| Part one Expression of miR-124in patients with epilepsy and a ratmodelObjective To investigate the expression pattern of miR-124in temporallobe tissue of intractable epilepsy patients and hippocampus of pilocarpineinduced rat models.Methods To detect the miR-124expression pattern in the temporal lobetissue of IE patients, surgically removed temporal neocortex of patients withTLE (n=10) and the control samples (n=6) from histologically normaltemporal lobes both were examined by quantitative real-time PCR(qRT-PCR). Meanwhile, adult male SD rats (n=48)were used for theseexperiments and were randomly divided into control group and epilepticgroup. The epileptic group were injected with pilocarpine hydrochloride toinduce the sustained seizures and killed at6H,1day,2days,3days,7daysfollowing the onset of SE. Control rats (n=8) were treated identically butreceived normal saline (NS) instead of pilocarpine. QRT-PCR was used toinvestigate the expression pattern of miR-124in hippocampus of differentgroups of pilocarpine induced rat models. Results1. Expression of miR-124in temporal neocortical of TLE patients:miR-124expression was downregulated in temporal neocortical of TLEpatients (P>0.05).2. Expression of miR-124in TLE rat models: In pilocarpine-induced ratmodels of epilepsy, miR-124dynamically decreased in a range of7days(P>0.05).Conclusion miR-124expression was downregulated in both intractableTLE patients and TLE rat models, thus, our results indicate that miR-124may be involved in epileptogenesis.Part two Effect of miR-124agomir on epileptic seizure activityObjective To investigate the in vivo effects of miR-124on pilocarpineinduced epileptic seizure of rat models by intracranial injection of chemicalsynthesized miR-124agomir.Methods Sprague-Dawley rats were randomly divided into controlgroup (NC group), agomir control group (injected with scrambled versionRNA)(AC group) and agomir group (AG group). The agomir group wasagain divided into three subgroups, A. microRNA agomir o.2nmol, B.microRNA agomir o.6nmol, C. microRNA agomir1.0nmol. Real-timereverse transcription PCR (qRT-PCR), and immunofluorescence were used to explore the interfere efficiency of intracranial injection of miR-124agomir in rat models. The rats behavioural changes (including:latency-induced seizure, seizure severity) were recorded during the epilepticinitial stage after intraperitoneal injection of lithium-pilocarprine in ratmodelsResults1. Following injection of agomir into hippocampus of rat, CY3conjugated miR-124agomir was observed in the dentate gyrus (DG) andCA3-CA1region of the rat hippocampus. Following injection of1.0nmolmiR-124agomir, endogenous miR-124level in the rat hippocampusincreased40~50%when compared with normal rats or scrambled miRNAagomir control injection rats (P>0.05).2. Following injection of agomir into hippocampus of rat, the statusepilepticus (SE) was induced by pilocarpine, and then we observed thatseven rats were kindled in NC group (n=8), eight rats were kindled in ACgroup (n=8), seven rats were kindled in0.2nmol AG group (n=8), five ratswere kindled in0.6nmol AG group (n=8) and four rats were kindled in1.0nmol AG group (n=8). There was no significant difference in the latencyof pilocarpine induced seizure between the0.2nmol injection group or0.6nmol injection group with AC groups (P>0.05). Following injection of1.0nmol miR-124agomir seizure severity and the latency-induced seizure weredelayed compared to the AC group (P>0.05). Conclusion1. Following injection of effective dose of miR-124agomir,endogenous miR-124level in the rat hippocampus increased40~50%whencompared with normal rats.2. Following injection of effective dose of miR-124agomir, pilocarpineinduced seizure severity and the latency-induced seizure was delayed whencompared with normal rats.Part three Potential role of miR-124in the epileptiform dischargesObjective To investigate the effects of miR-124on pilocarpineinduced epileptiform discharges of rat models by intracranial injection ofchemical synthesized miR-124agomir. Whole-cell