Abstracts

It is widely believed that mild insults produce apoptotic neurons, whereas severe insults produce necrotic neurons. We tested this hypothesis by subjecting adult rats to 60-min instead of 3-h lithium-pilocarpine-induced status epilepticus (LPCSE), and evaluated their brains 24 h afterward. Adult male Wistar rats (220-350 g) had skull screws implanted for EEG recording, and 3-5 d later lithium chloride, 3 mEq/kg was given i.p. The next day normal saline or pilocarpine, 30 mg/kg, was given i.p., and after 60 min of continuous clonic seizures, diazepam, 20 mg/kg, and phenobarbital, 25 mg/kg, were given i.p. to stop the seizures. Twenty-four h later rats were given an overdose of pentobarbital, and either had in situ transcardiac perfusion-fixation of their brains, with subsequent removal and processing for H [amp] E, TUNEL and active caspase-3 immunoreactivity (IR), or decapitation and rapid dissection of six brain regions (pooled from 4 rats) for DNA agarose gel electrophoresis. Thymuses of rats given normal saline or methamphetamine (MAP; 25 mg/kg i.p.) and killed 8 h later were used as controls for cellular apoptosis, active caspase-3 IR and DNA laddering. Twenty-four brain regions were assessed for the degree of neuronal death by H [amp] E and TUNEL stain and caspase-3 activation by immunohistochemistry, using a 0-3 grading scale. Data were analyzed with a 2-factor ANOVA and post-hoc t-tests based upon a Poisson distribution of the data. MAP-treated thymocytes showed morphological apoptosis, active caspase-3 IR and DNA laddering. Twelve of the 24 brain regions of SE rats (n=3) showed significant numbers of acidophilic neurons by H [amp] E stain, light-microscopic evidence of necrotic neurons. The mean damage score was 0.9 [plusmn] 0.1, or 10-25% damaged neurons, p=0.001. Seven brain regions had TUNEL-positive necrotic neurons, but only one region showed a significant difference from controls. None of the necrotic neurons had active caspase-3 IR, and none of the six brain regions studied showed DNA laddering. Scattered morphologically apoptotic neurons were found in control and SE groups, and will be reported separately. Twenty-four h after 60-min LPCSE, necrotic neurons are produced, as occurs after 3-h LPCSE. Although DNA fragmentation by TUNEL was found in 7 brain regions, there was no DNA laddering, as occurs after 3-h SE, probably because there were too few damaged neurons in the brain regions examined, and no caspase-3 activation in necrotic neurons. This suggests that in SE, a milder as well as a more severe insult produces caspase-3-independent necrotic neurons. (Supported by Department of Veterans Affairs.)