Total Retinal Ganglion Cell Research Articles

POU6F2, a risk factor for glaucoma, myopia and dyslexia, labels specific populations of retinal ganglion cells

Pou6f2 is a genetic connection between central corneal thickness (CCT) in the mouse and a risk factor for developing primary open-angle glaucoma. POU6F2 is also a risk factor for several conditions in humans, including glaucoma, myopia, and dyslexia. Recent findings demonstrate that POU6F2-positive retinal ganglion cells (RGCs) comprise a number of RGC subtypes in the mouse, some of which also co-stain for Cdh6 and Hoxd10. These POU6F2-positive RGCs appear to be novel of ON–OFF directionally selective ganglion cells (ooDSGCs) that do not co-stain with CART or SATB2 (typical ooDSGCs markers). These POU6F2-positive cells are sensitive to damage caused by elevated intraocular pressure. In the DBA/2J mouse glaucoma model, heavily-labeled POU6F2 RGCs decrease by 73% at 8 months of age compared to only 22% loss of total RGCs (labeled with RBPMS). Additionally, Pou6f2−/− mice suffer a significant loss of acuity and spatial contrast sensitivity along with an 11.4% loss of total RGCs. In the rhesus macaque retina, POU6F2 labels the large parasol ganglion cells that form the magnocellular (M) pathway. The association of POU6F2 with the M-pathway may reveal in part its role in human glaucoma, myopia, and dyslexia.

Impact of Acute Ocular Hypertension on Retinal Ganglion Cell Loss in Mice.

To assess the correlation between intraocular pressure (IOP) levels and retinal ganglion cell (RGC) loss across different fixed-duration episodes of acute ocular hypertension (AOH). AOH was induced in Thy1-YFP-H transgenic mice by inserting a needle connected to a saline solution container into the anterior chamber. Thirty-one groups were tested, each comprising three to five mice exposed to IOP levels ranging from 50 to 110mm Hg in 5/10mm Hg increments for 60/90/120minutes and a sham control group. The YFP-expressing RGCs were quantified by confocal scanning laser ophthalmoscopy, whereas peripapillary ganglion cell complex thickness was measured using spectral-domain optical coherence tomography. Changes in RGC count and GCCT were determined from values measured 30days after AOH relative to baseline (before AOH). In the 60-minute AOH groups, RGC loss varied even when IOP was increased up to 110mm Hg (36.8%-68.2%). However, for longer durations (90 and 120minutes), a narrow range of IOP levels (60-70mm Hg for 90-minute duration; 55-65mm Hg for 120-minute duration) produced a significant difference in RGC loss, ranging from <25% to >90%. Additionally, loss of YFP-expressing RGCs was comparable to that of total RGCs in the same retinas. Reproducible RGC loss during AOH depends on precise durations and IOP thresholds. In the current study, the optimal choice is an AOH protocol set at 70mm Hg for a duration of 90minutes. This study can assist in determining the optimal duration and intensity of IOP for the effective utilization of AOH models.

Characterization of the whole Advillin expressing retinal ganglion cell population in the rodent retina

Aims/Purpose: We have quantified and examined the topography of the total population of retinal ganglion cells (RGC) expressing Advillin (Advillin+RGCs), an actin‐binding protein expressed in somatic sensory neurons, in the retinas, as well as their projections to the brain, in control adult Advillin‐Cretg/+ mice (Zurborg et al. Mol Pain 2011, 7:66).Methods: Retinas from adult Advillin‐Cretg/+ Ai14fl/fl C57BL/6J mice (Frutos‐Rincón et al., Int J Mol Sci 2022, 23:2997) expressing tdTomato were prepared as whole‐mounts, labelled with antibodies recognizing Brn3a and melanopsin. The entire Advillin+RGC population was quantified and its retinal distribution analysed by nearest neighbour maps. The brains were serial coronal crysectioned and the axonal Advillin+RGC projections in the brain were anatomically analysed.Results: Advillin protein was mainly located in the somas and axons of a subset of RGCs. Total number of Advillin+RGCs was 1947 ± 157 (mean ± SD; n = 7), which constitutes 5% of the total RGC population. Approximately 17.5% (340 ± 76) and 0% (3 ± 1) of the Advillin+RGC population co‐expressed Brn3a or melanopsin proteins, respectively. The Advillin+RGC population was distributed across the entire retina, with a higher concentration in the temporal region. The axons of this RGC subpopulation project to the main retinal recipient regions in the brain, such as the lateral geniculate nucleus (LGN) and superior colliculus (SC). However, none of these axons project to the suprachiasmatic nucleus. These data indicates that Advillin+RGCs is not a subpopulation of ipRGCs.Conclusions: The Advillin protein, which is characteristic of sensory neurons, is also expressed in a specific RGC subpopulation of the adult mouse retina. A small percentage of Advillin+RGCs expressed the Brn3a transcription factor, while none expressed melanopsin. The axons of Advillin+RGCs project to the typical retino‐recipient areas in the brain, excluding the suprachiasmatic nucleus.

