Early and sequential recruitment of apoptotic effectors after focal permanent ischemia in mice

In experimental models of cerebral ischemia, cells within the damaged terri tory die by necrosis and by apoptosis that contributes to the expansion of the insult. Apoptotic machinery mobilizes intracellular processes such as i nduction of Bcl-2 family members, activation of the proteolytic cascade inc luding the caspases, and cleavage of caspase substrates, such as poly(ADP-r ibose) polymerase or PARP. Mitochondria play a pivotal role in controlling apoptosis by releasing cytochrome c and modulating redox state, both under the regulation of manganese superoxide dismutase (Mn SOD) via superoxide an ion detoxification. The implication and the kinetics of such events in apop tosis induced after focal permanent ischemia in mice remains to be studied. In a paradigm of ischemic insult induced by occlusion of the middle cerebr al artery (MCAO) in mice, we showed by immunohistochemistry a constitutive expression of caspase-3 that is enhanced after MCAO in neurons localized wi thin the infarcted zone. As a function of time intervals after MCAO, the cy tochrome c amount increased in the cytosolic fraction of ischemic cortical extracts. The kinetics of the release was in concordance with the expressio n of caspase-3 and the subsequent cleavage of PARP appearing before the int ernucleosomal fragmentation of DNA, the ultimate step of apoptosis. When th e apoptotic markers progressively appeared, no changes of Mn SOD activity o r Mn SOD expression were detected after MCAO. We can therefore speculate th at the recruitment of Mn SOD did not participate per se in the release of c ytochrome c elicited after permanent focal ischemia. (C) 2000 Published by Elsevier Science B.V. All rights reserved.