Abstracts

RATIONALE: We have shown that spontaneous neuronal apoptosis occurs in the adult rat brain. As in postnatal day 7 (P7) rats, apoptotic neurons are diffusely TUNEL-positive, except for the large chromatin clumps, and are easily recognizable by brightfield light microscopy. This appearance is caused by disruption of the nuclear membrane, confirmed by electron microscopy, and is contrary to the widespread belief that only nuclei are TUNEL-positive. We tested the hypothesis that the stress of status epilepticus, which produces necrotic neurons, increases the number of apoptotic neurons.
METHODS: Adult male Wistar rats had skull screws implanted 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 H & E, TUNEL, NeuN, and TUNEL/NeuN. Sections at the level of dorsal hippocampus (-3.3 mm from bregma) and ventral hippocampus (-5.60 mm from bregma) were examined. The numbers of TUNEL-positive neurons in the 2 brain sections from each rat were counted and summed, and the data were analyzed with t-tests.
RESULTS: Apoptotic neurons with the same appearance as in P7 rats were found in neocortex, amygdala, thalamus, piriform, perirhinal and entorhinal cortices, and hippocampal hilus and pyramidal cell layer of control and seizure rats. The total numbers of apoptotic neurons in the 2 brain sections examined in each rat were relatively few and were not increased significantly at either 6 h or 24 h following SE. At 6 h, the total number of apoptotic neurons was 29 [plusminus] 8 in controls and 54 [plusminus] 12 (mean [plusminus] SEM) in SE rats (p=0.17, n=3 for the control group and n=4 for the SE group). At 24 h, the total number of apoptotic neurons was 42 [plusminus] 9 in controls and 31 [plusminus] 11 in SE rats (p=0.48, n=3 for the control group and n=4 for the SE group).
CONCLUSIONS: Spontaneous neuronal apoptosis continues into adulthood, and has the same appearance as in P7 rats. Despite the stress of SE, which produces widespread neuronal necrosis in vulnerable brain regions, the numbers of spontaneously appearing apoptotic neurons in control rats are not increased in seizure animals. This suggests that neuronal apoptosis in the adult rat brain may not be influenced by, and may proceed independently of, a superimposed acute brain insult which induces neuronal necrosis.
Support: Dept. of Veterans Affairs.