The spatiotemporal pattern of somal and axonal pathology after perikaryal excitotoxic injury to retinal ganglion cells: A histological and morphometric study

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Experimental Neurology 211 (2008) 52 – 58 www.elsevier.com/locate/yexnr

The spatiotemporal pattern of somal and axonal pathology after perikaryal excitotoxic injury to retinal ganglion cells: A histological and morphometric study S.K. Saggu a,⁎, H.P. Chotaliya a , Z. Cai b , P. Blumbergs b , R.J. Casson a a b

Department of Ophthalmology and Visual Sciences, The University of Adelaide, SA-5000, Australia Neuropathology Division, Institute of Medical and Veterinary Science, Adelaide, SA-5000, Australia Received 24 September 2007; revised 12 December 2007; accepted 18 December 2007 Available online 8 January 2008

Abstract Objective: To describe the spatiotemporal pattern of somal and axonal pathologic changes after perikaryal excitotoxic injury to retinal ganglion cells in-vivo. Methods: 40 male Sprague–Dawley rats were killed at 0 h, 24 h, 72 h and 7 days after injecting 20 nM N-methyl-D-aspartate (NMDA) into the vitreous chamber of left eye. Saline-injected right eyes served as control. After perfusion fixation, the eyes and retrobulbar optic nerves from half of the animals in each group were embedded in paraffin and tissues from the other half embedded in resin. Paraffin-embedded eyes and resin-embedded proximal (intraorbital) and distal (intracranial) optic nerve segments were evaluated by light microscopy. Light microscopic photographs of proximal and distal optic nerve segments were compared using the following parameters: axon counts, axonal swellings and myelin changes. Results: Retinas showed cell loss in ganglion cell layer (GCL) and reduction in inner retinal thickness at 72 h after NMDA injection (p b 0.05), with changes becoming more advanced after 7 days (p b 0.001). The cell count in GCL correlated strongly with the axonal counts (R = 0.929, p b 0.001). Axon loss, axon swellings and myelin damage were seen in both proximal and distal segments of optic nerves 72 h post-NMDA exposure (p b 0.05), with changes increasing further at 7 days (p b 0.001). Pathological changes were more prominent in the distal segments (p b 0.05). Conclusion: Excitotoxic perikaryal injury causes an axonopathy, which is synchronous with the somal degeneration and which is most prominent in the distal portions of the axon, consistent with “dying-back like neuropathy”. © 2007 Elsevier Inc. All rights reserved. Keywords: Excitotoxicity; Retinal ganglion cells; Optic nerve; Histology; Dying-back neuropathy

Introduction Excitotoxicity describes the process of neuronal death caused by excessive or prolonged activation of receptors for excitatory amino acid neurotransmitters. A large body of evidence supports the notion that excitotoxicity plays a role in the pathogenesis of certain neurological diseases, including central nervous system (CNS) ischemia/stroke, Alzheimer's disease (AD), Huntington's disease, Parkinson's disease, HIV encephalopathy, motor neurone disease (MND) and glaucoma. ⁎ Corresponding author. Ophthalmology Network, Level-8, East Wing, Royal Adelaide Hospital, Adelaide, SA-5000, Australia. Fax: +61 8 8222 2741. E-mail address: [email protected] (S.K. Saggu). 0014-4886/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2007.12.022

(Bensimon et al., 1994; Doble, 1999; Reisberg et al., 2003; Hynd et al., 2004; Osborne et al., 2004; Johnston 2005; Van Damme et al., 2005; Casson, 2006a,b) Excitotoxic injury is classically considered as a somatodendritic neuronal insult; however, recently, evidence has accrued indicating that axonopathy is an early feature in several disorders which may involve excitotoxicity, including AD, MND and glaucoma. (Li et al., 2001; Fischer et al., 2004; Libby et al., 2005; Stokin et al., 2005) To our knowledge, the spatiotemporal pattern of axonal pathology after perikaryal excitotoxic injury has never been reported. The observed pathology may provide insight into the possible mechanisms of excitotoxic-associated axonal injury. The retina and optic nerve, as approachable regions of the CNS, provide a unique anatomical substrate to investigate the

