Neuroprotective Effect of PACAP Against NMDA-Induced Retinal Damage in the Mouse

Retinal excitotoxicity is one of the major causes of retinal ganglion cell (RGC) death in glaucoma. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic peptide with potent neuroprotective activity; however, whether it exerts such an effect in the retina and the mechanism by which RGCs are protected is still not well understood. In this study, we examined the effect of exogenous and endogenous PACAP on RGC death induced by N-methyl-d-aspartate acid (NMDA). The vitreous body of anesthetized adult male mice (C57/BL6J) was injected with NMDA (40?nmol in a 2???L saline solution). The number of RGCs decreased from days?1 to 7 after NMDA injection, and the number of dUTP end-labeling (TUNEL)-positive cells, an indicator of cell death, peaked at day?3. However, when PACAP38 (10^?8, 10^?10, 10^?12, 10^?14, or 10^?16M) was co-administered with NMDA, the 10^?10M dose resulted in significantly increased RGC survival at day?7, and a decrease in the number of TUNEL-positive RGCs at day?3. We next investigated the neuroprotective effect of endogenous PACAP using PACAP heterozygote(+/?) mice. Under normal circumstances, there was no significant difference in the number of RGCs in the PACAP(+/?) mice compared with their wild-type counterparts. However, the number of RGCs significantly decreased in the PACAP(+/?) mice 7?days after NMDA injection, relative to their wild-type counterparts. The number of TUNEL-positive RGCs peaked at day?1 in the PACAP(+/?) mice. These effects in the PACAP(+/?) mice were reversed by intravitreous injection of 10^?10M PACAP38. This suggests that exogenous PACAP is able to counteract NMDA-induced toxicity, and that endogenous PACAP exerts a neuroprotective effect in the retina.     

Critical neuroprotective roles of heme oxygenase‐1 induction against axonal injury‐induced retinal ganglion cell death

Although axonal damage induces significant retinal ganglion cell (RGC) death, small numbers of RGCs are able to survive up to 7 days after optic nerve crush (NC) injury. To develop new treatments, we set out to identify patterns of change in the gene expression of axonal damage‐resistant RGCs. To compensate for the low density of RGCs in the retina, we performed retrograde labeling of these cells with 4Di‐10ASP in adult mice and 7 days after NC purified the RGCs with fluorescence‐activated cell sorting. Gene expression in the cells was determined with a microarray, and the expression of Ho‐1 was determined with quantitative PCR (qPCR). Changes in protein expression were assessed with immunohistochemistry and immunoblotting. Additionally, the density of Fluoro‐gold‐labeled RGCs was counted in retinas from mice pretreated with CoPP, a potent HO‐1 inducer. The microarray and qPCR analyses showed increased expression of Ho‐1 in the post‐NC RGCs. Immunohistochemistry also showed that HO‐1‐positive cells were present in the ganglion cell layer (GCL), and cell counting showed that the proportion of HO‐1‐positive cells in the GCL rose significantly after NC. Seven days after NC, the number of RGCs in the CoPP‐treated mice was significantly higher than in the control mice. Combined pretreatment with SnPP, an HO‐1 inhibitor, suppressed the neuroprotective effect of CoPP. These results reflect changes in HO‐1 activity to RGCs that are a key part of RGC survival. Upregulation of HO‐1 signaling may therefore be a novel therapeutic strategy for glaucoma. © 2014 Wiley Periodicals, Inc.     

EphA5 and ephrin-A2 expression during optic nerve regeneration: a 'two-edged sword'

During development, gradients of EphA receptors (nasal(low)-temporal(high)) and their ligands ephrin-As (rostral(low)-caudal(high)) are involved in establishing topography between retinal ganglion cells (RGCs) and the superior colliculus (SC). EphA5-expressing RGC axons are repulsed by ephrin-A2-expressing SC neurones. In adult rats RGCs maintain graded EphA5 expression but ephrin-A2 expression is down-regulated in the SC to a weak gradient. At 1 month after optic nerve transection, EphA5 expression is reduced in the few remaining RGCs and is no longer graded; by contrast, SC ephrin-A2 is up-regulated to a rostral(low)-caudal(high) gradient. Here we examined expression in adult rat 1 month after bridging the retina and SC with a peripheral nerve graft, a procedure that enhances RGC survival and permits RGC axon regeneration. Double labelling with cell markers revealed preservation of a nasal(low)-temporal(high) EphA5 gradient in RGCs and establishment of a rostral(low)-caudal(high) ephrin-A2 gradient within neurones of the SC. The results suggest a potential for guidance cues to restore the topography of RGC axons in the SC. However, high ephrin-A2 levels were also found in astrocytes surrounding the peripheral nerve graft insertion site. The repulsive ephrin-A2 environment offers at least a partial explanation for the observation that only a limited number of RGC axons can exit the graft to enter target central nervous system tissue.     

