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We determined the effect of nilvadipine, a dihydropyridine-type calcium channel blocker, in preventing glutamate neurotoxicity in purified retinal ganglion cells (RGCs). RGCs were purified from dissociated rat retinal cells (postnatal days 6-8), using a modified two-step panning method, and cultured in serum-free medium containing neurotrophic factors and forskolin. RGC survival after exposure to glutamate (25 microM) with nilvadipine or other calcium channel blockers was measured by calcein-acetoxymethyl ester staining after 3 days in culture. Changes in the level of intracellular Ca(2+) ([Ca(2+)](i)) were measured with fura-2 fluorescence. Induction of apoptosis was evaluated using the TDT-dUTP terminal nick-end labeling technique. The neurotoxic effects of low doses of glutamate were blocked by a specific alpha-amino-3-dihydro-5-methylisoxazole-4-propionate-kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (20 microM). Simultaneous application of nilvadipine (1-100 nM) with glutamate protected against glutamate neurotoxicity in a dose-dependent manner. Calcium-imaging experiments showed that the glutamate-evoked [Ca(2+)](i) increase was significantly blocked by nilvadipine (P<0.001), but not nifedipine and diltiazem, in about 50% of RGCs. In addition, the application of nilvadipine significantly reduced glutamate-induced apoptosis (P<0.001). These findings suggest that nilvadipine may partly inhibit glutamate-induced apoptotic cell death by blocking calcium influx via voltage-dependent calcium channels in purified RGCs.

The purpose of this study was to determine whether timolol, an ocular hypotensive drug, has retinal neuroprotective effects in experimental in vitro and in vivo models. For in vitro studies, we used retinal neuron cultures from rat embryos and purified retinal ganglion cells (RGCs) from newborn rats. In the former, neurotoxicity was induced using 1 mM glutamate and cell viability was assessed. In RGCs, neurotoxicity was induced using 25 microM glutamate for 3 days and cell viability was assessed. For the in vivo study, we used a rat model of retinal ischemic injury induced by elevating intraocular pressure (IOP) by raising the hydrostatic pressure. The retinal damage was evaluated by counting the number of cells in the ganglion cell layer (GCL) and by examining the a- and b-waves in the electroretinogram (ERG). For the intraocular distribution study, 0.5% [3H]timolol was topically applied to rat eyes, and these were enucleated after various intervals and divided into parts. Each part was combusted and the radioactivity measured. Timolol (0.1 and 1 microM) markedly reduced the glutamate-induced neuronal cells in retinal neuron cultures and in RGCs. After ischemic-reperfusion, both the number of cells in the GCL and a- and b-waves in the ERG decreased; however, topically applied 0.5% timolol reduced these effects. Topically applied 0.5% timolol was detected at a concentration of approximately 1 microg/g wet tissue in retina-choroid at 30 min after its application. In conclusion, timolol was effective against retinal neuron damage both in vitro and in vivo. Furthermore, topically applied timolol reached the retina-choroid. These findings suggest that timolol has a direct neuroprotective effect against retinal damage., (Copyright 2002 Elsevier Science B.V.)

Purpose: Low concentrations of excitotoxic agents such as glutamate decrease survival of retinal ganglion cells (RGCs) and may be an important cause of RGC death in a variety of retinal diseases. Arachidonic acid (AA), an intercellular messenger in the central nervous system, has been reported to have multiple effects on glutamate receptors, including an inhibitory effect on non-N-methyl-D-aspartate (NMDA) receptors. The purpose of this study was to test the hypothesis that AA could protect RGCs from glutamate neurotoxicity., Methods: RGCs were purified from the rat retina on postnatal days 7 and 8 by a modified two-step panning method. Survival of RGCs after exposure to glutamate, with or without AA treatment, was measured after 3 days in culture. To visualize calcium signals, RGCs were loaded with a calcium indicator dye, fluo-3 acetoxymethyl ester, and the fluorescence was measured by laser scanning confocal microscopy. Electrophysiological effects of AA on non-NMDA ionotropic receptors were examined by using whole-cell patch clamp configurations., Results: Incubation of RGCs with 25 microM glutamate caused 60% loss of RGCs. This glutamate neurotoxicity was significantly ameliorated by low concentrations of AA. Concentrations of AA above 10 microM were toxic to RGCs. Calcium imaging showed that glutamate-, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- (AMPA) and kainate-induced intracellular calcium accumulation in these cells was reduced by AA. Electrophysiological recordings revealed that currents mediated by non-NMDA ionotropic receptors were inhibited by AA in a dose-dependent manner., Conclusions: Low concentrations of AA can reduce glutamate neurotoxicity to RGCs by the inhibition of non-NMDA ionotropic receptors. These results suggest that endogenous or exogenous AA may be used to protect RGCs from glutamate neurotoxicity and that AA may be one potential treatment for RGC loss in a variety of eye diseases, including glaucoma.

