Mutant EL Mouse Research Articles

Anticancer agent 6-MITC at extremely low dosage can control seizures by stabilizing membrane excitability system in mutant EL mice

Anticancer agent 6-MITC at extremely low dosage can control seizures by stabilizing membrane excitability system in mutant EL mice

New therapeutic approaches for epilepsies, focusing on reorganization of the GABAA receptor subunits by neurosteroids

Neurosteroids such as allopregnanolone (THP) act as positive allosteric modulators of gamma-aminobutyric acid (GABA)A receptors and have exerted anticonvulsant properties. However, their role in the regulation of epileptogenesis is unclear. It has been shown that circulating levels of THP fluctuate during development and seizure episodes. Furthermore, both chronic administration of THP and its withdrawal transiently increase expression of the alpha4 subunit of the GABAA receptor in the brain. The steroidogenic enzymes, 5-alpha-reductase (5aR) and 3-alpha-hydroxysteroid dehydrogenase (3aHSD) have been identified as well, indicating that various cell types are involved in the biosynthesis of neuroactive steroids in the brain. The purpose of the present study is to examine how GABAA receptor-modulating neurosteroids contribute to the epileptogenesis by using the epileptic mutant EL mouse. Male EL mice and control animals, DDY mice, were used. EL mice show secondary generalized seizures, which initiate primarily at the parietal cortex and generalize through the hippocampus. In the interictal period during development, changes of THP, 5aR, 3aSDH, and GABAA receptor alpha4, gamma2, and delta subunits were investigated by western blotting in the hippocampus. In EL mice, levels of the neurosteroid THP and the steroidogenic enzymes 5aR and 3aSDH significantly increased at 3 weeks of age, and rapidly decreased thereafter (5-10 weeks). The sharp withdrawal was observed before mice experienced frequent seizures. In contrast, GABAA alpha4, gamma2, and delta expressions were upregulated (3-8 weeks). In the brain of EL mice, positive neurosteroids such as THP were withdrawn before mice experienced repetitive seizures, which may likely be a trigger for ictogenesis and epileptogenesis. Furthermore, reorganization of the GABAA receptor subunits may lead to a hypersensitivity of the receptor to neurosteroids. Therefore, GABAA receptor-regulating neurosteroids may be a promising target for the development of novel antiepileptic agents.

Role of Cytokines during Epileptogenesis and in the Transition from the Interictal to the Ictal State in the Epileptic Mutant EL Mouse

Purpose Epileptic mutant EL mice show secondary generalized seizures. Seizure discharges initiate in the parietal cortex and generalize through the hippocampus. We have previously demonstrated an increase in the activity of inducible nitric oxide synthetase (iNOS) as well as a decrease in the activity of superoxide dismutase (SOD) in the hippocampus of EL mice, suggesting that cell toxic free radicals are increased in the brain of EL mice. In parallel with this, neurotrophic factors were significantly increased in the hippocampus of EL mice in earlier developmental stages before exhibiting frequent seizures. These findings were no longer present after frequent seizures, suggesting that these events may trigger ictogenesis. On the other hand, it is reported that limbic seizures rapidly induce cytokines and related inflammatory mediators. It remains to be seen, however, whether cytokines contribute to the transition from interictal to ictal state. The present study was designed to address this issue using EL mice. Methods EL mice at the age from 4 to 23 weeks and their control animal, DDY mice at the age of 10 and 20 weeks were used. Seizures were induced in EL mice once every week since 5 weeks. Cytokines, such as interleukin-1 alpha (IL-1a), interleukin 1-beta (IL-1b), IL-6, IL-1 receptor (IL-1r), IL-1 receptor antagonist (IL-ra) and tumor necrosis factor alpha (TNF-a) were examined by Western blotting in the ‘focus complex’ of brain (namely, in the parietal cortex and hippocampus) of EL mice in the interictal period at different developmental stages. In 15 week old EL mice, which show seizures once a week, these cytokines were similarly determined 5 min, 2 hr, 4 hr, 11 hr, 24 hr, 3 days and 6 days after the last seizure induced. Results A significant increase in the level of cytokines was observed in the brain of EL mice at any stages during development, compared with the level of cytokines in the brain of control DDY. Cytokines were increased predominantly before experiencing frequent seizures. In EL mice at the age of 15 weeks, the level of cytokines in the hippocampus was highest 6 days after seizures. In the parietal cortex, cytokines were most highly expressed 2 hr after seizures. The results indicate that cytokines were kept up-regulated until next seizures in the hippocampus, whereas they were transiently up-regulated immediately after seizures in the parietal cortex. Conclusion It is concluded that in the brain of EL mice, pro-inflammatory cytokines are increased progressively and periodically in association with the development and the seizure activity, respectively. A periodic increase of cytokines prior to the next seizure episode may play a role in triggering the ictal activity. Namely, alteration of region-specific cytokines may induce ictal activities from the interictal state. It is conceivable that inflammatory cytokines may work together with neuronal factors during epileptogenesis and in the transition from interictal to ictal state.

