Effect Of Ischemic Preconditioning On MicroRNAs Expression Profile In Hippocampus Of Rats With Cerebral Ischemia Reperfusion Injury

Objectives This thesis is to explore the pathophysiological effect and mechanism of cerebral ischemic pretreatment of focal cerebral ischemia reperfusion injury to rats.It also screens the differentially expressed tiny RNA(micro RNA)in hippocampus through gene chip technology and verifies the genes with marked differences through PCR.It,thus,tries to explain the intervention mechanism of cerebral ischemia pretreatment of cerebral ischemia reperfusion injury from the perspective of Genomics.Methods This experiment selected male SD rats as research objects through evaluation.Rats were randomly divided into three groups.They were cerebral ischemia-reperfusion group(I/R group),ischemic pretreatment group(CIP group),and sham-operation group(Sham group).Each group consisted of twenty-four rats.The I/R group employed ZeaLonga(modified)to block the middle cerebral artery of the rat's right brain and prepares the focal cerebral ischemia-reperfusion model.One day's perfusion was recovered after two hours' blocking.The CIP group utilized the ischemic pretreatment model established with the method of secondary thread thrombus of middle cerebral artery(MCAO).The thread thrombus briefly blocked the cerebral artery of the rat's right brain for ten minutes.After one day's perfusion,the blood flow was blocked again for two hours and then one day's perfusion again.The Sham group only isolated the rat's common carotid artery without blocking and special treatment of the control groups.When the rats were awake after operation,the neurobehavioral scores of SD rats in each group would be rated according to the Zea-Longa standard.SD rats of each group,which meet the selecting and rating standards.One day after reperfusion rats in each group,eight SD rats of it were selected.After anesthesia,the brain removed surgically was inserted in the brain mould to prepare the brain slices.The volume ratio of cerebral infarction was calculated by TTC(2,3,5-triphenylterazolium chloride)staining.And eight SD rats of it were divided into brain tissue(ischemic infarction zone,hippocampus region)to prepare paraffin section parallel HE staining.To observe the changes of neuron pathology and the distribution of black neuron in the ischemic infarction area of SD rats.High throughput microarray technique was used to measure the microRNA sequence in the hippocampus of rats in each group.The differentially expressed microRNAs were screened and the expression profile was established.Finally,the results of qRT-PCR(Real-time quantitative Polymerase chain reaction)for these microRNAs were verified.The differentially expression of microRNAs were analyzed by GO and KEGG patway analysis.We can infer the biological information of differentially expressed microRNAs and the possible signal pathways in the course of pathological changes.We deeply discussed the mechanism of differentially expressed genes up-regulated or down-regulated during cerebral ischemic preconditioning and cerebral ischemia-reperfusion.Results 1 Neurobehavioral score of SD rats in I/R group was(2.1250 � 0.339);Neurobehavioral score of SD rats in CIP group was(1.4583 � 0.411).There was no obvious neurologic dysfunction in SD rats in Sham group.Compared with the I/R group,the group of CIP significantly reduced the rat neurobehavioral score after ischemiareperfusion(P=0.035),and the difference was of statistical significance.The results suggest that ischemic preconditioning can significantly improve the motor dysfunction induced by right middle cerebral artery occlusion(MCAO)in rats.2 After TTC staining,the volume ratio of cerebral infarction in I/R group SD rats was(0.321 � 0.058);and thevolume ratio of cerebral infarction in CIP group was(0.264 � 0.032);Sham group rats showed no infarction in the brain.Compared with I/R group,the volume ratio of cerebral infarction in CIP group was significantly lower than that in I R group(P=0.021).It is indicated that ischemic preconditioning can significantly reduce the volume of cerebral infarction in SD rats caused by MCAO.3 Observation of paraffin sections of brain tissue under microscope: In Sham group,the cells of cerebral cortex and hippocampus of rats were arranged neatly,and no pathological changes were observed.The number of black neurons in ischemic cortex,basal ganglia and hippocampus of SD rats in I/R group increased significantly.In the CIP group,the number of neuron cells in the ischemic area of the SD rats was more and orderly than that of the I/ R group,the number of the black neuron cells was significantly reduced,and the morphology of the tissue was obviously improved.4 In this study,the high-throughput microarray chip technology was used to analyze the measured samples.Compared with the sham group,the results showed that the expression of 64 microRNAs in the CIP group was up-regulated,and the expression of 4microRNAs was down-regulated.Compared with the sham group,in CIP group,the expression of microRNAs was up-regulated and the expression of microRNAs was downregulated.After homogenous screening comparison between three groups of pairings,it was found that 5 microRNAs up-regulated,of which 4 were newly identified microRNAs.Taking rno-miR-3068-3p as an example,the results of qRT-PCR are verified.The results show that: the expression of rno-miR-3068-3p in the hippocampus after cerebral ischemia and reperfusion was up-regulated;Cerebral ischemic preconditioning down-regulated the expression of rno-miR-3068-3p(P<0.05).The results are consistent with the results of gene chip.5 GO clustering Analysis of differentially expressed microRNAs: In the pathophysiological process of brain injury,it is mainly involved in cation channels,positive and negative regulation of genes,protein kinase activity,transmembrane signal receptor activity.Molecular functions,such as voltage-gated channels,neuronal differentiation and projection,and the process of neurobiological processes.KEGG pathway and bioinformatics analysis showed that:In the adenylate-activated protein kinase(AMKP)regulatory network,rno-miR-3068-3p further participates in the regulation of cell energy homeostasis by acting on its transcription factors STRAD and TBC1D1.Thus,the control network model of rno-miR-3068-3p-Transcriptional Factors(STRAD and TBC1D1)and AMPK was constructed.Conclusions Cerebral ischemic preconditioning can induce cerebral ischemic tolerance and produce brain protection.Differential expression of microRNAs is involved in the pathophysiological process of cerebral ischemia-reperfusion injury and ischemic preconditioning.It played a biological role.The mechanism of the pathogenesis of ischemic stroke has been explained in the aspect of genomics.Figure 14;Table 4;Reference 128.