Lack of neuroprotection against experimental glaucoma in c-Jun N-terminal kinase 3 knockout mice

To determine if the absence of c-Jun N-terminal kinase 3 (JNK3) in the mouse retina would reduce retinal ganglion cell (RGC) loss in mice with experimental glaucoma. C57BL/6 mice underwent experimental intraocular pressure (IOP) elevation with a bead/viscoelastic injection into one eye. One-half of the mice were Jnk3 homozygous knockouts (KO) and were compared to wild type (WT) mice. IOP was measured under anesthesia with the TonoLab, axial length was measured post-mortem with calipers after inflation to 15 mmHg, and RGC layer counts were performed on retinal whole mount images stained with DAPI, imaged by confocal microscopy, and counted by masked observers in an image analysis system. Axon counts were performed in optic nerve cross-sections by semi-automated image analysis. Both WT and Jnk3^−/− mice had mean elevations of IOP of more than 50% after bead injection. Both groups underwent the expected axial globe elongation due to chronic IOP elevation. The absence of JNK3 in KO retina was demonstrated by Western blots. RGC layer neuron counts showed modest loss in both WT and Jnk3^−/− animals; local differences by retinal eccentricity were detected, in each case indicating greater loss in KO animals than in WT. The baseline number of RGC layer cells in KO animals was 10% higher than in WT, but the number of optic nerve axons was identical in KO and WT controls. A slightly greater loss of RGC in Jnk3^−/− mice compared to controls was detected in experimental mouse glaucoma by RGC layer counting and there was no protective effect shown in axon counts. Counts of RGC layer cells and optic nerve axons indicate that Jnk3^−/− mice have an increased number of amacrine cells compared to WT controls.     

Neuroprotective effects of recombinant human granulocyte colony-stimulating factor (G-CSF) in neurodegeneration after optic nerve crush in rats

The purpose of the present study was to investigate the effects of granulocyte colony-stimulating factor (G-CSF) on neurodegeneration of optic nerve (ON) and retinal ganglion cells (RGCs) in a rat model of ON crush. The ONs of adult male Wistar rats (150-180 g) were crushed by a standardized method. The control eyes received a sham operation. G-CSF (100 μg/kg/day in 0.2 ml phosphate-buffered saline) or phosphate-buffered saline (PBS control) was immediately administered after ON crush for 5 days by subcutaneous injection. Rats were euthanized at 1 or 2 weeks after the crush injury. RGC density was counted by retrograde labeling with FluoroGold application to the superior colliculus, and visual function was assessed by flash visual evoked potentials (FVEP). TUNEL assay, Western blot analysis and immunohistochemistry of p-AKT in the retina and ED1 (marker of macrophage/microglia) in the ON were conducted. 2 weeks after the insult, the RGC densities in the central and mid-peripheral retinas in ON-crushed, G-CSF-treated rats were significantly higher than that of the corresponding ON-crushed, PBS-treated rats (survival rate was 60% vs. 19.6% in the central retina; 46.5% vs. 23.9% in mid-peripheral retina, respectively; p < 0.001). FVEP measurements showed a significantly better preserved latency of the p1 wave in the ON-crushed, G-CSF-treated rats than the ON-crushed, PBS-treated rats (78 ± 9 ms in the sham operation group, 98 ± 16 ms in the G-CSF-treated group, and 174 ± 16 ms in the PBS-treated group; p < 0.001). TUNEL assays showed fewer apoptotic cells in the retinal sections in the ON-crushed, G-CSF-treated rats. p-AKT immunoreactivity was up-regulated in the retinas of the ON-crushed, G-CSF-treated rats at 1 and 2 weeks. In addition, the number of ED1-positive cells was attenuated at the lesion site of the optic nerve in the ON-crushed, G-CSF-treated group. From these results, we gather that administration of G-CSF is neuroprotective in the rat model of optic nerve crush, as demonstrated both structurally by RGC density and functionally by FVEP. G-CSF may work by being anti-apoptotic involving the p-AKT signaling pathway as well as by attenuation of the inflammatory responses at the injury site, as evidenced by less ED1-positive cell infiltration in the optic nerve.     

