Flow cytometric analysis of circulating microvesicles derived from myocardial Ischemic preconditioning and cardioprotection of Ischemia/reperfusion Injury in rats.
- Author:
Miao LIU
;
Yi-lu WANG
;
Man SHANG
;
Yao WANG
;
Qi ZHANG
;
Shao-xun WANG
;
Su WEI
;
Kun- wei ZHANG
;
Chao LIU
;
Yan-na WU
;
Ming-lin LIU
;
Jun-qiu SONG
;
Yan-xia LIU
- Publication Type:Journal Article
- MeSH: Animals; Cell-Derived Microparticles; metabolism; Coronary Vessels; pathology; Flow Cytometry; Heart Rate; Ischemic Preconditioning, Myocardial; Myocardial Infarction; physiopathology; Myocardial Reperfusion Injury; physiopathology; Myocardium; pathology; Phenotype; Rats
- From: Chinese Journal of Applied Physiology 2015;31(6):524-531
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles (MVs) from myocardial ischemic preconditioning (IPC) treated rats (IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion (I/R) injury in rats.
METHODSMyocardial IPC was elicited by three.cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending (LAD) coronary artery. Platelet-free plasma (PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFR PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 µm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure (MAP), heart rate (HR) and ST-segment of electro-cardiogram (ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin (HE) staining. The activity of plasma lactate dehydrogenase (LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining.
RESULTSTotal IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs (LMVs) and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (<1 Vm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats (P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR (P<0.01), decreased ST-segment and LDH activity (P < 0.05, P < 0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment (P < 0.01).
CONCLUSIONThe method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.