Retinal Ganglion Cell Quantification Research Articles

Retinal ganglion cells in a mouse model of Alzheimer's disease: A temporal study

Aims/Purpose: Alzheimer's disease (AD) is a degenerative neurological disorder characterized by the progressive loss of neurons and their synapses in the brain. Similar alterations have been observed in the retina even before they manifest in the brain. The retina has been proposed as a readily accessible biomarker for AD. In a previous work utilizing a murine model of AD (APPNL‐F/NL‐F), changes in retinal thickness, as measured by optical coherence tomography (OCT), were observed at 15 and 17 months. The objective of this study was to quantitatively assess the population of retinal ganglion cells (RGCs) in this same AD murine model over a specified time period and compare it with wild‐type (WT) mice.Methods: To quantitatively assess the population of retinal ganglion cells we used two study groups: the APPNL‐F/NL‐F model throughout the time period of (6, 9, 12, 17, and 20 months) and compared with wild‐type (WT) mice age matched (n = 30 in both groups). The quantification of retinal ganglion cells (RGCs) was conducted using immunohistochemical analysis with the Brn3a marker. This analysis was performed using a semi‐automatic algorithm implemented in the MatLab environment, which was developed by the Ramón Castroviejo Institute for Ophthalmic Research. For the analysis, the entire retina was considered and segmented into the peripapillary, intermediate, and peripheral zones.Results: A statistically significant decresase (p < 0.05) in the population of Brn3a + cells was observed in the APPNL‐F/NL‐F group compared to the WT group. This decreased was found in the total retina at 12 months, in the peripapillary zone at 9, 12, and 20 months, and in the intermediate zone at 12 months of the study.Conclusions: The APPNL‐F/NL‐F mouse model exhibits RGCs loss at 9, 12, and 20 months, mirroring the patterns observed in human cases. These findings suggest that the model holds promise for investigating and studying mechanisms related to RGC degeneration in Alzheimer's disease.

Sortilin Inhibition Protects Neurons From Degeneration in the Diabetic Retina.

To investigate the level and localization of the multifunctional receptor sortilin in the diabetic retina, as well as the effect of sortilin inhibition on retinal neurodegeneration in experimental diabetes. The localization of sortilin and colocalization with the p75 neurotrophin receptor (p75NTR) and Müller cell (MC) markers were determined using immunofluorescence on retinal sections from human patients with diabetes and streptozotocin-induced diabetic C57BL/6J male mice. In the diabetic mice, levels were further quantified using Western blot and quantitative PCR. Therapeutic studies were performed on diabetic mice using intravitreally injected anti-sortilin antibodies. Neuroprotection was evaluated in vivo by optical coherence tomography and by quantification of retinal ganglion cells (RGCs) in flat mounts. Increased levels of sortilin were observed in human and murine diabetic retinas compared with nondiabetic control retinas. Sortilin was highly localized to retinal MCs, and, notably, colocalization with p75NTR was only seen in diabetic retinas. A remarkable protective effect of sortilin inhibition on inner retinal cells was observed in diabetic mice. At eight weeks after diabetes induction, inner retinal thickness was reduced by 9.7% (-12.7%, -6.6%; P < 0.0001; n = 11-12) in the PBS-injected control group compared with the anti-sortilin injected group. Similarly, the count of RGCs was reduced by 20.5% (-30.8%, -10.2%; P = 0.0009) in the PBS-injected control group compared with the anti-sortilin-injected group. Sortilin is upregulated in the diabetic retina, and sortilin inhibition effectively protects against neuronal loss. Thus sortilin emerges as a novel pharmacological target in diabetic retinal neurodegeneration-an important early event in the pathogenesis of diabetic retinopathy.

Is the retinal senescent due to neuronal disfunction or neuronal death?