Commentary: Rethinking 10-2 visual fields in early diagnosis of glaucoma for a glided glaucoma practice: The right choice to pick up a feeble noise?

Commentary: Rethinking 10-2 visual fields in early diagnosis of glaucoma for a glided glaucoma practice: The right choice to pick up a feeble noise?

Effects of lighting environment on the degeneration of retinal ganglion cells in glutamate/aspartate transporter deficient mice, a mouse model of normal tension glaucoma

Lighting conditions may affect the development of retinal degenerative diseases such as macular degeneration. In this study, to determine whether the lighting environment affects the progression of degeneration of retinal ganglion cells (RGCs), we examined glutamate/aspartate transporter (GLAST) heterozygous (GLAST+/-) mice, a mouse model of normal tension glaucoma. GLAST+/- mice were reared under a 12-h light-dark cycle (Light/Dark) or complete darkness (Dark/Dark) condition after birth. The total RGC number in the Dark/Dark group was significantly decreased compared with the Light/Dark group at 3 weeks old, while the number of osteopontin-positive αRGCs were similar in both groups. At 6 and 12 weeks old, the total RGC number were not significantly different in both conditions. In addition, the retinal function examined by multifocal electroretinogram were similar at 12 weeks old. These results suggest that lighting conditions may regulate the progression of RGC degeneration in some types of glaucoma.

Possible antiapoptotic and neuroprotective effects of magnesium sulphate on retina in a preterm hypoxic-ischemic rat model

Background/aim The effects of systemic magnesium sulfate (MgSO4) on retina in preterm hypoxic-ischemic (HI) rat model are not known. Our aim was to investigate the effects of MgSO4 on retinal ganglion cell (RGC) count, retinal ganglion cell (RGC) apoptotic index, retinal vascular endothelial growth factor receptor-2 (VEGFR-2), and glial fibrillary acidic protein (GFAP) expressions in preterm HI rat model.Materials and methods Fifteen, postnatal day (PND) 7 rat pups were divided into 3 groups: 1. Sham-operated group, 2. HI group, and 3. MgSO4-treated HI group. The second and third groups underwent ischemia followed by exposure to hypoxia for 2 h (Vannucci model). The first and second groups received intraperitoneal saline and the third group received intraperitoneal MgSO4. On PND 10, eyes of the pups were evaluated for RGC count, apoptotic index, VEGFR-2, and GFAP expressions. Results In both HI and MgSO4-treated HI group, the mean total RGC counts were found to be significantly decreased. However, the mean total RGC count in the MgSO4-treated HI group was significantly higher than that of the HI group. The mean apoptotic index was found to be significantly increased in the HI group. Retinal VEGFR-2 and GFAP expressions were found to be significantly higher in the HI group.ConclusionsMagnesium sulfate preconditioning andtreatment in preterm HI rat model might diminish apoptosis, relatively preserve RGCs, and reduce retinal VEGFR-2 and GFAP expressions.

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.

The relation of individual variation in total retinal ganglion cell layer thickness to post-retinal anatomy

The relation of individual variation in total retinal ganglion cell layer thickness to post-retinal anatomy

Evidence of Hypoxic Glial Cells in a Model of Ocular Hypertension.