The PD-1/PD-L pathway is up-regulated during IL-12-induced suppression of EAE mediated by IFN-gamma

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), is mediated by autoantigen-specific T-helper1 (Th1) cells. IL-12, an inducer of Th1 cell development, exerts immunomodulatory effects in EAE. Programmed death-1 (PD-1) and PD-1 ligand (PD-L), new members of the B7 superfamily of costimulatory molecules, play a critical role in regulating EAE. Whether the interaction of IL-12 and the PD-1/PD-L pathway regulates EAE is unclear. We have previously shown that IL-12 suppresses EAE induced by MOG35-55 in C57BL/6 mice, but not in IFN-gamma-deficient mice, suggesting that IFN-gamma is required for the inhibitory effects of IL-12 on EAE. In the current study, PD-L1 expression is up-regulated following IL-12 treatment in wild-type mice, but not in IFN-(-deficient EAE mice. Similarly, IL-12 induces IFN-gamma and PD-L1 expression in cultured MOG-specific T cells from wild-type mice but not from IFN-gamma-deficient mice. Furthermore, PD-L1 expression increased specifically in CD11b+ antigen presenting cells (APCs) after IL-12 administration. These data suggest that one mechanism of IL-12 suppression of EAE is mediated by PD-1/PD-L signaling downstream of IFN-gamma induction in CD11b+ APCs. The regulation of PD-1/PD-L1 may have potential therapeutic effects for EAE and MS.     

Cooperative effects of bcl-2 and AAV-mediated expression of CNTF on retinal ganglion cell survival and axonal regeneration in adult transgenic mice

We used a gene therapy approach in transgenic mice to assess the cooperative effects of combining anti-apoptotic and growth-promoting stimuli on adult retinal ganglion cell (RGC) survival and axonal regeneration following intraorbital optic nerve injury. Bi-cistronic adeno-associated viral vectors encoding a secretable form of ciliary neurotrophic factor and green fluorescent protein (AAV-CNTF-GFP) were injected into eyes of mice that had been engineered to over-express the anti-apoptotic protein bcl-2. For comparison this vector was also injected into wildtype (wt) mice, and both mouse strains were injected with control AAV encoding GFP. Five weeks after optic nerve injury we confirmed that bcl-2 over-expression by itself promoted the survival of axotomized RGCs, but in contrast to previous reports we also saw regeneration of some mature RGC axons beyond the optic nerve crush. AAV-mediated expression of CNTF in adult retinas significantly increased the survival and axonal regeneration of RGCs following axotomy in wt and bcl-2 transgenic mice; however, the effects were greatest in the transgenic strain. Compared with AAV-GFP-injected bcl-2 mice, RGC viability was increased by about 50% (mean, 36 738 RGCs per retina), and over 1000 axons per optic nerve regenerated 1-1.5 mm beyond the crush. These findings exemplify the importance of using a multifactorial therapeutic approach that enhances both neuroprotection and regeneration after central nervous system injury.     

