The Effect Of NR2B On The Epileptogenesis Of In The Epileptic Model Rat Induced By Lithium Chloride-pilocarpine

PartⅠThe drug Control and Analysis of Lithium chloride-pilocarpine induced animal model of epilepsyObjective To search for the methods of reducing mortality and improving the success rate of modeling,when establishing of lithium chloride-pilocarpine chemical-induced epilepsy model.Methods In accordance with the use of the dose of lithium chloride(mg/kg) /the dose of pilocarpine(mg/kg)and antiepileptic agents,69 Wistar male rats were randomly divided into four groups:Ⅰ:125/10/chloral hydrate Group,Ⅱ:135/20/ chloral hydrate Group,Ⅲ:125/30 / chloral hydrate group,Ⅳ:135/20/diazepam group. Atropine 1 mg/kg intraperitoneal injection after Lithium chloride intraperitoneal injection within 18-20 h,then,half an hour later,pilocarpine given in accordance with different groups,ended it with diazepam or chloral hydrate after one hour of status epilepticus.Results Within 6.05 min±4.07 min,after the intraperitoneal injection of pilocarpine,rats developed the signs of cholinergic stimulation.Episodes of head and bilateral forelimb myoclonic movements with rearing and falling stared and progressed to SE at around 22.31 min±5.99 min after pilocarpine administration.The 30min of SE were characterized by long-lasting,sustained clonic seizure activity that reached Stage 4 on Racine,s scale for seizure severity.Following 1 hour after SE, antiepileptic agents administrated in according with different groups,rats didn't stop seizure activity within 30 min,which were injected half of the dosage.If necessary, antiepileptic agents repeated after eight hours.During the 4-20d of latent period, clinical symptom mainly show listlessness,decreased appetite,emaciation,but no spontaneous epileptic seizures performance.During the 21d-60d chronic phase,about 80%of rats developed spontaneous seizures performance,which associated episodes of focal epileptic activity,progressed to general tonic-clonic attack.They usually lasted for 20 sec-1 minute,then developed into a self-stop process.There were no statistical significance in the four groups at the onset of starting seizures and reaching the stage 4,5 and SE.To compare modeling success rate and mortality amongⅠ,Ⅱ,ⅢGroups,P＜0.05,GroupⅡof the highest modeling success rate(85%),the lowest mortality rate(23.5%).Between GroupⅡand GroupⅣ,P＞0.05,but the mortality suggested that effect with the chloral hydrate than that with diazepam,probably the samples were too small.The mortality of epileptic rats was positive linear correlation with the dose of pilocarpine.Conclusion In order to improve the modeling success rate and guarantee the number of survivors,the dose 135/20 mg / kg of lithium chloride-pilocarpine intraperitoneal injection combined with chloral hydrate should be recommended.PartⅡThe effect of ifenprodil on the characteristics of electroencephalogram recording in the epileptic model rat inducedObjective:To explore the characteristics and the effect of EEG(electroencephalogram)with the time,which Ifenprodil intervented LiC1-Pilo induced epilepsy in rats.Methods:Totally 90 Wistar male rats were randomly divided into three groups: control group,model group and Ifendilprol intervention group.To established animal model of epilepsy by intraperitoneal injection of Li-PILO(lithium-pilocarpine).To observate the characteristics of the EEG on 1^st,3^rd,7^th,15^th,30^thand 60^thdays after the SE(experimental status epilepticus),five rats at each time point.Results:There were no seizures in the control group.Contrasted with the rats in the model group,in the intervention group the latent periods of the seizures were longer,the frequency and the peak amplitude of the seizures were reduced,and the epileptic discharges exhibited a reduction,which indicated signification difference between the model and intervention group(P＜0.01),the characteristic EEG presented changes in the proportion and shape of kinds of bands at different times.During the latent period,the frequency and the peak amplitude of seizure were reduced suddenly in the intervention group and model group,the frequency and the amplitude gradually increased more in the intervention group than those in the model group,which in the intervention group normalized faster and sooner than those in the model group.Conclusion:1,In the model group,The epileptic discharges of EEG were characterized by reqular rhythm in the different phases.2,Ifenprodil has an anticonvulsant effect on the LiCl-Pilo induced epilepsy in rats,which reduced the the frequency gradually and the amplitude,normalized background waves faster and sooner than those in the model group.PartⅢThe expression of NR2B,GAP-43,GFAP and moss fiber sprouting(MFS)in hippocampus of the epileptic ratsObjective:To explore the mechanisms of the recurrent attacks of seizures and synapse reorganization through observing the expression of NR2B,GAP-43,GFAP and MFS in hippocampus of the epilepsia rats.Methods:Totally 140 Wistar male rats were randomly divided into two groups: control