K252a Inhibits BDNF-TrkB Signaling Pathway To Effect The Expression Of Arc In Electrical Stimulation Kindled Rats And Epilepsy

Objective Stimulate Sprague-Dawley Rat Amygdala to establish chronic Epilepsy Model, bydrug micro-injection technology,TrkB inhibitor k252a micro-injected into the hippocampus ofepileptic rats,delect active cytoskeleton protein(Arc) in the amygdala of chronic electricalignite the expression in the hippocampus of epileptic rats,explore of BDNF-TrkB signalingpathway between the relationship of the Arc expression and epilepsy in kindled rats ofamygdala stimulation.Methods1.Chronic rat amygdala kindled epilepsy model and drug injection:186healthy maleSprague-Dawley rats,the rats were randomly divided into blank control group,sham operationgroup,amygdala kindling group(kindling group alone,k252a injection group,DMSO controlgroup),(1)blank control rats(36) no treatment,non-implanted electrodes andcasing.(2)Electrode and casing implanted:referrence Paxinos&Watson atlas(electrode:leftamygdala:anterior fontanelle2.8mm,left side next to the open4.9mm,subdural8.6mm;casing:left hippocampus:anterior fontanelle5.6mm,left side next to the open4.5mm,subdural5mm),SR-5R stereotactic-assisted,implanted electrode and casing,recoveryafter7days follow-up experiment.sham-operated rats(36) implanted electrodes and casing,nopower to stimulate the lit.(3)Stimulus intensity from40uA,increase10uA each,stimulateinter-period≥90s,payment of electricity until after the EEG recording to≥3s,the rat after therelease of threshold.Ignite:day after the release threshold to1.5times the intensity to stimulateone,up to five days in a row leads to class V seizures deemed to ignite success.seizuresstrength of grading according to Racine.(4)Drud injection:k252a soluble in DMSO(dimethylsulfoxide),made of the spare of the1ul every solution containing10pmol k252a solution.k252a group ignited after the success of the administration,that is,since the sixdays,every day10minutes before stimulation5ul microinjector k252a inhippocampus,injection1ul each,injecton time of less than1min.Point in time the brains wereremoved10days after stimulation,DMSO control group injected DMSO,the same conditionsas the k252a group,simply ignite the group only five days follow-up to stimulate.2.Activityregulate cytoskeletal protein expression in the electrical stimulaton in the rat amygdalaepilepsy model:according to the experimental requirements at1h,3h,6h three time pointssub-group(n=6) detection of the Arc mRNA expression,3h,6h,12h three point subgroup(n=6)detected the expression of Arc protein.Ether anesthesia rats in each group according to thetime point,90rat brains were removed,rapid stripping of the left hippocampus,transferred to1.5ml EP tube,stored at-80℃，,90rats with PBS and paraformaldehyde after left ventricularperfusion-fixed brain,with4%formaldehyde fixed,sucrose dehydration,liquid nitrogen inOCT-embedded and stored at-80℃refrigerator,specimens by reverse transcriptionpolymerase chain reaction(RT-PCR),methods such as in situ hybridization andimmunohistochemical study of amygdala kindled epileptic rat hippocampus the Arc mRNAand protein expression.Results1.electrical stimulation of the rat left amygdala successfully established chronicelectrical kindling model:modeling ignite a success rate of93.6%,low frequency of normal ratEEG,usually at10HZ,low frequency,low energy,V level in rats attack increased EEGfrequency to30HZ,and energy has been greatly enhanced,detected in rat brain waves recordthe rats in each group EEG,k252a injected rats class V seizureduration(33.00±1.41s),significantlylower than kindling group(60.50±2.07),DMSO controlgroup(60.00±1.79s) rats class V duration of attact(p＜0.01).2.TO molecular biology: The Arcof positive cells percentage increasing at3h. Peaked at6h. Returned to baseline levels at12h.The Arc of positive cells percentage was significantly lower at3h,6h in k252a injection groupthan simple kinding group, DMSO control group(P<0.01).3h,6h,12h Arc expression of positive cells percentage was no significant difference (F=1.000, P>0.05). The expression ofArc positive cells percentage in each group at12h was no statistically significant (F=0.684,P>0.05). control group, sham group no significant difference (P>0.05). The resulted ofRT-PCR, hybridization showing: The expression of Arc mRNA at1h,3h significantlyincreased in simple kindling group and DMSO control group,returned to baseline levels at6h,the expression of Arc mRNA at1h,3h significantly lower in k252a injection group thansimple kinding group, DMSO control group (P<0.01). The defference of expressing of ArcmRNA at1h,3h,6h was no statistically significant in k252a injection group (P>0.05). Thedefference of expression of Arc mRNA at the6h between the groups was no statisticallysignificant (P>0.05). Control group, sham group no significant difference (P>0.05).Conclusions k252a can inhibiting BDNF-TrkB signaling pathyway inducing the expressionof Arc, to reduce the duration of seizure.