Modulation of GABA release by nitric oxide in the chick retina: Different effects of nitric oxide depending on the cell population

γ-Aminobutyric acid (GABA) is considered to be the most important inhibitory neurotransmitter in the central nervous system, including the retina. It has been shown that nitric oxide (NO) can influence the physiology of all retinal neuronal types, by mechanisms including modulation of GABA release. However, until now, there have been no data concerning the effects on endogenous GABA release of NO produced by cells in the intact retina. In the present study, we have investigated how NO production induced by drugs influences the release of endogenous GABA in cells of the intact retina of mature chicken. Retinas were exposed to different drugs that affect NO production, and GABA remaining in the tissue was detected by immunohistochemical procedures. A specific nNOS inhibitor (7-NI) reduced the number of GABA + amacrine cells and cells in the ganglion cell layer (GCL) by 33% and 41%, respectively. A GABA transporter inhibitor blocked this effect. L-arginine (100 μM), the precursor of NO, induced increases of 62% and 34% in the number of GABA + amacrine cells and GCL cells, respectively. A sodium (Na⁺)-free solution, 7-NI and a PKG inhibitor prevented the effect of L-arginine (100 μM). However, a higher concentration of L-arginine (1 mM) induced a 35% reduction in the number of GABA + cells by a Na⁺-dependent mechanism that was restricted to the GCL population. NMDA, which stimulates NO production, increased GABA release as indicated by 53% and 38% reductions in the number of GABA + amacrine cells and GCL cells, respectively. This effect was blocked by 7-NI only in GCL cells. We conclude that basal NO production and moderate NO production (possibly induced by L-arginine; 100 μM) inhibit basal GABA release from amacrine cells and GCL cells. However, NMDA or L-arginine (1 mM) induce a NO-dependent increase in GABA release in GCL cells, possibly by stimulating higher NO production.     

Parvalbumin-immunoreactive neurons in the inner nuclear layer of zebrafish retina

The purpose of this investigation is to characterize parvalbumin-immunoreactive (IR) neurons in the inner nuclear layer (INL) of zebrafish retina through immunocytochemistry, quantitative analysis, and confocal microscopy. In the INL, parvalbumin-IR neurons were located in the inner marginal portion of the INL. On the basis of dendritic stratification in the inner plexiform layer (IPL), at least two types of amacrine cells were IR for parvalbumin. The first one formed distinctive laminar tiers within s4 (PVs4) of the IPL, and the second within s5 (PVs5). The average number of PVs4 cells was 8263 cells per retina (n = 3), and the mean density was 1671 cells/mm². The average number of PVs5 cells was 1037 cells per retina (n = 3), and the mean density was 210 cells/mm². Quantitatively, 88.9% of anti-parvalbumin labeled neurons were PVs4 cells and 11.1% were PVs5 cells. Their density was highest in the midcentral region of the ventrotemporal retina and lowest in the periphery of the dorsonasal retina. The average regularity index of the PVs4 cell mosaic was 4.09, while the average regularity index of the PVs5 cell mosaic was 3.46. No parvalbumin-IR cells expressed calretinin or disabled-1, markers for AII amacrine cells, in several animals. These results indicate that parvalbumin-IR neurons in zebrafish are limited to specific subpopulations of amacrine cells and the expressional pattern of parvalbumin may not correspond to AII amacrine cells in several other animals. Their distribution suggests that parvalbumin-IR neurons are mainly involved in ON pathway information flow.     

Disruption of the complement cascade delays retinal ganglion cell death following retinal ischemia-reperfusion

Recent reports have indicated that components of the complement cascade are synthesized during the degeneration of retinal ganglion cells (RGC) in glaucoma. While complement deposition in the retina may simply serve to aid phagocytosis of damaged RGC, activation of the complement cascade can also contribute to neuronal loss in neurodegenerative diseases. This study was designed to determine if disruption of the complement cascade affects RGC survival in a murine model of retinal ischemia-reperfusion (I/R) injury. We induced retinal ischemia in the eyes of normal mice and mice with a targeted disruption of the complement component 3 (C3) gene. Tissue was harvested 7 and 21 days after induction of I/R and retinal complement synthesis was determined by quantitative PCR and immunohistochemical methods. RGC death and associated axon loss was evaluated through histological examination of the optic nerve and retina. Our data show that retinal I/R induces the expression and deposition of complement components. C3 deficient mice clearly exhibited reduced optic nerve damage and substantial preservation of RGC 1 week after I/R when compared to normal animals (p = 0.005). Three weeks after the ischemic event C3 deficient mice retained more RGC cell bodies although the degree of optic nerve damage was similar between both groups. These findings demonstrate that inhibition of the complement cascade delays optic nerve axonal and RGC degeneration in retinal I/R. It appears that injured RGC are targeted and actively destroyed through complement mediated processes. These results may have implications for the pathophysiology and clinical management of ischemic retinal conditions.     

