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 24h 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 24h after seizures. Apoptotic neurons from the retrosplenial cortex of postnatal day 8 rat pups served as positive controls. Six and 24h 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 24h 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 24h after 3-h seizures showed DNA laddering 24h 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.

Caspase-dependent programmed cell death pathways are not activated in generalized seizure-induced neuronal death.

Activation of the caspase-dependent cell death pathways has been shown in focal seizures, but whether this occurs in prolonged generalized seizures is not known. We investigated whether the initiator caspase in the extrinsic pathway, caspase-8, or the intrinsic pathway, caspase-9, is activated during the first 24 h following lithium-pilocarpine-induced status epilepticus, when neuronal death is maximal and widespread. The thymuses of rats given methamphetamine were used as positive controls for caspase-3-activated cellular apoptosis. Following methamphetamine treatment, caspase-9 but not caspase-8 was activated in thymocytes. However, 6 or 24 h following status epilepticus, none of 26 brain regions studied showed either caspase-8 or -9 activation by immunohistochemistry, western blotting and enzyme activity assays. Our results provide evidence against the activation of the extrinsic and intrinsic caspase pathways in generalized seizures, which produce morphologically necrotic neurons with internucleosomal DNA cleavage (DNA laddering), a programmed process. In contrast, there is increasing evidence that caspase-independent programmed mechanisms play a prominent role in seizure-induced neuronal death.

Caspase-3 is not activated in seizure-induced neuronal necrosis with internucleosomal DNA cleavage.

A caspase-3-activated DNase produces internucleosomal DNA cleavage (DNA laddering). We determined whether caspase-3 is activated by lithium-pilocarpine-induced status epilepticus in six brain regions with necrosis-induced DNA laddering. The thymuses of adult rats given methamphetamine or normal saline were used as controls for apoptosis. Some 6-8 h after methamphetamine treatment, thymocytes showed apoptosis by electron-microscopic examination, positive terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), DNA laddering, cleavage of caspase-3 into its active p17 subunit, active caspase-3 immunoreactivity, and a 25-fold increase in caspase-3-like activity. Six hours after SE, necrotic neurons by electron-microscopic examination in hippocampus, amygdala and piriform, entorhinal and frontal cortices showed no TUNEL and no DNA laddering. Twenty-four hours after seizures, most necrotic neurons were negative for TUNEL, some were positive, but all regions showed DNA laddering. However, 6 and 24 h after seizures, active caspase-3 immunoreactivity was negative, caspase-3-like activity did not increase, and western blot analysis failed to show the p17 subunit. In addition, 24 h after seizures,microdialytic perfusion of carbobenzoxy-valyl-alanyl-aspartyl (O-methylester) fluoromethylketone was not neuroprotective. Thus, caspase-3 is not activated in brain regions with seizure-induced neuronal necrosis with DNA laddering. Either caspase-activated DNase is activated by another enzyme, or a caspase-independent DNase is responsible for the DNA cleavage.