Nitric oxide mediates excitotoxic and anoxic damage in rat retinal ganglion cells cocultured with astroglia

Background: Nitric oxide has been implicated in the process of retinal ganglion cell death in glaucoma. Objective: To investigate the role of nitric oxide in mediating retinal ganglion cell death in a culture system that models glial-neuronal interactions at the level of the optic nerve head. Methods: Dissociated retinal ganglion cells from neonatal rats were plated on monolayers of astroglia and identified by retrograde labeling with the fluorescent marker 1.1-dioctadecyl-,3,3,3,tetramethylindocarbocyanine- perchlorate. Two days after dissociation, cocultures of retinal ganglion cells and glia were treated with graded concentrations of the nitric oxide synthase inhibitor N-nitro-L-arginine (NNA), and exposed to either anoxia for 1 to 24 hours or excitatory amino acids for 6 hours. Surviving retinal ganglion cells were counted with fluorescence microscopy and expressed as a percentage of retinal ganglion cells surviving in control cultures. Results: Cell survival after anoxia increased in a dose-dependent fashion with exposure to NNA. Mean +/- SD survival rate of retinal ganglion cells after 6 hours of anoxia was 57% +/- 10%, with NNA treatment compared with 31% +/- 3% without treatment (P<.01). When treated with excitatory amino acids, cell survival was 31% +/- 60% after administration of N-methyl D-aspartate, 500 mu mol/L, and 27% +/- 8% after administration of sodium glutamate, 500 mu mol/L. Survival was increased in cultures with exposure to NNA, 100 mu mol/L, to 53% +/- 11%, and 69% +/- 11%, respectively (P<.01). Conclusion: In this coculture of retinal ganglion cells and astroglia, reduction of the glial source of nitric oxide through nitric oxide synthase inhibition provided partial but significant protection against the lethal effects of anoxia and excitatory amino acids On retinal ganglion cells. Clinical Relevance: Neuroprotective agents may play a role in patients with glaucoma who have progressive visual field loss, despite satisfactory control of intraocular pressure. Inhibition of nitric oxide synthase at the level of the optic nerve head may contribute to a clinically significant level of neuroprotection.