A computerized analysis of the entire retinal ganglion cell population and its spatial distribution in adult rats

In adult albino (SD) and pigmented (PVG) rats the entire population of retinal ganglion cells (RGCs) was quantified and their spatial distribution analyzed using a computerized technique. RGCs were back-labelled from the optic nerves (ON) or the superior colliculi (SCi) with Fluorogold (FG). Numbers of RGCs labelled from the ON [SD: 82,818 ± 3,949, n = 27; PVG: 89,241 ± 3,576, n = 6) were comparable to those labelled from the SCi [SD: 81,486 ± 4,340, n = 37; PVG: 87,229 ± 3,199; n = 59]. Detailed methodology to provide cell density information at small scales demonstrated the presence of a horizontal region in the dorsal retina with highest densities, resembling a visual streak.     

Quantitative relations in the retinal ganglion cell layer of the rat: neurons, glia and capillaries before and after optic nerve section

Background: To study normal quantitative cellular relations and the effect of optic nerve section on neurons, glia and capillaries, morphometry was carried out on 24 whole-mount retinae of 12 rats. Methods: In the left eye the optic nerve had been sectioned 30 days before death; the right eyes served as controls. Using a cresyl violet stain, cells in the retinal ganglion cell layer were evaluated at three distances from the papilla (1.2, 2.4 and 3.6 mm). Results: Gradients for density of neurons, glial cells and capillary grid were all within a small range (center: mid:periphery=1.41–1.59: 1.29–1.33: 1.00). For all these distances we found a fairly constant ratio among the three histological parameters: 44.7–46.6 neurons and 2.3–2.6 glial cells were counted per capillary grid square (geometric model for the capillary meshwork). Thirty days after section of the optic nerve the capillary meshwork remained unaffected (96.2 grid squares/mm² before nerve section vs 94.7 grid squares/mm² after nerve section) while glial cells had more than doubled (238 vs 498 cells/mm²) and nearly half of all neurons had gone (4371 vs 2244 cell s/mm²). Size characteristics of amacrine cells were similar for all three eccentricities, whereas peripheral retinal ganglion cells tended to be considerably larger than central ones. Conclusions: Cresyl violet stain can be used to study quantitative changes of neurons, glial cells and capillary grid in the retinal ganglion layer of a single whole-mount retina. There is a remarkable degree of proportionality between the density of these cells over the whole normal retina.     

Displaced amacrine and ganglion cells in the newt retina

The inner nuclear layer and ganglion cell layer of the newt retina contain taurine- and GABA-accumulating cells which are located immediately adjacent to the inner plexiform layer. Transport studies with horseradish peroxidase (HRP) indicate the presence of displaced ganglion cells within the inner nuclear layer of the retina. These are more numerous at the periphery of the retina and constitute about 2.5% of the retinal ganglion cells. Autoradiography, combined with HRP-transport studies, indicate that the taurine-accumulating cells in the ganglion cell layer of the newt retina are not 'true' ganglion cells, but may be displaced amacrine cells. Counts of axons in the optic nerve compared with cell bodies in the ganglion cell layer, demonstrate that only 50-60% of the cells in the ganglion cell layer of the newt retina are truly ganglion cells.     

