Regulation Of MGE-derived Exosomes On Status Epilepticus And Epilepsy

PART I: ACQUISITION OF MGE-DERIVED EXOSOMESOBJECTIVE: To isolate and culture MGE from fetal mice and extract MGE-derived exosomes.Methods:1.Female and male mice were mated in cages by adult C57BL/6 mice,and the gestational age was calculated by detecting the pudendal embolism of female mice the next day.2.Fetal mice of E14.5d were dissected under a stereomicroscope,and MGE tissue regions were identified and isolated.GABA-ergic intermediate neuron precursor cells were cultured by primary cultured neurons.3.Fluorescence confocal technique was used to detect cell differentiation on day 0,day 1,day 3 and day 6,respectively.4.The exosomes secreted by cells in cell culture medium were extracted by ultrahigh speed centrifugation.5.The exosomes were identified by Western Blot combined with NTA and transmission electron microscopy.The experimental results are as follows:1.MGE tissues were successfully identified and separated under the microscope.2.On the 6th day of cell culture,obvious cell processes and synaptic connections were observed under the microscope.3.On the 6th day of cell culture,Nkx2.1,a specific marker of precursor cells,was co-localized with MAP2,GAD67,a dendritic marker of neurons.4.Teasaucer-like or spherical granules with diameters of 40-100 nm were observed under transmission electron microscopy.NTA results supported the above results.CD9 and TSG101 were positive for exosome-specific markers.The experimental conclusions are as follows:In vitro culture of the medial ganglion eminence of E14.5 day fetal rats could form obvious synaptic connections and differentiate into GABA neurons on the 6th day of culture.At this time,tea saucer-like or globular exosomes with complete membrane structure and activity could be isolated from the culture medium.PART II THE EFFECT OF MGE-DERIVED EXOSOMES ON LICL-PILO-INDUCED STATUS EPILEPTICUS AND EPILEPSYAIM: To investigate the effects of MGE-derived exosomes on pilo-induced status epilepticus and chronic epilepsy behavior.Methods:1.Stereotactic injection of exosome or PBS into hippocampus was used to establish pilocarpine-induced status epilepticus mice model and observe comparative behavior.2.After pilocarpine-induced status epilepticus,the hippocampus was injected with exosome or PBS maintenance therapy.The spontaneous seizures were monitored by video.Racine score was used to assess the seizure level,and latency,number of spontaneous seizures and duration of each seizure were counted.The experimental results are as follows:1.In pilocarpine-induced status epilepticus model,the latency of epilepticus persistence in mice injected with exosome into hippocampus was significantly longer than that in the control group(* P < 0.05,** P <0.01);there was no significant difference in the latency of epilepsy persistence between the control group and Nave group(P > 0.05).2.In pilocarpine-induced chronic epilepsy model,the number of spontaneous seizures in mice injected with exosome into hippocampus was significantly lower than that in control group,and the latency was significantly prolonged(* P < 0.05,** P < 0.01).There was no significant difference in the duration of each spontaneous seizure(P > 0.05);compared with Nave group,there was no significant difference in latency,frequency and duration of each seizure between the control group and Nave group(P > 0.05).The experimental conclusions are as follows:MGE-derived exosomes prolong the latency of pilocarpine-induced status epilepticus.Repeated exosome intervention during the development of chronic epilepsy can effectively prolong the latency of the first spontaneous seizure and reduce the number of spontaneous seizures.PART III: THE EFFECTS OF TSP-1 ON BEHAVIOUR AND FIELD POTENTIAL OF KAINIC ACID-INDUCED CHRONIC EPILEPSY MODEL AND PENTYLENETETRAZOL-INDUCED CHRONIC KINDLING MODELOBJECTIVE: Our team found that the expression of TSP-1 in serum exosomes of epilepsy patients was decreased.The expression of TSP-1 in brain exosomes of epilepsy patients and experimental animals was measured simultaneously.The results were consistent with those in blood.This part will discuss whether TSP-1 affects the onset of epilepsy.Methods:1.Intrahepatic viral injection: The expression of 5’-ga CGCATGACTG CAACAAGAA-3’ was down-regulated or up-regulated by inhibiting or enhancing the transcription level of target protein.Adult male C57BL/6mice were randomly divided into five groups.The mice were injected with TSP-1 RNA interference virus(LV-sh-TSP-1),Con-LV-sh-TSP-1,TSP-1overexpression virus(LV-TSP-1)and Con-LV-TSP-1(0.5ul each in unilateral hippocampus)by stereotaxis.The control group was injected with the same volume of physiological saline.。2.Establishment of kainic acid model: When the virus came into effect(10 days after virus injection),the right hippocampus of each group was injected with kainic acid(200 ng)to induce status epilepticus.The seizures of the model mice were monitored by behavioral video,and the local field potentials in the hippocampus were recorded after 4 weeks.3.On the basis of step 1,the mice in each group were kindled by intraperitoneal injection of a subthreshold dose of PTZ(35mg/kg)once every 48 hours,15 times in total.The classification of seizures during kindling,the total number of seizures above grade 4 and the latency of the first four-stage seizures were observed and compared.The experimental results are as follows:1.During the formation period of chronic spontaneous seizures(SRS)induced by kainic acid,the behavioral observation showed that compared with the empty virus group,the latency of the first spontaneous seizures in LV-sh-TSP-1 group was significantly shortened,and the total number of seizures was significantly increased;the latency of the first spontaneous seizures in LV-TSP-1 group was significantly prolonged,and the total number of seizures was significantly reduced.The results of local field potential recording in hippocampus showed that the total number and total time of seizure-like events(SLEs)in LV-sh-TSP-1 group increased significantly in 30 minutes compared with the empty virus group;the total number and total time of SLEs in LV-TSP-1 group decreased significantly,and the duration of single epileptic discharge did not change significantly,which was consistent with the behavioral performance.2.During the formation period of chronic spontaneous seizures induced by pentylenetetrazol,behavioral observation showed that the average seizure grade of LV-sh-TSP-1 group was higher than that of Con-LV-sh-TSP-1 group at each time point(* P < 0.05),the total number of seizures of stage 4 and above was significantly increased(** P < 0.01),the latency of the first stage 4 seizure was significantly shortened(*** P <0.001),and the LV-TSP-1 group showed the opposite result.3.After observing the behavioral and local field potentials,the hippocampal tissues of mice in each group were extracted.Western Blot was used to detect the persistent effect of the virus.Compared with the empty virus group,the expression level of TSP-1 protein in LV-sh-TSP-1group decreased,and the expression level of TSP-1 protein in LV-TSP-1group increased,which was consistent with the expression of behavioral and local field potentials(*P<0.05).The experimental conclusions are as follows:1.Stereotactic injection of virus into hippocampus can significantly change the expression level of TSP-1 protein in hippocampus,and its regulatory effect will continue until the end of the experiment.2.It was confirmed by two animal models that the change of TSP-1expression could affect seizures,reduce the expression of TSP-1,aggravate seizures,and increase the expression of TSP-1 to alleviate seizures during the development of epilepsy.