Neuoscience: Molecular Pathology and Epilepsy

Abstract

Abstract Yoshiya L. Murashima*, Jiro Suzuki*, and Mitsunobu Yoshii* *Department of Neural Plasticity, Tokyo Institute of Psychiatry, Tokyo, Japan . Purpose: We recently observed inducible nitric oxide synthase (iNOS) expression and decreased Cu, Zn-superoxide dismutase (Cu,Zn-SOD) activities in the hippocampus of epileptic mutant EL mice at age 30 weeks. In addition, DNA fragmentation was detected preferentially in region CA1 of the hippocampus and in the parietal cortex of the EL mouse brain. The results suggest that abnormalities in the region-specific iNOS isoenzyme activities and decreased Cu, Zn-SOD activities might produce spatially specific free radicals, leading to DNA fragmentation. However, neuronal cell loss is not found in the hippocampus. Furthermore, the immediate early gene (IEG), c-fos, also is expressed in the interictal period at age 30 weeks. These findings suggest that some protein cascades occur during epileptogenesis. In the present study, we used epileptic mutant EL mice to examine whether the altered equilibrium of the proapoptotic Bax and anti-apoptotic Bcl-2/Bcl-XL induces DNA fragmentation without cell loss. We also examined whether neurotrophic factors modulate the developmental process of epileptogenesis in EL mouse brain. Methods: Developmental changes of NOS isoenzymes were determined by using immunoblotting. Formalin-fixed mouse brains were stained by hematoxylin–eosin to examine cell loss in the tissue. IEG expression in the interictal period was analyzed with in situ hybridization by using the 35S x-ray emulsion method. Developmental changes of Bcl-2, Bcl-XL, and Bax were examined by immunoblotting in the hippocampus of EL mice compared with the control DDY mice. Alterations of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and fibroblast growth factor-2 (FGF-2) during development were investigated by immunoblotting. Results: Unexpectedly, the eNOS content in EL mouse brain was very low although eNOS appears to be responsible for NO that mediates an increase in local cerebral blood flow during focal seizures. Neuronal NOS, iNOS, and relatively lower concentration eNOS are essential to establish either ictogenesis or epileptogenesis. No cell loss was found in the hippocampus. Even in the interictal period, EL mice expressed the IEG, c-fos, continuously, preferentially in the parietal cortex and hippocampal CA1 when the seizure thresholds were very low. In the control DDY mice, very low steady-state levels of Bcl-2 and Bax were observed throughout development. In EL mice, the Bcl-2 and Bax levels increased predominantly after frequent seizures. The BDNF level in EL mice showed a significant increase during development, in the period of ictogenesis and epileptogenesis. Conclusions: The results indicate that in EL mice, the susceptibility of hippocampal neurons to DNA fragmentation increases after experiencing repetitive seizures during development. This effect probably is due to a change in the balance between the protective mechanism and inactivation of the proapoptotic pathway. BDNF may play a role in the ictogenesis and epileptogenesis by promoting abnormal synaptic plasticity.