Elsevier

Brain Research

Volume 1212, 30 May 2008, Pages 89-101
Brain Research

Research Report
Degenerative alterations in the visual pathway after NMDA-induced retinal damage in mice

https://doi.org/10.1016/j.brainres.2008.03.021Get rights and content

Abstract

In the present study, intravitreal injection of N-methyl-d-aspartate (NMDA) into the left eye induced retinal damage (decreases in the number of retinal ganglion cells) at 1 day after the injection. At 7 days after the injection, atrophy of the optic tract was observed on the contralateral side, but not on the ipsilateral side. Number of neuronal nuclear specific protein (NeuN)-immunostained neurons were decreased in the contralateral dorsal LGN (dLGN) and contralateral ventral LGN-lateral (vLGN-l) at 90 and 180 days, respectively, after the injection. Furthermore, expressions of glial fibrillary acid protein (GFAP) were increased in the contralateral dLGN and contralateral vLGN-l at 7 and 30 days, respectively, and those of brain-derived neurotrophic factor (BDNF) were increased in the contralateral dLGN at 30 and 90 days and in the contralateral vLGN-l at 7 and 30 days. All NeuN-positive neuronal cells exhibited BDNF, whereas only some GFAP-positive astroglial cells exhibited BDNF. However, the contralateral ventral LGN-medial (vLGN-m) and ipsilateral LGN displayed no significant differences related to NeuN, GFAP, or BDNF immunohistochemistry. Taken together, these results indicate that time-dependent alterations occurred after the NMDA injection along the retinogeniculate pathway (from retina to LGN), and that the degree of damage in the LGN was region-dependent. In addition, the increased activated astroglial cells and expressions of BDNF in the damaged regions may play some roles in the cell-survival process of the LGN.

Introduction

Glaucoma is an optic neuropathy resulting from the death of retinal ganglion cells (RGC). In clinical studies, a loss of more than 50% of RGC has been reported to induce visual field loss. However, the initial loss of RGC does not lead to visual field loss in humans (Quigley et al., 1989). Possibly, a compensatory action of the visual cortex may protect the visual field against such a decrease. However, there is recent evidence that the RGC death that occurs in glaucoma leads to neuronal degeneration within the lateral geniculate nucleus (LGN), the major relay center between eye and visual cortex (Yücel et al., 2000). Further, it was reported that time- and region-dependent morphological changes had occurred in LGN at 120 days after intraocular pressure (IOP) was elevated in rats (Wang et al., 2000). These reports suggest that the visual field loss induced by glaucoma may not result only from RGC loss, but also from neuronal degeneration in LGN. However, no previous investigation has been made of time-dependent alterations along the retinogeniculate pathway (i.e., retina, optic tract, and LGN) after retinal injury in mice. In addition, the possible pathophysiological mechanisms underlying neuronal cell death in LGN following RGC loss remain uncertain.

Excessive activation of glutamate receptors by glutamate released from injured RGC is implicated in the glaucomatous RGC death process (Osborne et al., 1999). Glutamate is the principal excitatory neurotransmitter within the central nervous system (CNS), and it has been found to be increased in the vitreous body in glaucoma (Dreyer et al., 1996). In contrast, this fact was not confirmed by Honkanen et al. (2003). However, in fact the toxic effects of elevated levels of glutamate are predominantly mediated by the overstimulation of ionotropic receptors. Overstimulation of the class of these receptors that respond specifically to the glutamate analog N-methyl-d-aspartate (NMDA) leads to an overload of intracellular Ca2+. Such elevations in Ca2+ elicit various cytotoxic biochemical reactions including the activation of nitric oxide (NO) synthase and the generation of reactive NO free radicals. Two other classes of ionotropic receptors, which respond to the agonists kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropianate (AMPA), respectively, can also mediate Ca2+ overload when overstimulated, but they are somewhat less permeable to this ion than the NMDA receptor (NMDAR). In fact, a single intravitreal injection of NMDA has been reported to damage the cells in GCL and the IPL without affecting the other retinal layers in rats 7 days after the injection (Akaike et al., 1998). Among the glutamate receptors, the NMDA receptor's role in cell death has been extensively studied: excessive doses of NMDA induce apoptotic cell death of RGC and amacrine cells in rat retina (Lam et al., 1999, Inomata et al., 2003). Furthermore, it has been reported that NMDAR positive cells are either RGC or amacrine cells (Jakobs et al., 2007). In addition, a previous study has shown that NMDAR-mediated neurotoxicity in the RGC is dependent on the influx of extracellular Ca2+ (Sucher et al., 1997). In fact, blockade of glutamate activity by modulation of its receptors has been advocated as an important strategy for neuroprotection in glaucoma, and memantine, an NMDAR antagonist, displays a neuroprotective effect in experimental glaucoma (Li et al., 2002, Yücel et al., 2006). In either case, the animal model employed in the present study (involving intravitreal injection of NMDA) exhibits high sensitivity and stability, as well as good reproducibility, and is widely used for investigating the mechanisms underlying neuronal cell death in the retina (Yoneda et al., 2001). We therefore used it to investigate time-dependent alterations in the murine LGN following NMDA-induced retinal damage.

Section snippets

NMDA-induced retinal damage

Intravitreal injection of NMDA at 40 nmol/eye decreased both the cell-count in the GCL and the thickness of the IPL in the retina, as compared with those in the non-treated control retina (Fig. 1). The cell-count in GCL was decreased to 85.3, 47.6, 38.5, 32.5, 29.8, and 29.6% of control at 1, 3, 7, 30, 90, and 180 days, respectively, after NMDA injection. The thickness of IPL was increased to 138.8% of control at 1 day after the NMDA injection (Fig. 1B and I), then time-dependently decreased to

Discussion

In the present study, unilateral intravitreal injection of NMDA induced neuronal damage that was detected, in turn, in RGC, the optic tract, and in neurons contralateral dLGN, and contralateral vLGN-l, the maximal extent of the neuronal damage in these tissues (versus control, non-treated mice) being about 70, 60, 18, and 10%, respectively. This suggests that the retinal damage induced by intravitreal injection of NMDA in mice lead to neuronal degeneration in the LGN connected to that

Animals

Male adult C57BL/6J mice weighing 20–32 g (Clea Japan, Inc. Fujimiya, Japan) were kept under lighting conditions of 12 h light and 12 h dark. All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved and monitored by the Institutional Animal Care and Use Committee of Gifu Pharmaceutical University.

NMDA injection

Mice were anesthetized with 3.0% isoflurane (Merck, Osaka, Japan) and maintained with 1.5% isoflurane in 70% N2O

Acknowledgments

This study was supported in part by Grants-in-Aid for exploratory research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (Nos. 18209053 and 18210101).

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