The Expression Of ICAM-1 And Therapeutic Benefit Of Intravenous Administration Of Bone Marrow Stromal Cells After Cerebral Ischemia In Rats

ObjectiveThe purpose of this study was to investigate the expression of ICAM - 1 and therapeutic benefit of intravenous administration of bone marrow stromal cells at 24 hours after the MCAO ( middle cerebral artery occlusion) for 1 hour and to study the protective effect of MSC against neural injury and the possible roles of alteration in the expression of ICAM - 1 after cerebral ischemia in rats.MethodsForty - five wistar rats were randomyly divided into three groups; Test group consisted of MCAO alone ( control group, n = 15 ) ; intravenous infusion of 1ml 3 × 106MSC at 24 hours after MCAO ( MSC group, n = 15) ; or infusion of lml saline at 24 hours after MCAO ( NS control group, n = 15 ). Firstly, transient ischemia of the middle cerebral artery occlusion ( MCAO) for 1 hour was induced by the suture occlusion technique . Briefly all rats were anesthetized with 10% chloral hydrate, 3.5 mg/kg intraperitoneally. A 2cm incision was made at the center of the neck and the right common carotid artery (CCA) , external carotid artery ( ECA) , and internal artery (ICA) were exposed. The CCA and ICA were clamped using clips. A 4. 0 silk suture was tied loosely at the origin ECA and ligated at the distal end of the ECA. A length (18. 5 to 20.0 mm) of nylon suture with its tip rounded by heating near a flame was introduced into theECA lumen through a small puncture. The nylon suture was gently advanced from the ECA into the ICA until it blocked the origin of the MCA. The silk suture around the ECA origin was tightened around the intraluminal nylon suture. After 1 hour, reperfusion was achieved by withdrawal of the suture. Secondly, Cultured bone marrow cells were obtained from an adult male rat given 5 - flu-orouracil , 150 mg/kg intraperitoneally to ablate mature blood cells and induce stem arid progenitor cells into cycle, using a syringe (1 ml) with phosphate -buffered saline ( PBS}, Fresh bone marrow was harvested aseptically from tibias and femurs. Then bone marrow was mechanically dissociated until a milky. E-rythrocytes were removed from bone marrow using 0. 84% NH4CL. Numbers of nucleated marrow cells were measured by a cytometer and 1×10~7 cells were seeded into 75 cm tissue culture flask. After 24 hours of incubation, MSC tightly adhered to plastic and were removed to fresh IMDM in new flasks. MSC were grown at 37℃ in a 5% CO2. To identify cells derived from MSC, bromode-oxyuridine was added to the medium at 24 hours before transplantation. Rats in the MSC group were injected with 3 × 10~6 BrdU marked MSC through vein systems at 24 hours after MACO. Rats in the NS control group were injected with 1 ml saline water at 24 hours after MACO. We done nothing to the rats in the control group . In all animals, modified Neurological Severity Scores ( NSS) test was performed before MCAO and 3, 7and 14 days after MCAO by an investigator who was blinded to the experimental groups. NSS is a composite of motor, sensory, reflex and balance tests. In the severity scores of injury, 1 score point is awarded for the inability to perform the test or for the lack of a tested reflex; thus the higher score, the more severe is the injury. Then we killed the rats at 3. 7. 14 days. Expression of ICAM - 1 and BrdU - reactive cells were confirmed respectively by immunohistoehemistry, microimage analysis system. Statistical analysis: all the data were analysis by SPSS11.0 statistical software. Data were analysis by ANOVA ( analysis of variance). All measurement data were expressed as the Mean±SEM. Significance was set at P <0.05.