AbstractThe continuous decline in vision due to aging is a common and irreversible degenerative process that is a consequence of multiple cumulative factors. Genetic heritage, behaviour, nutrition, and environmental factors are tangled along the course of life making aging a heterogeneously complex process. This contributes to make the inherent effects of aging difficult to evaluate in vision, and more so, when its outcome is often masked by other chronic ocular pathologies.Thus, investigating small mammals (rodents) under controlled conditions (food, environmental light, and temperature), we characterized the functional and anatomical changes in the aged retina. Full field electroretinograms in aged rodents displayed retinal function reduced by ~50% of the averaged response observed in juvenile ones. However, we did not observe any corneal or lens opacification that could provide any functional deficit. Therefore, based on the literature, we investigated whether neuronal loss in any of the different retinal layers could explain the reduced functionality. First, we estimated neuronal loss by examining the retinal thickness in vivo by Optical Coherence Tomography. As reported for aged mice and rats, we also observed a significant retinal thinning (&lt;20%) as measured by the total retinal thickness. However, this retinal thinning could not completely account for the ~50% reduction in retinal function.Consequently, we focused on the anatomical changes on aged retinas by quantifying two key neurons in the visual pathway: the photoreceptors, responsible for light capture, and the retinal ganglion cells (RGC), unique afferent neurons in the retina that project to the brain. First, Immunohistochemical analysis corroborated the retinal thinning; and second, quantitative analysis of Opsin and Brn3a (markers for photoreceptors and RGC respectively) demonstrated reduced densities in both cell populations as reported in other mammals. Interestingly, we found photoreceptor degeneration in the central retina of aged rodents. However, total quantification of RGCs in aged animals showed no significant differences compared to juvenile ones. By taking into account the growth of the retina over the lifetime of the rodents, the difference in retinal area provided an explanation to the observed retinal thinning and reduced RGC density across the retinal surface since volumetric analysis did not provide clear evidence of neuronal loss.Next, we assessed the glial state on aged brain and retina samples. In aged tissues, the microglial cells (Iba1) had ramified morphology, but quantitative analysis revealed a significant reduced dendritic arbor. Expression of GFAP in astrocytes and CD68, a lysosomal protein expressed on active phagocytic cells, showed a significant increase during healthy aging. Features that may contribute to a chronic parainflammation.Overall, these and previous data suggest that the reduced retinal function caused by aging may derive from neuronal disfunction, photoreceptor degeneration and imbalance of immune processes, including alterations in inflammation. Thus, the importance to better understanding of the aging processes become more crucial than ever before since our livespan is increasing over the years. Therefore, an early detection of these symptoms may help to identify healthier lifestyles to prevent vision loss, or to design new potential treatments to preserve or to restore vision.

Biological Correlations and Confounders for Quantification of Retinal Ganglion Cells by Optical Coherence Tomography Based on Studies of Outbred Mice.

PurposeDespite popularity of optical coherence tomography (OCT) in glaucoma studies, it's unclear how well OCT-derived metrics compare to traditional measures of retinal ganglion cell (RGC) abundance. Here, Diversity Outbred (J:DO) mice are used to directly compare ganglion cell complex (GCC) thickness measured by OCT to metrics of retinal anatomy measured ex vivo with retinal wholemounts and optic nerve histology.MethodsJ:DO mice (n = 48) underwent fundoscopic and OCT examinations, with automated segmentation of GCC thickness. RGC axons were quantified from para-phenylenediamine–stained optic nerve cross-sections and somas from BRN3A-immunolabeled retinal wholemounts, with total inner retinal cellularity assessed by TO-PRO and subsequent hematoxylin staining.ResultsJ:DO tissues lacked overt disease. GCC thickness, RGC abundance, and total cell abundance varied broadly across individuals. GCC thickness correlated significantly to RGC somal density (r = 0.58) and axon number (r = 0.44), but not total cell density. Retinal area and nerve cross-sectional area varied widely. No metrics were significantly influenced by sex. In bilateral comparisons, GCC thickness (r = 0.95), axon (r = 0.72), and total cell density (r = 0.47) correlated significantly within individuals.ConclusionsAmongst outbred mice, OCT-derived measurements of GCC thickness correlate significantly to RGC somal and axon abundance. Factors limiting correlation are likely both biological and methodological, including differences in retinal area that distort sampling-based estimates of RGC abundance.Translational RelevanceThere are significant—but imperfect—correlations between GCC thickness and RGC abundance across genetic contexts in mice, highlighting valid uses and ongoing challenges for meaningful use of OCT-derived metrics.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

