| Epilepsy, caused by a variety of causes, is a complex clinical syndrome. Epilepsy is a chronic disease characterized by recurrent episodic and central system dysfunction caused by abnormal discharge of neurons. Epilepsy is one of the chronic diseases which does harm to human health seriously. It has brought immeasurable heavy spirit and financial burden to society, families and individuals. Because of its high incidence and serious harm, it has gotten the most attention during the central nervous system disorders. In recent years, research in many aspects of epilepsy has made significant progress, but its pathogenesis is still not very clear.Gap junction, a special structure in cell membrane, constitute the only direct communication channel of energy, material and information exchange between adjacent cells. Information ions and small molecules can complete cell transferation passing through the channel. Gap junction plays an important role in the maintenance of electrical activity of neurons, the rapid synchronization of neurons and neuronal development, and also involved in second messenger delivery and integration of glial cell activity. Studies in recent years have found that gap junction plays a very important role in the process of formation and development of epilepsy. Connexin CX32 and CX43 are the main protein of the gap junctions between the central nervous systemal neurons and astrocyte, especially in the hippocampus. The relationships between CX32, CX43 and epilepsy have been concerned by scholars at home and abroad.Carbenoxolone is non-selective blocker of gap junction. It can connect directly with gap junction through derivatives, then the gap junction'conformation changes, leading to channel closure.ObjectiveIn this study, using lithium-pilocarpine epilepsy rats'model, we observe the rats'behavior, and investigate the expression of CX32, CX43 in the hippocampus region and the intervention effects induced by carbenoxolone. And carbenoxolone will be validated that its antiepileptic effect is more specific, and it can reduce the seizure latency and seizure levels, offerring new ideas to the development of antiepileptic drugs.Materials and methods1. Group:120 healthy 21-day-old SD rats, whatever female or male, with their weight ranges from 65 to 70 gram, were randomly divided into three groups:saline control group (n=40), epileptic group (n=40) and carbenoxolone treated group(n=40).2. Behavior observation:Rats in each group were observed immediately after the drug treatment, and the observation was continued to the rats were sentenced to death. Observed for at least 5 hours a day. Where a row out of three times more than gradeⅣattack, as fully ignited. Intensity of epileptic seizures refers to Racine 6 level (0-V) assessment method.3. Collection and preparation of specimen:Each group, at 24 hours,3 days,7 days,30 days respectively, were cut the chest, exposing the heart, cut the right atrial appendage after ether anesthesia, and then gave left ventricular perfusion of 9g/L saline and 40g/L paraformaldehyde using 20ml syringe connected to the 5th needle, until out flow of clear liquid from the right atrial appendage was observed, with liver white and the trunk harden up. Then brains were removed, and coronal slices were maken. And then gave coronal slices hematoxylin-eosin (HE) staining and connexin CX32, CX43 immunohistochemistry.4 Index measurement:1) Because of the characteristics of the model, behavioral observations were conducted in three stages:Acute phase:heavy attack rate, incubation period length;Quiescent:quiescent length;Spontaneous recurrent seizures period:the frequency of spontaneous recurrent seizures;2) HE staining:The sections was stained by HE and observed the pathological change under optical microscope.3) When the nucleuses were brown-yellow colored, expression of CX32 and CX43 were positive. Then we measured CX32 and CX43 absorbance value (OD) in hippocampal CA1 area using color pathological image analysis system. Each slice was measured from five horizons and chosen the mean at last. Simultaneously chose the corpus callosum OD value of the same slice as the background OD values, with the corrected absorbance values of COD (measured value-background value) for comparison and analysis, in order to avoid error caused by non-specific staining in the staining process.5 Statistical Analysis:Datas were described by mean±standard deviation. a=0.05 is the significance level. P value less than 0.05 indicate statistical significance. Using SPSS16.0 statistical package to analyze. Using t test to compare the couple groups, while using one-way ANOVA to compare the couple datas.Results1 Behavioral assessment:Scores of the acute severe elileptiform seizures (RacineⅣ-Ⅴlevel) in Lithium-pilocarpine group were significantly higher than that of the carbenoxolone treated group; and the latency in carbenoxolone treated group was prolonged significantly (P<0.05); while the control group had no seizures.2 HE staining:Compared with the control group, in the hippocampal of epileptic group, neurons were in a large number of demyelination; compared with epilepsy group, in the hippocampal of intervention group, neuron loss is not obvious and the number of neurons was increased significantly.3 Immunohistochemistry:Compared with control group, in the hippocampal of epileptic group, the expression of CX32 were in larger number significantly at the same time points (P<0.05). And the expression of CX32 in hippocampus of epileptic group was less than that of the intervention group at the same time points (P<0.05). The expression of CX43 in hippocampus increased gradually in epileptic groups, and increased with the time prolonged after SE, and there were significant differences when compared with the control groups at the same points (24h,3d,7d,30d, P< 0.05). The expression of CX43 in hippocampus of the intervention group was less than that of the epileptic group at the same time points (P< 0.05).Conclusions1 The expression of CX32 and CX43 increases dramatically after epileptic discharges, which indicates that CX32 and CX43 play an important role in the occurrence and development of epilepsy.2 As gap junction blocker, carbenoxolone has a clear antiepileptic effect.
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