SAD-B modulates epileptic seizure by regulating AMPA receptors in patients with temporal lobe epilepsy and in the PTZ-induced epileptic model

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the predominant mediators of glutamate-induced excitatory neurotransmission. It is widely accepted that AMPA receptors are critical for the generation and spread of epileptic seizure activity. Dysfunction of AMPA receptors as a causal factor in patients with intractable epilepsy results in neurotransmission failure. Brain-specific serine/threonine-protein kinase 1 (SAD-B), a serine-threonine kinase specifically expressed in the brain, has been shown to regulate AMPA receptor-mediated neurotransmission through a presynaptic mechanism. In cultured rat hippocampal neurons, the overexpression of SAD-B significantly increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Here, we showed that SAD-B downregulation exerted antiepileptic activity by regulating AMPA receptors in patients with temporal lobe epilepsy (TLE) and in the pentylenetetrazol (PTZ)-induced epileptic model. We first used immunoblotting and immunohistochemistry analysis to demonstrate that SAD-B expression was increased in the epileptic rat brain. Subsequently, to explore the function of SAD-B in epilepsy, we used siRNA to knock down SAD-B protein and observed behavior after PTZ-induced seizures. We found that SAD-B downregulation attenuated seizure severity and susceptibility in the PTZ-induced epileptic model. Furthermore, we showed that the antiepileptic effect of SAD-B downregulation on PTZ-induced seizure was abolished by CNQX (an AMPA receptor inhibitor), suggesting that SAD-B modulated epileptic seizure by regulating AMPA receptors in the brain. Taken together, these findings suggest that SAD-B may be a potential and novel therapeutic target to limit epileptic seizures.

Physical exercise in rats with epilepsy is protective against seizures: evidence of animal studies / Exercício físico em ratos com epilepsia como fator protetor contra crises epilépticas: evidencias de estudos em animais

People with epilepsy have been discouraged from participating in physical activity due to the fear that it will exacerbate seizures. Clinical and animal studies indicate a reduction of seizure frequency as well as decrease susceptibility to subsequently evoked seizures after an exercise program. Analyses from experimental studies of animals with epilepsy submitted to physical training programs were performed. In all studies the physical training was able to reduce the number of spontaneous seizures in rats with epilepsy. Seizure occurrence during exercise was relatively absent in the majority of studies. No death was found in animals with epilepsy during 1680 h of exercise. Based on these results it is plausible encouraging persons with epilepsy to non-pharmacological treatments and preventative measures such as physical exercise.

Pessoas com epilepsia têm sido desencorajadas da prática de atividade física por receio do exercício físico exacerbar as crises epilépticas. Estudos clínicos e em animais mostram uma redução da frequência de crises, assim como diminuição da susceptibilidade a crises subseqüentes após programa de exercício físico. Neste estudo realizamos uma análise de estudos experimentais de animais com epilepsia submetidos a programas de exercício físico. Em todos os estudos, o treinamento físico foi capaz de reduzir o número de crises espontâneas em ratos com epilepsia. A ocorrência de crises durante o exercício físico foi relativamente ausente na maioria dos estudos. Nenhuma morte foi encontrada em animais com epilepsia durante 1680 h de exercício físico. Baseados nestes resultados parece aceitável encorajar as pessoas com epilepsia a tratamentos não farmacológicos e medidas preventivas como o exercício físico.

The pilocarpine model of epilepsy: what have we learned?

The systemic administration of a potent muscarinic agonist pilocarpine in rats promotes sequential behavioral and electrographic changes that can be divided into 3 distinct periods: (a) an acute period that built up progressively into a limbic status epilepticus and that lasts 24 h, (b) a silent period with a progressive normalization of EEG and behavior which varies from 4 to 44 days, and (c) a chronic period with spontaneous recurrent seizures (SRSs). The main features of the SRSs observed during the long-term period resemble those of human complex partial seizures and recurs 2-3 times per week per animal. Therefore, the pilocarpine model of epilepsy is a valuable tool not only to study the pathogenesis of temporal lobe epilepsy in human condition, but also to evaluate potential antiepileptogenic drugs. This review concentrates on data from pilocarpine model of epilepsy.

