Exogenous Regulatory T Cells Can Reduce The Inflammatory Response In The Mice With Cerebral Hemorrhage And Improve Their Long-term Prognosis Of Neurological Function

Objective1. To Study the Cerebral protective effect of CD4+CD25+regulatory T cellstransplantation therapy on mice with cerebral hemorrhage.2. To observe the changes of inflammatory cytokines of the brain tissue in mice withcerebral hemorrhage after regulatory T cells transplantation therapy and to study the possi-ble mechanism of cerebral protective in the inflammatory response.Method1. Immunomagnetic beads two step extraction of adult healthy Kunming mice ofCD4+CD25+regulatory T cells. Autologous blood were injected to the left basal ganglia ofthe healthy male Kunming mice to establish experimental Cerebral hemorrhage mode-l.experimental animal were randomly divided into sham operation group, cerebral hemorr-hage group, PBS control group (cerebral hemorrhage+PBS intravenous injection with thevolume of Tregs), Tregs treatment group (Tregs IV). PBS, Tregs were intravenous infusionon2hours after the models were made. To measure the blood pressure and blood gas inarterial of mice of1hours before and2hours after operation. At six time points respecti-vely of1d,3d,7d,14d,21d,28d after the surgery, observe the neurological deficit and gotthe behavior score;brain water content was measured by dry and wet weight method on1d,3d,7d after surgey.2. Immunomagnetic beads two step extraction of adult healthy Kunming mice ofCD4+CD25+regulatory T cells. Autologous blood were injected to the left basal ganglia ofthe healthy male Kunming mice to establish experimental Cerebral hemorrhage model. Ex-perimental animal were randomly divided into sham operation group, cerebral hemorrhage group, PBS control group (cerebral hemorrhage+PBS intravenous injection with thevolume of Tregs), Tregs treatment group (Tregs IV). PBS, Tregs were intravenous infusionon2hours after the models were made. At three time points respectively of1d,3d,7d afterthe surgery, decapitate and get the fresh brain tissue to measure the expression levels ofinflammatory cells factor IL-6, TNF-α,IL-Iβ of the brain tissue homogenates by biotindouble antibody sandwich enzyme-linked immunosorbent assay (ELISA); decapitate andprepare brain paraffin sections of brain tissue to measure the expression of inflammatorycytokines IL-6,nuclear transcription factor NF-κB of perihematoma by immunohisto che-mistry staining fluorescence on the third day after the surgery.Result1. The blood pressure and blood gas analysis of arterial in each animal group of1hours and2hours after operation show no obvious difference.The sham operation grouphad no obvious neurological deficit; Except the sham operation group,another3groups’sneurobehavioral defects heavier before first three days, and worst on the first day, thengradually recovered; compared with the sham group, the differences were significant on1d,3d,7d,14d,21d after the surgery (P <0.05); Compared with the PBS group, the Tregstreatment group’s neurological deficit significantly reduced (P <0.05);The PBS groupshowed no significantly difference with the Cerebral hemorrhage group at each time points.The brain water content of cerebral hemorrhage group, PBS group and Tregs treatmentgroup obvious higher than the sham control group on1d,3d,7d after the surgery (P <005),and have the highest on the third day; The water content of brain tissue of Tregs group wasobviously lower than that of the PBS control group and cerebral hemorrhage group (P <005); cerebral hemorrhage group and PBS control group showed no significant difference ateach tome points.2.The inflammatory factor content of brain tissue in Cerebral hemorrhage group, PBScontrol group and Tregs treatment group were significantly higher than that in the shamoperation group on1d、3d、7d after the surgery(P <0.05), and had the highest expressionon the third day; But the content of inflammatory factor in Tregs treatment group weresignificantly lower than those of PBS control group and cerebral hemorrhage group (P <0.05).Compared with the sham operation group,the expression of inflammatory cytokineIL-6and transcription factor NF-κB in the cerebral hemorrhage group, PBS control groupand Tregs treatment group were significantly higher than those in the sham operation groupon the3th day after the surgery (P <0.05); But the Tregs’s expression was lower than that of the cerebral hemorrhage group, PBS control group. The difference was statistically sign-ificant (P <0.05).Conclusion1. CD4+CD25+regulatory T cells intravenous transplant by vein to mice withcerebral hemorrhage can reduce the brain edema, improve the long-term neurologicaldeficits and play a role in brain protection.2. CD4+CD25+regulatory T cells intravenous transplantation by vein to mice withcerebral hemorrhage can reduce the expression of inflammatory cytokines and nuclear fac-tor of the brain tissue around the hematoma, which may play a role in brain protection byreducing the inflammatory response, and be expected to provide a basic treatment toimprove the long-term function defect after cerebral hemorrhage through the infusion ofexogenous regulatory T cells.