Changes Of Neural Excitability In Medial Parabrachial Nucleus During Acute Epileptic Stage In Mice

Background: Temporal lobe epilepsy is one of the most common types of refractory epilepsy.The pilocarpine-induced epilepsy model is highly similar to human TLE epilepsy and has been widely used in TLE studies.Epilepsy is closely related to sleep disorders,and they are mutually reinforcing,creating a vicious circle,especially in TLE.Seizures are prone to occur during NREM sleep and rare during REM sleep.In normal conditions,patients with epilepsy have prolonged NREM sleep and shorten REM sleep,which promotes seizures.The parabrachial nucleus is the core brain structure of sleep regulation.After MPBN lesion by specific means,animals REM sleep was increased.This study focused on whether TLE will increase the excitability of medial parabrachial nucleus neurons,further shortening REM sleep and promoting seizures.Methods: This study was divided into three parts.To establish a TLE mouse model by pilocarpine.The isolated MPBN brain slices were prepared at 1 h and 3 d after successful modeling.Normal mice were considered as the control group.Whole-cell patch clamp technique was used to detect the intrinsic excitability of MPBN neurons.Immunofluorescence staining and Western blotting were used to detect the c-Fos and GFAP expression of MPBN neurons.Statistical analysis was performed using GraphPad Prism software.Using variance homogeneity test: If the variance is homogeneous,one-way ANOVA is used,and the LSD-t test is used to compare the means between groups;if the variance is not uniform,the Mann-Whitney test of nonparametric test is used,the difference is considered statistically significant,while P<0.05.Results: 1.About 80% of the mice after injection of pilocarpine produce chewing,nodding,wet dog-like shaking,and unilateral forelimb clonic behavior.About 28% ofthe mice who did not have seizures or below the criteria for epilepsy;the mortality rate was 16% and the final success rate was 56%.2.The results of patch clamp experiment showed that compared with the control group,the number of Ap discharges and the peak amplitude in EP 1h and EP 3d group were increased,the half width of action potential become narrowed,the maximum rise slope of action potential was increased,and the maximum decay slope was reduced.Those dates were significantly different from the control group(LSD-t-test,P<0.05).3.Immunofluorescence staining results: The positive staining area of GFAP in EP1 h group and EP3 d group was increased than the control group,with significant difference(LSD-t-test,P<0.001,P<0.001);The positive numbers of c–Fos in EP1 h group was increased significantly than the control group(LSD-t-test,P<0.001),There was no significant change between the EP3 d group and the control group(LSD-t-test,P>0.05).4.Western blotting results: The expression of GFAP protein in EP1 h group and EP3 d group were significantly higher than the control group(LSD-t-test,P<0.05,P<0.01);The expression of c-Fos protein in EP1 h group was significantly increased than the control group(LSD-t-test,P<0.001),and there was no significant change between EP3 d group and control group(LSD-t-test,P>0.05).Conclusion: 1.The mice were given intraperitoneal injection of pilocarpine 300 mg/kg,and the epilepsy model was successfully induced.2.In the acute phase of epilepsy,the excitability and activity of neurons in the medial parabrachial nucleus of the mice was enhanced.3.Astrocyte reactivity enhancement in the medial parabrachial nucleus of mice during acute epileptic stage.4.The acute phase of epilepsy may involve the medial paraventricular nucleus,which may shorten the REM phase and make the NREM phase relatively prolonged,leading to an increase in seizures during NREM sleep.