| Epilepsy, a brain dysfunctional syndrome caused by abnormal dischargeof neurons with many reasons, is characterized by temporary and recurrentdisorder of brain functions, which can be expressed in movement, sensation,consciousness, spirit, and other aspects of dysfunction. At present, there are atleast7million patients with epilepsy in our country. Among this population,approximately20%of epileptic patients do not respond adequately to theanti-epileptic drug, which remains a knotty problem for modern clinicalmedicine. Recurrent and long-term seizures not only seriously affected thepatients'life quality, but also brought immence burden and pressure to theirfamily and society.Currently, the surgery is mainly taken to treat the refractory epilepsy, butonly the onset of epileptic focus can be removed by the surgical excision, withno complete change to the neuronal hyperexcitability and abnormal functionof ion channel, while surgery itself have certain risks, such as anesthesiaaccidents, bleeding, infection, limb paralysis and so on. With this betterunderstanding of the pathogenesis of epilepsy and the development ofmolecular biology techniques, gene therapy to completely cure epilepsy bringsnew hope to the patient with epilepsy.The data shows that neuropeptide Y maily located in the cerebral cortex,hippocampus, thalamus, hypothalamus and brain stem in the central nervesystem, particularly in the hippocampus of the highest concentrations, NPYand its receptors had closely connection with the origination of epilepsy withbeen focused on widely. Because the complicated mechanism in differentepileptic model, the experimental results were always inconsistent or contradictory with each other due to the different animal models and methodof administration.In this study, neuropeptide Y was considered as the treated gene, andrecombinant adeno-associated viral was acted as vectors. After the NPY genewas transfered to the specific target in the brain tissue (In this study, thepreceding studies have demonstrated that rAAV2/1-NPY-EGFP in epilepticbrain tissue under pathological conditions can be expressed effectively. Theinjected approach to ventricle may be better than the hippocampus), the effectsof NPY overexpression on seizures in chronic epileptic rat induced by kainicacid were observed and further PCR, Immunohistochemistry, were used toanalyze the the biological mechanisms of NPY regulation to seizures.Objective: To observe the effects of gene transfection of rAAV2/1-NPY-EGFP on rat seizures, EEG and the expression of hippocampal p35, and toexplore its role in the pathogenesis of epilepsy, as well as the possible mecha-nism of the NPY gene therapy of chronic epilepsy.Methods: Altogether72healthy male Wistar adult rats were randomlydivided into a control group and a kainic acid (KA) treated group. Theepileptic models were established by the injection of KA1.5μl (0.4μg/μl)five times to hippocampus CA3area while the control rats were injected withan equal dose of saline. Then KA treated group were randomly divided intoKA group (n=24) and rAAV2/1-NPY-EGFP group (n=24).(In this study, thepreceding studies have demonstrated that rAAV2/1-empty-EGFP had nosignificant effect on the experimental results). The rat died in making modelsor didn't conform to the standard was rejected. Each group had24rats.rAAV2/1-NPY-EGFP group, in which10μl of rAAV2/1-NPY-EGFP(titer5×1011v.g./ml) were injected to ventricle in successful rats chronic model,while KA group were injected with an equal dose of saline. At2w and4wafter vector injection respectively, the seizure situation, the onset latency andEEG wave frequency and amplitude were observed. Then rats in3groupswere killed, hippocampal p35mRNA were detected with quantitative PCR andthe expression of p35protein were also done by Immunohistochemistry. Results:1The seizure degree in rats of rAAV2/1-NPY-EGFP groupgradually reduced with observation time; at4w post-injection, in rats ofrAAV2/1-NPY-EGFP group, the onset of symptoms and onset latencysignificantly reduced (P<0.05), EEG epileptic discharge frequency decreased(P<0.05), compared to rats of KA group.2There was significantly increasedof NPY expression at2w and4w post-injection in the KA group and therAAV2/1-NPY-EGFP group compared with the control group (P<0.05). At4wafter vector injection, expression of NPY mRNA and protein in rAAV2/1-NPY-EGFP group were higher than the KA group.3At2w and4w post-injection, hippocampal p35mRNA and protein expression in KA group weresignificantly increased compared with the control group with the significantdifference (P<0.05). The expression of mRNA and protein of hippocampalp35in rAAV2/1-NPY-EGFP group decreased significantly at4wpost-injection, compared to the KA group (P<0.05).Conclusion:1rAAV2/1-NPY-EGFP gene transfection can inhibitseizures, which provides strong support to the NPY gene therapy for clinicalrefractory epilepsy.2rAAV2/1-NPY-EGFP gene transfection inhibits theexpression of p35in epileptic rat hippocampus. NPY may play anti-epilepticand neuroprotective effects through downregulation to the expression ofhippocampal p35. |
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