Abstracts

The objective of this study 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 3-h lithium-pilocarpine-induced status epilepticus (LPCSE). 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.
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 an equivalent volume of saline i.p. After 3 h of status epilepticus (SE), diazepam (10 mg/kg) and phenobarbital (25 mg/kg) were given i.p. to stop the seizures (or after an equivalent period of time in controls). Rats were allowed to recover for 6 or 24 h, after they were euthanized with pentobarbital, their brains were removed rapidly on ice 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. Enzyme activity assays were also performed on the six brain regions 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.
The thymuses of rats given MAP 8 h previously showed 5-fold and 7-fold elevations of IETD-AFC (caspase-8) and LEHD-AFC (caspase-9) activity compared to saline-treated control thymuses (0.719[plusmn]0.121 vs 3.93[plusmn]0.548 units/[mu]g protein for IETD-AFC cleavage and 0.836[plusmn]0.066 vs 4.07[plusmn]0.444 units/[mu]g protein for LEHD-AFC cleavage, mean[plusmn]S.E.M.). On other hand, there was no difference between the control and SE groups in either IETD-AFC or LEHD-AFC activity in the six brain regions at either 6 h or 24 h following SE (0.493[plusmn]0.152 vs. 0.529[plusmn]0.197 units/[mu]g protein for IETD-AFC activity, and 0.362[plusmn]0.071 vs. 0.412[plusmn]0.074 units/[mu]g protein for LEHD-AFC activity in amygdala-piriform cortex, e.g., at 24 h).
Neither caspase-8 or caspase-9 is activated following LPCSE, further evidence that the seizure-induced programmed internucleosomal cleavage of DNA (DNA laddering) is a caspase-independent process.
[Supported by: The Department of Veterans Affairs.]