PurposeReoxygenation after hypoxia can increase reactive oxygen species and upregulate autophagy. We determined, for the first time, the impact of elevated IOP on hypoxia induction, superoxide accumulation, and autophagy in a bead model of glaucoma.MethodOcular hypertension was achieved with magnetic bead injection into the anterior chamber. Before mice were killed, they were injected with pimonidazole for hypoxia detection and dihydroethidium (DHE) for superoxide detection. Total retinal ganglion cells (RGCs) and optic nerve (ON) axons were quantified, total glutathione (GSH) was measured, and retinal and ON protein and mRNA were analyzed for hypoxia (Hif-1α and Hif-2α), autophagy (LC3 and p62), and SOD2.ResultsWith IOP elevation (P < 0.0001), the retina showed significantly (P < 0.001) decreased GSH compared with control, and a significant decrease (P < 0.01) in RGC density compared with control. Pimonidazole-positive Müller glia, microglia, astrocytes, and RGCs were present in the retinas after 4 weeks of ocular hypertension but absent in both the control and after only 2 weeks of ocular hypertension. The ON showed significant axon degeneration (P < 0.0001). The mean intensity of DHE in the ganglion cell layer and ON significantly increased (P < 0.0001). The ratio of LC3-II to LC3-I revealed a significant increase (P < 0.05) in autophagic activity in hypertensive retinas compared with control.ConclusionsWe report a novel observation of hypoxia and a significant decrease in GSH, likely contributing to superoxide accumulation, in the retinas of ocular hypertensive mice. The significant increase in the ratio of LC3-II to LC3-I suggests autophagy induction.

Melanopsin retinal ganglion cells are not labeled in Thy-1YFP-16 transgenic mice.

Retinal ganglion cells (RGCs) that express the photopigment melanopsin (mRGCs) are photosensitive and initiate the non-image-forming pathway, where the majority of their axons terminate in the suprachiasmatic nucleus (SCN). RGCs only make up approximately half of the cells in the ganglion cell layer of the retina; therefore, it is important to be able to distinguish them from other cell types. The transgenic Thy-1 YFP mouse line 16 (Thy-1 YFP-16) expresses yellow-fluorescent protein (YFP) in projection neurons, including RGCs. Our objective was to determine whether mRGCs are labeled with YFP in Thy-1 YFP-16 transgenic mice. Paraformaldehyde-fixed retinal wholemounts and frozen vertical sections were prepared from Thy-1 YFP-16 mice and fluorescently labeled with rabbit anti-melanopsin and guinea-pig anti-RNA binding protein with multiple splicing to identify mRGCs and total RGCs, respectively. Thy-1 YFP-16 mouse brains were sectioned coronally and imaged to view RGC axonal projections to the SCN. Confocal images of retinal preparations show that the majority (∼89%) of mRGCs are not YFP-positive in Thy-1 YFP-16 mice, where ∼11% expressed a weak fluorescent signal. In addition, there are almost no YFP-positive axons present in the SCN of coronal brain sections. We conclude that the majority of mRGC somas and axons are not labeled with YFP in the transgenic Thy-1 YFP-16 mouse line; therefore, this mouse model may not suitable for research involving mRGC visual pathways.

Regional Retinal Ganglion Cell Axon Loss in a Murine Glaucoma Model.

PurposeTo determine if retinal ganglion cell (RGC) axon loss in experimental mouse glaucoma is uniform in the optic nerve.MethodsExperimental glaucoma was induced for 6 weeks with a microbead injection model in CD1 (n = 78) and C57BL/6 (B6, n = 68) mice. From epoxy-embedded sections of optic nerve 1 to 2 mm posterior to the globe, total nerve area and regional axon density (axons/1600 μm2) were measured in superior, inferior, nasal, and temporal zones.ResultsControl eyes of CD1 mice have higher axon density and more total RGCs than control B6 mice eyes. There were no significant differences in control regional axon density in all mice or by strain (all P > 0.2, mixed model). Exposure to elevated IOP caused loss of RGC in both strains. In CD1 mice, axon density declined without significant loss of nerve area, while B6 mice had less density loss, but greater decrease in nerve area. Axon density loss in glaucoma eyes was not significantly greater in any region in either mouse strain (both P > 0.2, mixed model). In moderately damaged CD1 glaucoma eyes, and CD1 eyes with the greatest IOP elevation exposure, density loss differed by region (P = 0.05, P = 0.03, mixed model) with the greatest loss in the temporal and superior regions, while in severely injured B6 nerves superior loss was greater than inferior loss (P = 0.01, mixed model, Bonferroni corrected).ConclusionsThere was selectively greater loss of superior and temporal optic nerve axons of RGCs in mouse glaucoma at certain stages of damage. Differences in nerve area change suggest non-RGC responses differ between mouse strains.