recording techniques,qRT-PCR and western-bolt were used to investigate the role of miR-124inepileptogenesis and the potential antiepilepsy mechanism through the targetgene.Methods To evaluated weather the microRNAs can modifying theepileptic seizure and epileptiform discharge of rat models through activationof miRNAs with miRNA agomir, whole-cell patch clamp recordingtechniques were used to detect the frequency of spontaneous action potentialand the amplitude and frequency of spontaneous miniature EPSCs (mEPSC)and the amplitude of evoked EPSCs (eEPSCs) in CA1pyramidal cells. Immunohistochemistry, western-bloting and qRT-PCR were used to explorethe expression pattern of miRNA124and molecular of CREB pathway in thebrain tissue of rat models. Additonally, bioinformatic analysis of thepotential mRNA target and prediction target signaling pathway ofmicroRNA was used, then several protocols in molecular biology will betaken to explore the possible mechanism of the microRNA and the targetpathway to be involved in epileptogenesis.Results1. The adult rat brain slices prepared by this method, could withstandthe damage caused by ischemia and hypoxia in vitro, remainedelectrophysiological properties and adapted to the whole-cell patch clamprecording.2. Action potential: Following injection of effective dose of miR-124agomir and agomir control,we found significant differences in frequency ofspontaneous action potential between NC group, Pilo+AG group andPilo+AC group (P＜0.05). Effective dose of miR-124agomir usesignificantly decreased the frequency of spontaneous action potential whencompared with the agomir control injection group(P＜0.05).3. mEPSC: Following injection of miR-124agomir and agomircontrol,we found significant differences in frequency of mEPSC betweenNC group, Pilo+AG group and Pilo+AC group(P＜0.05). Effective dose ofmiR-124agomir injection significantly decreased the frequency of mEPSC when compared with the agomir control injection(P＜0.05). Followinginjection of miR-124agomir and agomir control, the cumulative amplitudedistributions of mEPSC amplitudes demonstrated a significant rightwardshift of Pilo+AC group compared with NC group and Pilo+AG group (P＜0.05)andnosignificantshiftofPilo+AGgroupcomparedwithNCgroup(P＞0.05).4. NMDAR EPSC:Following injection of miR-124agomir and agomircontrol,we found significant differences in amplitude of NMDA receptor-mediated-EPSC (NMDAR EPSC) between NC group, Pilo+AG group andPilo+AC group(P＜0.05). Effective dose injection of miR-124agomir usesignificantly decreased the amplitude of NMDAR EPSC when comparedwith the agomir control injection(P＜0.05).5. AMPAR EPSC:Following injection of miR-124agomir and agomircontrol,we found significant differences in amplitude of AMPA receptor-mediated-EPSC (AMPAR EPSC) between NC group, Pilo+AG group andPilo+AC group(P＜0.05). Effective dose injection of miR-124agomir usesignificantly decreased the amplitude of AMPAR EPSC when comparedwith the agomir control injection(P＜0.05).6. Using qRT-PCR and immunohistochemistry and western blotanalysis, we found that the intracranial injection of miR-124agomir canincrease the relative expression level of miR-124and decrease the relativeexpression level of pCREB, NMDAR1and GLUR1in hippocampus of a TLE rat model on24hours post-seizure.Conclusion1. Effective dose of miR-124agomir use significantly decreased thefrequency of spontaneous action potential when compared with the agomircontrol injection group(P＜0.05).2. Effective dose of miR-124agomir injection significantly decreasedthe mEPSC frequency and amplitudes when compared with the agomircontrol injection group(P＜0.05).3. Effective dose of miR-124agomir injection significantly decreasedthe amplitude of NMDAR EPSC and AMPAR EPSC when compared withthe agomir control injection group(P＜0.05).4. Pretreated of miR-124agomir can elevated the level of miR-124(P＜0.05)anddecreasethetotallevelofpCREB(P＜0.05)andsurfacelevelof NMDAR1(P＜0.05)in the hippocampus of rats. |
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