Developmental Program of Epileptogenesis in the Brain of EL Mice

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 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. 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 35S x-ray emulsion method. 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. 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.

Neuoscience: Molecular Pathology and Epilepsy

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.

Antiepileptic effects of allopurinol on EL mice are associated with changes in SOD isoenzyme activities.

We have investigated the potential antiepileptic action of superoxide dismutase (SOD) activities in the brain of the epileptic mutant EL mouse. EL mice which experienced frequent seizures (EL[s]) had abnormally low levels of SOD isoenzyme activity in the hippocampal area. Once epileptogenicity was established in these animals, activity of cyanide-sensitive Cu,Zn-SOD was maintained at significantly lower levels than in control mice. However, cyanide-insensitive Mn-SOD activity was not different from non-epileptic controls. In EL mice which had not experienced seizure provoking stimulations and exhibited no seizures (EL[ns]) there was moderately lower levels of SOD isoenzyme activities compared to controls. In spite of the low level of Cu,Zn-SOD activity in EL[s] mice, the Cu,Zn-SOD protein content was high in the hippocampus of these animals, suggesting that inactive Cu,Zn-SOD might be induced during development. After allopurinol (ALP) was given orally to EL[s] mice, Cu,Zn-SOD activities increased dramatically in the hippocampus and seizure activity was decreased. Even after 48 h, when antiepileptic action of ALP was lost, the SOD activity was maintained at the high level associated with initial ALP administration. EL[s] mice also showed DNA fragmentation in the hippocampal CA1 region and the parietal cortex, detected with in situ terminal transferase-mediated dUTP nick labeling with the aid of alkaliphosphatase or peroxidase. The degree of DNA fragmentation was less severe in EL[ns] mice. We propose that abnormalities in region specific Cu,Zn-SOD isoenzyme activity might produce free radicals, leading to DNA fragmentations and cell loss. This might contribute to hippocampal epileptogenesis in EL mice.

Role of nitric oxide in the hippocampus of epileptic mutant el mice

Exposure to ionizing radiation causes damage to living tissues; however, only a small number of agents have been approved for use in radiation injuries. Radioprotector is the primary countermeasure to radiation injury and none radioprotector has indeed reached the drug development stage. Repurposing the long list of approved, non-radioprotective drugs is an attractive strategy to find new radioprotective agents. Here, we applied a computational approach to discover new radioprotectors in silico by comparing publicly available gene expression data of ionizing radiation-treated samples from the Gene Expression Omnibus (GEO) database with gene expression signatures of more than 1309 small-molecule compounds from the Connectivity Map (cmap) dataset. Among the best compounds predicted to be therapeutic for ionizing radiation damage by this approach were some previously reported radioprotectors and baclofen (P < 0.01), a chemical that was not previously used as radioprotector. Validation using a cell-based model and a rodent in vivo model demonstrated that treatment with baclofen reduced radiation-induced cytotoxicity in vitro (P < 0.01), attenuated bone marrow damage and increased survival in vivo (P < 0.05). These findings suggest that baclofen might serve as a radioprotector. The drug repurposing strategy by connecting the GEO data and cmap can be used to identify known drugs as potential radioprotective agents.