A novel retinal ganglion cell quantification tool based on deep learning

Glaucoma is a disease associated with the loss of retinal ganglion cells (RGCs), and remains one of the primary causes of blindness worldwide. Major research efforts are presently directed towards the understanding of disease pathogenesis and the development of new therapies, with the help of rodent models as an important preclinical research tool. The ultimate goal is reaching neuroprotection of the RGCs, which requires a tool to reliably quantify RGC survival. Hence, we demonstrate a novel deep learning pipeline that enables fully automated RGC quantification in the entire murine retina. This software, called RGCode (Retinal Ganglion Cell quantification based On DEep learning), provides a user-friendly interface that requires the input of RBPMS-immunostained flatmounts and returns the total RGC count, retinal area and density, together with output images showing the computed counts and isodensity maps. The counting model was trained on RBPMS-stained healthy and glaucomatous retinas, obtained from mice subjected to microbead-induced ocular hypertension and optic nerve crush injury paradigms. RGCode demonstrates excellent performance in RGC quantification as compared to manual counts. Furthermore, we convincingly show that RGCode has potential for wider application, by retraining the model with a minimal set of training data to count FluoroGold-traced RGCs.

Mammalian Retinal Cell Quantification.

Normal retina and its cell layers are essential for processing visual stimuli, and loss of its integrity has been documented in many disease processes. The numbers and the axonal processes of retinal ganglion cells are reduced substantially in glaucoma, leading to vision loss and blindness. Similarly, selective loss of photoreceptors in age-related macular degeneration and hereditary retinal dystrophies also results in the compromise of visual acuity. Development of genetically modified mice has led to increased understanding of the pathogenesis of many retinal diseases. Similarly, in this digital era, usage of modalities to quantify the retinal cell loss has grown exponentially leading to a better understanding of the suitability of animal models to study human retinal diseases. These quantification modalities provide valuable quantifiable data in studying pathogenesis and disease progression. This review will discuss the immunohistochemical markers for various retinal cells, available automated tools to quantify retinal cells, and present an example of retinal ganglion cell quantification using HALO image analysis platform. Additionally, we briefly review retinal cell types and subtypes, salient features of retina in various laboratory animal species, and a few of the main disease processes that affect retinal cell numbers in humans.

Identification of Retinal Ganglion Cells from β-III Stained Fluorescent Microscopic Images.

Optic nerve crush in mouse model is widely used for investigating the course following retinal ganglion cell (RGCs) injury. Manual cell counting from β-III tubulin stained microscopic images has been routinely performed to monitor RGCs after an optic nerve crush injury, but is time-consuming and prone to observer variability. This paper describes an automatic technique for RGC identification. We developed and validated (i) a sensitive cell candidate segmentation scheme and (ii) a classifier that removed false positives while retaining true positives. Two major contributions were made in cell candidate segmentation. First, a homomorphic filter was designed to adjust for the inhomogeneous illumination caused by uneven penetration of β-III tubulin antibody. Second, the optimal segmentation parameters for cell detection are highly image-specific. To address this issue, we introduced an offline-online parameter tuning approach. Offline tuning optimized model parameters based on training images and online tuning further optimized the parameters at the testing stage without needing access to the ground truth. In the cell identification stage, 31 geometric, statistical and textural features were extracted from each segmented cell candidate, which was subsequently classified as true or false positives by support vector machine. The homomorphic filter and the online parameter tuning approach together increased cell recall by 28%. The entire pipeline attained a recall, precision and coefficient of determination (r2) of 85.3%, 97.1% and 0.994. The availability of the proposed pipeline will allow efficient, accurate and reproducible RGC quantification required for assessing the death/survival of RGCs in disease models.

