Proteomics Study Of Cerebrospinal Fluid In Cerebral Ischemia And Cerebral Ischemia-reperfusion

Objectives:1.The purpose of this study is to explore the dynamic changes of cerebral blood flow in the process of cerebral ischemia and cerebral ischemia-reperfusion.In addition,the proteomics of cerebrospinal fluid in cerebral ischemia and cerebral ischemia-reperfusion were also investigated.Methods:1.The models of cerebral ischemia and cerebral ischemia-reperfusion were established by thread embolism method.135 SD rats were randomly divided into 15 groups including sham group,ischemic 0.5h group,ischemic 2h group,ischemic 3h group,ischemic 4h group,ischemic 4.5h group,ischemic 6h group,ischemic 8h group,ischemic 0.5h reperfusion 2h group,ischemic 2h reperfusion 2h group,ischemic 3hreperfusion 2h group,ischemia-4h reperfusion 2h group,ischemia 4.5h reperfusion 2h group,ischemia 6h reperfusion 2h group,ischemia 8hreperfusion 2h group(9 rats in each group).2.In this experiment,the blood flow values of five brain regions were measured by laser speckle blood flow analyzer.Moreover,behavioral evaluation and TTC staining were performed on the models.3.The cerebrospinal fluid was extracted to conduct proteomic analysis.4.Combining the results of mass spectrometry,the penumbra and infarcted nuclei of cortex were selected to carry out Q-PCR experiments.Results:1.The cerebral blood flow value gradually decreased with the extension of ischemia time and reached the lowest value in 4.5 h.The cerebral blood flow value increased from 4.5h to 8h.2.Compared with ischemic 0.5h group,infarct volume increased significantly in ischemic 2h group,and the difference was statistically significant in infarct volume(p<0.01).When compared ischemic 2h group and ischemic 6h group,the infarct volume of ischemic 6h group increased significantly,and the difference was statistically significant in infarct volume(p<0.01).The ischemic 2h group had larger infarct volume than cerebral ischemia 2h reperfusion 2h group,the difference in infarct volume between the above groups has significant statistical significance(p<0.01).The behavioral evaluation scores of 0.5h group in Zea-Longa were significantly higher than thesham group,and the difference in behavioral evaluation scores was statistically significant in terms of neurological function(p<0.01).Compared with ischemic 0.5h group,Zea-Longa scores increased significantly in ischemic 2h group,and the difference was statistically significant in neurological function evaluation(p<0.01).Compared with ischemic 2h group,Zea-Longa scores in ischemic 6h group were obvious higher than ischemic 2h group,and the difference was statistically significant in nerve function evaluation(p<0.01).There was no significant change in Zea-Longa scores between cerebral ischemia 2h group and cerebral ischemia 2h reperfusion 2h group.3.The expression of AOAOG2KON6 protein in all ischemic groups was higher than the sham group.The protein that presented in cerebrospinal fluid(CSF)of sham group and disappeared in the CSF of cerebral ischemia group was D3ZS41(extracellular matrix protein 2,the encoding gene is Ecm2),which was verified by PCR.These proteins that didn’t exist in the CSF of sham operation group,and appeared in the CSF of operation group were Q5M842,A2NB82 and F1M5L5 through PCR test..The above protein changes were consistent with the results of the mass spectrum.The content of these proteins including P47942(the encoding gene is Dpys12),Q6LE95(the encoding gene is Kng1)and P09606(the encoding gene is Glu1)in cerebrospinal fluid increased after cerebral ischemia-reperfusion.The PCR verification showed that the content of F1LM84 protein(the encoding gene is Nid1),FILS40 protein(the encoding gene is Colla2),P02454 protein(the encoding gene is Collal),M9NW49 protein(the encoding gene is Igf2)and F1MAA7 protein(the encoding gene is Lamc1)decreased after cerebral ischemia and reperfusion.The results of protein content change were consistent with the results of the mass spectrum.Conclusion:1.The point-in-time of cerebral ischemia-reperfusion in rats is 4.5h.Reperfusion after 4.5h may aggravate brain injury.Cerebral ischemia-reperfusion after 4.5h did not increase cerebral blood flow.2.Proteomics results suggest that the diagnostic markers for early cerebral ischemia may exist in cerebrospinal fluid,such as A0A0G2K0N6 protein and D3ZS41 protein.