Cytochrome c Is Released from the Mitochondria of Vulnerable Hippocampal CA1 Neurons in Rats After Transient Global Cerebral Ischemia

Abstract

Release of cytochrome c from mitochondria to the cytosol is a critical step in apoptotic cell death after focal cerebral ischemia. The role of cytochrome c release on selective vulnerability and delayed death of hippocampal CA1 neurons after transient global ischemia is unknown and is the subject of this study. Global ischemia was induced by 10 min of bilateral common carotid artery occlusion and hypotension. Cytosolic expression of cytochrome c was evaluated by immunohisto- chemistry and Western blotting. Apoptosis after global ischemia was characterized by terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining and genomic DNA gel electrophoresis. Immunohistochemistry showed cytosolic cytochrome c-positive cells exclusively in the vulnerable CA1 subregion of the hippocampus as early as 2 h after ischemia. Double fluorescent immunostaining confirmed that CA1 neurons and a small number of astrocytes expressed cytochrome c. Western blot analysis revealed a band (15kDa) of cytochrome c in the cytosolic fraction, and a corresponding decrease in the mitochondrial fraction. A significant number of TUNEL-positive cells appeared only in the CA1 pyramidal cell layer of the hippocampus, and DNA gel electrophoresis showed a significant amount of DNA fragmentation 3–5 days after ischemia. Our data provide the first evidence that cytochrome c was released to the cytosol from mitochondria in CA1 neurons after global ischemia, and that the release preceded DNA fragmentation. These findings suggest that cytochrome c may be involved in the delayed death of hippocampal CA1 neurons in rats after transient global ischemia.