Influence of retinal NMDA receptor activity during autoimmune optic neuritis.

Autoimmune optic neuritis (AON), a model of multiple sclerosis-associated optic neuritis, is accompanied by degeneration of retinal ganglion cells (RGCs) and optic nerve demyelination and axonal loss. In order to investigate the role of N-methyl-d-aspartate (NMDA) receptors in mediating RGC degeneration, upstream changes in the optic nerve actin cytoskeleton and associated deterioration in visual function, we induced AON in Brown Norway rats by immunization with myelin oligodendrocyte glycoprotein. Subsequently, visual acuity was assessed by recording visual evoked potentials and electroretinograms prior to extraction of optic nerves for western blot analysis and retinas for quantification of RGCs. As previously reported, in Brown Norway rats RGC degeneration is observed prior to onset of immune cell infiltration and demyelination of the optic nerves. However, within the optic nerve, destabilization of the actin cytoskeleton could be seen as indicated by an increase in the globular to filamentous actin ratio. Interestingly, these changes could be mimicked by intravitreal injection of glutamate, and similarly blocked by application of the NMDA receptor blocker MK-801, leading us to propose that prior to optic nerve lesion formation, NMDA receptor activation within the retina leads to retinal calcium accumulation, actin destabilization within the optic nerve as well as a deterioration of visual acuity during AON.

Oral administration of the iron chelator deferiprone protects against loss of retinal ganglion cells in a mouse model of glaucoma

Glaucoma is a progressive neurodegenerative process affecting the retinal ganglion cells (RGCs) and the optic nerve. Oxidative stress has been implicated in glaucoma pathogenesis, and iron is a potent generator of oxidative stress. The oral iron chelator deferiprone (DFP) is protective against retinal degenerations associated with oxidative stress. To test whether DFP could be protective in glaucoma, we used microbead injections to induce elevated intraocular pressure (IOP) in a cohort of 3-month old C57BL/6J mice. One eye of each animal was injected with magnetic microbeads resulting in ocular hypertension for >7 weeks while the fellow eye was injected with saline and served as a normotensive internal control. While half of the cohort received oral DFP (1 mg/ml in the drinking water), the other half did not and served as controls. After 8 weeks, Brn3a immunolabeling of flat-mounted retinas was used for manual RGC quantification. Axon counts were obtained from thin sections of optic nerves using the AxonJ plugin for ImageJ. DFP administration was protective against RGC and optic nerve loss in the setting of elevated IOP. These results suggest that iron chelation by DFP may provide glaucoma neuroprotection.

Algorithms looking for patterns of cell loss in glaucoma models

SummaryWhole‐retinal mounts have proven an invaluable tool for the assessment of retinal health in rodent models of disease with well‐established protocols for labelling of a variety of retinal cell populations including Retinal Ganglion Cells (RGCs). Loss of RGCs is thought to play a central role in many ocular disorders such as glaucoma, and RGC preservation is an established therapeutic endpoint. Nevertheless, a limitation of this approach is the high variability in RGC density between central and peripheral rodent retina, which can complicate RGC quantification by sampling. Whilst automated whole‐retinal measures of RGC density have added scientific rigour to the quantification of RGC populations, this technique still discards a lot of useful information. This talk outlines recent developments in the extraction of additional information from retinal whole‐mounts and reviews future directions for this technique in developing our understanding of disease pathology.

Transcorneal electrical stimulation promotes survival of retinal ganglion cells after optic nerve transection in rats accompanied by reduced microglial activation and TNF-α expression

