Inhibition of caspase-9 activation and apoptosis is involved in ischemic preconditioning-induced neuroprotection in rat brain.

Objectives: Cerebral ischemic pre-conditioning (IPC) is capable of protecting hippocampal neurons from ischemia/reperfusion (I/R) injury. In the current study, we investigated the role of activated caspase-9 in the protective process induced by IPC and related it to cytochrome c release and apoptosis. Methods: I/R injury was induced by a four-vessel occlusion model in Wistar rats which were randomly divided into ischemia/reperfusion group (I/R), ischemic pre-conditioning + I/R group (IPC + I/R) and control group. Histologic changes in the pyramidal layer of the hippocampal CA1 region were determined by hematoxylin and eosin (H&E) staining. The relative proportion of apoptotic neurons in this area was assessed with TUNEL staining. The redistribution of cytochrome c and activation of caspase-9 were detected in the same area with immunohistochemistry and Western blotting respectively. Results: Compared to the I/R group, IPC increased the number of surviving neurons in the hippocampal CA1 region ( p < 0.001), markedly reduced the number of apoptotic pyramidal neurons (p < 0.001), inhibited the release of cytochrome c from mitochondria to cytoplasm (p < 0.001 for positively stained neurons) and decreased the amount of activated caspase-9 (p < 0.001). Discussion: These findings confirm that IPC is capable of protecting neurons from injury by apoptosis. The release of cytochrome c to the cytosol demonstrates that the mitochondrial pathway was involved, and the reduction in this release caused by IPC was clearly associated with reduced caspase-9 activation. Together, these results suggest that IPC protects neurons via action on the mitochondrial/caspase-9 pathway of apoptosis.