbara Wilhelm, Florian Gekeler. Pupil Research
Group, University of Tübingen, Germany, Tuebingen, Germany.
Purpose: The results presented here are related to the THAO study
(http://www.thao-project.com), dealing with acute mountain sickness
(AMS) and ophthalmological effects of hypobaric hypoxia after fast
ascent, both in regard to morphological and functional changes.
Methods: 14 participants (age 25-54yrs) intended to reach the
summit at Capanna Margherita (CM, Italy) within 24 hrs starting
from 1635m up to CM at 4559m. More than 14 days before exposure
to high altitude participants were not allowed to climb above 2000m.
During day 1 to 4 an almost complete ophthalmological assessment
including ERG, OCT, FLA, AF, and Pupillography was performed
twice a day. The pupillograph used was a Computer intergrated
Pupillograph (CiP by Amtech, Dossenheim Germany). The CiP
provides a 585nm LED light source resulting in corneal illuminance
of about 3lx. Stimulus length was set at 200ms. AMS was defined by
the Lake Louise and the AMS-c score.
Results: Ratios (value/value at baseline) of amplitude, latency,
velocity, initial diameter and relative amplitude were calculated and
plotted against day of condition. There was a significant result to
higher amplitude and higher velocity of the pupillary light reaction
and lower initial diameter in all participants in high altitude. The
changes in pupillary parameters did not correlate with the AMS score.
Conclusions: Tolerance and adaptation to exposure to high altitude
were different among participants. Results of pupillography showed
clearly the influence by high altitude after fast ascent, the changes
might be interpreted as indicators of an increased parasympathetic
tone. Acute mountain sickness was not predictable or clearly
detectable by means of pupillography.
Commercial Relationships: Tobias Peters, None; Andreas Schatz,
None; Max Schultheiss, None; Gabriel Willmann, None; Helmut
Wilhelm, None; Barbara Wilhelm, None; Florian Gekeler, None
Support: This study was supported by the Wilderness Medical
Society (WMS) with the Charles S. Houston Research Award.
Additional unconditional grants were provided by Novartis, Bausch
& Lomb, Alcon, Allergan, Ursapharm, IMS Gear and OptimaPharma.
The CIP was provided by AMTech, Dossenheim, Germany. The study
also received private donations by Martin Rohrbach, Till Schoeffel,
Ulrich Gekeler, Christina Fasser, Eduard Schatz und Norbert
Willmann. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
©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.