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effect of perikaryal excitotoxic injury on the axon. In this model, the neuronal cell bodies (retinal ganglion cell (RGC) cell bodies) and the toxic insult are physically isolated from the axons; hence, the observed axonal degeneration outside the eye is logically a consequence of the retinal injury. Here, we describe the spatiotemporal pattern of the light microscopic and morphometric changes in the retina and optic nerve after intravitreal NMDA injection. Materials and methods Animals Male Sprague–Dawley rats (n = 40) weighing 300–350 g (Animal House, Institute of Medical and Veterinary Sciences, Adelaide, South Australia) were kept at room temperature, with food and water available ad libitum. Adequate care was taken to minimise pain and discomfort for the animals used in this study and the experiments were conducted in accordance with the international standards on animal welfare. All experiments were approved and monitored by the Institute of Medical and Veterinary Science, Animal Ethics Committee (Ethics Approval No. 53/06). Excitotoxicity animal model Rats were anaesthetised using 2.5 L/min isoflurane in 2.5 L/min O2 and topical 0.4% benoxinate drops were instilled in both eyes. Perikaryal excitotoxic injury was induced by injecting 5 μl of 4 mM NMDA (Sigma-Aldrich, U.S.A) into the vitreous chamber of the left eye. The right eye was injected with 5 μl of sterile isotonic saline to serve as control. Animals were left to recover to be killed later at various time intervals: immediately, 24 h, 72 h and 7 days. The rats were killed humanely by cardiac perfusion under deep anaesthesia. The rats were anaesthetized by intraperitoneal injection of freshly prepared ketamine/xylazine solution (each 10 ml mixture contained 1.0 ml ketamine and 0.5 ml xylazine in 8.5 ml normal saline) in a dose of 0.1 ml/10 g body weight. 20 rats were killed by cardiac perfusion with 500 ml of 4% parafomaldehyde in 0.1 M phosphate buffer fixative and the eyes and the optic nerves dissected for paraffin processing. Although the paraffin sections provided excellent details of the retinal morphology and an opportunity for immunohistochemical analysis (not reported here), resin sections were necessary for axonal and myelin details; hence, a second set of 20 animals were killed by intracardiac perfusion with glutaraldehyde fixative solution (2.5% glutaraldehyde with 4% paraformaldehyde in 0.1 M Phosphate buffer, pH 7.4). Optic nerves were divided into four equal segments (1 to 4) from proximal (nearer to the eyeball) to distal (away from the eyeball i.e. nearer to the brain) with each segment measuring approximately 2 mm. Histological analysis of rat retina Paraformaldehyde perfused eyes were embedded in paraffin and sectioned. Seven sections (each section of 7 μm thickness)