Microglial activation plays a crucial role in the pathological processes of various retinal and optic nerve diseases. TNF-α is a pro-inflammatory cytokine that is rapidly upregulated and promotes retinal ganglion cells (RGCs) death after optic nerve injury. However, the cellular source of TNF-α after optic nerve injury remains unclear. Thus, we aimed to determine the changes of retinal microglial activation in a rat model of optic nerve transection (ONT) after transcorneal electrical stimulation (TES). Furthermore, we assessed TNF-α expression after ONT and evaluated the effects of TES on TNF-α production. Rats were divided into 2 control groups receiving a sham surgery procedure, 2 ONT+Sham TES groups, and 2 ONT+TES groups. The rats were sacrificed on day 7 or 14 after ONT. RGCs were retrogradely labelled by Fluorogold (FG) 7 days before ONT, one TES group and corresponding controls were stimulated on day 0, 4, and the second were stimulated on day 0, 4, 7, 10. Whole-mount immunohistofluorescence, quantification of RGCs and microglia, and western blot analysis were performed on day 7 and 14 after ONT. TES significantly increased RGCs survival on day 7 and 14 after ONT, which was accompanied by reduced microglia on day 7, but not 14. TNF-α was co-localized with ameboid microglia and significantly increased on day 7 and 14 after ONT. TES significantly reduced TNF-α production on day 7 and 14 after ONT. Our study demonstrated that TES promotes RGCs survival after ONT accompanied by reduced microglial activation and microglia-derived TNF-α production.

Pou4f2-GFP knock-in mouse line: A model for studying retinal ganglion cell development.

Pou4f2 acts as a key node in the comprehensive and step-wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2-green fluorescent protein (GFP) fusion protein expressed in RGCs. Co-localization of POU4F2 and GFP in the retina and brain of Pou4f2-GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild-type mice, the expression patterns of POU4F2 and POU4F1 and the co-expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2-GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2-GFP/GFP homozygote and wild-type mice. These results demonstrated that the development of RGCs in Pou4f2-GFP/GFP homozygote mice was the same as in wild-type mice. Thus, the present Pou4f2-GFP knock-in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs.

Quantitative measurement of retinal ganglion cell populations via histology-based random forest classification

The inner surface of the retina contains a complex mixture of neurons, glia, and vasculature, including retinal ganglion cells (RGCs), the final output neurons of the retina and primary neurons that are damaged in several blinding diseases. The goal of the current work was two-fold: to assess the feasibility of using computer-assisted detection of nuclei and random forest classification to automate the quantification of RGCs in hematoxylin/eosin (H&E)-stained retinal whole-mounts; and if possible, to use the approach to examine how nuclear size influences disease susceptibility among RGC populations. To achieve this, data from RetFM-J, a semi-automated ImageJ-based module that detects, counts, and collects quantitative data on nuclei of H&E-stained whole-mounted retinas, were used in conjunction with a manually curated set of images to train a random forest classifier. To test performance, computer-derived outputs were compared to previously published features of several well-characterized mouse models of ophthalmic disease and their controls: normal C57BL/6J mice; Jun-sufficient and Jun-deficient mice subjected to controlled optic nerve crush (CONC); and DBA/2J mice with naturally occurring glaucoma. The result of these efforts was development of RetFM-Class, a command-line-based tool that uses data output from RetFM-J to perform random forest classification of cell type. Comparative testing revealed that manual and automated classifications by RetFM-Class correlated well, with 83.2% classification accuracy for RGCs. Automated characterization of C57BL/6J retinas predicted 54,642 RGCs per normal retina, and identified a 48.3% Jun-dependent loss of cells at 35 days post CONC and a 71.2% loss of RGCs among 16-month-old DBA/2J mice with glaucoma. Output from automated analyses was used to compare nuclear area among large numbers of RGCs from DBA/2J mice (n = 127,361). In aged DBA/2J mice with glaucoma, RetFM-Class detected a decrease in median and mean nucleus size of cells classified into the RGC category, as did an independent confirmation study using manual measurements of nuclear area demarcated by BRN3A-immunoreactivity. In conclusion, we have demonstrated that histology-based random forest classification is feasible and can be utilized to study RGCs in a high-throughput fashion. Despite having some limitations, this approach demonstrated a significant association between the size of the RGC nucleus and the DBA/2J form of glaucoma.

