The Effects And Potential Mechanisms Of EphB3 On Epileptic Seizure And Epileptogeneisis

Epilepsy is one of common serious chronic neurological disease characterized by recurrent spontaneous seizures.Although a wide range of antiepileptic drugs were used in clinical,up to 20-30% of epilepsy patients still have recurrent seizures because of unknown epilepsy pathogenesis.Therefore,further studies of the epilepsy pathogenesis will be beneficial to the prevention and treatment of epilepsy.More and more studies showed that abnormal neural network plays an important role in the pathogenesis of epilepsy.Eph B/ephrin B is a kind of nerve axon guidance factors,which is one member of tyrosine family.Interaction with each other,they have an important role in neuron axon guidance,specific synaptic connections,neuron dendritic structure of central nervous system.To identify possible related subtypes of epileptogenesis,this study mainly studied the expression levels of different Eph B/ephrin B isoforms in the brain samples of TLE patients and rats.Through electrophysiology,molecular biology,ethology and morphological examination,we investigated the effects and potential mechanisms on epileptic seizure and epileptogeneisis in magnesium free induced hippocampal slices epilepsy model and two classical chronic epilepsy animal model intervened by functional recombinant fusion protein and lentivirus-mediated gene transfection technology,which would provide new ideas and new ways for epilepsy pathogenesis,and provide experimental evidence for seeking new clinical treatment targets and new antiepileptic antiepileptic drug development.Part one: The expression of Eph B/ephrin B in intractable temporal lobe epilepsy patients and epilepsy rats modelObjective: To identify the possible subtypes of Eph B/ephrin B related with epileptogeneisis,the expression of Eph B/ephrin B were detected in the brain tissues of refractory temporal lobe epilepsy patients and epilepsy rat induced by lithium chloride-pilocarpine,Method: 1.Twenty intractable TLE temporal neocortex samples and control twenty histologically normal temporal neocortex samples were selected randomly from brain tissue bank of neurosurgery of Xinqiao Hospital,the second Affiliated Hospital of the Third Military University.2.SD adult male rats were randomly divided into control group and model group(n = 10 per group),model rats were given an intraperitoneal lithium chloride-pilocarpine.3.The expression of Eph B/ephrin B in the epilepsy brain tissues were detected by RT-q PCR,and screened the subtypes which mRNA expression were consistent in brain tissues,then verified their protein expression by Western blot and immunohistochemistry.Followed their functional protein phosphorylation level were detected by Western blot,and double-labeled immunofluorescence was utilized to determine its cell distribution.Results: 1.The EphB3 and ephrin B3 RNA expression were up-regulated in the intractable TLE patients and rats(p<0.05),while the Eph B1/2/4/6 and ephrin1/2 mRNA expression were inconsistent between TLE patients and rats.2.Western blot and immunohistochemistry verified the EphB3 and ephrin B3 protein expression were also up-regulated in the TLE patients and rats(p<0.05).3.Functional p-EphB3 protein level increased in the epilepsy specimens(p <0.05),while p-ephrin B protein level did not change(p > 0.05).4.Double-labeled immunofluorescence showed that EphB3 was expressed in the cell membrane and cytoplasm of neurons.Conclusion: Up-regulated expression of EphB3 and p-EphB3 in the brain of intractable TLE patients and lithium chloride-pilocarpine induced epilepsy rats suggested that EphB3 might be involved in the epileptogeneisis.Part two: The effects of EphB3 functional changes on neuronal excitability of rat hippocampal slicesObjective: In order to investigate the effects of EphB3 on epileptic seizures,we assessed the effects of EphB3 functional changes on neuronal excitability by whole cell patch clamp technique in the hippocampal slices epilepsy model induced by magnesium free artificial cerebrospinal fluid(ACSF).Method: Healthy male SD rats were decapitated after anesthesia,the brain tissues were removed immediately and were produced into hippocampal slices,based on the normal ACSF perfusion,brain slices was induced epilepsy model in the magnesium-free ACSF.Then EphB3 activity regulator functional recombinant fusion proteins(inhibitor EphB3-Fc,agonist ephrin B3-Fc)and control Fc were added to the perfusate of magnesium-free ACSF,and the neuronal