Manganese superoxide dismutase deficiency exacerbates cerebral infarction after focal cerebral ischemia/reperfusion in mice - Implications for the production and role of superoxide radicals

Background and Purpose-Superoxide anion radicals (O-2(.-)) are implicated in ischeinia/reperfusion injury, although a direct relationship has not been elucidated. Recently, a specific method of hydroethidine (HEt) oxidation by O-2(.-) was developed to detect O2(.-) production in a variety of experimental brain injury models. To clarify the role of O-2(.-) in the mechanism of ischemia/reperfusion, we investigated O-2(.-) production after ischemia/reperfusion and ischemia/reperfusion injury in mutant mice deficient in mitochondrial manganese superoxide dismutase (MnSOD) and in wild-type littermates. Methods-Ischemia/reperfusion was performed for 60 minutes using intraluminal suture blockade of the middle cerebral artery in the mutant or wild-type mice. We evaluated fluorescent kinetics of HEt or ethidium, the oxidized form of HEt, in brains after an intravenous injection of HEt, followed by measurement of cellular O-2(.-) production using specific HEt oxidation by O-2(.-) before and after ischemia/reperfusion. Furthermore, we compared O-2(.-) production and subsequent infarct volume in the mice using triphenyltetrazolium chloride after ischemia/reperfusion. Results-HEt oxidation to ethidium is primarily a result of mitochondrially produced O-2(.-) under physiological conditions. Cerebral ischemia/reperfusion produced O-2(.-) prominently in neurons shortly after reperfusion, followed by a delayed increase in endothelial cells. A deficiency in MnSOD in mutant mice increased mitochondrial O-2(.-) production and exacerbated cerebral infarction, worsening neurological deficits after ischemia/reperfusion. C Conclusion-These results suggest that mitochondrial O-2(.-) production may be a critical step underlying the mechanism of ischemia/reperfusion injury and that MnSOD may protect against ongoing oxidative cell death after ischemia/reperfusion.