No Morphological Evidence of Apoptosis Following Mild to Severe Episodes of Four-Vessel-Occlusion Ischemia in Rats

Summary

Severe, but brief forebrain ischemia in rat leads to a loss of hippocampal CA1 neurons 24–72 h post-ischemia. Whereas the ultrastructural features of this cell loss have been repeatedly described as necrotic, recent biochemical studies implicate apoptosis. In this study, we examined the consequences of 5-min and 15-min periods of four-vessel-occlusion (4-VO) ischemia using light and electron microscopy. Because slower maturation follows the 5-min insult, it is possible that such injury might have morphological features of apoptosis. We also examined whether caspase inhibition by z-VAD and z-DEVD would reduce injury following 10 min of ischemia. In the first experiment, CA1 cells were examined under light and electron microscopy following 5 (14-day survival) or 15 min (7-day survival) of 4-VO ischemia in rats. In the second experiment, CA1 injury (7-day survival) was quantified in rats occluded for 10 min and given an immediate 320 ng i.c.v. injection of z-VAD, z-DEVD or 3% dimethylsulfoxide (DMSO) as a control. Five minutes and 15 min of ischemia resulted in 78% and 81% CA1 damage at 14 days and 7 days, respectively. Ultrastructural examination revealed extensive necrosis, with pre-lethal signs of injury in many of the surviving neurons, including dilated organelles, e.g., mitochondria, rough endoplasmic reticulum, and numerous vacuoles in the cytoplasm and nucleus. Extensive gliosis was also noted. No qualitative differences were noted between the groups and no evidence of apoptosis was observed. In the second experiment, z-VAD (76%±14% dead) and z-DEVD (72%±17% dead) failed to reduce injury compared with control (84%±11% SD). Thus, no morphological or pharmacological evidence of apoptosis was evident following transient forebrain ischemia that produced either typical or more delayed CA1 injury.