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were recovered from the region of optic disc and mounted on glass slides coated with poly-L-lysine (Menzel GmbH & Co., Braunschwerg). Three randomly selected deparaffinised sections from each animal were stained with haematoxylin and eosin (H&E) and examined by light microscopy. Retinal damage was evaluated as described previously. (Yoneda et al., 2003) Morphometric evaluations were carried out under a light microscope equipped with a digital DP12 Olympus camera (Olympus, Japan). To reduce sampling errors, 3 adjacent, but, non-overlapping light microscope images (at 200× magnification) were photographed from both posterior (200 μm from the optic disc), and peripheral (3 mm from the optic disc) retinas. Cell counts in the GCL (number of cells in the ganglion cell layer counted along 300 μm length of retina) and the inner retinal thickness (IRT, from the junction of outer nuclear and outer plexiform layer to the inner limiting membrane along a vertical line) were measured on photographs (267 × 200 mm) in a masked fashion by a single experienced histologist. Histological analyses of optic nerve pathway Eyeballs, intraorbital and intracranial optic nerves from glutaraldehyde perfused rats were further fixed by immersion in the same fixative for 24 h at 4°C. Tissues were then placed in 2% osmium tetroxide in saline for overnight and washed with cacodylate buffer at room temperature. Subsequently, they were dehydrated in graded alcohols and embedded in epoxy resin for transverse (segments 1 and 3) and longitudinal sectioning (segments 2 and 4). 0.5 μm transverse sections were cut on an ultramicrotome, mounted on glass slides, and stained for myelin using 1% toluidine blue. A morphological survey of transverse and longitudinal sections of the optic nerve was done using a light microscope equipped with a digital camera (Olympus, Japan). Ten images (each image representing a microscope field of 3630 µm2) from transverse sections of each segment, representing approximately 25% of the total nerve cross-section, were obtained under oil immersion lens (at 1000× magnification) for quantitative analysis. Optic nerve pathology was analysed using different parameters. Firstly, total numbers of normal axons remaining in the section were counted.(Chauhan et al., 2006) Secondly, a method modified from Garthwaite et al was used to quantify axonal swelling. (Garthwaite et al., 1999) Garthwaite et al reported that the density of axons having an average internal diameter above 2.5 µm (about average in control rats) was the optimal index of damage.(Garthwaite et al., 1999) In preliminary analyses with the current model, we found that more subtle changes could be quantified if the benchmark was increased towards the maximum diameter detected in averaged control sections, about 5 μm. Therefore, all axons with more than 5 μm internal diameter were counted to quantify axonal swelling. Lastly, myelin changes were analysed: all myelin, which appeared thickened, separated, or clumped was counted. The data for each parameter was expressed as mean of normal axon count, axonal swellings and myelin changes per 3630 µm2 (± S.E.M.) All the counts were performed in a masked fashion by a single observer.

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Statistical analysis Retinal and optic nerve images from all 5 animals in each group were analysed. For the retinas, three images were taken from each posterior and peripheral regions of all three retinal sections of each eye of the animal. Data obtained for each region was averaged for each eye, and thus, two datasets were available for the central and peripheral retina for quantitative analysis of cell counts in the GCL and IRT. For optic nerves, counts were obtained from all ten selected fields for total normal axons, axonal swellings and myelin damage. Analyses were performed using a commercially available statistical software package (SPSS for Windows, version 13.0, SPSS Inc., Chicago, IL). Differences in means were analysed using an ANOVA and a posthoc Tukey test, and correlations were determined with a Pearson coefficient. A p value b 0.05 was considered statistically significant. Results

saline injection at any time point (p N 0.05). Compared with the saline injection, NMDA did not produce any significant change in the cell counts both immediately and after 24 h of injection (p N 0.05 in posterior and peripheral retinas). However, a significant loss of cells was observed in the GCL in both regions after 72 h (p b 0.05). In comparison to the control eyes, 51% of cells remained in the posterior region and 44% cells remained in the peripheral region. The cell count decreased at 7 days in both regions (p ≤ 0.001), with only 23% and 35% cells remaining in the posterior and peripheral retinas (see Fig. 1 and Table 1). Inner retinal thickness IRT in the posterior and peripheral regions of NMDAinjected retinas was not significantly different from controls at 24 h of injection (p N 0.05). A significant reduction in the IRT occurred in both regions at 72 h (p = 0.002 for posterior and p = 0.012 for peripheral retina). 7 days after NMDA injection there was a further reduction in the IRT in both regions (p b 0.05) (see Fig. 1 and Table 1).