Abnormalities in SOD Isoenzyme Patterns and DNA Fragmentation During Development of the Epileptic Mutant EL Mouse

Purpose: Several lines of evidence suggest that free radicals and their scavenger enzyme superoxide dismutase (SOD) are involved in long‐term potentiation (LTP) and epileptogenesis in the CA1 field of the hippocampus. In rats with kainate‐induced epilepsy and tottering mice (a model for petit ma1 seizures), “apoptosis” has been observed in neurons. Free radicals in the brain have been suggested to facilitate the apoptosis. The EL mouse, a mutant model of epilepsy, shows secondarily generalized seizures. In the mutant mice, the susceptibility to seizures gradually increases during development and through the experience of repetitive seizures. The parietal cortex in EL mice plays an important role in the seizure initiation, and the hippocampus shows a high electrical excitability during generalization of the seizures. In this study, we examined SOD isoenzyme activities and protein contents in the parietal cortex and hippocampus of EL mice during development. To investigate the region‐specific targets of free radicals in EL mice, the spatial specificity of DNA fragmentation also was studied. Methods: SOD activities were determined by using the method of Fridovich (1991). Total SOD activity included cyanide‐insensitive Mn‐SOD and cyanide‐sensitive Cu, Zn‐SOD. After coincubation with 1 mM cyanide, the activities of Mn‐SOD were assayed. Cu, Zn‐SOD activities were obtained from total SOD activities by subtracting Mn‐SOD activities. Mn‐SOD and Cu, Zn‐SOD contents were assayed with a commercially available enzyme‐linked immunosorbent assay (ELISA) kit (Boehringer). DNA fragmentation was detected in EL mice by using in situ terminal transferase‐mediated deoxyuridine triphosphate (dUTP) nick‐labeling detected with alkaline phosphatase and peroxidase. Results: EL mice which had experienced frequent seizures and established epileptogenicity, EL[s], revealed abnormal SOD isoenzyme activity patterns characterized by a remarkable decrease in the hippocampus. Once epileptogenicity was established, SOD activities, especially cyanide‐sensitive Cu, Zn‐SOD activities, were maintained at low levels. Despite the conspicuous decrease in Cu, Zn‐SOD activities, the protein contents of this isoenzyme were increased in the hippocampus. Cyanide‐insensitive Mn‐SOD activities were not changed. On the other hand, EL mice that had not experienced seizure‐provoking stimulations and exhibited no seizures, EL[ns], showed a moderate decrease in their SOD isoenzyme activities. In EL[s] mice, there was a fragmentation of DNA in the CAI field of the hippocampus and the parietal cortex, whereas in EL[ns], the DNA fragmentation was restricted to the parietal cortex and was much less obvious. Conclusions: The results revealed a region‐specific decrease in the Cu, Zn‐SOD activity and an increase in the regional free radical concentration in the EL brain during epileptogenesis. The discrepancy between enzyme activity and protein content might indicate the induction of an inactive Cu, Zn‐SOD isoenzyme during development. We speculated that region‐specific abnormalities in the activity of Cu, Zn‐SOD isoenzyme, presumably due to a disorder of Cu metabolism, produce spatially specific free radicals, which in turn could cause DNA fragmentation. This process might contribute to the common pathway for epileptogenesis.

GABAergic Abnormalities and IEG Expression Might Be Involved in the Formation of Abnormal Plasticity in Epileptic Mutant EL Mice.

GABAergic Abnormalities and IEG Expression Might Be Involved in the Formation of Abnormal Plasticity in Epileptic Mutant EL Mice.

Antiepileptic Effects of Allopurinol Involved in Hippocampal Specific SOD (Superoxide Dismutase) Induction in the Mutant El Mouse.

Antiepileptic Effects of Allopurinol Involved in Hippocampal Specific SOD (Superoxide Dismutase) Induction in the Mutant El Mouse.

