Immunohistochemical nitrotyrosine distribution in neonatal rat cerebrocortical slices during and after hypoxia

Abstract

The peroxynitrite contributions to hypoxic damage in brain slices that arise from N-methyl- d-aspartate (NMDA) receptor activation were studied by following the temporal-spatial course of nitrotyrosine (NT) formation during six conditions: hypoxia ( pO 2<5 mmHg) with or without 10 μM MK-801 treatment; with exposure to 10, 100 and 1000 μM NMDA; and no treatment (control). In each experiment, twenty 350-μm thick cerebrocortical slices, obtained from the parietal lobes of ten 7-day-old Sprague–Dawley rats, were metabolically recovered and allowed to respire in a well-oxygenated perfusion system. Thirty minutes exposures to hypoxia or NMDA were followed by 2 h of oxygenated reperfusion. MK-801 administration began 15 min prior to hypoxia and was discontinued during reperfusion. Anti-NT serum immunohistochemistry stains in 20-μm frozen sections of slices taken during oxygenated reperfusion, after hypoxia or NMDA exposure, were positive in both neurons and endothelial cells. NT-positive neurons were detected sooner after hypoxia than after NMDA exposure, suggesting that mechanisms of superoxide generation were different in both groups. After hypoxia and even more so after NMDA exposure, more intense NT-positive staining was observed in endothelial cells than in neurons. Cell damage after hypoxia was attenuated by MK-801. MK-801 decreased post-hypoxia counts of NT-stained endothelial cells by 78.5% ( p<0.001) and NT-stained neurons by 54.1% ( p<0.05). Our findings suggest that NMDA receptor activation in hypoxic brain slices is associated with increased post-hypoxic peroxynitrite production that contributes to acute neuronal death and endothelial cell injury. Peroxynitrite injury to endothelial cells, caused either by increased peroxynitrite from within or from increased vulnerability to peroxynitrite from without, might play an important role in hypoxic–ischemic brain injury and NMDA-induced brain injury.