Electron microscopic evidence against apoptosis as the mechanism of neuronal death in global ischemia

It has been repeatedly claimed that neuronal death in the hippocampal CA1 s ector after untreated global ischemia occurs via apoptosis. This is based l argely on DNA laddering, nick end labeling, and light microscopy. Delineati on of apoptosis requires fine structural examination to detect morphologica l events of cell death. We studied the light and ultrastructural characteri stics of CA1 injury after 5 min of untreated global ischemia in gerbils. To increase the likelihood of apoptosis, some ischemic gerbils were subjected to delayed postischemic hypothermia, a treatment that mitigates injury and delays the death of some neurons. In these gerbils, 2 d of mild hypothermi a was initiated 1, 6, or 12 hr after ischemia, and gerbils were killed 4, 1 4, or 60 d later. Ischemia without subsequent cooling killed 96% of CA1 neu rons by day 4, whereas all hypothermia-treated groups had significantly red uced injury at all survival times (2-67% loss). Electron microscopy of isch emic neurons with or without postischemic hypothermia revealed features of necrotic, not apoptotic, neuronal death even in cells that died 2 months af ter ischemia. Dilated organelles and intranuclear vacuoles preceded necrosi s, Unique to the hypothermia-treated ischemic groups, some salvaged neurons were persistently abnormal and showed accumulation of unusual, morphologic ally complex secondary lysosomes. These indicate selective mitochondrial in jury, because they were closely associated with normal and degenerate mitoc hondria, and transitional forms between mitochondria and lysosomes occurred . The results show that untreated global ischemic injury has necrotic, not apoptotic, morphology but do not rule out programmed biochemical events of the apoptotic pathway occurring before neuronal necrosis.