| Objects: Inflammation plays a central role in the pathogenesis of cerebral ischemia. Local activation of microglia astrocytes and macrophages, as well as leukocyte infiltration are the committed step of inflammatory processes in the central nervous system(CNS). After cerebral ischemia, inflammation develops as a consequence of 2 processes: activation of microglia and resident perivascular/parenchymal macrophages, and mobilization and infiltration of peripheral inflammatory cells. Selectin which mediate leukocyte recruitment following cerebral ischemia contributes to neuronal damage. Selectin is a member of vascular adhesive molecular family. Selectin can mediate the inflammation reaction in the cerebral ischemia reperfusion injury,and induce cerebral injury of the ischemia region. L-selectin is expressed on kinds of leukocytes and is cell surface marker of leukocyte.The article explore leukocyte infiltration and the expression and effect of L-selectin following focal cerebral ischemia in rat.Inflammatory response in focal cerebral ischemia has been studied in animal models by histological, immunohistochemical, and biochemical methods.These studies are the time-consuming methods which required to evaluate the effective inhibition of inflammatory cell infiltration. Compared with these methods, Flow cytometry is a well-established method for quantitative and qualitative analysis of inflammatory cells in the cerebral ischemia reperfusion injury. It has the rapid, precise and accurate characteristic.Materials and Methods: We use the mechanical dissociation to prepare the brain cell suspension.The method is a simple process that requires no special instrumentation, and is sufficient to isolate inflammatory cell from the brain tissue sample. Mouse monoclonal antibodies specific for rat surface markers in Flow cytometry are as follows:FITC- Mouse Anti Rat CD62L ,PE- Mouse Anti Rat CD45(a leukocyte specific markers). FITC, PE conjugated isotype standards were used as isotype controls.The model of focal cerebral ischemia in rats were established by Suture-occluded method. 66 male Rats were randomly divided into sham- operated group, focal cerebral ischemia group and artificial synthetic E-selectin-treating group,whose arteries were occlusive for 2 hours. All groups were further divided into 5 sectors according to there perfusion time (2h, 6h, 12h, 24h and 48h) to test leukocyte infiltration, the expression of L-selectin and the effect of artificial synthetic E-selectin following focal cerebral ischemia in rat by flow cytometry.Results: There aren't expression of L-selectin and CD45 in sham- operated group. For focal cerebral ischemia group, L-selectin is detected as early as 2h, peak at 6h, significantly decrease at 24h and 48h; CD45 gradually increase after 12h reperfusion. For the artificial synthetic E-selectin-treating group, the decrease of L-selectin is comparatively lower than the focal cerebral ischemia group at various times(P<0.05); the decrease of CD45 is comparatively lower than the focal cerebral ischemia group at 12h, 24h and 48h(P<0.05).Conclusions:The upregulated expressions of L-selectin enhance adhesion between leukocytes and cerebral microvascular endothelial cells, and involved in the pathologic process of cerebral ischemia reperfusion injury. Leukocyte infiltration appear slightly in the earlier period of cerebral ischemia reperfusion injury, start to increase between 12h and 24h of reperfusion, and then gradually increase. The artificial synthetic E-selectin can significantly inhibit expressions of L-selectin and adhesion between leukocytes and cerebral microvascular endothelial cells. The artificial synthetic E-selectin may reduce leukocyte infiltration to protect against cerebral ischemia reperfusion injury. Flow cytometric analysis of ischemic rat brain is feasible and provides a reliable and rapid assay to assess neuroinflammation in experimental models of brain ischemia reperfusion injury.
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