A freely available semi-automated method for quantifying retinal ganglion cells in entire retinal flatmounts

Glaucomatous optic neuropathies are characterized by progressive loss of retinal ganglion cells (RGCs), the neurons that connect the eye to the brain. Quantification of these RGCs is a cornerstone in experimental optic neuropathy research and commonly performed via manually quantifying parts of the retina. However, this is a time-consuming process subject to inter- and intra-observer variability. Here we present a freely available ImageJ script to semi-automatically quantify RGCs in entire retinal flatmounts after immunostaining for the RGC-specific transcription factor Brn3a. The blob-like signal of Brn3a-immunopositive RGCs is enhanced via eigenvalues of the Hessian matrix and the resulting local maxima are counted as RGCs. After the user has outlined the retinal flatmount area, the total RGC number and retinal area are reported and an isodensity map, showing the RGC density distribution across the retina, is created. The semi-automated quantification shows a very strong correlation (Pearson's r ≥ 0.99) with manual counts for both widefield and confocal images, thereby validating the data generated via the developed script. Moreover, application of this method in established glaucomatous optic neuropathy models such as N-methyl-D-aspartate-induced excitotoxicity, optic nerve crush and laser-induced ocular hypertension revealed RGC loss conform with literature. Compared to manual counting, the described automated quantification method is faster and shows user-independent consistency. Furthermore, as the script detects the RGC number in entire retinal flatmounts, the method allows detection of regional differences in RGC density. As such, it can help advance research investigating the degenerative mechanisms of glaucomatous optic neuropathies and the effectiveness of new neuroprotective treatments. Because the script is flexible and easy to optimize due to a low number of critical parameters, it can potentially be applied in combination with other tissues or alternative labeling protocols.

Functional and anatomical assessment of retinal ganglion cells in glaucoma

Purpose The aim of the study was to assess the ability of retinal ganglion cell (RGC) complex measurements obtained by optical coherence tomography (OCT) to detect glaucoma, to measure RGC loss by pattern electroretinogram (PERG), and to correlate the results of PERG with OCT for glaucoma detection. Patients and methods Fifty glaucoma suspects (100 eyes), 70 glaucoma patients (140 eyes), and 50 age-matched controls (100 eyes) were examined. Participants had clear ocular media, no or mild refractive errors, and no concomitant ocular or systemic diseases. Superior and inferior ganglion cell complex thickness and retinal nerve fiber layer (RNFL) thickness were measured by means of OCT. The amplitude and latency of PERG were elicited by counter-phase checks. Results The mean RNFL thickness and mean RGC thickness were reduced in glaucoma patients compared with glaucoma suspects and controls. The mean RNFL thickness was 88 ± 9.9 µm in early glaucoma, 94 ± 12 µm in glaucoma suspects, and 99 ± 15.5 µm in controls. The total RGC thickness was 55 ± 6.2 µm in early glaucoma, 64.9 ± 3.8 µm in glaucoma suspects, and 65 ± 4.1 µm in controls. The mean PERG amplitude was decreased in both glaucoma suspects and glaucoma patients compared with controls. In glaucoma suspects, PERG amplitude did not correlate significantly with both RNFL thickness and RGC thickness, whereas in glaucoma patients PERG amplitude was positively correlated with both RNFL and RGC thickness. Conclusion OCT can assess the RGC anatomically, whereas PERG can functionally evaluate RGC loss. Lack of anatomical–functional relationship in glaucoma suspects suggests that at this stage PERG losses appear to affect primarily the retinal/optic nerve function. In glaucoma patients, PERG reflects both dysfunction and loss of ganglion cells.

Oxygen-induced retinopathy induces short-term glial stress and long-term impairment of photoentrainment in mice