Hypoxia-induced activation of N-methyl-D-aspartate receptors causes retinal ganglion cell death in the neonatal retina

It is well established that hypoxia causes excess accumulation of glutamate in developing neural tissues. This study aimed to elucidate the mechanism by which glutamate can cause retinal ganglion cell (RGC) death through the N-methyl-D-aspartate (NMDA) receptors (NR) in the developing retina. One-day-old Wistar rats were exposed to hypoxia for 2 hours and then killed at different time points. Normal age-matched rats were used as controls. NR1, NR2A-D, and NR3A messenger RNA and protein expression showed significant increases over control values, notably at early time points (3 hours to 7 days) after the hypoxic exposure, and immunoexpression of NR1, NR2A-D and NR3A on retinal ganglion cells (RGCs) was enhanced in hypoxic rats and this was confirmed in cultured hypoxic RGCs. Ca(2+) influx in cultured RGCs was increased after hypoxic exposure, and the intracellular Ca(2+) concentration was suppressed by MK-801. Mitochondrial permeability transition pore opening, mitochondrial/cytosolic cytochrome c, and cytosolic caspase-3 expression levels were significantly increased in the hypoxic RGCs. These increases were reversed by MK-801, suggesting that the NMDA receptor subunits in the retina respond rapidly to the hypoxia-induced glutamate overload that leads to the cascade of events that result in RGC death.     

Migration of phagocytotic cells and development of the murine intraretinal microglial network: an in vivo study using fluorescent dyes

This work was undertaken to study whether retinal ganglion cell (RGC) death, which occurs during postnatal development of the mouse retina could aid in assessing the topological and chronological pattern of microglial cell migration. The study was conducted from postnatal day 0 (P0) to adulthood. The fluorescent dyes Fluorogold (FG) or (4-[4-didecylaminostyryl]-N-methylpyridinium iodide (4Di-10ASP) used in this study, were transported retrogradely to the RGC soma when either dye was injected into the superior colliculus (SC) at P0. Some of these labeled RGCs die due to natural apoptosis during this stage of development and are phagocytosed by microglial cells, which move to the site of RGC death, to become labeled with the same dye. The retinas were examined to quantify the microglial cells from P5 to adulthood. In addition, the reaction of microglia to optic nerve crush was studied in adult animals. Both dyes labeled RGCs in the contralateral retina and a few RGCs in the retina ipsilateral to the injected SC. The density of labeled RGCs decreased by 22% between P5 and P7. During this phase, microglial cells become visible as they ingested the fluorescent detritus of the dying RGCs. Microglial cells were evenly distributed across the entire retinal surface and migrated to the outer plexiform layer. Migrating microglia consecutively altered their morphology from the amoeboid to the ramified form. In terms of intracellular storage of the dyes, resident microglial cells retained the fluorescent dye 4Di-10ASP over a period of 12 months. In contrast, FG was completely transferred from the RGCs and microglial cells to intramural cells (pericytes) of the retinal capillaries after 10 months. This resulted in delineation of the entire intraretinal vascular network. Finally, resident retinal microglial cells were also activated by injury to the adult optic nerve and phagocytosed degenerating neurons. Retinal microglial cells can be monitored with vital fluorescent dyes while they migrate across the retina and establish their intra-retinal network. It is possible to label microglia with lipophilic dyes and they remain labeled for a long time. In addition, intramural pericytes can be labeled by slow release of FG from RGCs and microglial cells. The findings suggest that ingested fluorescent dyes having different properties can be used to study different populations of retinal cells in vivo.     

Math5 (Atoh7) gene dosage limits retinal ganglion cell genesis

The basic helix-loop-helix factor Math5 (Atoh7) is critical for the determination of retinal ganglion cell (RGC) fate in mice. Recently, genome-wide association studies have identified the ATOH7 locus as a major determinant of variation in the human optic disc area, which is directly correlated with the RGC number. These studies suggest that the level of Math5 expression may determine the ultimate number of RGCs. To test this hypothesis, we systematically compared optic nerve area and RGC axon number in C57BL/6J congenic Math5+/- and +/+ mice at young adult and neonatal ages by transmission electron microscopy. Optic disc area and RGC abundance were not significantly different in adults, but heterozygotes had thinner optic nerves and 25-30% fewer RGCs at birth than wild-type littermates (P<0.05). Our results suggest that Math5 dosage is important for the genesis, but not the ultimate number, of RGCs. Our findings highlight the importance of ganglion cell culling as a compensatory mechanism for retinal homeostasis, and support a quantitative role for Math5 in RGC specification.     

