Mitochondrial injury has been implicated in ischemic neuronal injury. Mitochondria, producing adenosine triphosphate by virtue of electron flow, have been shown to be both the sites of superoxide anion (O2-) production and the target of free radical attacks. We evaluated these mechanisms in an in vivo cerebral ischemia model, using mutant mice with a heterozygous knock-out gene (Sod2 -/+) encoding mitochondrial manganese superoxide dismutase (Mn-SOD). Sod2 -/+ mice demonstrated a prominent increase in O2- production under normal physiological conditions and in ischemia, as evidenced by specific oxidation of a fluorescent probe, hydroethidine, reflecting decreased activity of Mn-SOD. A mitochondrial viability assay that used rhodamine 123, which is accumulated by transmembrane potential of viable mitochondria, demonstrated accelerated development of mitochondrial injury. This rapid progress of ischemic injury resulted in exacerbation of infarct size and hemisphere enlargement, causing advanced neurological deficits but without altering DNA fragmentation induction. The present study suggests that O2- overproduced in a mitochondrial compartment, when uncoupled from antioxidant defenses, induces impairment of mitochondrial function and causes exacerbation of cerebral infarction after ischemia.

Apoptotic neuronal cell death has recently been associated with the development of infarction after cerebral ischemia. In a variety of studies, CuZn-superoxide dismutase (CuZn-SOD) has been shown to protect the brain from ischemic injury. A possible role for CuZn-SOD-related modulation of neuronal viability is suggested by the finding that CuZn-SOD inhibits apoptotic neuronal cell death in response to some forms of cellular damage. We evaluated this possibility in the model of transient focal cerebral ischemia in mice bearing a disruption of the CuZn-SOD gene (Sod1). Homozygous mutant (Sod1 Ϫ/Ϫ) mice had no detectable CuZn-SOD activity, and heterozygous mutants (Sod1 ϩ/Ϫ) showed a 50% decrease compared with wild-type mice. Sod1 Ϫ/Ϫ mice showed a high level of bloodbrain barrier disruption soon after 1 hr of middle cerebral artery occlusion and 100% mortality at 24 hr after ischemia. Sod1 ϩ/Ϫ mice showed 30% mortality at 24 hr after ischemia, and neurological deficits were exacerbated compared with wildtype controls. The Sod1 ϩ/Ϫ animals also had increased infarct volume and brain swelling, accompanied by increased apoptotic neuronal cell death as indicated by the in situ nick-end labeling technique to detect DNA fragmentation and morphological criteria. These results suggest that oxygen-free radicals, especially superoxide anions, are an important factor for the development of infarction by brain edema formation and apoptotic neuronal cell death after focal cerebral ischemia and reperfusion. Key words: CuZn-superoxide dismutase; focal cerebral ischemia; blood-brain barrier; Evans blue extravasation; neuronal apoptosis; TUNEL; oxidative stressOxygen-free radicals are believed to be involved in the pathogenesis after cerebral ischemia and reperfusion. During cerebral ischemia, a number of events that predispose the brain to the formation of oxygen-free radicals may occur (Siesjö, 1984;McCord, 1985). After reperfusion, these events can set off a cascade of other biochemical and molecular sequelae such as the xanthine-xanthine oxidase reaction and phospholipase activation, leading to free-radical production (Gaudet and Levine, 1979;Chan et al., 1984;Siesjö, 1984;McCord, 1985). Among these oxygen-free radicals, superoxide anion (O 2 Ϫ ), being directly toxic to neurons (Fridovich, 1986;Patel et al., 1996), may initiate a free radical-mediated chain reaction causing additional CNS damage (Saugstad and Aasen, 1980;Chan, 1994).One of the manifestations of CNS damage after cerebral ischemia is the formation of brain edema caused by the breakdown of the blood-brain barrier (BBB). CuZn-superoxide dismutase (CuZn-SOD), a cytosolic protein, prevents vasogenic brain edema after several kinds of injuries Kinouchi et al., 1991;Shukla et al., 1993), suggesting that O 2 Ϫ is an important factor for BBB disruption. Another manifestation of CNS damage is the direct injury of neuronal cells, including excitatory events that are induced by glutamate release after cerebral ischemia. Glutamate elevates cytosolic free calcium (Ca 2ϩ ) (...

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in

Background and Purpose-Superoxide anion radicals (O 2 ·Ϫ ) 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 ·Ϫ was developed to detect O 2 ·Ϫ production in a variety of experimental brain injury models. To clarify the role of O 2 ·Ϫ in the mechanism of ischemia/reperfusion, we investigated O 2 ·Ϫ 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 ·Ϫ production using specific HEt oxidation by O 2 ·Ϫ before and after ischemia/reperfusion. Furthermore, we compared O 2 ·Ϫ 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 ·Ϫ under physiological conditions. Cerebral ischemia/reperfusion produced O 2 ·Ϫ 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 ·Ϫ production and exacerbated cerebral infarction, worsening neurological deficits after ischemia/reperfusion. Conclusion-These results suggest that mitochondrial O 2 ·Ϫ 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.