Plasma Exosomal MiR-122-5p And MiR-300-3p As Potential Markers For Transient Ischaemic Attack In Rats

Objective: Ischemic stroke has become the main cause of disability and the second commonest cause of death in the world,which causes a serious threat to human health and quality of life.Treatments of ischemic stroke are quite limited,intravenous thrombolysis or arterial thrombectomy are the most effective treatments in the superacute phase according to the international evidence based guideline,however,only a few patients benefited due to the restricted time window.Differentiation of transient ischemic attack(TIA)from ischaemic stroke within the time window is the key to improve the ratio of intravenous thrombolysis or arterial thrombectomy.Although TIA may be diagnosed within this window,the latest imaging technologies are complex and costly.Serum markers,which are non-invasive,rapid and economic,are used for diagnosis and prognosis of various diseases.Exosome-derived miRNA markers for TIA are unknown.Methods: We examined focal brain ischaemia produced by occlusion of the middle cerebral artery(MCAO for 5 min,10 min,2 hours in rats,and shame group as the control.We evaluated the ischaemic injury of the MCAO based on H&E and TUNEL staining.Plasma/ cerebrospinal fluid(CSF)exosomes and exosomal RNA were isolated via the relative kits.And to ensure the exosomes isolated from the kits were suitable for the subsequent experiments,their morphology,size and purity were examined through transmission electron microscopy,Nanosight analysis and Western blot.Exosomal mi RNAs with CSF and plasma were identified by deep sequencing and quantitative real-time polymerase chain reaction(q RT-PCR).One-way analysis of variance followed by the Games-Howell post hoc test was used for difference analysis.The areas under the curve(AUC)of the receiver operating characteristic(ROC)curve were used to evaluate the diagnostic accuracy of these mi RNAs for TIA in rats.Results: We successfully isolated exosomes via the related kits,and exosomes from rat CSF showed round or oval vesicles,the mode size was 123.7 nm;exosomes isolated from rat plasma showed the mode size was 148.1 nm.Moreover,all of the isolated exosomes were positive for HSP70 and CD63,confirmed by Western blotting.In summary,the exosomes extracted using the related kits expressed normal characteristics of exosomes.Rno-miR-122-5p and rno-miR-300-3p were selected through deep sequencing and qRT-PCR.Plasma exosomal rno-miR-122-5p was significantly downregulated in 10 min ischaemic rats compared with shame group and 5 min group.Plasma exosomal rno-miR-300-3p was significantly upregulated in 5 min ischaemic rats compared with shame group,10 min and 2 hour groups.Plasma and CSF levels of these miRNAs were correlated.ROC analysis showed high AUC values for rno-miR-122-5p(0.960)and rno-miR-300-3p(0.970)in the 10 min and 5 min groups,respectively,compared with shame group.Conclusions: Exosomes were isolated from plasma/CSF of rats successfully.We successfully simulated TIAs through the middle cerebral artery occlusion model of rats(ischemia 5 min and 10 min).Plasma exosomal rno-miR-122-5p and rno-miR-300-3p may be potential blood-based TIA biomarkers.