Effects Of Adoptive Transfer Of Regulatory T Cells On Cerebral Vasospasm And Early Brain Injury After Subarachnoid Hemorrhage

BeckgroundsSubarachnoid hemorrhage is a kind of clinical syndrome，which is caused by ruptureof the blood vessels at the bottom of the brain or on the surface of the brain,and the blooddirectly flows into the subarachnoid space. Epidemiological investigation shows that SAHaccounts for about10%of acute brain stroke,and most of SAH is caused by rupture of theaneurysm. Aneurysmal SAH is a kind of extremely severe disease, and its morbidity andmortality is very high.The major population of the SAH is the population of40-60years old.The incidence of SAH is some10/100,000,and about12%of the SAH patients died beforethey were sent to the hospital and given any treatments,about40%of the SAHpatients died in the first month after the first bleeding.And about30%of the survivals’sexistence depend on others because of their neurological impairments.Besides,50%ofSAH patients lived with cognitive dysfunction in a relatively long period, and the cognitiveimpairments is almost impossible to recover to the level before. Although the diagnostictechniques and surgical treatments have been greatly improved, the clinical outcomes ofSAH patients is still not ideal. The research works found that cerebral vasospasm and earlybrain injury are the main factors which induces the high Mortality and high morbidity ofSAH patiens.CVS appeared at the third day after SAH, peaked at6thto8thday after SAH,and persisted for2or3weeks.CVS is one of the most severe complications of SAH,whichcould induces se-vere cerebral ischemia, leads to serious neurological dysfunc-tion,andeven death.In the past,most of the researchers thought that CVS is the main factorsleading to high mortality and poor outcomes,so,the researchers put their emphasis on CVS.Although our acknowle dge of the pathogenesis of CVS has a significant enhancement,thetreatments targeting th-ese mechanisms did not significantly improve the outcomes ofSAH patients.In recent years,the study found that EBI is closely associated with theoutcomes of SAH patiens.EBI is the brain tissue damage occurred in the first72hous after SAH.some researchers put for-wards a challenging view:the influence of EBI on theoutcomes of SAH is greater than that of the CVS. The pathophysiology of EBI includes theglobal cerebral ischemia injury caus-ed by rapid increase of intracranial pressure and sharpdrop of cerebral perfusion pressure，the activation of apoptosis，the destruction of the bloodbrain barrier, brain edema and im-mune inflammation responses.Based on ouracknowledge of the mechanisms of the CVS and EBI.Finding out a safe,effective and easyto implement measurements is the key point to improve the outcomes of SAH patients. Thegovernments of at home and abroad have contribute much manpower and materialresources to the study of CVS and EBI after SAH, but the safe and effective treatments isstill limited.In recent years,some researchers found that the immune inflammatory responsesmediated by activation of the immune system play an important role in the diseases of thecentral nervous system.Researchers at home and abroad found that significant and sustain-ed immune inflammatory responses exist in the spastic cerebral arteries and brain parenchychymal.After the rupture of the aneurysm,blood is released into the subarachnoid space andthe internal materials such as hemoglobin, bilirubin were released into subarachnoid space,which lit up the immune inflammatory response.After the first bleeding,there are a series ofimmune inflammatory response,including the neutrophils, macrophages and other immunecells infiltration into the arterial wall, immunoglobulin and complement deposition,vascularendothelial cells swelling and degeneration apoptosis, blood vessels thickening and so on,and these immune inflammatory response keep associated with the delayed cerebral ische-mia.Besides,In SAH rat brain tissue,some genes associated with inflammation and itsproducts has significant change.Although having many potential side-effects,the drugs,suchas steroids and immunosuppressant, aiming to reduce the immune inflammatory response,shows positive effects on CVS and EBI.Therefore, Immune inflammatory response couldbe a promising therapeutic targets. Inhibiting excessive immune inflammatory response andpreventing inflammatory cell activation, infiltration, and inflammatory cytokine productionare expected to become a new effective way to treat the CVS and EBI after SAH.ObjectivesFirstly,to investigate the inhibiting effects of the adoptive transfer of Regulatory Tcells on cerebral vasospasm after subarachnoid hemorrhage.Secondly, to discuss the protective effects of the adoptive transfer of Regulatory Tcells on early brain injury after subarachnoid hemorrhage.Thirdly,to work out the drugs which could improve the outcome of the SAH patients or treat the SAH patients from the research---we use the method of adoptive transfer ofthe Tregs to investigate the