The role of nitric oxide in reoxygenation injury of brain microvascular endothelial cells

Object. The role of nitric oxide (NO) in reperfusion injury of the brain is still controversial. The authors demonstrate that NO injures the brain capillary endothelial cells in a reoxygenation state by the formation of peroxynitrite. Materials and Methods. 1) Rat brain capillary endothelial cells (BCECs) were isolated by a two-step enzymatic purification method. The BCECs were identified by the presence of factor VIII. 2) Anaerobic cell preparations were achieved by purging under nitrogen gas with 0.5% CO2 at 37 degreesC for 20 minutes and then reoxygenating in an incubator. To ascertain the degree of BCEC injury after anoxia and reoxygenation, lactate dehydrogenase (LDH) release was measured. The production of NO was measured by the Griess method. 3) The cell-protective effects of superoxide dismutase (SOD), NG-nitro-L-arginine (L-NAME), and S-Methyl-ITU (i-NOS blocker) were studied. Results. 1) Both L-NAME and SOD protected the cells from reoxygenation injury. The protective effect of L-NAME was dose-dependent. S-Methyl-ITU did not protect the cells. 2) The NO production after anoxia/reoxygenation was blocked by L-NAME. Conclusion. NO from the BCEC can injure the cells themselves through the formation of peroxynitrite under anoxia/reoxygenation conditions. Increased NO production after anoxia can be attributed to the induction of e-NOS.