Mild as well as severe insults produce necrotic, not apoptotic, cells: Evidence from 60-min seizures

We tested the hypothesis that mild insults produce apoptotic, and severe insults necrotic, cells by subjecting adult Wistar rats to 60-min instead of 3-h generalized seizures. Rats' brains were evaluated 6 and 24 h later for evidence of neuronal necrosis by light and electron microscopy, the presence of TUNEL staining and active caspase-3 immunoreactivity, and for evidence of DNA laddering 24 h after seizures. Apoptotic neurons from the retrosplenial cortex of postnatal day 8 rat pups served as positive controls. Six and 24 h after seizures, 16 and 15 brain regions respectively out of 24 showed significant numbers of acidophilic neurons by hematoxylin and eosin stain. Three brain regions had significant numbers of TUNEL-positive neurons 24 h after seizures. No neurons showed active caspase-3 immunoreactivity. Acidophilic neurons were necrotic by electron-microscopic examination. Ultrastructurally, they were shrunken and electron-dense, with shrunken, pyknotic nuclei and swollen mitochondria with disrupted cristae. Nuclei did not contain the irregular chromatin clumps found after 3-h seizures. None of the six brain regions studied ultrastructurally that show DNA laddering 24 h after 3-h seizures showed DNA laddering 24 h after 60-min seizures, probably because there were too few damaged neurons, although the lack of chromatin clumping might have been a contributing factor. Following seizures, a mild as well as a severe insult produces caspase-3-negative necrotic neurons. These results do not support the hypothesis that mild insults produce apoptotic, and severe insults, necrotic, cells.