Study On The Mechanism Of VNS Improving Learning And Memory Ability In Epileptic Rats

Objective: Epilepsy is a common neurological disorder,and studies tracking the phenotype of patients with epilepsy have found that not only do seizures cause short-lived violent convulsions and loss of consciousness,but that recurrent seizures also cause significant structural changes in the medial temporal lobe nuclei,such as the hippocampus and amygdala,leading to cognitive and emotional impairment.Currently,medication is the mainstay of clinical treatment for epilepsy,while surgical removal of the epileptogenic focus is usually the only way to reduce seizures in refractory epilepsy.Vagus Nerve Stimulation(VNS)is an adjunctive treatment for inoperable refractory epilepsy that reduces seizures by applying continuous electrical stimulation to the vagus nerve(neck).Some studies have demonstrated that VNS has significant effects on cognitive function in addition to its anti-seizure effects.However,the mechanisms underlying the improvement of patients’ learning and memory abilities by VNS for epilepsy are not yet clear.In this study,we established a rat model of epilepsy and observed the improvement of learning and memory ability of epileptic rats after VNS treatment and explored the possible mechanism of action.Methods: 1.Grouping: Adult male Sprague-Dawley(SD)rats were divided into 5 groups: Control group(n=8),epilepsy group(n=8),VNS group(EPI+VNS)(n=10),antagonist intervention VNS group(EPI+ PR+VNS)(Propranolol,PR,Beta-adrenergic receptor antagonist)(n=5),agonist treatment for epilepsy group(EPI +ISO)(Isoproterenol,ISO,beta-adrenergic receptor agonist)(n=3).2.Animal model: lithium chloride-Pilocarpine epileptic rat model.3.Operation: VNS implantation and drug tube implantation.4.VNS:(1)Chronic stimulation: Rats in EPI group,EPI+VNS group and EPI+ PR+VNS group were subjected to VNS,and each rat was stimulated once a day for 2 hours for 2 weeks(EPI group was stimulated without electricity).Conditioned fear experiment and WB experiment were performed after the end of stimulation.(2)Acute stimulation: The rats in Control group,EPI group and EPI+VNS group were subjected to VNS,and each rat was stimulated for only 2 hours before the in vitro field potential test.5.Behavioral detection indicators: The changes in learning and memory ability of each group were observed and compared through Fear Conditioning Test(FCT).6.Semi-quantitative analysis of protein: Western Blotting(WB)was used to detect the expression of synaptic plasticity related proteins related to learning and memory,such as β2-adrenergic receptor,β2-ARs),protein kinase A(PKA).7.Electrophysiological detection indicators: LTP changes of rats in Control group,EPI group and EPI+VNS group were detected by the experiment of Long-term potentiation(LTP).Results: 1.Behavioral science: FCT showed that the episodic fear memory of the EPI group was significantly reduced compared with the NC group,the episodic fear memory of the EPI+VNS group was significantly enhanced compared with the EPI group,and the episodic fear memory of the EPI+VNS+PR group was significantly reduced compared with the EPI+VNS group.2.Protein quantitative analysis: the protein expressions of β2-ARs and PKA in the EPI group were significantly lower than those in the Control group;The protein expressions of β2-ARs and PKA in the EPI+VNS group were significantly higher than those in the EPI group.The protein expressions of β2-ARs and PKA in the EPI+VNS+PR group were significantly lower than those in the EPI+VNS group.The expression of β2-Ars protein in the EPI+ISO group was significantly higher than that in the EPI group.3.In vitro field potential experiment: LTP level of the EPI group was significantly decreased compared with the Control group;LTP levels in the EPI+VNS group were higher than those in the EPI group.Conclusions: 1.In the epileptic rat model of lithium chlorine-Pilocarpine,the learning and memory ability of epileptic rats was significantly reduced through behavioral observation,and the learning and memory ability of epileptic rats could be improved to some extent by continuous 2-week treatment with VNS.2.In the epileptic rat model of lithium chlorine-pilocarin,when β2-adrenergic receptor antagonist was injected into hippocampal CA1 region of epileptic rats,the improvement effect of VNS on learning and memory ability of epileptic rats was blocked,and the expression levels of β2-Ars and PKA protein were significantly decreased in the WB experiment.It is suggested that VNS may improve the learning and memory ability of epileptic rats by acting on β2-ARs.3.After VNS treatment,the expression of β2-ARs and PKA proteins in hippocampus of epileptic rats was significantly increased,which had a certain regulatory effect on synaptic plasticity,and thus improved the learning and memory ability of epileptic rats.4.In vitro field potential test,LTP level in EPI group was significantly decreased compared with that in Control group,and LTP level in epileptic rats was significantly increased after 2 hours of acute VNS treatment,suggesting that VNS regulates synaptic plasticity and enhances memory consolidation in rats.