Retina-specific T cells provide neuroprotection in a mouse model of glaucoma (BA6P.140)

Abstract Glaucoma is a disease that leads to degeneration of retinal ganglion cells (RGC) and is potentially blinding if left untreated. Based on the concept of “beneficial autoimmunity”, we examined whether naïve, retina-specific T lymphocytes can provide neuroprotection to retinal ganglion cells in an optic nerve crush injury glaucoma model. Purified T cells from mice that express the transgenic R161H TCR for the retinal antigen IRBP, as well as eGFP (R161H-eGFP), were adoptively transferred into wild type (WT) mice. Also, irradiated bone marrow (BM) chimeras were stably engrafted with &amp;lt;10% circulating R161H-eGFP cells. Recipient mice were subjected to retrograde fluorogold labeling for quantification of RGC prior to an optic nerve crush injury. Retinas were imaged 4 or 7 days later for quantification of RGCs and donor R161H-eGFP T cells were detected by confocal microscopy. In both types of recipients, T cell influx into the eye and neuroprotection were apparent, but differed in kinetics. In adoptively transferred mice, R161H T cells were detected in the eye 4 days post injury and were associated with 27% less RGC loss compared to controls infused with normal polyclonal eGFP+ T cells (p&amp;lt;0.05). In the BM chimeric mice, major influx of R161H-eGFP cells occurred at 7 days post injury and was associated with a 16% statistically significant reduction in RGC loss. We conclude that naïve retina-specific T cells can mediate neuroprotection of retinal ganglion cells.

The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina

There are few neurochemical markers that reliably identify retinal ganglion cells (RGCs), which are a heterogeneous population of cells that integrate and transmit the visual signal from the retina to the central visual nuclei. We have developed and characterized a new set of affinity-purified guinea pig and rabbit antibodies against RNA-binding protein with multiple splicing (RBPMS). On western blots these antibodies recognize a single band at 〜24 kDa, corresponding to RBPMS, and they strongly label RGC and displaced RGC (dRGC) somata in mouse, rat, guinea pig, rabbit, and monkey retina. RBPMS-immunoreactive cells and RGCs identified by other techniques have a similar range of somal diameters and areas. The density of RBPMS cells in mouse and rat retina is comparable to earlier semiquantitative estimates of RGCs. RBPMS is mainly expressed in medium and large DAPI-, DRAQ5-, NeuroTrace- and NeuN-stained cells in the ganglion cell layer (GCL), and RBPMS is not expressed in syntaxin (HPC-1)-immunoreactive cells in the inner nuclear layer (INL) and GCL, consistent with their identity as RGCs, and not displaced amacrine cells. In mouse and rat retina, most RBPMS cells are lost following optic nerve crush or transection at 3 weeks, and all Brn3a-, SMI-32-, and melanopsin-immunoreactive RGCs also express RBPMS immunoreactivity. RBPMS immunoreactivity is localized to cyan fluorescent protein (CFP)-fluorescent RGCs in the B6.Cg-Tg(Thy1-CFP)23Jrs/J mouse line. These findings show that antibodies against RBPMS are robust reagents that exclusively identify RGCs and dRGCs in multiple mammalian species, and they will be especially useful for quantification of RGCs.

Quantitative Analysis of Retinal Ganglion Cell Survival with Rbpms Immunolabeling in Animal Models of Optic Neuropathies

