The Effects Of Neuropeptide Y Gene Transfection Using Recombinant Adeno-associated Virus Vector On Seizure In Rat Model Induced By Kainic Acid And On The Expression Of Tau Protein In The Rat Hippocampus With Chronic Epilepsy

Epilepsy, a chronic brain nervous system disease caused by abnormaldischarge of neurons with many reasons, is characterized by temporary andrecurrent epileptic seizures. It has bring great distress to people’s lives becauseof its recurrent and long-term seizures. Temporal lobe epilepsy remainsamongst the most common and drug refractory of neurological disorders. Alarge number of studies have shown that genetic factors is very important inthe pathogenesis of epilepsy, refractory epilepsy is associated with Multi-drugresistance genes, so genetic research is the hot spot of the current domesticand international epilepsy research.Currently the foreign epilepsy therapy researches are focused on theapplication of antiepileptic drug therapy, vagus nerve stimulation and surgerytreatment, although the treatment methods and the effects of treatment makesome progress comparing with before, but there is still a considerable portionof refractory epilepsy can’t be effectively controlled, which makes domesticand foreign experts seek a newer solution. Gene therapy might become analternative treatment for drug refractory epilepsy patients.Gene therapy is a new way of disease treatment which developed sincethe1980s. It treats diseases by carrying normal gene or therapeutic genes intotarget cells, to correct genetic defects or synthesis protein of treatmentfunction. The Recombinant adeno-associatedviral (rAAV) vectors is the carrierwhich has no pathological reaction of human host in common viral vectors forhuman gene therapy. Recombinant adeno-associatedviral (rAAV) vectors inparticular show promise for gene therapy of neurological disorders due to theirneuronal tropism, lack of toxicity, and stable persistence in neurons, which results in robust, long-term expression of the transgene.Neuropeptide Y (NPY), neuropeptide tyrosine, is a peptide substancewhich composed of36amino acids. NPY widely located in the central nervesystem and Peripheral nervous system, particularly in the hippocampus of thehighest concentrations, which has anticonvulsive effect. Now as the importantneurotransmitter which gene therapy of epilepsy rats. Tau protein is a kind ofphosphorous glycoprotein which is closely related to microtubule assembly.Phosphorylated tau protein is the product of nerve fiber damage formation.In this study, The specification of epileptic models were established bythe Adeno-associated virus gene transfection technology, neuropeptide Y wasconsidered as the treated gene, and recombinant adeno-associated viral wasacted as vectors. After the NPY gene was transfered to the specific target inthe brain tissue (In this study, the preceding studies have demonstrated thatrAAV2/1-NPY-EGFP in epileptic brain tissue under pathological conditionscan be expressed effectively. The injected approach to ventricle may be betterthan the hippocampus), the effects of NPY overexpression on seizures inchronic epileptic rat induced by kainic acid were observed and Westernblotting were used to analyze the the biological mechanisms of NPYregulation to seizures.Objective: To discuss the effects of gene transfection of rAAV2/1-NPY-EGFP on KA-induced rat seizures, EEG and the expression of hippocampalP-tau protein, and to explore its role in the pathogenesis of epilepsy, as well asthe possible mechanism of the NPY gene therapy of chronic epilepsy.Methods: Altogether72healthy male Wistar adult rats were randomlydivided into three groups (n=24) control group KA group and NPY group. Todetermine the center of hippocampal CA3area on the right side of thehippocampus injection targets which refered the George Painos’s rat brainstereotaxic atlas. The epileptic models were established by the injection of KA2μl (0.4μg/μl) five times to the right side of the hippocampus CA3area everythree days. rAAV2/1-NPY-EGFP group, in which10ul ofrAAV2/1-NPY-EGFP (titer5×1011v.g./ml) were injected to ventricle in successful rats chronic model, while KA group were injected with an equaldose of saline.(In this study, the preceding studies have demonstrated thatrAAV2/1-empty-EGFP had no significant effect on the experimental results).The control group in the hippocampal CA3district and intracerebroventricularinjection of the same amount of saline. We observed the seizure situation, theonset latency and EEG wave frequency and amplitude at2w and4w aftervector injection. Then all the rats were killed, hippocampal total proteinandP-tau protein were detected with Wesrtern blotting.Results:1Behavior Observation Compared with KA group, scale andlatency of each seizure onset in rats of rAAV2/1-NPY-EGFP group have nosignificant difference at2w (P＞0.10). Compared with KA group, the scale ofseizure in rats of rAAV2/1-NPY-EGFP group significantly reduced at4w(P<0.05); latency of seizure onset significantly increased at4w (P<0.05), EEGepileptic discharge frequency and wave amplitude decreased (P<0.05) at4w.The control group had no seizures.2Western blot result compared with thecontrol group, KA group and NPY group rats at2weeks and4weeksphosphorylation of P-tau protein expression was significantly increased (P<0.05); Compared with the KA group NPY group rats at4w hippocampusphosphorylated P-tau protein expression levels were significantly lower(P<0.05).Conclusion:1rAAV2/1-NPY-EGFP gene transfection significantlyreduces scale of seizure onset and prolongs latency of seizure onset inKA-induced rat model.2rAAV2/1-NPY-EGFP gene transfection may playanti-epileptic and neuroprotective effects through inhibits the expression ofP-tau in epileptic rat hippocampus of KA-induced rat model.