Abstracts

RATIONALE: The objective of this study was to determine if activated caspase-3, the central executioner caspse in both the intrinsic mitochondrial pathway and the extrinsic Fas ligand receptor-activated extrinsic pathway, is present in TUNEL-positive morphologically apoptotic neurons in adult rats, and if prolonged seizures increase the number of these activated caspase-3-positive apoptotic neurons, compared to control rats.
METHODS: Adult male Wistar rats had skull screw implantations for EEG recording, and 3 d later they were given lithium chloride, 3 mEq/kg i.p. The next day they were given 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 which they underwent transcardiac brain perfusion-fixation, their brains were removed and left hemispheric slices were embedded in paraffin and cut into 6-[mu]m-thick coronal sections, which were stained with TUNEL and a polyclonal antibody to the active p17 fragment of caspase-3 (CM1 antibody). Sections were counterstained with methyl green (TUNEL) and hematoxylin (CM1 antibody). Sections at the level of caudate-putamen (-0.3 mm from bregma), dorsal hippocampus (-3.3 mm from bregma) and ventral hippocampus (-5.60 mm from bregma) were examined. The numbers of TUNEL-positive and CM1 antibody-positive apoptotic neurons in the 3 brain sections from each rat were summed, and the data were analyzed with 3-factor repeated-meaures ANOVA and post-hoc t-tests.
RESULTS: The total numbers of TUNEL-positive apoptotic neurons in the 3 brain sections examined in each rat were significantly increased 6 h following SE (46 [plusminus] 13 in controls and 78 [plusminus] 15 following SE, mean [plusminus] SEM, p[lt]0.05, n=3 and 4 respectively), but not 24 h following SE (44 [plusminus] 10 in controls and 35 [plusminus] 10 after SE, p=0.39, n=5 and 6 respectively), and the number 6 h after SE was also higher than 24 h after SE (p[lt]0.01). These results must be confirmed with larger numbers of control and SE rats 6 h after SE. At both 6 h and 24 h, the total number of CM1 antibody-positive apoptotic neurons were substantially fewer in number than the TUNEL-positive neurons, and the numbers of these neurons did not differ between control and SE rats at 6 h (1 [plusminus] 1 in controls and 16 [plusminus] 2 in SE rats, p=0.27, n=3 and 4 respectively), and at 24 h (0.4 [plusminus] 0.2 in controls and 1.5 [plusminus] 0.7 in SE rats, p=0.92, n=5 and 6 respectively).
CONCLUSIONS: Active caspase-3 immunoreactivity is found in very few of the naturally occurring apoptotic neurons in adult rat brain, and SE does not increase the number of neurons showing caspase-3 activation. This indicates that either caspase-3 is activated in only a small fraction of apoptotic neurons, or, more likely, that caspase-3 activation occurs transiently in these neurons. Our results provide further evidence that neuronal apoptosis in the adult rat brain may not be influenced by the superimposed stress of SE, which produces widespread neuronal necrosis without caspase-3 activation, but with the DNA laddering which occurs in programmed cell death.
[Supported by: The Department of Veterans Affairs.]