Abstracts

RATIONALE: To determine if caspase-3 is activated by lithium-pilocarpine-induced status epilepticus (SE) in brain regions with necrosis-induced internucleosomal DNA cleavage (DNA laddering). A caspase-3-activated DNase (CAD) has been shown to produce DNA laddering. METHODS: Lithium chloride (3 mEq/kg) was injected i.p. in adult Wistar rats 12 h before pilocarpine (30-60 mg/kg) i.p., to induce SE. SE was stopped after 3 h with diazepam and phenobarbital i.p. Rats were killed either 6 or 24 h after SE with an overdose of pentobarbital. Their brains were removed, and dorsal and ventral hippocampus, entorhinal cortex, amygdala/piriform cortex and neocortex were rapidly dissected out and processed to measure the amount of DEVD-AFC cleavage activity, which reflects endogenous caspase-3-like activity. Thymuses of adult rats given methylamphetamine (MAT), which produces apoptotic thymocytes and DNA laddering, or saline i.p. were used as controls for DEVD-AFC cleavage. The results for DNA gel electrophoresis and light- (LM) and electron-microsopic (EM) histology 24 h after SE have been reported previously. RESULTS: 24 h after SE, the TUNEL-negative acidophilic neurons found by LM in hippocampus, amygdala and piriform, entorhinal and frontal cortices are necrotic by EM, and DNA laddering occurs in all five regions. 6 h after MAT treatment, thymocytes showed DNA laddering and a 23-fold increase in DEVD-AFC cleavage compared to controls (1.13 0.05 vs. 0.046 0.008 units/mg protein, mean SEM, p=0.01, n=3). However, in the five brain regions with necrotic neurons and DNA laddering 24 h after SE, DEVD-AFC cleavage did not increase at either 6 or 24 h after SE in any of the five brain regions. For example, 6 h after SE in dorsal hippocampus, DEVD-AFC cleavage was 0.030 0.012 vs. 0.050 0.024 units/mg protein in controls vs. SE respectively (p=0.34, n=3). 24 h after SE, DEVD-AFC cleavage was 0.039 0.008 vs. 0.039 0.006 units/mg protein in controls vs. SE (p=1.0, n=3). CONCLUSIONS: Caspase-3 is not activated in brain regions with SE-induced neuronal necrosis with DNA laddering. A caspase-independent DNase must be responsible for SE-induced, 180-base pair DNA cleavage.