Intra and postischemic mild hypothermia : Effects on inducible nitric oxide synthase and peroxynitrite in experimental stroke
Background: Mild hypothermia is protective even when delayed hours after stroke onset. Because metabolic depletion and glutamate release occur early after ischemia onset, other mechanisms may underlie the protection, particularly when cooling is delayed. Inflammation and free radicals mediate delayed ischemic injury. We examined whether mild hypothermia alters induction and activity of inducible nitric oxide synthase (iNOS, found primarily in inflammatory cells) and peroxynitrite generation. Methods: Rats were subjected to transient middle cerebral artery occlusion for 2 hours. Mild hypothermia began immediately (intraischemic, 33OC-I, n=8) or 2 h (delayed, 33OC-D, n=8) after ischemia onset during the reperfusion phase. Hypothermia was maintained for 2 h. Normothermic animals were maintained at 37OC (n=8). Brains were examined 1 and 3 days later for infarction, NOS activity (NADPH diaphorase), iNOS induction and peroxynitrite (nitrotyrosine) production. Results: Mild hypothermia attenuated infarct size in both groups compared to normothermia (33OC-I: 26.8±7.7%, 33OC-D: 30.4±4.8%, 37OC: 70.8±3.3%, P<0.001). iNOS-positive cells appeared 1 d after ischemia in the infarct area and increased in number by 3 d, but NOS activity was only seen at 3 d. Nitrotyrosine-positive cells also increased by 3 d. Positive cells in all cases appeared microglia/monocyte/macrophage-like with ameboid morphology. Mild hypothermia significantly decreased NADPH diaphorase, iNOS and peroxynitrite densities at 3 d, but not 1 d. NADPH diaphorase-positive cells per hemisphere were: 37OC: 1028±68, 33OC-I: 152±12, 33OC-D: 101±1 (P<0.001). Densities of iNOS-positive cells were: 37OC: 1265±83/mm2, 33OC-I: 435±93/mm2, 33OC-D: 688±59/mm2 (P<0.001). Densities of nitrotyrosine-positive cells were: 37OC: 585±14/mm2, 33OC-I: 214±23/mm2, 33OC-D: 106±12mm2 (P<0.001). No differences in any of the parameters were seen at 1 d. Conclusion: Mild hypothermic neuroprotection is related to attenuation of iNOS and peroxynitrite in inflammatory cells at 3 d but not earlier. This is evident even when cooling is delayed by 2 h.