A administração sistêmica do potente agonista muscarínico pilocarpina em ratos promove alterações comportamentais e eletrográficas que podem ser divididas em três períodos distintos: (a) período agudo o animal evolui progressivamente para o status epilepticus, que perdura por até 24h; (b) período silencioso, caracterizado pela normalização progressiva do comportamento e do EEG e pode ter uma duração de 4 a 44 dias; período crônico, aparecimento de crises epilépticas espontâneas e recorrentes (SRSs). As características das SRSs observadas nos animas durante o período crônico são semelhantes às crises parciais complexas dos seres humanos e recorrem de 2-3 vezes por semana/animal. Além disso, o modelo de epilepsia induzido pela pilocarpina é válido não somente para se estudar a patogênese da epilepsia do lobo temporal em humanos como também para se testar a viabilidade de drogas antiepilépticas. Esse artigo de revisão aborda diversos aspectos do modelo de epilepsia induzido pela pilocarpina.

Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death

Intrahippocampal administration of kainic acid (KA) induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 ± 8 min in saline controls (N = 4) which increased to 369 ± 71 and 322 ± 63 min in animals receiving herbimycin A (1.74 nmol, N = 4) and K252a (10 pmol, N = 4), respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90 percent of cell loss in KA-treated animals) in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

Effect of aerobic physical exercise in pinealectomized animals submitted to the pilocarpine model of epilepsy

OBJECTIVE: To better clarify the positive effects of physical exercise in the epilepsy, we analyzed the effect of the pinealectomy in animals with temporal lobe epilepsy (TLE) induced by pilocarpine submitted to an aerobic physical program. MATERIAL AND METHODS: Forty adults Wistar rats were used: 1) PX + CHRONIC - Pinealectomized animals (PX) with TLE (CHRONIC) without exercise (n = 9); 2) PX + CHRONIC + EXERCISE - submitted to an aerobic physical exercise program (n = 5); 3) CHRONIC - without exercise (n = 8); 4) CHRONIC + EXERCISE (n = 8); 5) CTRL - control without exercise (n = 5); 6) CTRL + EXERCISE (n = 5). The physical exercise program consisted of 1 hour of treadmill, 5 days/week, during 30 days, at 60 percent VO2max. The Nissl and neo-Timm methods were used. RESULTS: The pinealectomy increased the frequency of seizures in animals with epilepsy. It was observed a reduction of the neuronal death and mossy fiber sprouting in the animals with epilepsy submitted to an aerobic physical exercise program. However, the physical exercise program did not modify the frequency of the seizures in the pinealectomized animals.

OBJETIVO: Buscando elucidar os efeitos positivos do exercício físico aeróbio na epilepsia, analisamos a influên-cia da pinealectomia em animais com epilepsia do lobo temporal (ELT) induzida por pilocarpina e submetidos a um programa de exercício físico. MATERIAL E MÉTODOS: Quarenta ratos Wistar adultos foram usados: 1) PX + CRÕNICO - pinealectomizados (PX) com ELT (CRÕNICO) sem exercício (n = 9); 2) PX + CRÕNICO + EXERCíCIO - submetidos a um programa de exercício físico aeróbio (n = 5); 3) CRÕNICO - sem exercício (n = 8); 4) CRÕNICO + EXERCíCIO (n = 8); 5) CTRL - controle sem epilepsia, sem exercício (n = 5); 6) CTRL + EXERCíCIO (n = 5). O programa de exercício físico consistiu de corrida em esteira, 5 dias/semana (30 dias) a 60 por cento VO2max. Os métodos de Nissl e neo-Timm foram utilizados. RESULTADOS: A pinealectomia aumentou a freqüência de crises em animais com epilepsia. Foi observada uma diminuição da morte neuronal e do brotamento de fibras musgosas em animais com epilepsia, submetidos ao programa de exercício físico. Entretanto, este programa não alterou a freqüência de crises em animais pinealectomizados.

