Abstract W MP78: Small Changes in Intracellular pH Block Ischemic and Excitotoxic Cell Death by Suppressing Neuronal Superoxide Production

Tissue acidosis has long been recognized as a crucial variable affecting outcome after brain ischemia, with mild acidosis (pH 7.0 - 6.6) providing robust neuroprotection. pH in ischemic tissues is reduced by anaerobic glucose metabolism, and normalized by reperfusion. Superoxide produced by neuronal NADPH oxidase (NOX2) has recently been identified as a major source of oxidative stress in ischemia and excitotoxicity. Given that NOX2 in other cell types is highly sensitive to intracellular pH, we evaluated the possibility that pH effects on neuronal NOX2 activity mediate the neuroprotective effects of mild acidosis. In cultured neurons, reducing medium pH from 7.2 to 7.0 produced a comparable 0.2 pH-unit intracellular acidification and completely prevented NMDA-induced NOX2 activity, superoxide production, and cell death. These effects were observed even when extracellular calcium was elevated to overcompensate for reduced NMDA-induced calcium influx at the lowered pH. Intracellular H + produced by NOX2 is be extruded to permit continued NOX2 activity. Neurons treated with an inhibitor of Na + /H + exchange, cariporide showed a 0.2- 0.3 unit intracellular acidification (with no change in extracellular pH). Cariporide- treated neurons also showed no superoxide formation and no cell death in response to NMDA, with no change in NMDA-induced calcium influx. To evaluate this effect in vivo, mice were given stereotaxic striatal injections NMDA. NMDA induced superoxide production and subsequent neuronal death, both of which were blocked by co-injection with cariporide. Superoxide production and neuronal death were similarly blocked in mice with reduced expression of Na + /H + exchanger isoform 1 protein (NHE-1 +/- mice). NHE-1 +/- mice also exhibited much less superoxide production and neuronal death after forebrain ischemia-reperfusion. These findings identify a mechanism by which mild acidosis improves outcomes in ischemia and excitotoxicity. The findings may also help explain why superoxide production is greater in reperfused than non-reperfused brain, and why both NOX2 inhibitors and NMDA receptor antagonists are more neuroprotective in reperfused than non-reperfused brain.