group and model group.To established animal model of epilepsy by intraperitoneal injection of Li-PILO(lithium-pilocarpine).The expression of NR2B, GAP-43,GFAP and the neuronal survivor in hippocampus were investigated by immunohistochemistry and hematoxylin and eosin stain and MFS in hippocampus by Timm staining method.Results:1,NR2B expression was increased in each area of hippocampus at 1h after SE, reached peak level at 6h,began to decrease to 7d,then gradually increased again to 2m.NR2B expression in the Area CA1 was higher than that in Area CA3.The expression in the dentate gyrus was the lowest.2,The regular patterns in the time and situs of GFAP expression were similar to the NR2B expression.Glial nodules were observed in Area CA1 during 30d∽60d. There was negative correlation by spearman analysis between the survivors of neuron and the optical density of GFAP in CA1,the same as the survivors of neuron and the NR2B expression.(respective coefficient correlation r=-0.826,-0.73,P=0.000＜0.01).3,GAP-43 protein expression decreased during the acute phase,reached the lowest level in the CA1 and CA3 at 2d,in dentate gyrus at 7d,then decreases.4,MFS gradually increased after 7d of SE,correlated negatively with the survivors of neuron in the hilus.The optical density of GAP-43 protein expression related with losses of neuron in the acute phase,with MFS in the chronic phase.MFS increased gradually with recurrent seizure.There was statistics difference in the model and control group after 7d of SE(P＜0.01).Conclusion:NR2B receptor was involved in the epileptogenesis.To lessen neuronal necrosis and glial proliferation,to inhibit NR2B expression and MFS and formation of abnormal neural networks,those provided a new way to clinic epilepsy treatment and offering a direction of medicine development.PartⅣThe Effect of Ifenprodil on synapse formation in the hippocampus with epilepsia rat modelObjective:To explore the effect of Ifenprodil on synapse reorganization in the hippocampus as well as neuroprotective effect in the epilepsia rat model.Methods:Totally 210 Wistar male rats were randomly divided into three groups: control group,model group and Ifendilprol intervention group.The control and model group rats were intraperitoneal injected with physiological saline for 1 week,the intervention group was pretreated with Ifendilprol for 1 week before the animal model of epilepsy was established.After the epilepsia model was established,the drug went on injection.The rats were decapitated at diffence time point in according with protocol.The expression of NR2B,GAP-43,GFAP,MFS and the neuronal survivor in hippocampus were investigated in hippocampus of each group.Results:1,the neuronal survivor:There existed statistical difference in CA1 at every time point between the model group and intervention group(CA1:p＜0.05,at 1h and 15d after SE;p＜0.01,at other time points.The Area hilus:P＞0.05,at 1h,6h,2d,7d;P＜0.05,at 15d;P＜0.01,at 30d and 60d).2,NR2B expression:NR2B expression pattern was similar between the intervention group and model group,which in the Area CA1 was higher than that in Area CA3.The expression in the dentate gyrus was the lowest.There was no statistical difference in CA1 at 15d and in CA3 at lh and 15d after SE between them, reversely,at other time points in CA1 and CA3(P＜0.01).3,GFAP expression:GFAP expression pattern was similar between the intervention group and model group.Glial nodule wasn't formed in CA1 and CA3. There was no statistical difference in the hilus at 1h and in CA1 at 2d after SE between them,reversely,at other time points(The hilus:P＜0.05 at 15d,30d;P＜0.01, at other time points)(CA1:P＜0.05 at 7d;P＜0.01,at other time points).4,GAP-43 protein expression:The GAP-43 protein expression pattern intervention group was opposite to that in model group.There was no statistical difference at 60d in CA3,reversely,at other time points in CA3(P＜0.01)and at each time point in the hilus(P＜0.05,at 30d;P＜0.01,at other time points).There appears quadratic curve between the optical density of GAP-43 protein in inner molecular stratum of dentate gyrus and the number of survival neuron in the hilus.5,The result of Timm staining:There was no statistical difference about moss fiber sprouting at all of time points in hilus and CA3 between intervention group and model group.MFS mark and the number of neuron survivor negatively correlated by spearman analysis in hilus in the two groups,respectively.(respective coefficient correlation r=-0.791,-0.891,P=0.000＜0.01).Conclusion:1.Ifenprodil mitigated the neuron damage and then reduced the nerve cell death. during the recurrent seizures.2.Ifenprodil could have the function of restraining the seizure and significantly reduce the expression of NR2B.3.Ifenprodil inhibited the expression of GFAP,reduces the glial cells hyperplasia and inhibits formation of the Glial nodule.4.Ifenprodil promotes the expression of GAP-43 protein,which should be further studied on the anti-experimental epilepsy action of ifenprodil and its mechanism.5.Ifenprodil had no inhibition effect on MFS.