Retinopathy of prematurity is a serious potentially blinding disease of pre-term infants. There is extensive vascular remodeling and tissue stress, but data concerning alterations in retinal neurons and glia, and long-term functional sequelae are still incomplete. ROP was induced using the oxygen-induced retinopathy (OIR) mouse model. Postnatal day 7 (P7) 129SVE mice were exposed to hyperoxia (75 ± 0.5 % oxygen) for 5 days, and then returned to normoxia to induce OIR. Exposed animals were euthanized at 5 (P17-OIR) and 14 days (P26-OIR) after return to normal air, together with corresponding age-matched control mice (P17-C and P26-C respectively) raised only in room air. Their retinas were examined by immunohistochemistry using a battery of antibodies against key glial and neuronal proteins. A further group of OIR mice and controls were examined at 10 weeks of age for their ability to re-entrain to changing 12 h light/12 h dark cycles, assayed by wheel-running actimetry. In this protocol, animals were subjected to three successive conditions of 300 lux, 15 lux and 1 lux ambient light intensity coupled with 6 hours of jetlag. Animals were euthanized at 4 months of age and used in immunoblotting for rhodopsin. Compared to P17-C, immunohistochemical staining of P17-OIR sections showed up-regulation of stress-related and glutamate-regulatory proteins in astrocytes and Müller glial cells. In contrast, glial phenotypic expression in P26-OIR retinas largely resembled that in P26-C. There was no loss in total retinal ganglion cells (RGC) at either P17-OIR or P26-OIR compared to corresponding controls, whereas intrinsically photosensitive RGC showed significant decreases, with 375 ± 13/field in P26-OIR compared to 443 ± 30/field in P26-C (p < 0.05). Wheel actimetry performed on control and OIR-treated mice at 4 months demonstrated that animals raised in hyperoxic conditions had impaired photoentrainment at low illuminance of 1 lux, as well as significantly reduced levels of rhodopsin compared to age-matched controls. OIR leads to transient up-regulation of retinal glial proteins involved in metabolism, and partial degeneration of intrinsically photosensitive RGC and rod photoreceptors. OIR affects circadian photo-entrainment at low illuminance values, possibly by affecting the rod pathway and/or intrinsically photosensitive RGC input to the circadian clock. This study hence shows that retinopathy of prematurity affects light-regulated circadian behavior in an animal model, and may induce similar problems in humans.

Axotomy-induced retinal ganglion cell death in adult mice: Quantitative and topographic time course analyses

The fate of retinal ganglion cells after optic nerve injury has been thoroughly described in rat, but not in mice, despite the fact that this species is amply used as a model to study different experimental paradigms that affect retinal ganglion cell population. Here we have analyzed, quantitatively and topographically, the course of mice retinal ganglion cells loss induced by intraorbital nerve transection. To do this, we have doubly identified retinal ganglion cells in all retinas by tracing them from their main retinorecipient area, the superior colliculi, and by their expression of BRN3A (product of Pou4f1 gene). In rat, this transcription factor is expressed by a majority of retinal ganglion cells; however in mice it is not known how many out of the whole population of these neurons express it. Thus, in this work we have assessed, as well, the total population of BRN3A positive retinal ganglion cells. These were automatically quantified in all whole-mounted retinas using a newly developed routine. In control retinas, traced-retinal ganglion cells were automatically quantified, using the previously reported method (Salinas-Navarro et al., 2009b). After optic nerve injury, though, traced-retinal ganglion cells had to be manually quantified by retinal sampling and their total population was afterwards inferred. In naïve whole-mounts, the mean (±standard deviation) total number of traced-retinal ganglion cells was 40,437(±3196) and of BRN3A positive ones was 34,697(±1821). Retinal ganglion cell loss was first significant for both markers 5 days post-axotomy and by day 21, the last time point analyzed, only 15% or 12% of traced or BRN3A positive retinal ganglion cells respectively, survived. Isodensity maps showed that, in control retinas, BRN3A and traced-retinal ganglion cells were distributed similarly, being densest in the dorsal retina along the naso-temporal axis. After axotomy the progressive loss of BRN3A positive retinal ganglion cells was diffuse and affected the entire retina. In conclusion, this is the first study assessing the values, in terms of total number and density, of the retinal ganglion cells surviving axotomy from 2 till 21 days post-lesion. Besides, we have demonstrated that BRN3A is expressed by 85.6% of the total retinal ganglion cell population, and because BRN3A positive retinal ganglion cells show the same spatial distribution and temporal course of degeneration than traced ones, BRN3A is a reliable marker to identify, quantify and assess, ex-vivo, retinal ganglion cell loss in this species.

