A Preliminary Study On Biomarkers Of Cerebral Ischemia Attack

BACKGROUND: Repeated episodes of transient cerebral ischemia(TIA)have an ischemic encephalopathy rate of approximately 50% within 48 hours,and the risk factor is relatively large,which is a threat to ischemic encephalopathy.It is not easy to distinguish TIA from ischemic encephalopathy by imaging examination in the thrombolysis time window,and blood markers have the advantages of being economical and non-invasive,and can be used for the diagnosis of diseases.Therefore,the primary objective of this study was to find effective plasma exosome miRNAs as a blood marker for the diagnosis of TIA.Therefore,this study is mainly to find a rapid and effective exosome miRNA as a blood marker for the diagnosis of TIA and promote thrombolytic therapy.METHODS: First,rat middle cerebral artery occlusion(MCAo,sham operation group,ischemia 5 min group,ischemia 10 min group,ischemia 2 h group)was used to simulate rat TIA model,and plasma and cerebrospinal fluid samples of rats after modeling were collected,and the pathological changes of neuronal cells in the brain tissue of rats after ischemia were evaluated by HE staining and TUNNEL staining.Secondly,exosomal total RNA was extracted from plasma and cerebrospinal fluid by kit,and the expression of exosome miRNA in rat plasma was detected by deep sequencing,and miRNAs with significantly differential expression were screened.Third,in this study,real-time quantitative polymerase chain reaction(PCR)and digital PCR(ddPCR)were used to detect miRNA expression levels in exosomes in plasma and cerebrospinal fluid,respectively,and exosome miRNAs with the same plasma and cerebrospinal fluid expression trends were selected.And the selected miRNAs were analyzed by bioinformatics.Finally,the area under the curve(AUC)of the receiver operating characteristic(ROC)curve was determined to assess the diagnostic value of miRNA for TIA in rats.RESULTS: First,TUNEL staining results showed that fewer tunel-positive cells were observed in brain tissue sections of the 5-minute ischemia group,while the number of tunel-positive cells was more in the 10-minute ischemia group than in the 5-minute ischemia group,while tunel-positive cells were the most important cells in the brain tissue of 2 hours ischemia group.The positive rate after TUNEL staining at different ischemic time was also different,and the positive rate in the 10-minute ischemia group and 2 hours ischemia group was higher than that in the 5-minute ischemia group and the sham operation group.The results of HE staining indicated that the shape,size and structure of the neurons on the brain tissue sections of the sham operation group were normal;some of the neuronal cell atrophy began to appear in the 5 min ischemia group,with eosinophilic necrosis,atrophic necrosis,nuclear pyknosis,and deep staining;In the 10-minute ischemia group,the number of atrophied neurons increased,the nucleus condensed,the nucleolus disappeared,the number of atrophied neurons increased,and the neuronal cell structure was disordered;a large number of neuronal cell necrosis,fibrinolysis,neuronal cell fibrosis,and interstitial edema were observed in the 2 hours ischemia group.Second,the average particle size of the exosome sample and the main peak of the particle size were all consistent with the particle size distribution of the Exosome in the Exosome particle size range(20 nm-200 nm)by transmission electron microscopy and Nano-ZS,and the positive expression rates of exosome surface marker specific protein markers CD63 and CD81 by flow cytometry were all above 95%.Third,high-throughput sequencing was used to detect the expression of total RNA in rat plasma exosomes,and rno-mi-450b-5p was screened;the expression level of rno-mi-450b-5p in cerebrospinal fluid and plasma samples was verified by PCR,and the results indicate that the expression trend of rno-miR-450b-5p is consistent in both samples.Bioinformatics analysis indicated that exosomes rno-miR-450b-5p may be involved in the development of cerebral ischemia.Third,high-throughput sequencing was used to detect the expression of total RNA in rat plasma exosomes.Rno-mi-450b-5p was screened and the expression level of rno-mi-450b-5p in cerebrospinal fluid and plasma samples was verified by PCR.This indicates that rno-miR-450b-5p is consistent in the expression trends in both samples.Bioinformatics analysis indicated that exosomes rnomiR-450b-5p may be involved in the development of cerebral ischemia.Finally,the ROC curve was prepared,and the AUC area of the exosome rno-miR-450b-5p in the sham operation group and the ischemic 10 minute group was calculated to be(0.880).Therefore,the exosomes rno-miR-450b-5p are of high value for the diagnosis of rat TIA.Conclusion: Plasma exogenous rno-miR-450b-5p has a high diagnostic value and may become a therapeutic target for rat TIA.