Developmental Program of Epileptogenesis in the Brain of EL Mice

Abstract

Summary: Purpose:We recently observed inducible nitric oxide synthetase (iNOS) expression and decreased Cu, Znsuperoxide 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 CA1 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 35 S 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, however, 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-fos continuously, preferentially in the parietal cortex and hippocampal CA1. 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-NT-3-FGF-2-Abnormal plasticity.EL mouse is an inbred mutant strain that has been used as an animal model of secondarily generalized seizures (1,2). Several lines of evidence indicate that seizure discharges are initiated in the parietal cortex and then generalized through the hippocampus (3). These findings have been substantiated by the histochemical and biochemical analyses of glucose utilization (4) and inhibitory neurotransmissions by γ -aminobu...

Antiepileptic effects of levetiracetam related to the regulation of cell cycle reentry in the parietal cortex of EL mouse brain

“…In parallel, anti-apoptotic Bcl-2 is also overexpressed from 8 to 19 weeks of age. Expression of anti-apoptotic Bcl-X L peaks at 5 and 24 weeks of age, corresponding to early period of ictogenesis and late period of epileptogenesis, respectively [4,5,6]. There is an altered equilibrium between pro-apoptotic Bax and anti-apoptotic Bcl-2, which are associated with increased DNA fragmentation without cell loss [6].…”

“…Neurotrophic factor-3 (NT-3) shows a peak expression from 8 to 12 weeks of age, which covers the early period of epileptogenesis. In addition, fibroblast growth factor-2 (FGF-2) shows a linear increase in expression with age and reaches a maximum level at 24 weeks, which covers the late period of epileptogenesis [4,5,6]. On the other hand, seizure activity also induces expressions of neurotrophic factor mRNA and protein [7,8] and causes cell loss [9].…”

“…In addition, various cell death-related proteins are overexpressed during early stages of development. Pro-apoptotic Bax is overexpressed from 8 to 19 weeks of age, which covers the period of epileptogenesis [4,5,6]. In parallel, anti-apoptotic Bcl-2 is also overexpressed from 8 to 19 weeks of age.…”

“…In this model, DNA fragmentation in the hippocampus CA1 and the parietal cortex [1,2] has been demonstrated using in situ terminal transferase-mediated dUTP Nick labeling [1,3]. However, in the hippocampus and the parietal cortex of EL mice, neuronal cell loss is not found despite occurrence of frequent seizures during development [4,5]. In addition, various cell death-related proteins are overexpressed during early stages of development.…”

“…Brain-derived neurotrophic factor (BDNF) shows biphasic overexpression during 8 to 12 weeks as well as around 20 weeks of age, which cover the period of epileptogenesis [4,5]. Neurotrophic factor-3 (NT-3) shows a peak expression from 8 to 12 weeks of age, which covers the early period of epileptogenesis.…”