Melanopsin retinal ganglion cells are resistant to neurodegeneration in mitochondrial optic neuropathies

Mitochondrial optic neuropathies, that is, Leber hereditary optic neuropathy and dominant optic atrophy, selectively affect retinal ganglion cells, causing visual loss with relatively preserved pupillary light reflex. The mammalian eye contains a light detection system based on a subset of retinal ganglion cells containing the photopigment melanopsin. These cells give origin to the retinohypothalamic tract and support the non-image-forming visual functions of the eye, which include the photoentrainment of circadian rhythms, light-induced suppression of melatonin secretion and pupillary light reflex. We studied the integrity of the retinohypothalamic tract in five patients with Leber hereditary optic neuropathy, in four with dominant optic atrophy and in nine controls by testing the light-induced suppression of nocturnal melatonin secretion. This response was maintained in optic neuropathy subjects as in controls, indicating that the retinohypothalamic tract is sufficiently preserved to drive light information detected by melanopsin retinal ganglion cells. We then investigated the histology of post-mortem eyes from two patients with Leber hereditary optic neuropathy and one case with dominant optic atrophy, compared with three age-matched controls. On these retinas, melanopsin retinal ganglion cells were characterized by immunohistochemistry and their number and distribution evaluated by a new protocol. In control retinas, we show that melanopsin retinal ganglion cells are lost with age and are more represented in the parafoveal region. In patients, we demonstrate a relative sparing of these cells compared with the massive loss of total retinal ganglion cells, even in the most affected areas of the retina. Our results demonstrate that melanopsin retinal ganglion cells resist neurodegeneration due to mitochondrial dysfunction and maintain non-image-forming functions of the eye in these visually impaired patients. We also show that in normal human retinas, these cells are more concentrated around the fovea and are lost with ageing. The current results provide a plausible explanation for the preservation of pupillary light reaction despite profound visual loss in patients with mitochondrial optic neuropathy, revealing the robustness of melanopsin retinal ganglion cells to a metabolic insult and opening the question of mechanisms that might protect these cells.

Compensatory and transneuronal plasticity after early collicular ablation

Plasticity within the visual system was assessed in the quokka wallaby following unilateral superior collicular (SC) ablation at postnatal days (P) 8-10, prior to the arrival of retinal ganglion cell (RGC) axons. At maturity (P100), projections were traced from the eye opposite the ablation, and total RGC numbers were estimated for both eyes. Ablations were partial (28-89% of SC remaining) or complete (0-5% of SC remaining). Projections to the visual centers showed significant bilateral (P < 0.05) increases in absolute volume. Minor anomalous projections also formed within the deep, surviving non-retino-recipient layers of the ablated SC and via a small bundle of RGC axons recrossing the midline to innervate discrete patches in the SC contralateral to the lesion. Total absolute volume of projections did not differ between partial and complete ablations; moreover, values did not differ from normal (P > 0.05). Compared with normal, total RGC numbers were significantly (P < 0.05) reduced in the eye opposite the ablation but increased (P < 0.05) in the other eye. Consequently, the sum of the two RGC populations did not differ from normal (P > 0.05). As in rodents, the visual system in quokka compensates following injury by maintaining a set volume of arborization but does so by forming only minor anomalous projections. Furthermore, increased RGC numbers in the eye ipsilateral to the lesion indicate that compensation occurs transneuronally, thus maintaining total numbers of projecting neurons. The implication is that the visual system acts in concert following unilateral injury to maintain set values for RGC terminal arbors as well as their cell bodies.

Topographic and Morphologic Analyses of Retinal Ganglion Cell Loss in Old DBA/2NNia Mice

To evaluate the relationship between retinal ganglion cell (RGC) size, density distribution, and survival in senescent DBA2/NNia mice that develop pigmentary glaucoma. To evaluate the validity of nearest neighbor distance (NND), a measure of focal density for surviving RGCs in the retina, as a method to quantify RGC loss in mice. Fifteen-month-old DBA2/NNia mice were labeled retrogradely with fluorogold. Retinas were flat mounted and imaged in their entirety using an epifluorescence microscope with a motorized stage. Digital maps of the retinal wholemounts were constructed to automatically count and establish spatial coordinates for RGCs over the entire retina. RGC size and NND were determined from these maps. RGC counts in the group of 15-month-old DBA/2NNia animals ranged from 22,330 to 92,157 cells per retina. Mean RGC cell size per retina ranged from 22.35 to 35.64 microm2 and correlated linearly with total RGC counts. NND distribution histograms were compared for retinas with variable degrees of RGC loss. The distribution of NNDs in each retina was skewed toward larger distance values in more affected retinas. In partially damaged retinas, areas with severe pathology coincided with areas of maximal loss of large RGCs, and areas of preserved RGCs correlated with larger cell sizes. Damaged retinas have a smaller mean cell size, indicating preferential loss of larger RGCs or size reduction of surviving cells. NND analysis of the RGC population in a retina is a useful measure of glaucomatous RGC loss. The skewed NND distribution of surviving RGCs and the finding that RGC loss correlates with a shift/amplitude change in the mode of the histogram and its tail suggests two different patterns of RGC loss possibly attributable to different pathologic processes in glaucomatous DBA/2 mice.