immune inhibiting effects of the Tregs on the SAH rat models.Methods1. Choose healthy Wistar rats, male and female accounts for50%to50%, weightingbetween300-350g.These rats were randomly divided into four groups: control group, SAH+PBS group, SAH+spleen cells group, SAH+regulatory T cells group.Rat SAH modelwas established by the cisterne magna double injection of autologus arterial hemolysates.We inject normal saline, PBS,Spleen cells and Treg cells into the femoral vein of the rats ofcontrol group、the SAH+PBS group、the SAH+SP group、the SAH+Tregs group separately.2. with the help of the stereotactic instrument, to make a skull window on the parietalbone of the rat in the four groups.Use the doppler blood flow meter to measure the regionalcerebral blood flow at three time point,and the time points are before the SAH modelestablished,6hours after the SAH model established and12hours after the SAH modelestablished.3days after induction,after cardiac perfusion，we isolate the rat stem and makeit into frozen section,then,we use hematoxylin-eosin staining to observe morphologicalchanges of the basilar artery.we measured and compared the differences of the basilarartery diameter of the rat model in the four groups and the thickness changes of the basilarartery among the four groups.3. one day after induction,we isolated the cerebral cortex tissue of the rats brain andextracting protein.then,we use Western blot method to detect the expression level of matrixmetalloproteinases9in the cerebral cortex of the rats in different groups. One day afterinduction,we use the dry and wet method to measure and compare the moisture content ofthe rats brain in the four groups.4. three days after induction,we use Nissl’s staining to observe and measure thedensity of the neuron cells in Hippocampal CA1area of the rats in the four groups.Cellapoptosis and cell injury in the cerebral cortex were detected by TUNEL method.theprotein expressions of active caspase-3in the rats cerebral cortex of the four groups wereassessed by the methods of immunofluorescence and western blot.Results1.(1)6h and12h after induction,the rCBF of the rat in the SAH+PBS group、SAH+SPgroup、 SAH+Tregs group,compared with that before the modelinduction,significantly reduced(p＜0.01);Compared with the rCBF of the rats in SAH+PBSgroup、SAH+SPgroup,the rCBF of the rats in SAH+Tregs group is evidently increased(p＜0.05).(2)The BA diameter of the rats in the SAH+PBS group、SAH+SP group、 SAH+Tregs group is obviously decreased(p＜0.05)，and the wall thinkness of BA issignificantly increased(p＜0.05);Compared with that of the rats in the SAH+PBS group、SAH+SP group,the BA diameter of the rats in the SAH+Tregs group is distinctlyincreased(p＜0.01),the wall thinkness of BA is significantly decreased(p＜0.01).2.(1)By dry and wet method, moisture content of the rats in SAH+PBS group、SAH+SP group、SAH+Tregs group,compared with that of the rats in control group,isevidently increased(p＜0.05);compared with that of rats in SAH+PBS group、SAH+SPgroup,the moisture content of the rats in SAH+Tregs group is significantly decreased(p＜0.01).By western blot,the expression level of MMP-9in the cerebral cortex of the rats inSAH+PBS group、 SAH+SP group、 SAH+Tregs group is obviously increased(p＜0.05);compared with that of rats in SAH+PBS group、SAH+SP group,the expression levelof MMP-9in the cerebral cortex of the rats in SAH+Tregs group is significantlydecreased(p＜0.05).3.(1)By Nissl’s staining,3days after model induction, Compared with that of rats incontrol group,the density of the neuron cells in Hippocampal CA1area of the rats in theSAH+PBS group、SAH+SP group、SAH+Tregs group is significantly decreased(p＜0.05);Compared with that of rats in SAH+PBS group、SAH+SP group group,the density of theneuron cells in Hippocampal CA1area of the rats in the SAH+Tregs group is increased(p＜0.05).(2)By TUNEL method, compared with that of rats in control group,number of cellapoptosis in the cerebral cortex of rats in the SAH+PBS group、SAH+SP group、SAH+Tregs group is increased obviously (p＜0.05);compared with that in the SAH+PBSgroup、SAH+SP group, number of cell apoptosis in the cerebral cortex of rats in theSAH+Tregs group is decreased (p＜0.05).(4) By the methods of immunofluorescenceand western blot, the protein expressions level of active caspase-3in cerebral cortex of ratsin the SAH+PBS group、SAH+SP group、SAH+Tregs group is significantly increased(p＜0.01);Compared with that in SAH+PBS group、SAH+SP group, the protein expressionslevel of active caspase-3in cerebral cortex of rats in the SAH+Tregs group is decreased(p＜0.05)Conclusions1. The adoptive transfer of regulatory T cells could reduce the degree of CVS afterSAH and prevent pathological change of arterial wall after SAH.2. The adoptive transfer of regulatory T cells could reduce the degree of brain swelling and decrease the expression level of MMP-9in the cerebral cortex of SAH ratsmodel.3. The adoptive transfer of regulatory T cells could prevent the cells of cerebral cortexof rats SAH model from apoptosis.