Expression Of CD8~+T Lymphocytes In The Ischemia-reperfusion Brain Tissue In Rats

Objectives:Applying the method of immunohistochemical staining, using middle cerebralartery occlusion (MCAO) model,observe the expression of CD8＋T cells in ischemia-reperfusion tissue at different time points to investigate the relationship between CD8＋T cells and ischemic brain injury.Methods:35healthy adult male SD (Sprague-Dawley) rats, body weight250-300g, arerandomly divided into7groups:sham operation group,cerebral ischemia-reperfusion24hours group,3days group,5days group,7days group,10days group and14daysgroup,5for each group.Refer to Zea-Longa patching method,establish middle cerebralartery occlusion (MCAO) model for all model groups.sham operation group rats are thesame as model groups but not patching.Refer to Zea Longa5-point scale,score all modelgroups rats in24hours after they come round from the anaesthetic.Select the rats whosescores are1-3and write down the scores.Then kill the rats to get their brains when itreaches the reperfusion point.Brains are cut into2-2.5mm coronary slices from thebeginning to the end.Place the slices in4%formaldehyde solution for at least24hoursand keep in dark place.Wash brain tissue with running tap water overnight,dehydrationby graded ethanol, vitrification by dimethylbenzene, wax immersion (62degrees) andparaffin embedding. Make paraffin section whose thickness is4um by paraffin slicingmachine. HE staining shows pathological morphology of ischemia-reperfusion tissue;Immunohistochemical s-p assay shows the expression of CD8molecular. Under lightmicroscope(200times) select five overlap of vision randomly in the target area of eachsection (mainly ischemic cortex) and count positive cells respectively(Characteristics ofpositive cells is that the cell membrane is stained brownish yellow). The average of5vision is positive cell count results. Photograph the section under light microscope by digital camera.Results:1.Pathological observation of ischemia-reperfusion brain tissue: the shamoperation group is normal brain tissue whose structure is order and distinct.You can seenormal morphology of neurons and glial cells. At ischemia-reperfusion3days thestructure of ischemic cortical tissue is disorder, cortex thickening, light staining, a largenumber of neurons are degenerated; At reperfusion7days, the number of normalmorphological neurons in the ischemic cortex significantly reduce, the line betweencytoplasm and nucleus is blurred and the nucleus is pyknotic and dissolved. Vacuoles ofdifferent sizes appear between nerve cells. Lots of vacuoles appear in central necroticarea, honeycomb, where nerve cells almost disappear; The view of reperfusion10daysis similar to7days.2.The expression of CD8＋cells by immunohistochemical staining: almost noexpression in the sham operation group. At ischemia-reperfusion24hours there are asmall number of CD8＋cells in ischemic cortex; at reperfusion3days numerous CD8＋cells distribute diffusely in ischemic penumbra. The positive cells are round and a slightdifference in size, some gathered around the vessels. As the extension of time, thenumber of CD8＋cells increases gradually. During reperfusion7-10days it is up to apeak, and the infiltration position gradually goes to the central necrotic area; Atreperfusion14days the number decreases slightly.3.At high magnification CD8positive cell count: the sham operation group is2.2±0.5/HP; ischemia-reperfusion24hours,3days,5days,7days,10days and14daysgroup respectively is6.4±1.6/HP,18.5±1.1/HP,21.8±2.3/HP,26.4±1.8/HP,25.9±2.2/HP and23.7±1.2/HP. Each model group, the number of positive cells issignificantly higher than that in the sham group (P <0.05).Conclusions:1.At ischemia-reperfusion24hours CD8＋T cells begin to express, during7-10days reach a peak, then decrease;2.A large number of CD8＋T cells express in the ischemia-reperfusion braintissue, and are mainly distributed in the ischemic penumbra;3.CD8＋T cells are involved in the inflammatory response of the ischemic braintissue.