Developmental program of epileptogenesis in the brain of EL mice

Purpose: We recently observed inducible nitric oxide synthetase (iNOS) expression and decreased Cu, Zn-superoxide dismutase (Cu, Zn-SOD) activities in the hippocampus of epileptic mutant EL mice at the age of 30 weeks. In addition, the immediate early gene (IEG) c-fos is unusually expressed in the interictal period, suggesting activation of protein cascades associated with the epileptogenesis. Furthermore, DNA fragmentation has been detected preferentially in the hippocampus CAI and the parietal cortex of EL mouse brain. It remains to be seen, however, how these abnormalities are related to the DNA fragmentation, and whether neuronal cell loss is involved. The present study was designed to address these issues. Methods: NOS isoenzymes, pro- (Bax) and antiapoptotic factors (Bcl-2, Bcl-XL), and neurotrophic factors (brain-derived neurotrophic factor, BDNF; neurotrophin-3, NT-3; fibroblast growth factor-2, FGF-2) were determined by immunoblotting in the EL mouse brain at various developmental stages. Hematoxylin-eosin staining was applied to the formalin-fixed brains to examine the cell loss in the tissue. IEG expression in the interictal period was analyzed by in situ hybridization by using the S-35 x-ray emulsion method. Results: nNOS was the major component of NOS in the hippocampus of either EL or control DDY mice. In EL mice, how-ever, iNOS was detectable at the age of 10 weeks, at which the animals usually experience the first seizures. eNOS, which appears in DDY brain, could scarcely be identified. Even in the interictal period, EL mice expressed c-fios continuously, preferentially in the parietal cortex and hippocampal CAI. In DDY mice, very low steady-state levels of Bcl-2 and Bax remained constant throughout development. In EL mice, these Bcl-2 and Bax levels were increased even before experiencing frequent seizures. BDNF in EL mice markedly increased temporarily during ictogenesis and epileptogenesis in their early periods. Unexpectedly, no cell loss was found in the hippocampus. Conclusions: DNA fragmentation without cell loss found in EL mouse brains appears to result from initial activation and later inactivation of the apoptotic process. Neurotrophic factors may play a role in the ictogenesis and the epileptogenesis during the early development. These gene expressions closely related to the periods critical for ictogenesis and epileptogenesis may be of particular importance in the development of antiepileptic drugs (AEDs) with novel mechanisms. Key Words: Epileptic mutant-EL mouse-Ictogenesis-Epileptogenesis-c-fos-DNA fragmentation-Bcl-2-Bcl-XL-Bax-BDNF-NT3-FGF-2-Abnormal plasticity.