SIGMA-1 RECEPTOR STIMULATION PROTECTS PURIFIED RETINAL GANGLION CELLS FROM ISCHEMIC INSULT THROUGH THE PHOSPHORYLATION OF EXTRACELLULAR SIGNAL REGULATED KINASE 1/2

Purpose (a): Sigma-1 receptor activation and mitogen-activated protein kinases (MAPKs) have been shown to have neuroprotective roles in protecting retinal ganglion cells (RGCs) from cell death. The purpose of this study was to determine if sigma-1 receptor stimulation with pentazocine could promote neuroprotection under conditions of ischemia through the phosphorylation of extracellular signal regulated kinase (pERK)1/2. Methods (b): Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy 1.1 antibody. RGCs were cultured for 7 days before subjecting the cells to an ischemic insult (0.5% oxygen in glucose-free medium) for 6 hours. During the ischemic insult, RGCs were treated with pentazocine (sigma-1 receptor agonist) with or without BD1047 (sigma-1 receptor antagonist). In other experiments primary RGCs were treated with pentazocine, in the presence or absence of PD98059 (ERK1/2 inhibitor). Cell survival/death was assessed by staining with the calcein-AM/ethidium homodimer reagent. Levels of pERK1/2, total ERK1/2, and beta tubulin expression were determined with immunoblotting and immunofluorescence. Results (c): RGCs subjected to an ischemic insult demonstrated more than a 40% increase in cell death, compared to untreated controls. RGCs maintained under ischemia also showed a 50% decrease in expression of pERK1/2 (p<0.05). Cell death was attenuated when RGCs were treated with pentazocine under ischemic conditions and levels of pERK1/2 were increased more than 60% (p<0.05), compared to untreated RGCs subjected to ischemia. Treatment with BD1047 abrogated the pentazocine neuroprotection effects, and also attenuated the increase in levels of pERK1/2 (p<0.05). Finally, treatment with PD98059 also reversed the pentazocine mediated neuroprotective effects on RGCs, and abolished the expression of pERK1/2 (p<0.05). Conclusions (d): These results establish a direct relationship between sigma-1 receptor stimulation and neuroprotective effects under ischemia through the involvement of the MAPK/ERK1/2 pathway in purified RGCs. These findings support a role for sigma receptor agonists as potential neuroprotective agents.