Fetal growth restriction induced by chronic placental insufficiency has long-term effects on the retina but not the optic nerve

PURPOSE: Reduced birth weight is associated with an increased risk of visual impairments. This study was undertaken to determine whether prenatal exposure to a chronic compromise sufficient to cause fetal growth restriction (FGR) results in long-term alterations to the retina and optic nerve. METHODS: FGR was induced by umbilicoplacental embolization (UPE) in two cohorts of pregnant ewes from (1) 120 days of gestation (dg) until 140 dg and (2) 120 dg until term ( approximately 147 dg). Control fetuses were not subjected to UPE. The structure and neurochemistry of the retina and number and structure of ganglion cell axons were assessed in near-term (140 dg) and adult animals (2.3 years). RESULTS: In near-term FGR fetuses compared with control fetuses there were significant reductions (P < 0.05) in the outer plexiform layer (OPL), the photoreceptor inner and outer segment layers, the inner nuclear layer (INL) in the central retina and the outer nuclear layer (ONL) in the peripheral retina, and the diameter of ganglion cell axons in the optic nerve, with a proportional reduction in the thickness of myelin sheaths. In FGR animals compared with the control at 2.3 years, there were significant reductions (P < 0.05) in the total thickness of the retina, the thickness of the photoreceptor outer segment layer and the INL and the number of tyrosine hydroxylase-immunoreactive (TH-IR) dopaminergic amacrine cells. Axonal diameter and myelin sheath thickness in the optic nerve were not different (P > 0.05) between groups. CONCLUSIONS: Chronic placental insufficiency in late gestation results in long-lasting effects on specific retinal components, including photoreceptor outer segments and TH-IR amacrine cells. Other alterations observed at term, including reductions in growth and myelination of optic nerve axons, do not persist, suggesting delayed rather than permanently compromised development. Alterations persisting into adulthood could affect visual function.     

Evaluation of Fluoro-Jade C as a marker of degenerating neurons in the rat retina and optic nerve

Detection of neuronal death is an essential requirement for researchers investigating retinal degeneration. Fluoro-Jade C (FJC) is a novel, fluorescent dye that has been successfully used to label degenerating neurons in the brain, but its effectiveness in the eye has not been ascertained. In the current study, we determined the efficacy of FJC for detection of neuronal degeneration in the retina and optic nerve in various paradigms of injury. N-methyl-d-aspartate (NMDA) and kainic acid-induced excitotoxicity, optic nerve transection, and bilateral occlusion of the common carotid arteries (BCCAO) were performed using standard techniques. Rats were killed at various time points and the retinas with optic nerves attached were removed for tissue processing prior to labelling for FJC, for DNA fragmentation by TUNEL or for immunohistochemical analysis. Retinas from RCS rats of different ages were also analysed. After excitotoxicity-induced injury, cell bodies and dendrites within the ganglion cell and inner plexiform layers were specifically labelled by FJC within 6 h, a time point comparable to the appearance of TUNEL-positive nuclei and to reductions in mRNA levels of retinal ganglion cell-specific proteins, but in advance of alterations in some immunohistochemical markers. The number of FJC-labelled cell bodies in the retina declined over time as cell loss proceeded, although dendritic staining remained prominent. Colocalisation of FJC with TUNEL and with immunohistochemical neuronal markers was achieved. FJC was successful at identifying somato-dendritic degeneration following ischemia induced by BCCAO, but surprisingly, not after optic nerve transection. FJC visualised photoreceptor degeneration in the RCS rat, albeit less effectively than with the TUNEL assay, and was also effective for imaging and quantifying degenerating axons in the optic nerve after multiple injuries. In addition to labelling degenerating neurons, however, FJC also bound non-specifically to astrocytes and to blood cells in unperfused rats. Since the ganglion cell layer is adjacent to astrocytes within the nerve fibre layer, caution is needed when using FJC as a quantitative tool for detecting ganglion cell death.     

Human intraretinal myelination: Axon diameters and axon/myelin thickness ratios

Purpose:

Human intraretinal myelination of ganglion cell axons occurs in about 1% of the population. We examined myelin thickness and axon diameter in human retinal specimens containing myelinated retinal ganglion cell axons.

Materials and Methods:

Two eyes containing myelinated patches were prepared for electron microscopy. Two areas were examined in one retina and five in the second retina. Measurements were compared to normal retinal and optic nerve samples and the rabbit retina, which normally contains myelinated axons. Measurements were made using a graphics tablet.

