Temporal profile of cytochrome c and caspase-3 immunoreactivities and TUNEL staining after permanent middle cerebral artery occlusion in rats

Although apoptotic pathways play important roles in ischemic neuronal injur y, exact mechanism of apoptotic enzyme cascade has not been fully studied. Immunohistochemical stainings for cytochrome c and caspase-3, and histochem ical staining for a terminal deoxynucleotidyl-transferase (TdT)-mediated dU TP-biotin nick end-labeling method (TUNEL) were examined in a rat model of permanent middle cerebral artery (MCA) occlusion. Cytochrome c was strongly induced in neurons of the ischemic penumbra from 3 h after MCA occlusion, and caspase-3 began to be induced in the same area from 3 h with a peak at 8 h. Neuronal cells in MCA area became TUNEL positive at delayed time, reac hing a peak at 24 h. Thus, the peak of induction of cytochrome c preceded t hat of caspase-3, and these two peaks were also precedence of the peak of D NA-fragmentation. Western blot analysis showed cytosolic expression of cyto chrome c from mitochondria. This study demonstrated 1. Rapid release of cyt ochrome c from mitochondria to the cytosol, mainly in neurons of the cortex at 3 h after ischemia. 2. Subsequent peaks of caspase-3 and TUNEL in this order. These temporal profiles suggest a serial cascadic activation of apop totic pathways in neuronal death after permanent MCA occlusion of rats.