Primary Study On Anti-epilepsy Effects And Michanism Of Resverotral In Kainic Acid-induced Epilepsy Rat

ObjectiveThe rats were induced temporal lobe epilepsy(TLE) by Kainate acid(KA) injected into the center site of CA3 region of right hippocampus with the stereotaxic technology.The following chronic pharmacological experiments were carried on this model.Three levels experiments(behavior monitoring,intracalvarium electroencepholography recording and histological analyses) were used to evaluate the efficiency and analyse the mechanism of resveratrol(Res) in anti-epilepsy field.Methods1.KA-induced TLE.Adult male Wistar rats(220-260 g and clean stage) were used in the experiments.Under chloral hydrate(350 mg/kg,i.p.) anesthesia,rats were placed on the stereotaxic apparatus and 2.5μl KA(0.04μg/μl) was slowly injected(about 10 min) to CA3 region of right hippocampus(4.0 mm posterior to bregma,4.4 mm lateral to the midline,3.8 mm below dura).The needle was left for 3min and the rats' scalps were sutured.The behavioral progression of KA-induced seizures was scored according to Racine's standard classification.Those rats that could reached at least the class 4 or 5 seizures were used in this study.The same volumes of normal saline to the same site were injected in the control group.2.Grouping.All the rats in the experiments were divided randomly into the following five groups:Normal Saline(NS) group,KA and Vehicle Solution(VS) group,KA and Sodium Valproate(VPA,300mg/kg/d) group,KA and low dosage of Res(LRes, 15mg/kg/d) group,and KA and high dosage of Res(HRes,30mg/kg/d) group.The drugs were filled into stomach once a day after the first onset of seizure,and continuance ten days.3.Behavior monitoring.Two weeks after KA injection,the spontaneous seizures number of rats was recorded in 2 monthes(8 h/d,5 d/week,40 h/week) by video camera.4.Intracalvarium electroencepholography recording(IEEG).After the behavior monitoring,under chloral hydrate(350 mg/kg,i.p.) anesthesia,two electrodes of stainless steel for recording electrode and one for reference electrode were stereotaxically implanted in the right and left of CA3 region and the dura of prefrontal lobe,respectively.Data were collected by an electroencepholography recording amplifier for 120min.Thirty minutes of EEG data were randomly chosen for analysis. The number of epileptic-form waves,such as sharp wave and spike wave,was calculated.5.Nissl and Timm stainings.After the EEG recording,rats were deeply anesthetized by an overdose chloral hydrate and perfused transaorticly with the modified fixation procedure.Then,the brain was removed and postfixed overnight immersed in 30% sucrose at 4℃for 2-3 d.The hippocampus was cut in 20μm thin for histological analyses.Nissl staining evaluated the degenerating neurons of CA1,CA3 and dentate gyrus.Timm staining evaluated mossy fiber reorganization in the inner molecular layer of dentate gyrus that accompanied epileptogenesis.6.Statistical analysis.The difference of spontaneous seizures,the number of epileptic waves in 30 min IEEG and the extent of mossy fiber sprouting(MFS) were compared between the groups.The SPSS 13.0 software was performed in all statistical analyses. Values were expressed as mean±SEM.Results1.The results of behavior:In the NS group,no rats appeared spontaneous seizures.In VS,VPA,LRes and HRes groups,9 of 12,1 of 7,1 of 7 and 4 of 6 rats appeared spontaneous seizures,respectively. 2.The results of IEEG:In LRes(n=7,P＜0.01) and VPA(n=6,P＜0.01) group,the frequency of epilepitic discharge was significantly less than that of in VS group,and the inhibiting effect on epileptic discharge was no different.3.The results of histology:Lots of neurons were lost in the CA1,CA3,and hilus region detected by Nissl staining in VS group.Administration of VPA just markedly protected neurons against KA-induced neuron death in CA1 region(n=7,P＜0.05). Administration of low dosage of Res markedly protected neurons in CA1 region as well as CA3a region,but did not protected neurons in CA3b and hilus regions(n=7,P＜0.05).Administration of high dosage of Res had no significant protection.Timm staining shows that mossy fibers invaded the granule cell layer and the third of inner molecular layer.But in NS group,neither neurons lost nor mossy fiber sprouting(MFS) appeared.The MFS were significantly inhibited in LRes group(n=7,P＜0.05),which was better than that of in VPA group(n=7,P＜0.05).However,the MFS were not inhibited in HRes group.Conclusion1.VPA can protect the pyramid cells in CA1 region against KA-induced neuronal cell death.As well as inhibit the MFS in TLE animal model.2.Low dosage of Res markedly reduced the severity of KA-induced neuronal cell death in CA1 and CA3a regions,and the MFS is also inhibited in TLE animal model.3.Low dosage of Res has the therapeutic effect the epilepsy generation and progression on the KA-induced TLE animal model...