Results:

Mean axon diameter of myelinated axons at all locations were significantly larger than unmyelinated axons (P ≤ 0.01). Myelinated axons within the patches were significantly larger than axons within the optic nerve (P < 0.01). The relationship between axon diameter/fiber diameter (the G-ratio) seen in the retinal sites differed from that in the nerve. G-ratios were higher and myelin thickness was positively correlated to axon diameter (P < 0.01) in the retina but negatively correlated to axon diameter in the nerve (P < 0.001).

Conclusion:

Intraretinally myelinated axons are larger than non-myelinated axons from the same population and suggests that glial cells can induce diameter changes in retinal axons that are not normally myelinated. This effect is more dramatic on intraretinal axons compared with the normal transition zone as axons enter the optic nerve and these changes are abnormal. Whether intraretinal myelin alters axonal conduction velocity or blocks axonal conduction remains to be clarified and these issues may have different clinical outcomes.     

The relative time course of axonal loss from the optic nerve of the developing guinea pig is consistent with that of other mammals.

The relative timing of a number of events during the development of the visual system has recently been suggested to be consistent across a number of mammalian species (Dreher & Robinson, 1988). Some conflicting reports, however, had suggested that the precocial guinea pig might represent an exception to the generalized scheme. A quantitative study was thus carried out on the development of the optic nerve and retina of the guinea pig. Consistent with the prediction of a stable relative time course of mammalian visual development, axons and growth cones were found in the optic stalk from the 24th postconceptional day (40% of the period from conception to eye opening--the cecal period), the peak number of axons was observed on the 32nd postconceptional day (56% of the cecal period), and the phase of rapid axonal loss extended to the 39th and 42nd postconceptional days (68-74% of the cecal period). The number of axons in the adult optic nerve (117,000) represented about 37% of the peak number of axons. Additional observations indicated that during development of the optic nerve the mean axonal diameter increased approximately threefold from 0.31 microns to 1.06 microns. As in other mammals studied so far, myelination was first noted after the period of rapid axonal loss and continued until in the adult 97% of axons were found to be myelinated. In the retina, the presence of pyknotic profiles in the ganglion cell layers extends throughout the periods of loss of the optic nerve axons. Finally, the presence of pyknotic profiles in the amacrine sublayer suggest that in the guinea pig, as in other mammalian species, there is a loss of displaced amacrine cells as well as ganglion cells from the ganglion cell layer.     

The effect of experimental glaucoma and optic nerve transection on amacrine cells in the rat retina

PURPOSE: To detect alterations in amacrine cells associated with retinal ganglion cell (RGC) depletion caused by experimental optic nerve transection and glaucoma. METHODS: Intraocular pressure (IOP) was elevated unilaterally in 18 rats by translimbal trabecular laser treatment, and eyes were studied at 1 (n = 6), 2 (n = 5), and 3 (n = 7) months. Complete optic nerve transection was performed unilaterally in nine rats with survival for 1 (n = 4) and 3 (n = 5) months. Serial cryosections (five per eye) were immunohistochemically labeled with rabbit anti-gamma-aminobutyric acid (GABA) and anti-glycine antibodies. Cells in the ganglion cell and inner nuclear layers that labeled for GABA or glycine were counted in a masked fashion under bright-field microscopy. Additional labeling with other RGC and amacrine antigens was also performed. RGC loss was quantified by axon counts. RESULTS: Amacrine cells identified by GABA and glycine labeling were not significantly affected by experimental glaucoma, with a mean decrease of 15% compared with bilaterally untreated control cells (557 +/- 186 neurons/mm [glaucoma] versus 653.9 +/- 114.4 neurons/mm [control] of retina; P = 0.15, t-test). There was no significant trend for amacrine cell counts to be lower in eyes with fewer RGCs (r = -0.39, P = 0.11). By contrast, there was highly significant loss of GABA and glycine staining 3 months after nerve transection, both in the treated and the fellow eyes (P < 0.0001, t-test). However, there was a substantial number of remaining amacrine cells in transected retinas, as indicated by labeling for calretinin and calbindin. CONCLUSIONS: Experimental glaucoma causes minimal change in amacrine cells and their expression of neurotransmitters. After nerve transection, neurotransmitter presence declines, but many amacrine cell bodies remain. Differences among optic nerve injury models, as well as effects on "untreated" fellow eyes, should be recognized.     

