The Study Of Antiepileptic Mechanism Of Mannitol Mediated By GABA

Objective:To evaluate the application of4-AP model established on SD rat in vivo and in vitro and investigate an improved and optimized scheme of the model.Method:1. In vivo:Healthy male SD rats (200±20g) were divided randomly into3groups:4-AP, penicillin and pilocarpine group.4-AP group were divided randomly into five groups according to the dosage and concentration ratio:1)dose:5mg/kg, concentration:1mg/ml group;2) dose:5mg/kg, concentration:0.67mg/ml group;3) dose:2.5mg/kg, concentration:0.67mg/ml group;4) dose:2.5mg/kg, concentration:0.5mg/ml group;5) dose:2mg/kg, concentration:0.5mg/ml group, each group of6. Epilepsy was induced by intraperitoneal injection of these three kinds of drugs, the behavior performance was observed and epileptiform discharges were recorded with electroencephalogram (EEG) for6hours.2. In vitro:Healthy male SD rat (50-100g) hippocampal slices were prepared, spontaneous epileptiform discharges were induced by superfusing slices with ACSF-containing4-AP(100μM) and detected with patch clamp technology.Results:1. In vivo:1) In dose of5mg/kg, concentration of1mg/ml and dose of5mg/kg, concentration of0.67mg/ml4-AP groups, the successful rates of the model were16.7%and mortality rates were83.3%; In dose of2.5mg/kg and concentration of0.67mg/ml4-AP group, the successful rate was50%and mortality rate was50%; In dose of2.5mg/kg and concentration of0.5mg/ml4-AP group, the successful rate was100%; In dose of2mg/kg and concentration of0.5mg/ml4-AP group, the successful rate was0%. While in penicillin and pilocarpine groups, the successful rates were83.3%and66.7%, the mortality rates were16.7%and33.3%.2) In dose of2.5mg/kg and concentration of0.5mg/ml4-AP group, the average latent period was3.25±0.56seconds, the average time to reach Racine3was27.38±1.55seconds, and the seizure often lasted for2±0.4hours; in penicillin group the average latent period was12.83±1.83seconds, the average time to reach Racine3was32.40±0.95seconds; in pilocarpine group, the average latent period was21.67±2.94seconds, the average time to reach Racine3was46.92±0.75seconds.3) There appeared obvious epileptiform waves in EEG recording after4-AP, penicillin and pilocarpine were injected, the amplitude of slow wave in pilocarpine group was the highest and in penicillin group was the lowest.2. In vitro:Burst frequences of action potential increased after4-AP was perfused.Conclusion:1) The in vivo model induced by4-AP at the dose of2.5mg/kg and concentration of0.5mg/ml can simulate maximumly the epileptic seizures of human in behavior performance and epileptiform discharges recorded with EEG.2) The in vitro model induced by4-AP discharged steadily. It’s a mature chemical epilepsy model and well suitable for epilepsy research in vivo and in vitro. Objective:To explore the antiepileptic mechanism of mannitol through detecting the expression of GAD65and GABAARal in4-AP epileptic rats.Method:70healthy male SD rats (200±20g) were divided randomly into7groups:1)4-AP group;2) control group (intraperitoneal injection of isodose saline);3)mannitol intervention group1(a single injection of mannitol into the saphenous vein,3min after4-AP injection);4) mannitol intervention group2(a single injection of mannitol into the saphenous vein,15min after4-AP injection);5)mannitol intervention group3(two injections of mannitol into the saphenous vein,15min and6.25h after4-AP injection);6) mannitol intervention group4(two injections of mannitol into the saphenous vein,2h and8h after4-AP injection);7)4-AP+saline group (two injections of saline into the saphenous vein,15min and6.25h after4-AP injection), each of10. The behavior performance was observed and the epileptiform discharges were recorded with EEG for2hours after the treatment of drugs. After72hours, the expression of GAD65and GABAARα1of hippocampus was observed with SABC immune histochemistry, detected with RT-PCR technology, and tested with Western blot technology.Results:1. The successful rate of the model was88.3%and mortality rate was4.3%.2. The result of immune histochemistry showed, compared with control group, the expression level of GAD65and GABAARα1in other groups elevated significantly (P<0.05), and mannitol intervention group3was the highest (P<0.05), there were no statistical differences between other groups.3. The result of RT-PCR showed, compared with control group, the expression level of GAD65and GABAARα1in other groups elevated significantly (P<0.05), and mannitol intervention group3was the highest (P<0.05), there were no statistical differences between other groups.4. The result of Western blot showed, compared with control group, the expression level of GAD65 and GABAARα1in other groups elevated significantly (P<0.05), and mannitol intervention group3was the highest (P<0.05), there were no statistical differences between other groups.Conclusion:Mannitol mediated the up-regulation of GAD65and GABAARal expression level and was associated with antiepileptic effects. Objective:To explore the antiepileptic mechanism of mannitol on brain slices in vitro.Method:Healthy male SD rat (50-100g) rat hippocampal slices were prepared, blinded patch clamp technology was applied to record:1)Interictal bursts/min of CA3neurons in4-AP and high K+solution model treated with mannitol.2) The influence of mannitol on LTPGABA in high-frequency electrical stimulation model.Results:1. In4-AP model, interictal bursts/min was25.8±7.5before mannitol was perfused and reduced to15.4±0.7after mannitol was perfused, dropped by40%(P<0.05), but reversed to23.9±1.3after mannitol was washout (P>0.05).2. In high K+solution model, interictal bursts/min was28.4±3.8before mannitol was perfused and reduced to18.2±3.1after mannitol was perfused, dropped by36%(P<0.05), but reversed to24.6±4.3after mannitol was washout (P>0.05).3. In the presence of mannitol, LTPGABA failed to be induced.Conclusion:Mannitol plays a role in antiepileptic effect through reducing interictal bursts/min and increasing GABAergic release.