Marking Of Rat EPCs And Changes In Circulating Rat EPCs After TBI

Objective:1.EPCs were identified by double-labeling cells with antibodies against CD34 and CD133,set up the protocol to mark EPCs in rat's circulating.2. With the Fluid-percussion brain injury(FPI)rat models,based on the Flow cytometer and double-antibody(CD34/CD133)mark technology,monitor the changes in rats circulating EPCs after traumatic brain injury.3.The white blood cells and platelets of the TBI rats were also counted with the Sysmex K-1800,so as to analyze the correlations between the EPCs and platelets,white blood cells.Methods:1.45 weight and age matched male Wister rats of 300-350g(from Academy of Military Medical of China)were divided into four groups.The rat model of traumatic brain injury was performed with the fluid-perfusion instrument and little animal stereotaxis equipment(Tianjin Neurology Institute).We made the skull incision(φ=3mm)at the cross of 2mm to the sagittal suture lateral and 3mm to the bregma posterior in order to avoid injuring the brain stem and the motor area,and the dura must be intact.We just performed anesthesia on 10 rats of sham operation group as control.And exerted on the rats of experimental groups at the different degree of fluid-perfusion(150mv,300mv,400mv),based on which we divided the rats into group of light(10 rats),moderate(10 rats)and severe(15 rats)brain injury.In these groups,choosed one rat for the MR and another for HE stain randomly.Among these rats,1 rat of the light group was ruled out for inflammation at 72h,1 rat of the moderate group was dead at 48h after injury,and in the severe group,2 rats were dead at 24h after injury,and 4 rats were dead at 48h after injury,one of these 4 rats was ruled out for inflammation.The experiments conformed with the Guide for the Care and Use of Laboratory Animals published in China.Rats were anesthetized with 3ml/kg intraperitoneal 10%chloral hydrate.2.Isolation of Rats Monocytes and Fluorescent Staining of Rats EPCs,1 ml of blood was subjected to Ficoll density gradient centrifugation with Histopaque-10831(Sigma-Aldrich)at 2400rpm for 20 min at 20℃for the isolation of the peripheral mononuclear cells.The isolated cells were washed with phosphate-buffered saline(PBS,pH 7.2)and resuspended in 200μl of PBS supplemented with 0.5%of bovine serum albumin(BSA)and 2 mM of EDTA by centrifugation at 2400rpm,2000rpm and 1500rpm three times.The cells were then labeled with the PE(R-phycoenythrin)-conjugated monoclonal CD34 antibody(Santa Cruz)and FITC(Fluorescein Isothiocyanate)-conjugated CD34 monoclonal antibody(Santa Cruz)or for 10 min at room temperature.3. Fluorescence-Activated Cell Sorter Analysis:FACS analysis(BD FACS Aris^TM,USA) was performed.Rats EPCs were analyzed for CD34-PE(Santa Cruz,USA)and CD133(Santa Cruz,USA),the latter was detected with a FITC-conjugated secondary antibody(Rabbit Anti-Goat IgG,Zhongshan Goldenbridge Biotech,Beijing).The stained cells were washed with PBS/BSA and suspended in 1%PFA (paraformaldehyde)then analyzed by flow cytometry.Isotype-matched antibodies served as controls in every experiment(Santa Cruz,USA).Positive cells were determined for their fluorescent intensity exceeding the background staining from isotype controls.For double fluorescence detection,cells were first gated for their CD34 positivity and then for CD133 staining.Double-stained cells were analyzed.4. Counting of Rat Peripheral Blood Cells,0.1ml of blood was subjected to the rat white blood cell and platelet counting(Sysmex,Japan).Result:1.With the antibodies CD34/CD 133 double-antibody-stained,using FCM, succeeded in marking the rat's EPCs in circulating;2.After fluid percussion brain injury,EPCs in rat's circulating follows a two phases trail initially lower at 3h after brain injury,and increase at 6h after brain injury,eventually to a level higher than that of control group.This bi-phase change in circulating EPCs is in clear contrast to the sham operation group;3.Changes in EPCs level are not correlated with those of platelets and white blood cells.Conclusion:1.EPCs exist in the peripheral blood,FCM can be used to identify EPCs for angiogenesis study.2.With the antibodies CD34/CD133 double-stained, using FCM,succeeded in marking the rat's EPCs in circulating,there are EPCs in rat's circulating.3.After the brain fluid-percussion injury in rats,EPCs in rat's circulating follows a two phase trail.4.Changes in EPCs level are correlated well with those of platelets but not white blood cells.5.The EPCs levels are initially decreased in the first 3h.6.The Rat EPCs changes are not correlated with platelets and peripheral leukocytes counts. In summary,we have demonstrated a dual-phase change in circulating early EPCs in TBI rats.These results showed some evidence for our clinical research,and may provide some data for the next research.