Cell counts in RGC layer Morphological examination of optic nerve The average cell count in the GCL was significantly higher (p b 0.05) in the posterior retina (16.5 ± 0.3) than the peripheral retina (9.9 ± 0.12). Cell counts in both regions did not change after

24 h after NMDA injection, no structural changes were observed in any portion of the optic nerve. The axons and myelin

Fig. 1. Progressive changes in the retina after NMDA injection in rat as seen in H&E stained paraffin sections. Images are of the posterior retina (200 μm from the optic disc). No changes are seen in the retinal morphology after saline injection at 0 h (A), 24 h (B), 72 h (C) and 7 days (D). NMDA-injected retinas at 0 h (E), 24 h (F) also show a normal morphological appearance. A substantial decrease in the number of cells in GCL and the thickness of the inner retina is seen at 72 h post-NMDA exposure (G). A further reduction in the GCL cell count and IRT is obvious at 7 days of NMDA injection (H). Bar = 50 μm. INFL = inner nerve fibre layer; GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer.

S.K. Saggu et al. / Experimental Neurology 211 (2008) 52–58 Table 1 The table compares the cell count in ganglion cell layer (GCL) and the inner retinal thickness in the posterior and peripheral retinas of the saline-injected and NMDA-injected rat eyes Retinal changes Time points

NMDA-injected (experimental) eyes

Saline injected (control) eyes Posterior retina

Peripheral retina

Posterior retina

Cell count in ganglion cell layer/standard unit length 0h 16.5 ± 0.3 9.9 ± 0.12 16.4 ± 0.5 24 h 14.5 ± 0.9 9.8 ± 1.16 13.6 ± 0.16 72 h 12.9 ± 0.6 9.8 ± 0.8 6.6 ± 0.7 ⁎⁎ 7 days 14.5 ± 0.4 11.3 ± 0.4 3.4 ± 0.4 ⁎⁎

Peripheral retina 10.3 ± 0.2 10.3 ± 0.8 4.5 ± 0.3 ⁎ 4.0 ± 0.4 ⁎⁎

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total nerve calibre seen in both regions. In the proximal segment [see Fig. 2 (A3, C3) and Table 2], the interfascicular septa became more prominent and fascicle size reduced. Each fascicle contained a reduced number of axons compared to normal. Compared to changes at 72 h, myelin changes and densely staining axoplasm became more prominent features. The number of axonal swellings was reduced compared to the 72 h time point. More glial cells were seen dispersed between remaining axons, which were identified as astroglial and microglial cells under electron microscope. The distal optic nerve exhibited similar but more pronounced atrophic changes, and more axonal swelling and myelin disruption were seen in this region [see Fig. 2 (B3, D3) and Table 2]. Correlation of retinal and optic nerve damage

Inner retinal thickness 0h 93.5 ± 0.5 24 h 95.5 ± 6.1 72 h 95.9 ± 2.5 7 days 85.7 ± 3.0

66 ± 0.7 75.5 ± 7.6 72.5 ± 4.0 70.1 ± 3.6

93.1 ± 0.8 94.3 ± 3.4 72.2 ± 3.1 ⁎ 58.3 ± 5.3 ⁎⁎

71.7 ± 1.6 72.9 ± 4.0 50.5 ± 2.9 ⁎ 53.5 ± 3.5 ⁎

The cell count represents the number of cells per 300 μm retinal length. Not much difference is observed between the control and experimental groups at 0 and 24 h. In comparison to the controls, NMDA-injected eyes show a significant decrease in the GCL cell count and thickness of the inner retina at 72 h postNMDA injection. Both the changes became highly significant at day 7. Each value represents mean ± S.E. NMDA = N-Methyl-D-Aspartate. ⁎ p b 0.05 vs. Control fellow eyes (ANOVA with posthoc Tukey test). ⁎⁎ p b 0.001 vs. Control fellow eyes (ANOVA with posthoc Tukey test).

