| Epilepsy is one of the most prevalent neurologic disorders worldwide. 20% to 30% of epilepsy is intractable based on the current development of anti-epileptic drugs. Therefore, a new method for the treatment of patients with epilepsy is imminent. Recent studies suggest that, in addition to conventional chemical synapses, the "non-synaptic" mechanism plays an important role in the pathogenesis of epilepsy and synchronization of neurons. Gap junctions between neurons may be the structural basis of seizures. modulation of Cx36 expression may be a useful treatment for epilepsy.Objective: The purpose of our study was to investigat the effect of connexin 36-associated(Cx36) channel and its blockers on the process of epilepsy, and its mechanisms associated with alternation of cytoskeleton. Methods: In this study, kainic acid(KA) was injected into the rat amygdala to induce an epilepsy model. 228 adult male Wistar rats were divided into four groups of six animals per group, including the CONT, SHAM, KA, and the intervention-carbenoxolone, quinine, quinidine(CBX, QN, QND) groups. The KA group were divided into five subgroups including the 3 hours, 6 hours, 24 hours, 3 days and 1 week groups to investigated the time dependent changes in electroencephalographic activities, pathogenesis and connexin(Cx)36 protein expression. We examined the anticonvulsant properties of carbenoxolone, quinine, and quinidine on epileptic discharge and the expression of Cx36.Expression of microtubule associated protein-2(MAP-2) and synaptophysin(SYP) were assessed via immunofluorescent and immuno-histochemical examination to investigated the potential anti-epileptogenic effect of neuronal Cx36 gap-junction blockage via its inhibitory effect on expression of MAP-2 and SYP. Results:(1)The appearance of epileptic discharges started 71.8± 23.7s after KA administration. Spike frequency and amplitude of epileptiform activity reached the maximum level at 30±5.2 min. The maximum level of spike frequency and amplitude of epileptiform activity was 13.9±0.3 Hz and 198 ± 14.3m V respectively. Immunoblot analysis and immunofluorescence immunohistochemistry showed the Cx36 protein expression level in hippocampus increased significantly in the KA 6h group relative to the sham group, while it decreased significantly in the KA 3d and KA 7d group. Haematoxylin and eosin staining showed that the neuronal cell death was obvious in CA3 in hippocampus 6h after KA injection. A significant cell loss and glial cell proliferation progressively increased from 24 h, 3d to 7d.(2)30 min after KA injection, spike frequency and amplitude were noticeably and reversibly reduced in Cx36 blocker pretreated animals. At the 90 min time point, carbenoxolone, quinine, and quinidine significantly decreased the spike amplitude but not frequency. Cx36 protein level significantly increased in Cx36 blocker pretreated animals relative to control. There were no significant differences among the three drug treatment groups in spike frequency, spike amplitude, or level of Cx36 expression.(3)Expression of MAP-2 and SYP was significantly increased after KA administration in sham group compared with control group. Expression of MAP-2 and SYP was significantly decreased in CBX, QN, and QND groups compared with sham group. These provide novel evidence for key roles of MAP-2 and SYP over-expression in the modulation of neuronal plasticity, hyperexcitability of the hippocampal neuronal network, and persistent seizure discharge. Conclusion:(1)KA injected into the rat amygdala was a successful model of epilepsy. Cx36 may play an important role in the epileptic process.(2)The specific gap junction blockers may be efficacious for the treatment of epilepsy by preventing the propagation and synchronization of epileptiform activity.(3)The reversal of increased MAP-2 and SYP expression after the administration of Cx36 channel blockers indicates their potential role in anti-epileptogenic treatment and the critical need for further investigations of these compounds. |
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