Adenovirus-mediated expression of ciliary neurotrophic factor (CNTF) rescues axotomized rat retinal ganglion cells but does not support axonal regeneration in vivo

Rat optic nerve (ON) transection leads to mainly apoptotic cell death of about 85% of the retinal ganglion cell (RGC) population within 14 days after lesion. In the present study, we tested the effect of adenovirally delivered CNTF (Ad-CNTF) on survival and regeneration of axotomized adult RGCs in vivo. Single intravitreal Ad-CNTF injection led to stable CNTF mRNA and protein expression for at least 18 days and significantly enhanced RGC survival by 155% when compared to control animals 14 days after ON transection. ON stump application of Ad-CNTF also resulted in an increased number of surviving RGCs. Ad-CNTF injection led to better preservation of intraretinal RGC axons but did not support regeneration of axotomized RGCs into a peripheral nerve graft. Thus, adenovirus-mediated neurotrophic factor supply is a suitable approach for reducing axotomy-induced RGC death in vivo and may constitute a relevant strategy for clinical treatment of traumatic brain injury.     

Global and Regional Damages in Retinal Ganglion Cell Axon Bundles Monitored Non-Invasively by Visible-Light Optical Coherence Tomography Fibergraphy

Retinal ganglion cells (RGCs) exhibit compartmentalized organization, receiving synaptic inputs through their dendrites and transmitting visual information from the retina to the brain through the optic nerve. Little is known about the structure of RGC axon bundles extending from individual RGC somas to the optic nerve head (ONH) and how they respond to disease insults. We recently introduced visible-light optical coherence tomography fibergraphy (vis-OCTF), a technique for directly visualizing and analyzing mouse RGC axon bundles in vivo. In this study, we validated vis-OCTF''s ability to quantify RGC axon bundles with an increased number of RGCs using mice deficient in BCL2-associated X protein (BAX^−/−). Next, we performed optic nerve crush (ONC) injury on wild-type (WT) mice and showed that the changes in RGC axon bundle width and thickness were location-dependent. Our work demonstrates the potential of vis-OCTF to longitudinally quantify and track RGC damage at single axon bundle level in optic neuropathies.SIGNIFICANCE STATEMENT Nearly all clinical and preclinical studies measure the retinal nerve fiber (RNFL) thickness as the sole indicator of retinal ganglion cell (RGC) damage without investigating RGC axon bundles directly. We demonstrated visible-light optical coherence tomography fibergraphy (vis-OCTF) to directly quantify global and regional RGC axon bundle organizations in vivo as a new biomarker for RGC health. We validated in vivo vis-OCTF measures using both confocal microscopy of the immunostained flat-mounted retina and numerical simulations. Vis-OCTF for monitoring RGC axon bundle organization has the potential to bring new insight into RGC damage in optic neuropathies.     

Expression of Hsp27 in retinal ganglion cells of the rat during postnatal development

The small heat shock protein Hsp27 has been shown to protect neurons from apoptosis. We have recently shown the expression of Hsp27 in a subset of injured adult retinal ganglion cells (RGCs), a response that is muted by the administration of brain-derived neurotrophic factor. This work has suggested a role for Hsp27 in the long-term survival of RGCs following injury. The purpose of this study was to investigate the expression of Hsp27 during postnatal retinal development, based on Hsp27's role as a neuronal survival factor and on its up-regulation in the adult injured retina. Expression of Hsp27 in the developing retina was examined at various times postnatally (between P0 and P24) by using immunohistochemical techniques. We report that Hsp27 expression peaks in the ganglion cell layer between P6 and P12 and is not detected at earlier (P0-P3) or later (P15-P24) times. Double labeling of the Hsp27-positive cells with Fluorogold applied to the superior colliculus confirmed that Hsp27-positive cells in the ganglion cell layer are RGCs. We have shown developmentally regulated expression of Hsp27 in RGCs of the postnatal rat. The retinal expression of Hsp27 correlates temporally with innervation of the tectum by late-born RGCs and with onset of spontaneous retinotectal activity. We propose that the expression of Hsp27 may play an important role in retinal development during a critical period of RGC functional connectivity with the superior colliculus.     

