Observation Of The Curative Effect Of Human Neural Stem Cell Transplantation Combined With Immunosuppressants On Ischemic Stroke In Mice

Background:Stem cell transplantation therapy is widely used in regenerative repair medicine.However,when stem cells are used in ischemic stroke,due to the particularity of the brain’s immune system,there is still controversy as to whether stem cell transplantation of different species needs to use immunosuppressive agents to treat stroke.Purpose:To evaluate the effect of human neural stem cells（hNSCs）transplantation in a mouse model of ischemic stroke and reperfusion,and explore the effect of combined immunosuppressant use on the efficacy of ischemic stroke.Method:The ischemia-reperfusion model of C57B6/l mice（ischemia 45min）was made by the suture method to simulate ischemic stroke.The C57B6/l mice through behavioral training were divided into group A:hNSCs transplantation group（n=8）;Group B:hNSCs+cyclosporin A transplantation group（n=9）;Group C:cerebral ischemia model control group（cell solvent therapy for cerebral ischemia model,n=8）.Using hNSCs prepared from fetal brain tissue that was spontaneously aborted at 12 weeks of gestation,the hNSCs was injected into the striatum and cerebral cortex of the injured side of mice with ischemia-reperfusion injury through stereotactic injection,and the striatum and cortex of the injured side were 5×10 ⁵ cells（6μL cell suspension）.One week and four weeks after transplantation,the movement balance function of mice in different transplantation groups was evaluated through behavioral experiments such as the rotating rod experiment and the corner experiment.Three days after modeling and 4 weeks after transplantation of hNSCs,the mice were examined by MRI T₂ imaging,and ITK-SNAP3.8 software was used to calculate the cerebral infarct volume of mice.After 4weeks of transplantation,the brain was perfused and taken,paraffin sectioned,histopathological and immunofluorescent staining,Label the implanted hNSCs with anti-human cytoplasmic protein antibody,label astrocytes with anti-glial fibrillary acidic protein antibody,and label neurons with anti-tubulin-Ⅲ-antibody to observe the survival,distribution and differentiation of implanted cells.Result:（1）In the rotating rod experiment,the results of group A and group B were significantly better than group C in the first and fourth weeks after transplantation,and the difference was statistically significant（p<0.05）.There was no statistically significant difference between group B and group A（p>0.05）.（2）In the corner experiment,the number of right turns of group A and group B in the first and fourth weeks after transplantation was significantly lower than that of the cerebral ischemia model control group,the difference was statistically significant（p<0.05）,and there was no difference between group B and group A（p>0.05）.（3）The infarct volume of group A,B and C 4 weeks after transplantation was smaller than that 3 days after modeling.In the 4th week after transplantation,the infarct volume of group A and group B was smaller than that of the control group（p<0.05）,the infarct volume of group B was smaller than that of group A,and the difference was statistically significant（p<0.05）.（4）The hNSCs in group B were in the transplantation site.The survival rate near the point was better than that of group A（p<0.05）.（5）In the observation,it was not found that the use of immunosuppressive agents had a significant effect on the migration,distribution and differentiation of hNSCs implanted in the brain of mice.In the two treatment groups A and B,most of the implanted hNSCs still gathered at the transplantation site nearby,a small part migrated around.In both treatment groups A and B,part of the implanted hNSCs differentiated into astrocytes,and a small part differentiated into neurons.Conclusion:hNSCs transplantation can promote the recovery of motor balance after ischemic stroke in mice,and the use of cyclosporin A has no significant effect on the recovery of motor balance after ischemic stroke in mice.The transplantation of hNSCs therapy can protect the ischemic penumbra,thereby reducing the infarct volume of cerebral ischemic stroke in mice,and the combined use of immunosuppressive agents can promote this protective effect.When hNSCs are used to treat ischemic stroke in mice,the combined use of immunosuppressive agents can increase the survival rate of transplanted hNSCs.