Purpose: Low concentrations of excitotoxic agents such as glutamate and nitric oxide decrease survival rates of purified retinal ganglion cells (RGCs). In the retina, RGCs are ensheathed by retinal Müller glial (RMG) cell processes. The purpose of this study was to determine whether RMG cells could protect RGCs from these excitotoxic injuries., Methods: RGCs were purified from 7- or 8-day-old Long Evans rats and cultured on polylysine/laminin-coated coverslips in serum-free medium for 2 days. The coverslips were then moved to dishes containing either confluent RMG monolayers or no glial cells in glutamate-free medium. Some dishes with confluent RMG cells were exposed to D,L-threo-beta-hydroxyaspartate (THA), a blocker of glutamate uptake. Three days after exposure to various concentrations of glutamate or the NO donor, 2, 2'-(hydroxynitroso-hydrazino)bisethanamine, survival rates of RGCs were measured by calcein-acetoxymethyl ester staining. Glutamate concentrations in the medium were measured using amino acid analysis., Results: Without RMG cells, the application of increasing concentrations (5-500 microM) of glutamate caused a dose-dependent increase in RGC death after 3 days. The neurotoxic effects of glutamate were blocked in the RMG cell cocultures, even when there was no direct contact between the cell types. The protective effect of RMG cells was weakened by THA treatment. NO also had toxic effects on RGC. RMG cells prevented this toxicity but only when in direct contact with the RGCs., Conclusions: RMG cells can protect RGCs from glutamate and NO neurotoxicity. We suggest that functional disorders of glutamate uptake in RMGs might be one of the etiologies of glaucoma.

Purpose: To determine whether low concentrations of glutamate induce cell death in purified rat retinal ganglion cells (RGCs)., Methods: Rat retinal ganglion cells were purified from dissociated retinal cells by a modified two-step panning method and were cultured in serum-free medium containing neurotrophic factors and forskolin. Survival of RGCs after exposure to glutamate, with or without glutamate receptor antagonists, was measured by calcein-acetoxymethyl ester staining after 3 days in culture. To visualize calcium signals, RGCs were loaded with the calcium indicator dye, fluo-3 acetoxymethyl ester, and fluorescence was measured by laser scanning confocal microscope. Electrophysiological properties of RGCs were examined by using the whole-cell, patch-clamp technique., Results: The application of increasing concentrations (5-500 microM) of glutamate caused a dose-dependent increase in RGC death after 3 days in culture. Neurotoxic effects of low doses of glutamate were totally blocked by a specific alpha-amino-3-dihydro-5-methyl-isoxazol-4-propionic acid-kainate (AMPA-KA) receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), but not by a specific N-methyl-D-aspartate receptor antagonist, 2-amino-5-phosphonovalerate (APV). In addition, calcium imaging and patch-clamp recordings showed that intracellular calcium accumulation and glutamate-evoked inward currents were completely blocked by DNQX but not by APV., Conclusions: Low doses of glutamate can activate AMPA-KA receptors in RGCs, which causes increases in intracellular calcium and decreases in cell survival. This is the first report to show the functional role of calcium-permeable AMPA-KA receptors in cultured RGCs.