Fate of multipotent neural precursor cells transplanted into mouse retina selectively depleted of retinal ganglion cells

In some parts of the CNS, depletion of a particular class of neuron might induce changes in the microenvironment that influence the differentiation of newly grafted neural precursor cells. This hypothesis was tested in the retina by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult female mice and examining whether intravitreally grafted male neural precursor cells (C17.2), a neural stem cell (NSC)-like clonal line, become incorporated into these selectively depleted retinae. In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection. Cells were injected intravitreally 6-48 h after SC ablation (neonates) or 0-7 days after ON injury (adults). Cells were also injected into non-RGC depleted neonatal and adult retinae. At 4 or 8 weeks, transplanted cells were identified using a Y-chromosome marker and in situ hybridisation or by their expression of the lacZ reporter gene product Escherichia coli beta-galactosidase (beta-gal). No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis (n = 16). Donor cells were however stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6-31% of surviving cells were found in the RGC layer (GCL). These NSC-like cells were also present in intact retinae, but on average, there were fewer cells in GCL. In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for beta-III tubulin, a protein highly expressed by, but not specific to, developing RGCs. Targeted rapid RGC depletion thus increased cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype.     

Efficient estimation of retinal ganglion cell number: a stereological approach

Retinal ganglion cells (RGCs) are the only output neurons of the retina, and their degeneration after damage to the optic nerve or in glaucoma is a well established system for studying apoptosis in the central nervous system. Frequently used procedures for assessing RGC number in retinal flat mounts suffer from two problems: RGC densities are not uniform across retinal flat mounts, and density measures may therefore not reflect total number, and flat mounts do not allow efficient use of tissue. To overcome these problems we developed a stereological method for efficiently assessing RGC number in cryostat sections of the retina. We empirically demonstrate that only approximately 1:20 sections need be assessed to accurately estimate the total number of RGCs in the rat retina, providing ample tissue for additional studies in the same retina and saving considerably on more exhaustive sampling strategies. Using this method, we estimate that there are 86,282+/-4759 RGCs in the normal Brown Norway rat retina. These counts match well with estimates of axon counts in optic nerve. In a pilot study of experimental glaucoma, we determined a reduction of RGCs to 53,862+/-4272 (p<0.05). The current technique should prove advantageous to assess neuroprotective strategies in these experimental models.     

Retinal ganglion cells in normal hamsters and hamsters with novel retinal projections. I. Number,distribution, and size

We examined the number, spatial distribution, and size of ganglion cells in the retinae of normal Syrian hamsters and hamsters with retinal projections to the auditory and somatosensory nuclei of the thalamus, induced by neonatal surgery. As revealed by retrograde filling with horseradish peroxidase, there are about 64,600 contralaterally projecting retinal ganglion cells (RGCs) and 1,700 ipsilaterally projecting RGCs in the retinae of normal adult hamsters. Contralaterally projecting RGCs are distributed throughout the retina and have two local density peaks located within a central streak of high RGC density that is oriented approximately along the nasal-temporal axis. RGC density falls above and below the central streak, with a steeper gradient towards the upper retina. Ipsilaterally projecting RGCs are diffusely distributed within a crescent at the inferotemporal retinal periphery and are most dense at the internal border of the crescent. The soma diameter of contralaterally projecting RGCs ranges from 6 to 25 μm; the diameter distribution is unimodal, with a peak in the 10–13 μm range and is skewed toward smaller values, with an elongated tail towards higher values. Contralaterally projecting RGCs tend to be smaller in regions of higher density. Ipsilaterally projecting RGCs tend to be larger than contralaterally projecting RGCs both globally and within the temporal crescent, and their size distributions tend to be less regular and less well related to local density. The retinae of neonatally operated hamsters with novel retinal projections to the auditory and somatosensory systems contain about one-fourth the normal number of contralaterally projecting RGCs, whose relative density distribution is approximately normal despite the drastic reduction of absolute RGC density. The range and distribution of RGC soma diameters are similar in normal and neonatally operated hamsters, and, in operated as in normal hamsters, contralaterally projecting RGC somata tend to be smaller in regions of higher density. Our results in normal hamsters suggest a role for intraretinal mechanisms in the determination of RGC size. Our findings in neonatally operated hamsters suggest that, despite the reduced number of RGCs in these animals, the same types of RGCs are found in the retinae of normal and neonatally operated hamsters. © 1995 Wiley-Liss, Inc.     

