Changes In Number Of Peripheral Blood Endothelial Progenitor Cells In Old Rat After Acute Ischemia

Objective:1.To study the method of abstractionand labeling for EPC(endothelial progenitor cell) in peripheral blood of old rat by double-labeling cells with antibodies against EPC surface antigens CD34 and CD133.2.To handle the focal cerebral ischemic(FCI) model of rat,the model was induced by middle cerebral artery occlusion(MCAO).3.To detect and count EPCs in peripheral blood doubled-labeling with antibodies against surface antigens CD34 and CD133 by Flow cytometer.To investigate the change in number of peripheral blood endothelial progenitor cells in the middle cerebral artery occlusion(MCAO) rats.To study the role of EPC in pathphysiology during the development of acute cerebral ischemia.Method:1.We had got fifty Wistar old male rats(550±50g,18 months) which were been divided randomly into 3 groups which were permanence ischemic group(n=20),ischemic reperfusion group(n=20),sham operation group(n=10). Permanence ischemic and ischemic reperfusion groups were induced by modified Zea-Longe' MCAO.2.We got 1ml of blood of rat from venous plexus Behind the eyeball in angulus oculi medialis by micro-tube(D=1mm) when rat was awake.Blood were be got from each rat at the time before operation and the time after operation of 3h,24h,3d,5d,7d,10d.Each 6 rats were be took from permanence ischemic group and ischemic reperfusion group at 24h of postoperative,whose brains were be used for TTC dyeing and pathology.3.Rats' mononuclear cells in peripheral blood were obtained by Ficoll density gradient centrifugation.4.The cells were then labeled with the PE(R-phycoenythrin)-conjugated polyclonal CD34 antibody(Santa Cruz) and FITC(Fluorescein Isothiocyanate)-conjugated CD133 polyclonal antibody(Santa Cruz).FACS analysis(BD FACS Aris^TM) was performed to detect and count EPC. Rats' EPC were be analyzed for double-labeling with CD34-PE(Santa Cruz) and CD133(Santa Cruz).It was observed to syudy the changes of EPC number in peripheral blood of rats after ischemia.Result:1.There were EPCs in rat's peripheral blood which can be succeeding double- stained with antibodies CD34/CD133.2.There was not marked change in the number of EPC in peripheral blood at 3h of postoperative in Permanence ischemic and ischemic reperfusion group(P＞0.05).After that,the level of EPC was fall-off in the two groups and decreased to the lowest at 24h of postoperative(P＜0.01),and the number of EPC in Permanence ischemic group was lower significantly than what is in ischemic reperfusion group.The level of EPC in the two groups recovered gradually after the 3rd day of postoperative.3.The level of EPC in ischemic reperfusion group upgraded obviously on the 5th day of postoperative(P＜0.01),and reached normal level on the 7th day of postoperative(P＞0.05).The level of EPC in Permanence ischemic group was still lower than normal level on the 7th day of postoperative (P＜0.01),and was to achieve normal on the 10th day of postoperative(P＞0.05).Conclusion:1.There were EPC in rat's peripheral blood which can be characterized by the expression of 2 markers,CD133and CD34.2.Cells are labeled with fluorescent antibodies to EPC surface antigens and subsequently counted with a flow cytometer.3.MCAO to rats is convenient and less-damaged,the model is succeeded.4.It is feasible to use flow cytometer to detect and count EPC of rat's peripheral blood.In our study,there was no direct-fluorescently-labeled antibody to CD133.In order to elevate precise of future study,direct fluorescence-labeled antibody will be needed.5.The number of EPC in peripheral blood decreased significantly on the 24h after occlusion in acute cerebral ischemic rat,and it can recovery to normal level along with time.6.There was a marked lower in the number of EPC of permanence ischemic group in which EPC needed a longer time to achieve normal.What manifest the level of EPC may be correlated with the severity of ischemic.