Objective
Our objective was to determine if either caspase-9 or caspase-8, upstream cysteine proteases in the intrinsic mitochondrial and Fas death receptor extrinsic caspase-dependent pathways respectively, is activated following lithium-pilocarpine-induced status epilepticus (LPCSE).
Background
Seizure-induced neuronal death is morphologically necrotic, but involves programmed processes such as internucleosomal DNA cleavage (DNA laddering). It is controversial whether caspase-dependent pathways are activated following SE. We have shown that caspase-3, the central downstream executioner caspase in both pathways, is not activated by LPCSE, but the lack of downstream activation of caspase-3 does not rule out initial activation of either pathway.
Methods
Adult male Wistar rats were given lithium chloride, 3 mEq/kg i. p. The next day they received either pilocarpine, 30–60 mg/kg i.p., or saline i.p. After 3 h of LPCSE, diazepam (10 mg/kg) and phenobarbital (25 mg/kg) were given i.p. to control rats or to stop the seizures. Rats recovered for 6 or 24 h, after which they were killed with pentobarbital, and their brains were removed and dissected into the six brain regions we have shown exhibit necrotic neurons and DNA laddering: dorsal and ventral hippocampus, amygdala and piriform cortex, entorhinal cortex and neocortex. The thymuses of rats given saline or methamphetamine (MAP) were used as negative and positive controls for caspase-8 and -9 activation, by IETD-AFC and LEHD-AFC cleavage assays for enzyme activity respectively (n=3 in each group), and by assessing immunoreactivity for active caspase-9 and caspase-8. Enzyme activity assays and immunohistochemistry were also performed on the six brain regions (pooled from 4 brains) of control and SE rats with 6 h and 24 h recovery periods. The enzyme activity data was analyzed with 3-factor, repeated-measures ANOVA and post-hoc t-tests.
Results
The thymuses of rats given MAP 8 h previously showed an 8-fold elevation of LEHD-AFC (caspase-9) activity compared to saline-treated control thymuses (0. 0248 0.0183 vs. 0.194 0.0418 units/g protein, mean SEM, p<0.05), but no increase in IETD-AFC (caspase-8) activity (0.0979 0.00814 vs. 0.159 0.0264 units/g protein, p=0.09). There was no difference between the control and SE groups in either LEHD-AFC or IETD-AFC activity in the six brain regions at either 6 h or 24 h following SE (0.0138 0.00925 vs. 0.00502 0.00226 units/g protein for LEHD-AFC activity in amygdala-piriform cortex, e.g., at 24 h [p=0.99]). Thymus tissue showed active caspase-9 immunoreactivity but no caspase-8 immunoreactivity. None of the six brain regions in control and SE groups showed active caspase-9 or caspase-8 immunoreactivity 6 h or 24 h after SE.
Conclusions
Neither caspase-8 or caspase-9 is activated following LPCSE, further evidence that seizure-induced neuronal necrosis with DNA laddering is a caspase-independent programmed process.