action potential(AP)frequencies were detected by the whole-cell patch-clamp before and after the intervention(n = 6 per group).Results: Neuronal AP frequency was evidently enhanced in the magnesium-free ACSF(p <0.05);EphB3-Fc(10nm and 100nm)reduced the magnesium-free induced neuronal AP frequency(p <0.05);ephrin B3-Fc(10nm and 100nm)increased magnesium-free induced neuronal AP frequency(p <0.05);while Fc(10nm and 100nm)had no effect on the magnesium-free induced neuronal AP frequency(p> 0.05).Fusion proteins(EphB3-Fc,ephrin B3-Fc 10nm)and control Fc had no effect on neuronal AP frequency in normal ASCF environment(p> 0.05).Conclusion: Hippocampal slices epilepsy model could be induced by magnesium-free ASCF.Inhibited EphB3 activity could reduce the magnesium-induced neuronal excitability,activated EphB3 could increase magnesium-induced neuronal excitability,which suggested EphB3 functional changes could affect epileptic seizures.Part three: The effects of EphB3 functional changes on ethology of epilepsy rats and related Rho GEF gene expressionObjective: In order to further clarify the impact of EphB3 on epileptic seizures and epileptogeneisis.we observed the ethology of rats,mossy fiber sprouting(MFS)of hippocampal dentate gyrus,dendritic spine density of inner molecular layer(IML)and related Rho GEF gene expression induced by EphB3 functional changes on two classic animal chronic epilepsy models.Method: Healthy male SD rats were randomly divided into three large groups including functional recombinant fusion protein intervention,lithium chloride-pilocarpine induced epilepsy model and pentylenetetrazole kindling epilepsy model after a week recovery of embedded intracerebroventricular catheter.Functional recombinant fusion protein intervention group was randomly divided into four sub-groups including ephrin B3-Fc group,EphB3-Fc group,control Fc group and normal control PBS group(n = 5 per group),ephrin B3-Fc,EphB3-Fc,Fc and PBS were injected through the lateral ventricle catheter respectively and continuous intervented for a week.In the 7th,14 th,30th and 60 th day after intervention,the rats decapitated after anesthesia and the brains were removed immediately,EphB3 and p-EphB3 proteins expression in hippocampus of each group were detected by Western blot.Epilepsy model group was randomly divided into four sub-groups including model control(PBS)group,model + Fc control group,model + ephrin B3-Fc group and model + EphB3-Fc group(n = 10 per group),fusion proteins(Fc,ephrin B3-Fc and EphB3-Fc)and sterile PBS were injected through the lateral ventricles catheter respectively with the same volume,and establish model after a week of continuous intervention.We observed the acute seizure classes,acute latency and spontaneous recurrent seizures times of chronic period in Li Cl-pilocarpine model,and observed the daily seizure classes and the time of fully kindled in pentylenetetrazole model.MFS in dentate gyrus of epilepsy rats were detected by Timm staining,the density of dendritic spines in IML of epilepsy rats were detected by Golgi staining,related Rho GEF genes(Kalirin,Intersectin1 and Intersectin2)expression in hippocampus of epilepsy rats were detected by RT-q PCR and Western blot.Results: 1.EphB3-Fc decreased p-EphB3 protein expression of hippocampus in a long time(7-60d)(p <0.05),ephrin B3-Fc increased p-EphB3 protein expression of hippocampus in a long time(7-60d)(p <0.05),while the Fc had no effect on the expression of p-EphB3 protein of hippocampus(p > 0.05).2.In Li Cl-pilocarpine model,EphB3-Fc decreased the acute seizure classes,prolonged acute latency and reduced spontaneous recurrent seizures times of chronic period(p <0.05),ephrin B3-Fc increased the acute seizure classes,shortened the acute latency and increased spontaneous recurrent seizures times of chronic period(p <0.05).At the same time,in the pentylenetetrazol kindlinged epilepsy rats,EphB3-Fc reduced seizure classes(11-33d)and prolonged seizure latency(p <0.05),ephrin B3-Fc increased the seizure classes(9-29d)and shortened seizure latency(p <0.05).while Fc had no effect on he above ethology of Li Cl-pilocarpine and pentylenetetrazol induced epilepsy rats(p> 0.05).3.EphB3-Fc reduced the degree of dentate gyrus MFS and decreased the density of dendritic spines in IML of epilepsy rats,ephrin B3-Fc aggravated the degree of dentate gyrus MFS and increased the density of dendritic spines in IML of epilepsy rats(p <0.05),Fc had no effect on the degree of dentate gyrus MFS and the density of dendritic spines