Intravitreal injections of neurotrophic factors and forskolin enhance survival and axonal regeneration of axotomized β ganglion cells in cat retina

Some retinal ganglion cells in adult cats survive axotomy for two months and regenerate their axons when a peripheral nerve is transplanted to the transected optic nerve. However, regenerated retinal ganglion cells were fewer than 4% of the total retinal ganglion cell population in the intact retina. The present study examined the effects of intravitreal injections of neurotrophic factors (brain-derived neurotrophic factor, ciliary neurotrophic factor, basic fibroblast growth factor, glial cell-derived neurotrophic factor, neurotrophin 4), first on the survival of axotomized cat retinal ganglion cells within 2 weeks, and then on axonal regeneration of the retinal ganglion cells for 2 months after peripheral nerve transplantation. We tested first enhancement of the survival by one of the factors, and then one or two of them supplemented with forskolin, which increases intracellular cAMP. Single injections of 0.5 μg or 1 μg brain-derived neurotrophic factor, 1 μg ciliary neurotrophic factor, or 1 μg glial cell-derived neurotrophic factor significantly increased total numbers of surviving retinal ganglion cells; 1.6–1.8 times those in control retinas. Identification of retinal ganglion cell types with Lucifer Yellow injections revealed that the increase of surviving β cells was most conspicuous: 2.5-fold (brain-derived neurotrophic factor) to 3.6-fold (ciliary neurotrophic factor). A combined injection of 1 μg brain-derived neurotrophic factor, 1 μg ciliary neurotrophic factor, and 0.1 mg forskolin resulted in a 4.7-fold increase of surviving β cells, i.e. 50% survival on day 14. On the axonal regeneration by peripheral nerve transplantation, a combined injection of brain-derived neurotrophic factor, ciliary neurotrophic factor, and forskolin resulted in a 3.4-fold increase of β cells with regenerated axons. The increase of regenerated β cells was mainly due to the enhancing effect of neurotrophic factors on their survival, and possibly to a change of retinal ganglion cell properties by cAMP to facilitate their axonal regeneration.

Total Retinal Nitric Oxide Production is Increased in Intraocular Pressure-elevated Rats

Nitric oxide (NO) is a well-known vaso-dilator but its regulation in the retina is unclear. This study was conducted to quantify total NO production and retinal ganglion cell (RGC) loss in an experimental glaucoma model. Three quarters of the peri-limbal/episcleral drainage vessels and anterior angle (right eyes) of Sprague-Dawley rats were thermally blocked using laser irradiation, while the left eyes served as controls. We measured the intraocular pressure (IOP) of both eyes using a digital tonometer (Tonopen) 21, 28 and 35 days after the laser treatment. After 35 days, we determined the total NO level in retinas and remaining ocular tissues for the laser-treated and control eyes using a spectro-photometric assay. The viable RGC numbers were also determined by counting the cell bodies stained retrogradely by fluoro-gold. The laser treatment significantly increased the IOP 2.0-2.6x throughout the whole periodof measurements (P<0.0001). The mean total RGC number decreased significantly from 98725+/-5383 (+/-S.E. (M.)) to 69276+/-5592, or 29.8% reduction, in the laser-treated eyes after 35 days (P=0.008). The mean total NO level in the laser-treated retina was significantly increased by 2.4x compared with controls (P=0.016), but no significant difference was found in the eyecups (P>0.05). Laser treatment resulted in significant IOP elevation and RGC loss, suggesting that thermal coagulation of the perilimbal region may provide an alternate protocol for glaucoma study. NO level was increased by two-fold in the retina but not in other ocular tissues. Since NO is capable of producing powerful peroxynitrite anions and hydroxyl radicals, elevated level of NO has a potential role in glaucoma.