retained normal morphology in the proximal and distal segments of the optic nerve [see Fig. 2 (A1, B1, C1, D1) and Table 2]. 72 h after NMDA injection After 72 h, all five rats in this group began to show changes in the optic nerve. In the proximal optic nerve segment [see Fig. 2 (A2, C2) and Table 2], although most fibres retained normal morphology, a number of axonal swellings appeared. Most of the swollen axons exhibited clearing or clumping/fragmentation of axoplasm with many surrounded by normal appearing myelin. Only a few swollen fibres developed myelin changes such as thickening or separation of the myelin lamellae in comparison to the normal controls. Together with the clear swollen axons, some fibres showed dense staining with the toluidine blue. Hyperdense appearance of these fibres was either due to intense staining of the myelin bodies formed due to complete loss of axoplasmic structure or due to the increased density of axoplasm associated with or without myelin changes. No changes were observed in the appearance of the interfascicular region and glial cells. In comparison to the proximal optic nerve, the distal segment [see Fig. 2 (B2, D2) and Table 2] showed more prominent changes. Axonal swellings were more frequent and more marked. Myelin changes were more evident. The interfascicular region showed no obvious changes. Number and distribution of glial cells appeared normal. 7 days after NMDA injection 7 days after NMDA injection, both proximal and distal segments of optic nerve exhibited atrophic changes, with decrease in

The number of axons in the distal optic nerve showed a very strong positive correlation with the cell count in GCL in the retina (correlation coefficient R = 0.928, p b 0.001). There was a linear relationship between these two parameters. Moreover, a strong correlation was observed between the loss of cells in GCL and the number of axonal swellings (R = − 0.876, p b 0.001) as well as myelin changes (R = − 0.928, p b 0.001) [see Fig. 3]. Discussion In the current study, we have used intravitreal injections of NMDA in rats and investigated the effect on the RGC axons outside the eye, both intraorbital and intracranial. The retina and optic nerve, as approachable regions of the CNS, provide a unique anatomical substrate to investigate the effect of perikaryal excitotoxic injury on the axon. In this model, the neuronal cell bodies (the RGCs) and the toxic insult are physically isolated from the axons; hence, the observed axonal degeneration outside the eye is logically a consequence of the retinal injury, rather than a secondary effect caused by excitotoxic injury to axonalassociated glial tissue. The most striking findings from the current study were the synchronous pathological changes in the retina and distal optic nerve, indicating that axonal pathology is an early feature of perikaryal excitotoxic injury. Furthermore, at a given time point, the observed changes (axon swelling, axon loss and myelin changes) were more prominent in the distal portions of the axon, consistent with a dying-back-type axonal degeneration. In the classical Wallerian degeneration following axotomy, there is a degradation of the axon and its associated myelin distal to the point of transection. Although well studied, the spatiotemporal pattern of typical Wallerian degeneration following axonal trauma remains controversial, suggesting that in most cases there are early uniform and synchronous changes throughout the severed segment. However, in certain types of neuronal injury, a “Wallerian-like” degeneration occurs and a “dying-back” process is seen, in which the distal axon is the first region to be affected and the degeneration spreads in a centrifugal manner (Schlamp et al., 2006). Recently, it has been shown that axonal defects in mouse models of AD precede known disease-related pathology by more than a year. (Stokin et al., 2005) Similar axonal defects in

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Fig. 2. Progressive changes in the the rat optic nerve after NMDA injection as seen in toluidine blue stained resin sections. The top row shows transverse sections of the proximal optic nerve segments at 24 h (A1), 72 h (B1) and 7 days (C1). The second row demonstrates transverse sections of the distal optic nerve at 24 h (A2), 72 h (B2) and 7 days (C2). The third row contains photographs of the longitudinal sections of proximal optic nerves at 24 h (A3), 72 h (B3) and 7 days (C3). The last row shows longitudinal sections of distal optic nerves at 24 h (A4), 72 h (B4) and 7 days (C4). Both proximal (A1, A3) and distal (A2, A4) optic nerve segments at 24 h show nerve fibres of various sizes with each nerve showing pale axon surrounded by the thick compact myelin. The degenerative changes begin to appear at 72 h with the appearance of axonal swellings (black arrowheads), hyperdense axons (black arrows) and some myelin disruption (white arrowheads) in both proximal (B1, B3) and distal optic nerves (B2, B4). It can be seen that the distal segment shows more swollen axons than the proximal. At day 7, a significant increase in the number of hyperdense axons (blackarrows) is demonstrated in both the proximal (C1, C3) and distal optic nerves (C2, C4). At this time, a substantial length of the hyperdense axon (series of small black arrows) can be seen in the longitudinal sections (C3, C4). Bar = 10 μm.