To investigate whether a recently described retinal ganglion cell (RGC) marker Rbpms (RNA binding protein with multiple splicing) could be used for RGC quantification in various models of RGC degeneration. Optic nerve crush, excitotoxicity, and elevated intraocular pressure (IOP) rat models were used. Topographic analysis of Rbpms immunolabeling was performed on retinal wholemounts. Retrograde labelings with Fluorogold (FG) and III β-tubulin immunohistochemistry were compared. In the optic nerve crush model, 37%, 87%, and 93% of Rbpms-positive cells were lost 1, 2, and 4 weeks, respectively. Significant loss of Rbpms-positive cells was noted 1 week after intravitreal injection of 12, 30, and 120 nmol N-methyl-d-aspartate (NMDA), whereas coinjection of 120 nmol of NMDA along with MK-801 increased the cell number from 10% to 59%. Over 95% of Rbpms-positive cells were FG- and III β-tubulin-positive after injury caused by optic nerve crush and NMDA injection. In rats with elevated IOP, induced by trabecular laser photocoagulation, there was a significant loss of Rbpms-positive cells compared with that of contralateral controls (P = 0.0004), and cumulative IOP elevation showed a strong linear relationship with the quantification of RGCs by Rbpms immunolabeling and retrograde labeling with FG. More than 99% of the remaining Rbpms-positive cells were double-labeled with FG. Rbpms can reliably be used as an RGC marker for quantitative evaluation in rat models of RGC degeneration, regardless of the nature and the location of the primary site of the injury and the extent of neurodegeneration.

Caspase inhibitors protect against NMDA-mediated retinal ganglion cell death

Apoptosis is a major mechanism of cell death in glutamate-induced excitotoxicity and caspases as the executors of apoptosis play an important role in the development of various central nervous system and eye diseases. We studied the involvement of certain caspases in excitotoxic retinal ganglion cell death, which was experimentally induced in Brown Norway Rats by application of the glutamate receptor agonist N-methyl-D-aspartate (NMDA). Animals were injected intravitreally with one of six caspase inhibitors (against caspases 1, 3, 4, 6, 8 and 9). Seven hours later, NMDA or phosphate-buffered saline as a control was injected intravitreally into the respective eyes. The neuroprotective potential against NMDA toxicity was assessed by retinal ganglion cell quantification. Additionally, wholemount TUNEL was performed. Statistical analysis revealed significant neuroprotective effects for the inhibitors of caspases 3, 6, 8 and 9, but not for those of caspases 1 and 4. The inhibitors of caspases 6 and 9 showed greater neuroprotective potential than those of caspases 3 and 8, although cell death was not entirely averted in any case. Results of ganglion cell counts were confirmed for the most pronounced treatment groups using wholemount TUNEL. Excitotoxic retinal ganglion cell death after NMDA injection is mediated mainly through apoptosis, whereby extrinsic as well as intrinsic pathways of caspase activation play a role.

Chronic and Acute Models of Retinal Neurodegeneration TrkA Activity Are Neuroprotective whereas p75NTR Activity Is Neurotoxic through a Paracrine Mechanism

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75(NTR). During retinal injury, endogenous NGF, TrkA, and p75(NTR) are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75(NTR) affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75(NTR) agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75(NTR) action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α(2)-macroglobulin. Antagonists of p75(NTR) inhibit TNF-α and α(2)-macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.

RNA binding protein with multiple splicing: a new marker for retinal ganglion cells.

To characterize expression of the RNA binding protein (RBPMS) in the retina as a specific marker for retinal ganglion cells (RGCs). Optic nerve transection (ONT) was performed on adult male Wistar rats. Retrograde RGC labeling was performed with FluoroGold (FG) applied to the cut surface of the optic nerve. RBPMS mRNA and protein expression in the retina was analyzed by in situ hybridization and immunohistochemistry, respectively. The expression of RBPMS in various rat tissues was analyzed with semiquantitative RT-PCR. RBPMS mRNA and protein expression was localized primarily to irregularly shaped cells in the ganglion cell layer of the retina. Quantitative analysis showed that almost 100% of RGCs labeled by FG were also RBPMS-positive, irrespective of their location relative to the optic nerve head. Approximately 94% to 97% of RBPMS-positive cells were also positive for Thy-1, neurofilament H, and III beta-tubulin. In 2-week ONT retinas, the remaining few RGCs were weakly stained with RBPMS compared with intact RGCs in control retinas. Outside the retina, expression of RBPMS was observed in the heart, kidney, liver, and lungs. No expression was detected in any neuronal tissues except the retina. The data indicate that in the retina RBPMS is selectively expressed in RGCs and therefore could serve as a marker for RGC quantification in normal retinas and for estimation of RGC loss in ocular neuropathies.