in IML of epilepsy rats(p > 0.05).4.In the hippocampus of epilepsy rats,functional fusion proteins had no effect on the mRNA expression of Intersectin1 and Intersectin2(p> 0.05).EphB3-Fc reduced the mRNA and protein expression of Kalirin,ephrin B3-Fc increased the mRNA and protein expression of Kalirin(p <0.05),while Fc had no effect on the mRNA and protein expression of Kalirin(p > 0.05).Conclusion: 1.Functional recombinant fusion proteins(ephrin B3-Fc and EphB3-Fc)could affect the function of EphB3 in a long time(7-60d).2.EphB3 functional changes could affect the acute seizure classes,the length of latency and spontaneous seizure frequency of chronic phase of epilepsy rats.3.EphB3 functional changes could regulate the MFS in hippocampal dentate gyrus and the density of dendritic spines in IML of epilepsy rats.4.Kalirin may be the potential downstream target of EphB3.Part four: EphB3 participate in epileptic seizures and epileptogenesis through Kalirin pathwayObjective: To explore the possible mechanisms of EphB3 in epileptic seizures and epileptogenesis,we observed the effects of ephrin B3-Fc on ethology of rats,MFS of hippocampal dentate gyrus and dendritic spine density of IML after silenced Kalirin by lentivirus-mediated Kalirin-sh RNA in two classic chronic epilepsy animal models,and to clarify whether EphB3 participate in epileptic seizures and epileptogenesis through Kalirin pathway.Method: Kalirin protein expression was detected in TLE patients and rats by Western blot.Healthy male SD rats were randomly divided into three groups including control group,LV-GFP group and LV-Kalirin-sh RNA group,normal saline,LV-GFP and LV-Kalirin-sh RNA were injected respectively through the lateral ventricle catheter with the same volume.In the 3th,7th,14 th,30th and 60 th day after intervention(7th day group n=10,the remaining groups n=5),part of the LV-Kalirin-sh RNA intervented rats were decapitated after anesthesia and the brains were removed immediately,the green fluorescence expression of rat hippocampal and cortical was observed under the fluorescence microscopy,Kalirin protein expression of rat hippocampus was detected by Western blot.Another portion of rats(n =thirty)were modeled induced by Li Cl-pilocarpine and pentylenetetrazole after a week of intervention(n=5 per group),the ethology of rats were observed,EphB3 and p-EphB3 protein of rats hippocampus expression were detected by Western blot.The remainder rats(n = thirty)were continuous intervened for a week by injection of ephrin B3-Fc through the lateral ventricles catheter,and establish model by Li Cl-pilocarpine and pentylenetetrazole(n=5 per group),then the ethology,MFS in dentate gyrus and the density of dendritic spines in IML of epilepsy rats were observed.Results: 1.Kalirin protein expression was increased in TLE patients and rats(p <0.05).2.Green fluorescence of lentiviru was widely distributed in the cerebral cortex and hippocampus of rats,Kalirin protein expression was decreased from 7th days to 60 th days after injection of LV-Kalirin-sh RNA(p <0.05).3.LV-Kalirin-sh RNA decreased the acute seizure classes,prolonged acute latency and reduced the spontaneous recurrent seizures times of chronic period in Li Cl-pilocarpine epilepsy model(p <0.05),also reduced seizure classes(9-35d)and prolonged seizure latency in the pentylenetetrazol induced epilepsy rats(p <0.05).LV-Kalirin-sh RNA had no effect on EphB3 and p-EphB3 protein expression in hippocampus of epilepsy rats(p> 0.05).4.Silenced Kalirin could decrease the acute seizure classes,acute latency and reduce spontaneous recurrent seizures times of chronic period in Li Cl-pilocarpine epilepsy model and reduce seizure classes(11-29d)and seizure latency in the pentylenetetrazol kindlinged epilepsy rats induced by ephrin B3-Fc(p <0.05).5.Silenced Kalirin reduced the degree of dentate gyrus MFS and decreased the density of dendritic spines in IML of epilepsy rats induced by ephrin B3-Fc(p <0.05).Conclusion: 1.Upregulated Kalirin protein expression in TLE patients and rats.2.Silenced endogenous Kalirin could reduce the acute seizure classes,prolong latency and reduce spontaneous recurrent seizures times in chronic period of epilepsy rats.3.Silenced endogenous Kalirin could reversible the ethology changes and morphological changes in hippocampal neural circuits of epileptic rats induced by EphB3 activation.4.EphB3 participate in epileptic seizures and epileptogenesis through regulating Kalirin pathway and affecting hippocampal neural circuits reconstruction.