Induction of Bis, a Bcl-2-binding protein, in reactive astrocytes of the rat hippocampus following kainic acid-induced seizure

The expression of Bis (also called Bag-3), a Bcl-2-binding protein, was investigated in the rat kainic acid (KA) model of temporal lobe epilepsy. Western blot analysis showed a significant increase in the expression levels of Bis protein in the hippocampus following the systemic administration of KA. Bis immunoreactivity increased preferentially in the CA1 and CA3 regions, as well as in the hilar region of the dentate gyrus. Experiments with double immunofluorescence revealed that, in KA-administered rats, the cells expressing Bis were GFAP-expressing reactive astrocytes. The increase in Bis immunoreactivity was accompanied by increased Bcl-2 in reactive astrocytes in the striatum radiatum, whereas Bcl-2 immunoreactivity in pyramidal neurons was not affected. These results of the co-expression of Bis and Bcl-2 in reactive astrocytes in this seizure model suggest that Bis might modulate the glial reaction under excitotoxic brain injury, probably by interacting with Bcl-2.

c-JUN Expression and Apoptotic Cell Death in Kainate-Induced Temporal Lobe Epilepsy

Following kainate (KA)-induced epilepsy, rat hippocampal neurons strongly ex-press immediate early gene (IEG) products, i.e., c-FOS and c-JUN, and neural stress protein, HSP72. Prolonged expression of c-JUN and c-FOS 48 hr after cerebral ischemia has been underwent delayed neuronal death. However, it is not yet clear whether IEGs actually assume the essential roles in the cell death process or simply as a by-product due to external stimuli because of the prolonged expression of c-FOS, more than one week, on intact CA2 neurons of the hippocampus in a KA-induced epilepsy model. This study investigated the relationships between prolonged expression of c-JUN and hippocampal neuronal apoptosis in a KA-induced epilepsy model. Epileptic seizure was induced in rats by a single microinjection of KA (1g/l) into the left amygdala. Characteristic seizures and hippocampal neuronal injury were developed. The expression of c-JUN was evaluated by immunohistochemistry, and neuronal apoptosis by in situ end labeling. The seizures were associated with c-JUN expression in the hippocampal neurons, of which the level showed a positive correlation with that of apoptosis. Losses of hippocampal neurons, especially in the CA3 region, were partly caused by apoptotic cell death via a c-JUN-mediated signaling pathway. This is thought to be an important component in the pathogenesis of hippocampal neuronal injury via KA-induced epilepsy.

Calcium/Calmodulin Kinase II Activity of Hippocampus in Kainate-Induced Epilepsy

This study investigated calcium/calmodulin kinase II (CaMKII) activity related to long-standing neuronal injury of the hippocampus in kainate (KA)-induced experimental temporal lobe epilepsy. Epileptic seizure was induced by injection of KA (1 g/L) dissolved in phosphate buffer (0.1 M, pH 7.4) into the left amygdala. Clinical seizures, histopathologic changes and CaMKII activity of the hippocampus were evaluated. Characteristic early limbic and late seizures were developed. Hippocampal CaMKII activity increased significantly 4 and 8 weeks after intra-amygdaloid injection of KA, when late seizures developed. The histopathologic changes of the hippocampus included swelling of neuronal cytoplasm with nuclear pyknosis and loss of neurons in CA3 during this period. The increased activity of CaMKII may correlate with appearance of distant damage in the hippocampus. The above results indicate that intra-amygdaloid injection of KA produces excitatory signals for ipsilateral CA3 neurons in the hippocampus and that subsequently increased levels of CaMKII in postsynaptic neurons induce neuronal injury via phosphorylation of N-methyl-D-aspartate type glutamate receptor.