Potentiation of oxygen-glucose deprivation-induced neuronal death after induction of iNOS

Background and Purpose: Previous studies have shown that brain ischemia and other insults can induce a marked increase in inducible nitric synthase (iNOS) expression in astrocytes and some immune cells, but the biological significance of this of this phenomenon has not been elucidated. The purpose of the present study was to determine whether this induction of astrocyte iNOS alters neuronal vulnerability to severe hypoxic insults. Methods: Astrocytic iNOS was induced by exposure of murine cortical cultures to interferon gamma in combination with either interleukin-1β or lipopolysaccharide. Cultures were exposed to combined oxygen-glucose deprivation.The extracellular concentration of glutamate was measured by high-performance liquid chromatography. N-Methyl-D-aspartate (NMDA) receptor activity was assessed by measurement of ⁴⁵Ca²⁺ influx: neuronal dearth was assessed by morphological examination and quantitated by measurement of lactate dehydrogenase efflux to the bathing medium. Results: In murine neocortical cell cultures containing neurons and astrocytes, neuronal injury induced by combined oxygen-glucose deprivation was not reduced by the addition of the nitric oxide-synthase inhibitors N(G)-nitro-L-arginine or L(G)-nitro-arginine methyl ester. However, after induction of astrocyte iNOS activity with interferon gamma plus lipopolysaccharide or interleukin-1β, oxygen-glucose deprivation-induced neuronal injury was markedly enhanced and nitric oxide synthase inhibitors became protective. This iNOS-mediated potentiation was associated with a large increase in both extracellular glutamate accumulation and ⁴⁵Ca²⁺ influx into neurons. The potentiation could be blocked by MK-801 but not CNQX, suggesting critical involvement of NMDA receptor activation. Conclusions: These results support the idea that nitric oxide production mediated by induced astrocytic iNOS can potentiate NMDA receptor-mediated neuronal death consequent to hypoxic-ischemic insults.