Nitric Oxide Synthase Inhibition Delays Axonal Degeneration and Promotes the Survival of Axotomized Retinal Ganglion Cells

Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated in neuronal cytotoxicity following trauma to the central nervous system. The aim of the present study was to examine the role of NO in mediating axotomy-induced retinal ganglion cell (RGC) death. We observed increases in iNOS expression by microglia and Müller cells in the retina after optic nerve transection. This was paralleled by the induced expression of constitutive NOS (cNOS) in RGCs which do not normally express this enzyme. In order to determine if NO is cytotoxic to axotomized RGCs, the nonspecific NOS inhibitors Nomega-nitro-L-arginine (NOLA) or N-nitro-L-arginine methyl ester (L-NAME) were delivered to the vitreous chamber by intraocular injections. Both NOLA and L-NAME significantly enhanced RGC survival at 7, 10, and 14 days postaxotomy. The separate contributions of iNOS and cNOS to RGC degeneration were examined with intraocular injections of the specific iNOS inhibitor L-N(6)-(I-iminoethyl)lysine hydrochloride or the specific cNOS inhibitor L-thiocitrulline. Our results suggest that cNOS plays a greater role in RGC degeneration than iNOS. In addition to enhancing RGC survival, NOS inhibitors delayed the retrograde degeneration of RGC axons after axotomy. We conclude that NO synthesized by retinal iNOS and cNOS plays a major role in RGC death and retrograde axonal degeneration following axotomy.     

The majority of infiltrating CD8 T lymphocytes in multiple sclerosis lesions is insensitive to enhanced PD-L1 levels on CNS cells

Central nervous system (CNS) cells locally modulate immune responses using numerous molecules that are not fully elucidated. Engagement of programmed death-1 (PD-1), expressed on activated T cells, by its ligands (PD-L1 or PD-L2) suppresses T-cell responses. Enhanced CNS PD-1 and PD-L1 expression has been documented in inflammatory murine models; however, human CNS data are still incomplete. We determined that human primary cultures of astrocytes, microglia, oligodendrocytes, or neurons expressed low or undetectable PD-L1 under basal conditions, but inflammatory cytokines significantly induced such expression, especially on astrocytes and microglia. Blocking PD-L1 expression in astrocytes using specific siRNA led to significantly increased CD8 T-cell responses (proliferation, cytokines, lytic enzyme). Thus, our results establish that inflamed human glial cells can express sufficient and functional PD-L1 to inhibit CD8 T cell responses. Extensive immunohistochemical analysis of postmortem brain tissues demonstrated a significantly greater PD-L1 expression in multiple sclerosis (MS) lesions compared with control tissues, which colocalized with astrocyte or microglia/macrophage cell markers. However, more than half of infiltrating CD8 T lymphocytes in MS lesions did not express PD-1, the cognate receptor. Thus, our results demonstrate that inflamed human CNS cells such as in MS lesions express significantly elevated PD-L1, providing a means to reduce CD8 T cell responses, but most of these infiltrating immune cells are devoid of PD-1 and thus insensitive to PD-L1/L2. Strategies aimed at inducing PD-1 on deleterious activated human CD8 T cells that are devoid of this receptor could provide therapeutic benefits since PD-L1 is already increased in the target organ.     

Upregulation of CREM-1 Relates to Retinal Ganglion Cells Apoptosis After Light-Induced Damage In Vivo

Previous studies have shown activation of cyclic AMP response element-binding protein (CREB) family is involved in the retinal ganglion cells (RGCs) protection. However, the function of cyclic AMP response element modulator-1 (CREM-1), one member of the CREB family, is still with limited acquaintance. To investigate whether CREM-1 is involved in RGCs death, we performed a light-induced retinal damage model in adult rats. Upregulation of CREM-1 was observed in retina after light-induced damage by performing western blot. Immunofluorescent labeling indicated that upregulated CREM-1 was localized mainly in the RGCs. We also investigated co-localization of CREM-1 with active-caspase-3 and TUNEL (apoptotic markers) in the retina after light-induced damage. In addition, the expression patterns of B cell lymphoma/leukemia-2 and Bcl-2 associated X protein were parallel with that of CREM-1. Collectively, we hypothesized upregulation of CREM-1 in the retina was associated with RGCs death after light-induced damage.