the early stages of AD in humans have also been reported. (Stokin et al., 2005) These axonal defects consisted of swellings that accumulated abnormal amounts of microtubule-associated and molecular motor proteins, organelles, and vesicles. (Stokin et al., 2005) Impairing axonal transport by reducing the dosage of a kinesin molecular motor protein enhanced the frequency of axonal defects and increased amyloid-β peptide levels and amyloid deposition (Stokin et al., 2005). A comprehensive spatiotemporal analysis of disease progression in SOD1G93A mice, a well-described animal model of MND, found that motor neurone pathology clearly commenced

at the distal axon and proceeded in a dying-back fashion. (Fischer et al., 2004) Human autopsy findings of MND also support this pattern of disease progression (Fischer et al., 2004). Excitotoxicity may be involved in the pathogenesis of glaucoma.(Casson, 2006a,b) The two strongest lines of evidence relate to (1) the link between the role of excitotoxicity in ischaemia and the role of ischaemia in glaucoma, (Flammer, 1994; Chung et al., 1999; Flammer et al., 2002; Grieshaber and Flammer, 2005) and (2) the attenuating effect of anti-excitotoxic treatment in experimental glaucoma (Chaudhary et al., 1998; Hare et al., 2004) (the results of a long-term clinical trial are

S.K. Saggu et al. / Experimental Neurology 211 (2008) 52–58 Table 2 The table shows progressive changes in the proximal and distal optic nerve segments in the control and experimental eyes Optic nerve changes Time points

Saline injected (control) eyes Proximal optic nerve

NMDA-injected fellow (experimental) eyes Distal optic nerve

Total axon count/standard unit area 0h 1738.60 ± 36.9 1722.00 ± 12.2 24 h 1736.40 ± 20.6 1711.40 ± 24.9 72 h 1739.00 ± 40.2 1738.00 ± 19.6 7 days 1742.80 ± 27.0 1746.60 ± 15.9

Proximal optic nerve

Distal optic nerve

1738.60 ± 22.9 1751.40 ± 18.3 1710.80 ± 16.6 1728.00 ± 10.2 1486.00 ± 30.0 ⁎ 1315.20 ± 38.6 ⁎ 1075.40 ± 31.5 ⁎⁎ 968.20 ± 21.2 ⁎⁎

Number of swollen axons/standard unit area 0h 0.20 ± 0.2 0.20 ± 0.2 1.00 ± 0.5 24 h 0.60 ± 0.2 0.60 ± 0.4 0.40 ± 0.2 72 h 1.00 ± 0.3 1.20 ± 0.2 42.60 ± 4.1 ⁎⁎ 7 days 1.00 ± 0.0 1.80 ± .02 22.80 ± 2.3 ⁎

0.20 ± 0.2 0.80 ± 0.4 49.80 ± 4.6 ⁎⁎ 43.80 ± 3.8 ⁎⁎

Number of myelin changes/standard unit area 0h 0.60 ± 0.2 0.40 ± 0.2 0.60 ± 0.4 24 h 1.00 ± 0.3 1.40 ± 0.2 1.80 ± 0.4 72 h 1.80 ± 0.3 2.60 ± 0.5 137.00 ± 15.0 ⁎ 7 days 3.60 ± 0.5 3.40 ± 0.5 300.40 ± 43.5 ⁎

