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 ) (...

Transient global cerebral ischemia resulting from cardiac arrest is known to cause selective death in vulnerable neurons, including hippocampal CA 1 pyramidal neurons. It is postulated that oxygen radicals, superoxide in particular, are involved in cell death processes. To test this hypothesis, we first used in situ imaging of superoxide radical distribution by hydroethidine oxidation in vulnerable neurons. We then generated SOD1 transgenic (Tg) rats with a five-fold increase in copper zinc superoxide dismutase activity. The Tg rats and their non-Tg wild-type littermates were subjected to 10 min of global ischemia followed by 1 and 3 d of reperfusion. Neuronal damage, as assessed by cresyl violet staining and DNA fragmentation analysis, was significantly reduced in the hippocampal CA 1 region, cortex, striatum, and thalamus in SOD1 Tg rats at 3 d, as compared with the non-Tg littermates. There were no changes in the hippocampal CA 3 subregion and dentate gyrus, resistant areas in both SOD1 Tg and non-Tg rats. Quantitative analysis of the damaged CA 1 subregion showed marked neuroprotection against transient global cerebral ischemia in SOD1 Tg rats. These results suggest that superoxide radicals play a role in the delayed ischemic death of hippocampal CA 1 neurons. Our data also indicate that SOD1 Tg rats are useful tools for studying the role of oxygen radicals in the pathogenesis of neuronal death after transient global cerebral ischemia.

Recent in vitro cell-free studies have shown that cytochrome c release from mitochondria is a critical step in the apoptotic process. The present study examined the expression of cytochrome c protein after transient focal cerebral ischemia in rats, in which apoptosis was assumed to contribute to the expansion of the ischemic lesion. In situ labeling of DNA breaks in frozen sections after 90 minutes of middle cerebral artery (MCA) occlusion showed a significant number of striatal and cortical neurons, which were maximized at 24 hours after ischemia, exhibiting chromatin condensation, nuclear segmentation, and apoptotic bodies. Cytosolic localization of cytochrome c was detected immunohistochemically in the ischemic area as early as 4 hours after 90 minutes of MCA occlusion. Western blot analysis of the cytosolic fraction revealed a strong single 15-kDa band, characteristic of cytochrome c, only in the samples from the ischemic hemisphere. Western blot analysis of the mitochondrial fraction showed a significant amount of mitochondrial cytochrome c in nonischemic brain, which was decreased in ischemic brain 24 hours after ischemia. These results provide the first evidence that cytochrome c is being released from mitochondria to the cytosol after transient focal ischemia. Although further evaluation is necessary to elucidate its correlation with DNA fragmentation, our results suggest the possibility that cytochrome c release may play a role in DNA-damaged neuronal cell death after transient focal cerebral ischemia in rats.