Axon deviation in the human lamina cribrosa

AIMS—To examine the course taken by individual retinal ganglion cell axons through the human lamina cribrosa.
METHODS—Retinal ganglion cell axons were labelled using the retrograde tracer horseradish peroxidase applied directly to the optic nerve in two normal human eyes removed during the course of treatment for extraocular disease.
RESULTS—A majority of axons took a direct course through the lamina cribrosa but a significant minority, in the range 8-12%, deviated to pass between the cribrosal plates in both central and peripheral parts of the optic disc.
CONCLUSIONS—It is postulated that these axons would be selectively vulnerable to compression of the lamina cribrosa in diseases such as glaucoma in which the intraocular pressure is increased.

Keywords: retina; optic nerve; glaucoma; lamina cribrosa     

Up-regulation of cytochrome oxidase in the retina following optic nerve injury

The purpose of this study is to investigate the cytochrome oxidase (COX) activity in the retina and optic nerve following an optic nerve injury. The optic nerve crush of one eye was carried out in Balb/c mice. A semi-quantitative RT-PCR method was then adopted to evaluate the mRNA expression of cytochrome oxidase subunit 1 (COX1) in the retina after surgery. Up-regulation of COX1 mRNA in the retina was detected by RT-PCR at 24 hr following the optic nerve injury. Total retinal mitochondrial mass measured by fluorescent intensity of MitoTracker green was not altered following the injury. COX histochemistry performed on cryostat sections showed an elevated enzyme activity of COX in the retina and in the optic nerve. In the retina, elevation of the COX activity was observed in the retinal ganglion cell layer and the overlying nerve fibre layer. The increase of COX activity began from 24 hr after injury, peaked around day 3, and maintained up to 1 week after the operation. In the optic nerve, increase of COX activity was observed in regions distal to the crush line and distributed either randomly or in a cone shape. In conclusion, both the expression of COX1 mRNA in retina and the activity of COX in inner plexiform layer and retinal ganglion cell layer were elevated following optic nerve injury without affecting total retinal mitochondrial mass. These findings suggested that one of early responses in the retina and in the optic nerve after the optic nerve injury is to scale up the energy production.     

Dendritic differentiation in the periphery of the growing zebrafish retina

In the retina of teleost fish, new ganglion cells are generated from a circumferential peripheral growth zone at the edge of the eye throughout life. Addressing the question how new cells are fitted into the existing retina, we investigated newly formed ganglion cells in the zebrafish retina morphologically, by tracing them from the cut optic nerve with rhodamine dextran. We identified proliferating cells by antibody detection against proliferating cell nuclear antigen. In addition, newly formed bipolar cell and amacrine cell dendrites were investigated by antibodies against protein kinase C (PKC) and choline-acetyl-transferase (ChaT), respectively, and analyzed in sections or wholemount preparations using confocal microscopy. In retinal sections we observed that ganglion cell dendritic branches in the inner plexiform layer were in close apposition to dividing cells. In the periphery of retinal wholemounts, we detected rhodamine traced ganglion cells adjacent to the growth zone, extending dendrites in proximity to the growth zone, typically branching off in opposite directions running parallel to the retinal rim over more then 100 μm. Ganglion cells with similar dendritic branching patterns were not found in more central retinal areas. Similarly, the dendrites of ChaT-positive amacrine cells showed a preference for running parallel to the circumference in the periphery. Dendritic branches of PKC-positive bipolar cells did not show similar preferred orientation. The change in shape of the dendritic tree with distance from the periphery was studied for the Ma type ganglion cell. The data are consistent with the idea that existing ganglion cells might control differentiation of new ganglion cells. Moreover, ganglion cells with specific branching patterns towards the retinal periphery undergo a restructuring of their dendritic trees.     

