Role Of Endogenous Regulatory T Cells In Cerebral Vasospasm After Subarachnoid Hemorrhage And Brain Injury

Objective1.Establish CD4+CD25+regulatory T cells deletion model in mice to inve-stigate the effects of CD4+CD25+regulatory Tcells on neurological function afterdeletion.2.Through the blood-brain barrier permeability measured brain water cont-ent determination,determination of regional cerebral blood flow and TUNE-L,caspase-3was observed,and to explore CD4+CD25+regulatory T cells in the d-eletion of subarachnoid hemorrhage after early brain injury affected.3.Explore deleted CD4+CD25+regulatory T cells in the brain tissue inflam-mation.4.Detected by immunofluorescence cortical areas of inflammatory cytokin-es IL-6,GFAP levels by western blot detection of brain tissue levels of IL-6,andfurther explore the CD4+CD25+regulatory T cells in the deletion of brain tissueinflammation subarachnoid hemorrhage affected.MethodsCreating and grouped models:male mice (25-30g),were randomly divided i-nto sham group,SAH group,Treg cells remove the group and PBS injectiongroup.Intravascular plug wire method with established mouse model of subara-chnoid hemorrhage,laser Doppler flowmetry records changes in blood flo-w.Sham operation group in the puncture process,only resistance is felt,but notthe vascular puncture.Tregs group of models subject to deletion mice by intrape-ritoneal injection0.20mg CD25specific antibody (PC61),to build Treg cellde-ficient mice(Treg cell-depleted mice).PBS by intraperitoneal injection,intrap-eritoneal injection and0.20mg CD25antibodies specific volume of phosphate buffer.Determine the extent of bleeding:the mice were sacrificed,carried blee-ding score.After the mice were sacrificed and the brain was removed quick-ly,camera basilar Ikebe bleeding,according to Sugawara and other reports,carrie-d bleeding grade determination.Neurological score:Each randomly selected sixneurological science score,using the scoring system Garcia total score5-18poi-nts.Detection of cerebral edema:six mice in each group were ran-domly selectedin72hours after subarachnoid hemorrhage,anesthesia, decapit-ated and thebrain were sacrificed,quickly weighed,then put in the oven and bake80℃for48h,weighed.Calculated according to the formula:brain water content=(wet wei-ght－wet weight)/wet weight×100%for each experimental group were compared BBB permeability detection:first establish a standard curv-e.Then,eachgroup of10mice were randomly selected,one hour prior to measurement miceby10%chloral hydrate (0.16ml) after intraperitoneal injection of anesthesia,mice were exposed to the right femoral vein in each group,Evans Blue injectedvia the right femoral vein mice,1h saline perfused through the left ventricle,right atrium until clear liquid flows out,after decapitated,cut the middle line,thepreparation of brain tissue homogenates,then determined after further detectedin brain tissue in Iraq Evans blue content and compared.Cerebral vasospasmdetection:6mice were divided randomly into experimental groups at72hoursafter its death,take the brain,paraffin sections, hematoxylineosin staining wereobserved in each group changes in mouse basilar artery diameter size and wallthickness,and compared.Nissl staining:six mice in each group were randomly se-lected,72h mice were sacrificed after brain surgery,made of frozen sections wereNissl stained with toluidine blue,mice in each group were observed corticalneurons Nissl shape and the number of bodies,and compared.TUNEL assay:PBSgroup,sham operation group,SAH model group,Tregs deleted mice,after makingover the depth of anesthesia,the left ventricle rapidly after craniotomy brainperfusion fixation,placed40g/L poly fixed in formaldehyde solution for24hour-s,embedded in paraffin,serial coronal sections lines,thickness5μm,each block10brain.Staining procedure,strict accordance with the instructions provided bythe U.S.Roche in situ apop-tosis detection kit.Finally, the application of laserfluorescence microscope system operating system,scan positive reaction.Imageanalysis system to capture images,each slice randomly selected five high-powerfield (×20), the count of TUNEL positive neurons.Determination of the cortex IL6,and distribution of GFAP expression by immunofluorescence.Determinationof brain tissue IL-6,TNF-α levels by enzyme-linked immunosorbent assay,and t-he groups were compared. Cortical areas by western blot detection of inflamma-tory cytokines IL-6levels.Results1.In terms of the degree of bleeding,no bleeding sham group;In terms of thedegree of bleeding,no bleeding sham group;Blood in SAS were seen in all othergroups with no statistical significance.2.neurological score,the sham group,PBS injection group score was18points,set the model group compared with sham group score was statisticallysignificant(P<0.05),Tregs delete groups compared to PBS injection group scorestatistically significant(P<0.05),SAH group were significantly different com-pared(P<0.05) and Tregs delete a group score.3.Determination of blood-brain barrier permeability and brain edema:increased permeability of the blood brain barrier in mice after SAH,brain water content increased;Tregs delete group than SAH group compared to the BBB permeability was significantly increased(P<0.05),brain water content was significantlyincreased(P<0.05).4.Basilar artery morphology detection:basilar artery after subarachnoid he-morrhage in mice significantly spasm after spasm of Tregs delete more signific-ant(P<0.05).5.Nissl staining:after subarachnoid hemorrhage in mice the number of neur-ons in the cortex containing Nissl bodies decreased significantly after the red-uction of Tregs delete more significant(P<0.05).6.Detectionand TUNEL assay of apoptosis proteins:apoptosis after suba-rachnoid hemorrhage in mice cortex cells increased the number of apoptotic cel-ls compared with Tregs deleted after SAH,there was a significant and signific-ance(P<0.05)7.Determination immunofluorescence cortex IL-6,astrocytes:model group,PBS group,Treg cells remove the group increased cortical expression levels, Tr-egs delete group compared to the previous two,the increase is more significant(P<0.05).8.Enzyme-linked immunosorbent assay(ELISA)determination of brain tissue IL-6and TNF-α levels:SAH in brain tissue of mice IL-6and TNF-α ex- pression levels have increased,Treg cells compared to delete group there was s-tatistically significant(P<0.05).9.Western blot detection of cortical areas IL-6:model group,brain tissue P-BS group,Treg cells remove the group’s increased expression levels of IL-6,Tregcells were deleted with the model group,compared to the sham group was significant(P<0.05).Conclusion1.This study successfully constructed a mouse model of subarachnoid hem-orrhage and Tregs deleted mouse model and found that Tregs will increase after-deleting cerebral vasospasm,cerebral edema,blood-brain barrier permeability,apoptosis,and also aggravate the inflammation of brain tissue damage.2.Mice after SAH have neurological damage in mice may increase after de-leting Tregs motor,sensory damage functions.