Superoxide during reperfusion contributes to caspase-8 expression and apoptosis after transient focal stroke.
OBJECTIVE
Reactive oxygen species produced during reperfusion may play a detrimental role in focal cerebral ischemia (FCI). We examined the protein expression of caspase-8, which plays a major role in both Fas-dependent and cytochrome c-dependent apoptotic pathways after FCI with or without reperfusion. Caspase-8 expression after transient FCI was compared between wild-type and transgenic mice that overexpress the cytosolic antioxidant copper/zinc superoxide dismutase (SOD1).


METHODS
Adult male CD-1 mice were subjected to 1 hour of FCI and reperfusion or to permanent FCI by intraluminal blockade of the middle cerebral artery. DNA fragmentation was evaluated by genomic DNA gel electrophoresis. Caspase-8 expression was analyzed by Western blot.


RESULTS
Caspase-8 was significantly induced 4 hours after transient FCI and remained at an increased level until 24 hours, whereas it was not modified after permanent FCI. Genomic DNA gel electrophoresis showed DNA laddering in a pattern similar to that seen in apoptosis, with a small amount of background smear 24 hours after transient FCI, whereas 25 hours of permanent FCI resulted in less DNA laddering with a strong background smear. Caspase-8 induction was significantly reduced in SOD1 transgenic mice compared with wild-type mice 4 hours after transient FCI.


CONCLUSIONS
The results suggest that increased reactive oxygen species production during reperfusion may contribute to the induction of caspase-8, thereby exacerbating apoptosis after FCI.
Morita-Fujimura
Y
Y
Department of Neurosurgery, Program in Neurosciences, Stanford University School of Medicine, Stanford, CA, USA.
Fujimura
M
M
Yoshimoto
T
T
Chan
P H
PH
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D001921:Brain / Q000098:blood supply / Q000378:metabolism; D002545:Brain Ischemia / Q000150:complications / Q000378:metabolism*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000096:biosynthesis*; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation; D018450:Disease Progression; D004790:Enzyme Induction / Q000187:drug effects; D002546:Ischemic Attack, Transient / Q000150:complications / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008928:Mitochondria / Q000201:enzymology; D015424:Reperfusion; D020521:Stroke / Q000150:complications / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors
D003574:Cytochrome c Group; D013481:Superoxides; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases