Protective Effect Of CD4~+CD25~+Regulatory T Cells On White Matter By Transplanting Into Cerebral Ischemia Stroke Rats

Objective1. Construction a treatment model of CD4~+CD25~+regulatory T cells onischemia/reperfusion rats by intravenous transplantation,To investigate the effects oninfarct volume and neurofunctional outcome after focal brain ischemia/reperfusion injuryin rat model by transplanting CD4~+CD25~+regulatory T cells.2. To research the impact of CD4~+CD25~+regulatory T cells on the white matterinjury after ischemia by observating the indicators of white matter histology of cerebralischemia/reperfusion rats after transplanting CD4~+CD25~+regulatory T cells.Methods1. In this study, Adult male Sprague-Dawley rats weighted between275g-300g wereused as experimental animals. All of the rats were randomly divided into: normal controlgroup(co), sham operation group(sham) and surgical group. In the surgical group, all of theanimals were subjected to middle cerebral artery occlusion and subsequent reperfusion(MCAO/R) operation with modified Zealonga method.The surgery group was divided intothree subgroups: Tregs transplantation group(Treg group),2×106Tregs were transplantedinto rats through the femoral venous, construction a treatment model ofCD4~+CD25~+regulatory T cells by transplantation.Splenic cell group(SP group),2×106splenic cells were also transplanted into rats through the femoral venous; while in the PBSinfusion group(PBS group),equal volume of PBS was injected through the femoral venous.Neurological function was also assessed on the first day and the third day after the surgery.The brains were removed after72hours, the infarct volume was assessed by using TTCstaining and a computerized image analysis system, Then analysis the infarct volume andneurological function score in the experimental group with relevant statistical method.2. In this study, Adult male Sprague-Dawley rats weighted between275g-300g wereused as experimental animals. All of the rats were randomly divided into: normal control group(co), sham operation group(sham) and surgical group. In the surgical group, all of theanimals were subjected to middle cerebral artery occlusion and subsequent reperfusion(MCAO/R) operation with modified Zealonga method.The surgery group was divided intothree subgroups,Tregs transplantation group(Treg group),2×106Tregs were transplantedinto rats through the femoral venous,construction a treatment model ofCD4~+CD25~+regulatory T cells by transplantation. Splenic cell group(SP group),2×106splenic cells were also transplanted into rats through the femoral venous; while in the PBSinfusion group(PBS group),equal volume of PBS was injected through the femoralvenous. The brains were removed after72hours, preparation of brain tissue paraffinsections, observating the Oligedendrocyte(OLs) and the axon which is labeled by axonsmyelin basic protein(MBP) and Neurofliment200(NF200) in the ischemic hemisphere byusing immunofluorescence staining.Then analysis the number of the OLs and thefluorescence intensity of MBP and NF200using related statistical methods.Results1. In the Tregs transplantation group, the infarct volume and the ratio of the ischemichemisphere volume account for the contralateral hemisphere volume were significantlydecreased than the SP group and PBS group(p<0.01). In24h、72h neurological scoreexperiments, the Zealonga score in the Treg group was significantly lower than the SPgroup and PBS group (p<0.01), The modified Neurological Severity Score (mNSS) in theTreg group was also significantly lower than the SP group and the PBS group (p<0.05).2. The related indicators of white matter after cerebral ischemia detected byImmunofluorescence microscopy showed: In the surgical group, the striatum which is atthe border zone of the infarct core in the ischemic hemisphere,The fluorescence intensityof MBP and NF200were significantly lower compared with ipsilateral hemisphere innormal control group and sham group(p<0.05).The fluorescence intensity of aboveindicators in the Treg group were higher than SP group and PBS group(p<0.05).Observating the OLs in the corpus callosum in ischemic hemisphere, the number in thesugical group was lower than the control group (p<0.01). the number in the Treg group washigher compared with SP group and PBS group (p<0.01).Conclutions1. It can be successfully construct a treatment model of CD4~+CD25~+regulatory T cellson ischemia/reperfusion rats by Intravenous transplantation2. Transplantion of Tregs after focal brain ischemia/reperfusion injury may reducingbrain infarct volume and improve neurological function outcome in a rat model. 3. CD4~+CD25~+regulatory T cells transplantion by intravenous can reduce the damagecaused by ischemia on oligodendrocytes and axons, reducing white matter injury aftercerebral ischemia in rats.