These studies have addressed the role of
caspase-3 activation in neuronal death after
cerebral ischemia in different animal models. The authors were unable to show activation of
procaspase-3 measured as an induction of
DEVDase (
Asp-Glu-Val-Asp) activity after focal or transient forebrain
ischemia in rats.
DEVDase activity could not be induced in the cytosolic fraction of the brain tissue obtained from these animals by exogenous
cytochrome c/dATP and Ca2+. However, the addition of
granzyme B to these cytosolic fractions resulted in a significant activation of
DEVDase, confirming that the conditions were permissive to analyze proteolytic cleavage of the
DEVD-AMC (7-amino-4-methyl-coumarin) substrate. Consistent with these findings, zVal-
Ala-Asp-fluoromethylketone administered after focal
ischemia did not have a
neuroprotective effect. In contrast to these findings, a large increase in
DEVDase activity was detected in a model of hypoxic-
ischemia in postnatal-day-7 rats. Furthermore, in postnatal-day-7 animals treated with
MK-801, in which it has been suggested that excessive apoptosis is induced, the authors were unable to detect activation of
DEVDase activity but were able to induce it in vitro by the addition of
cytochrome c/dATP and Ca2+ to the cytosolic fraction. Analysis of
cytochrome c distribution did not provide definitive evidence for selective
cytochrome c release in the permanent focal
ischemia model, whereas in the transient model a small but consistent amount of
cytochrome c was found in the cytosolic fraction. However, in both models the majority of
cytochrome c remained associated with the mitochondrial fraction. In conclusion, the authors were unable to substantiate a role of mitochondrially derived
cytochrome c and
procaspase-3 activation in
ischemia-induced cell death in adult brain, but did see a clear induction of
caspase-3 in neonatal
hypoxia.