The spatial and temporal expression patterns of netrin receptors, DCC and neogenin, in the developing mouse retina

Recently it has been demonstrated that the guidance of retinal ganglion cell (rgc) axons through the optic disc is dependent on the DCC/netrin-1 axonal guidance system. To gain further insight into the function of the netrin receptors, DCC and Neogenin, in retinal development we have studied the expression patterns of these receptors in the embryonic mouse retina. Neogenin mRNA was restricted to a single neural cell type, the rgc. However, strong Neogenin mRNA expression was observed in the extending fiber cells of the developing lens suggesting a role for Neogenin in the migration events shaping the early lens. Our studies demonstrated that DCC mRNA was expressed at high levels in chains of closely opposed neurons as they migrated towards the emerging mantle layer in the early retina (E12.5-E13.5) suggesting a role for DCC in the migration of neurons out of the ventricular zone. DCC protein expression was high on rgc axons as they actively navigated through the optic disc into the optic nerve. At birth, when the majority of rgc axons had projected through the optic disc, DCC protein was no longer detectable on the distal axonal segments within the optic nerve despite significant DCC protein expression on the proximal axonal membranes in the nerve fiber layer. These observations suggest that a localized down-regulation of DCC protein occurs on projecting axonal membranes once the DCC guidance function is no longer required. We also demonstrated that DCC mRNA and protein were expressed by amacrine cells and Müller glial cells while DCC mRNA was detected in horizontal cells. Taken together, these expression patterns suggest a role for DCC in axon outgrowth and/or pathfinding for a variety of retinal neurons and in the migration of newly born neurons within the developing retina.     

Increase in endothelin B receptor expression in optic nerve astrocytes in endothelin-1 induced chronic experimental optic neuropathy

The purpose of this study was to determine whether endothelin B (ETB) receptor levels in the optic nerve are related to retinal ganglion cell (RGC) loss in a model of chronic endothelin-1 (ET-1) induced optic neuropathy. RGCs of adult Brown Norway rats were first retrogradely labeled with fluorochrome from the superior colliculi. An osmotic minipump was surgically implanted 7 days later to deliver 10⁻¹¹ M (n = 9), 10⁻⁹ M (n = 12) or 10⁻⁷ M (n = 9) ET-1 to the retrobulbar optic nerve for 28 days. RGC survival was expressed as the ratio of RGC counts in experimental versus control eyes in wholemounted retinas. Optic nerves were used for either ETB western blot analysis (n = 24) or immunohistochemistry (n = 6) for ETB and glial fibrillary acidic protein (GFAP) to localize astrocytes. ETB expression was higher in the experimental nerve compared to the fellow untreated control nerve in 19 (79%) of the 24 animals with a mean increase of 16.7 ± 4.5% in densitometric analyses of the immunoblots. Experimental nerves showed stronger labeling for both ETB and GFAP compared to control nerves. ETB-positive cells almost completely co-localized with GFAP-positive cells in both experimental and untreated control nerves, however, ETB expression was stronger in the astrocyte soma and proximal processes, while GFAP was expressed more strongly in the distal processes. There was a weak relationship between RGC loss and increase in ETB expression (r = −0.417, p = 0.076). There is an upregulation of ETB expression in optic nerve astrocytes in ET-1 induced chronic optic neuropathy causing RGC loss.     

Organization of the inner retina following early elimination of the retinal ganglion cell population: effects on cell numbers and stratification patterns.

The present study has examined the effects of early ganglion cell elimination upon the organization of the inner retina in the ferret. The population of retinal ganglion cells was removed by optic nerve transection on the second postnatal day, and retinas were subsequently studied in adulthood. Numbers of amacrine and bipolar cells were compared in the nerve-transected and nerve-intact retinas of operated ferrets, while stratification patterns within the inner plexiform layer were compared in these and in normal ferret retinas. Early ganglion cell elimination was found to produce a 25% reduction in the population of glycine transporter-immunoreactive amacrine cells, and 18 and 15% reductions in the populations of parvalbumin and calbindin-immunoreactive amacrine cells, respectively. GABAergic amacrine cells were also reduced by 34%. The number of calbindin-immunoreactive displaced amacrine cells, by contrast, had increased in the ganglion cell-depleted retina, being three times their normal number. Other amacrine and bipolar cell types were unaffected. Despite these changes, the stratification patterns associated with these cell types remained largely intact within the inner plexiform layer. The present results demonstrate a class-specific dependency of inner retinal neurons upon the ganglion cell population in early postnatal life, but the ganglion cells do not appear to provide any critical signals for stratification within the inner plexiform layer, at least not after birth. Since they themselves do not produce stratified dendritic arbors until well after birth, the signals for stratification of the bipolar and amacrine cell processes should arise from other sources.