0.20 ± 0.2 1.20 ± 0.6 226.00 ± 11.7 ⁎⁎ 471.80 ± 21.5 ⁎⁎

It represents total number of axons, number of swollen axons and number of myelin changes per standard unit area and each value represents mean ± S.E/ 3630 μm2. In comparison to the controls, the number of axonal swellings and disrupted myelin increase significantly in both proximal and distal optic nerve segments at 72 h and 7 days of NMDA injection. The total number of axons remaining in the nerve decreases at these time points. In comparison to the proximal, the distal optic nerve segments show more axon loss, presence of more swollen axons and damaged myelin at 72 h and 7 days after NMDA injection. NMDA = N-Methyl-D-Aspartate. ⁎ p b 0.05 vs. Control fellow eyes (ANOVA with posthoc Tukey test). ⁎⁎ p b 0.001 vs. Control fellow eyes (ANOVA with posthoc Tukey test).

nearing completion). In human glaucoma, the optic neuropathy is presumed to start at the level of lamina cribrosa, (a primary axonopathy) as the RGC axons exit the eye, with secondary retrograde degeneration of the RGC somata. Recently, evidence form a mouse model of glaucoma has supported this theory; (Schlamp et al., 2006) however, the spatiotemporal pattern of distal axonal degeneration has never been reported. This study identified that intravitreal injection of NMDA causes severe, progressive, selective damage to the inner retina, evident as a decreased cell count in the GCL and reduced IRT. Counts in the GCL included both RGCs and displaced amacrine cells. No obvious abnormality was detected in the outer layers of the retina. Previous reports have shown that the degree of retinal damage depends on the dose of intravitreal excitotoxin. (Dreyer et al., 1994; Lam et al., μ) With 20 nM of NMDA used in the current study, loss of 45–55% cells in the GCL was seen after 72 h and only 25–35% cells remained in the GCL a week after injection. The thinning of the inner retina is believed to be due to the loss of dendritic tree of the RGCs and possibly amacrine cells injured as a result of excitotoxin exposure. Both the posterior and peripheral retina showed degenerative changes. After 7 days, the posterior retina was damaged more (approximately 75% decrease in cell count in GCL and 30% reduction in IRT) than the peripheral retina (65% cell loss in

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GCL and 24% reduction in IRT). The observed spatial variability in retinal responsiveness may be due to difference in the type and distribution of RGCs in different regions of the retina. No attempt was made to identify regional differences in the retinal structure or classify RGCs according to their size, type or distribution. However, there is an evidence that, in albino rats, the posterior retina has a comparatively higher concentration of small-sized RGCs. (Schober and Gruschka, 1977; Siliprandi et al., 1992) Although, the small RGCs are known to be less vulnerable to excitotoxic injury (Siliprandi et al., 1992), their higher cell density in the posterior retina (N 6000/mm2) may make this region more susceptible to injury than the peripheral retina (1000–1500/mm2).(Fukuda, 1977) Using a 20 nM intravitreal injection of NMDA in the adult male albino Lewis rats, Lam et al. reported 50% and 25% reduction in GCL cell count in the posterior and peripheral retinas, respectively, after seven days.(Lam et al., 1999) In the present study with adult male Sprague–Dawley rats, around 75% and 65% cell loss was found in these regions after one week. This difference may be due to interspecies differential susceptibility to NMDA. The degree of optic nerve degeneration correlated strongly with the retinal damage, suggesting that the axon damage and myelin disruption in the optic nerve is related to the somal injury. It is possible that the axonal degeneration simply represents a “withering away” type pathology due to the toxic insult to the soma. However, the synchronous changes observed in the distal portions of the axon suggest the involvement of a positive trigger. The possible involvement of the Wallerian degeneration associated ubiquitin proteosome system and the effect of this injury on axonal transport will be the topic for further research. In conclusion, we have demonstrated that perikaryal excitotoxic injury of the RGC causes synchronous somal and axonal degeneration, with distal portions of the axon showing more

Fig. 3. Correlation between the retinal damage and number of axons in the optic nerve of experimental animals. Scatter plot shows correlation between cell count in GCL (mean of posterior and peripheral retinas) and number of optic nerve axons in distal segment (R = 0.929. p b 0.001). Solid line drawn through datapoints shows best-fitting function (along with 95% confidence limits: interrupted line).

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