Circulating Microparticles Change After Stroke Onset And Microparticle-implicated Protection Of Remote Ischemic Preconditioning Against Cerebral Ischemia

Cerebrovasular embolism,one of the major causes of human disability and mortality,presented with the tendency of ascending occurrence year by year.Ischemic preconditioning(IPC)belongs to the general definition of ischemic preconditioning(IPC),in which the application of short episodes of sublethal ischemia protects against a later episode of lethal ischemia.When IPC treatment is performed to an organ or tissue remote from the ischemic target organ,such as mesenteric vasculars,renal,limb,this kind of IPC can be specifically termed as remote ischemic preconditioning(RIPC).RIPC has been proven of its efficiency in alleviating the damage of infarction of target organs afterwards.Microparticles(MPs)are microvesicles bledding from cell membranes via budding or exocytosis,the formation and release of which increase significantly upon stimulation of cells.It has been reported in clinical studies that the count and subpopulation of MPs within circulating blood extremely altered under condition of various diseases,including cardiovascular diseases and metabolic syndrome.It was also inidicated that MPs,especially platelet-derived microparticles,released under ischemic environment activated survival pathways and enhanced angiogenesis,which may support post-ischemia tissue rehabilitation.We previously found that MPs from occluded artery stimulated eNOS-based salvage signaling pathway in vitro and neovascularization in vivo.The current researches majorly studied the relation between MPs and stroke to see if they had altered bioactive capacities after stimulation of ischemia.Also a recently widely used non-invasive protective method RIPC,which has been reported of its relationship with MPs,was studied to see if MPs are implicated in its protection against ischemic damage.This study mainly contained the following two parts:Part Ⅰ Circulating microparticles from clinical stroke patients stimulates endothelial cells through eNOS-based signaling pathwayObjective:To study if circulating microparticles of stroke patients hold biological properties different from those within healthy control subjects,as well as their effect on endothelial cells,in order to find out whether these post-stroke MPs induced endothelial dysfunction or protection against ischemic damage.Methods and results:Blood samples were collected from patients at onset of stroke and 5 days after treatment to analyze the subpopulation and count of MPs of the two time points.MPs were extracted by ultracentrifugation and were administrated to EA.hy926 endothelial cells to see if eNOS-based signaling pathway can be activated by MPs of either time point.It was found out that MPs at onset of stroke significantly promoted endothelial cells migration and transwell behavior.Besides,the middle cerebral occlusion(MCAO)model of mice was established to study whether injection with MPs from stroke patients or healthy subjects would alleviate or aggregate the ischemic damage.The staining results of post-ischemia brain indicated that MPs from patients somehow reduced the ischemic area can be resulted by injection of MPs of patients at onset of stroke.Conclusion:Compared with MPs from healthy control and post-treatment patients,MPs at stroke onset significantly activate eNOS signaling pathway,accompanied with signiciant promotion of migration and transwell of endothelial cells,also such MPs may perform beneficial effect against ischemic damage in MCAO mice model.Part Ⅱ Microparticles are implicated in remote ischemic preconditioning against stroke in a rat MCAO modelObjective:To demonstrate whether protection of remote ischemic preconditioning against ischemic organ has anything to do with microparticles.We tested the hypothesis that remote ischemic conditioning can attenuate cerebral stroke in a rat MCAO model by microparticles.Methods and results:Animal experiments were performed with male Sprague-Dawlay rats,which were randomly separated in donor and recipient groups.For donor groups,rats received RIPC treatment in hind limbs(5 min of occlusion by clapping to transiently stop blood flow,followed with 5 min of reperfusion,repeat such 5 min-5 min cycles for 3 times).After that,blood was drawn from abdominal aorta for extracting post-RIPC treatment MPs.While for the recipient groups,rats received the post-RIFC MPs 30 min pervious to MCAO-surgery through femoral vein.The MCAO lasted for 90 min,followed with 24 hour of reperfusion.Then infracted area and edema level were confirmed with T2-weighted magnetic resonance imaging(MRI)and TTC staining.Results:Significant increase of PMP,EMP and total count of MPs was observed in circulation after RIPC treatement.For post-MCAO rats treated with RIPC or MP transfusion,it was observed that PMP increased significantly under the latter condition.MP transfusion to post-MCAO rats also confirmed that the protective effect can be transferred,although compared with the rats received MCAO as well as RIPC,the ischemic damage was still significantly more severe.The neurological severity scores assessing the post-stroke behavior of rats were estimated for differently-treated rats,however,with no significant difference to be found.Conclusion:RIPC induced an significant increment of PMPs and EMPs in circulating blood.Compared with rats only received MCAO surgery,the R-MP injected rats were detected to hold significant increase of PMP in circulating blood.R-MP transfusion reduced area of ischemic damage in some degree,however,no significant difference was found among the three differently-treated groups in neurological behavior scores.The increase of MP may confer at least part of the remote protective effect against MCAO injury.