Regional excitatory and inhibitory amino acid levels in epileptic El mouse brain

Inbred mutant El mice are highly susceptible to convulsive seizures upon "tossing" stimulation. The levels of excitatory (e.g. glutamate and aspartate) and inhibitory amino acids [e.g. gamma-amino-butyrate (GABA)] were examined in discrete regions of stimulated El mice [El(+)], non-stimulated El mice [El(-)] and ddY mice, which do not have convulsive disposition. In comparison with ddY, a general increased levels of aspartate, glutamate, glutamine, and taurine were detected in brain regions of El(-). The levels of GABA and glycine were almost the same in ddY and El(-). Compared to El(+), the levels of aspartate, glutamate, glutamine, and GABA in El(-) were either the same or higher. In the case of taurine and glycine, the levels in El(-) were either the same or lower than El(+). Alanine is special in that El(-) have a higher level than El(+) in hippocampus but lower in cerebellum. Furthermore, while marked changes were registered in several brain regions, none of the amino acids investigated showed any significant differences in the hypothalamus of three different groups of mice.

Depressed convulsions by diazepam and its effects on brain monoamines and amino acids in El mice

The effect of diazepam on inbred mutant El mice, which develop convulsive seizures after repeated sessions of being tossed up, was examined. Acute administration of diazepam (32 mg/kg i.p.) completely inhibited the convulsions. At that time, the dopamine level was increased in the cortex and hippocampus, and the norepinephrine level in the cerebellum was decreased. 5-Hydroxytryptamine levels were not changed. As for amino acids, the glutamine level increased and the levels of GABA, glutamic acid, aspartic acid, alanine and other amino acids were not changed.

Characteristics of primary cultured neurons from embryonic mutant El mouse cerebral cortex

To elucidate the differences between neurons of epileptogenic animals and those of normal animals, cellular characteristics of neurons of mutant strain El mice which are highly susceptible to seizures were investigated using immunocytochemical techniques. In neurons of 3-day primary cultures, the control ddY mouse neurons showed dividing stages in about 0.2% of neurofilament (NF)-positive neurons, whereas no dividing neurons were observed among the NF-positive El mouse neurons. In 7-day cultures, localization of GD 3 ganglioside in the proliferating control ddY mouse neurons was observed, but there was no GD 3 ganglioside in the mutant El mouse neuron. The content of GD 3 ganglioside detected by high-performance thin-layer chromatography of El mouse cultured cells was ca. 1/4 of that of ddy mice. These findings suggest that neurons of the El mouse are differentiated earlier than those of the control ddY mouse.

Local cerebral glucose utilization in epileptic seizures of the mutant El mouse

The neural pathway and the focus of the epileptic seizure of mutant E1 mice were studied by the [ 14C]2-deoxyglucose technique. In the mice with full tonic-clonic seizure, the entire neocortex and hippocampus showed a remarkably increased metabolic rate. By contrast, a lower metabolic rate was evident in the thalamic nuclei, central gray, locus coeruleus and some interstitial nuclei. The epileptic focus in E1 mice presumably exist in the hippocampus .

Abnormal plastic phenomena of sensory-precipitated epilepsy in the mutant El mouse

The processes of development of seizures of the El mouse, an inbred mutant strain susceptible to epilepsy, were investigated by long-term clinical observations and EEG recordings. The phenomena of extinction, reinforcement, and an aftereffect of the antecedent occurrence of seizures were observed. Seizures of El mice were elicited by repetition of natural sensory stimulation, which consisted of observation and tossing procedures. The seizure thresholds were lowered with a repetition of stimuli, and the incidence of seizures became higher, attaining 100%. Clinical manifestations started with abortive forms several weeks after the mice were initially exposed to stimulation, and concluded with typical general convulsions and wild running. Paroxysmal discharges appeared in the EEG as isolated spikes, first from restricted parietal areas; subsequently spike and wave complexes were extedded over wide areas of the brain. Even during the interictal period isolated spikes sometimes could be seen in the EEGs of the El mice most susceptible to seizures. After a long period without stimulation, the thresholds were raised, or extinction of seizures was observed in mice with fully developed seizures. Reinforcement stimulation was necessary for seizures of El mice to occur constantly. During recovery of the occurrence of seizures, their incidence increased sooner than in the previous developmental period. In general, the El mouse seizure might represent an abnormal plastic phenomenon associated with repetition of sensory stimuli in addition to genetic predisposition and ontogenetic development.