Manganese Superoxide Dismutase Deficiency Exacerbates Cerebral Infarction After Focal Cerebral Ischemia/Reperfusion in Mice

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Abstract
Background and Purpose — Superoxide anion radicals (O 2 ·− ) are implicated in ischemia/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 O 2 ·− 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. 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.