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Expression And Mechanism Of TMEM230 In Mouse Chronic Epilepsy Model

Posted on:2022-07-23Degree:MasterType:Thesis
Country:ChinaCandidate:L L LiFull Text:PDF
GTID:2504306512995109Subject:Neurology
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Objective: Expression of TMEM230 in mice with epilepsy and its possible mechanisms involved in epilepsy formation..Methods: Healthy male C57BL/6 mice(body weight 20-25g)were randomly divided into normal control group,PTZ ignited epilepsy group(regular daily injection of PTZ35mg/kg,),and Ka-induced epilepsy group(unilateral injection of 1nmol of Ka in hippocampus stereolocation,).Weatem blot,immunohistochemistry and immunofluorescence were used to detect the increased expression of TMEM230 in the cortex and hippocampus of PTZ ignition and Ka-induced epilepsy mice.In order to further confirm whether TMEM230 is involved in the seizure of epilepsy,we injected LV-TM230 and LV-SHTM230 into the CAL region of the unilateral hippocampus of mice through hippocampal stereotaxization and microtutule,and observed the effects of TMEM230 overexpression and gene knockdown on the seizure of mice.Two weeks after the construction of lentivirus,PTZ ignition model(n=8)and Ka-induced mouse chronic epilepsy model(n=8)were constructed.The epileptic seizure score was quantified with the Racine standard scale.The intracranial electrophysiological microline array was implanted in the ipsilateral hippocampus and fixed on the skull with dental cement.The incubation period and frequency of spontaneous recurrent epilepsy in 1 month were observed by video monitoring.TMEM230 was found to be associated with epileptic seizures.In PTZ ignition model,the co-expression of TMEM230 with neurons and astrocytes was observed by immunofluorescence technique,and the co-expression of TMEM230 with VGLUT1 and VGAT was observed in primary neurons.Finally,immunoprecipitation was used to detect the co-expression of TMEM230,VGLUT1 and VGAT in the mouse hippocampus.Results:Western blot analysis showed that basic expression of TMEM230 was detected in the hippocampal tissues of normal mice.Elevated levels of TMEM230 were found in the cortex and hippocampus of mice with PTZ ignition and Ka-induced epilepsy.TMEM230 was correlated with epileptic seizures.Racine score was used for behavioral evaluation after PTZ injection the next day,and those with Racine score of grade 4 or above were considered epileptic seizures.The epileptic seizure score of LV-TM230 treated mice was significantly higher than that of control group(P <0.01).The epileptic score of LV-SHTM230 mice was significantly lower than that of control group(P <0.05).In the Ka-induced epilepsy model,the latency of LV-TM230 and LV-SHTM230 were(4.8±1.5)d and(8.8±1.5)d respectively.The control group were(5.8±1.5)d and(5.8±1.3)d,respectively.The incubation period of the overexpression group was shorter than that of the control group,and the difference was statistically significant(P<0.01).The incubation period of the knockout group was longer than that of the control group,and the difference was statistically significant(P<0.05).The number of SRS seizures in LV-TM230 treated mice was(15.8±2.6)in 4 weeks,the number of SLE in LV-TM230 treated mice was(5.2±1.5)times,and the number of SLE in control group was(9.8±1.9)times,and the number of SLE in control group was(10.4±3.2)times,respectively.The difference was statistically significant(P<0.05).The protein content of TMEM230 detected in the Ka-induced epilepsy group was compared with that in the normal control group,and the difference was statistically significant(P <0.05).The protein content of TMEM230 detected in PTZ ignited epilepsy group was compared with that of normal control group,and the difference was statistically significant(P <0.01).In addition,TMEM230 was co-expressed with neurons,but not with astrocytes,in the hippocampus of epileptic mice by immunohistochemical and immunofluorescence techniques.Through immunoprecipitation,it was found that VGLUT1 and anti-TMEM230 antibodies had co-immunoprecipitation,while VGAT and anti-TMEM230 antibodies had no co-immunoprecipitation.Conclusion: TMEM230 is involved in the chronic formation of epilepsy in mice,and the mechanism may be related to its interaction with VGLUT1.
Keywords/Search Tags:Epilepsy, TMEM230, Glutamate vesicle transporter 1, Glutamic acid, γ-aminobutyric acid
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