A Kawasaki1, Y Otori, C J Barnstable. 1. Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06520-8061, USA.
Abstract
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: 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.
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