The Pilot Study Of The Therapy For TBi Immerged With Seawater In Rats By The Transplantation Of NSCs Combined With GABAergic Neurons

Objective:1.To investigate the effect of blocking Hesl on gamma aminobutyric acid(GABA)neurons' differentiation in human neural stem cells(NSCs)and find a convenient and efficient method for GABAergic cells' differentiation.2.Modified the model of traumatic brain injury(TBI)immerged with seawater,so as to simulate the characteristic of TBI immerged with seawater more veritably.3.To observe the change of pathology on neuro-inflammation in regional brain tissues and their excitation,assembly,distribution of microglia in TBI insults with seawater immersion rats.4.To investigate the therapeutic effect for the neural function defect of TBI immerged with seawater's rats by stereotaxic transplanting NSCs combined with GABAergic neurons into the injured rats.Material and method:During the differentiation of GABA neurons with human NSCs,the control cultured medium were the common differentiated medium added 10%BSA,and the experimental cultured mediums were identical with the control excepted for the anti-Hes1.Anti-GABA immunofluorescence staining was carried out when most of the cells were differentiated.Modified the needle pellet which was used for making the model of TBI,and changed the mothed of rats immerged with seawater by fixing the tail end of the enema bag above the surrounding of the damage.To assess the new model,the effect on the neural function defect was observed on different time of immerged seawater(30 min,45min and 60min).When the model of serious TBI was made,the regional brain tissues were immerged with seawater for 45 minutes.The brain tissues were harvested at 24h,48h,72h,and 96h after immerged with seawater.Then the tissues were investigated by hematoxylin-eosin(HE)and immune-histochemical staining.Observe the change of pathology on neuro-inflammation in regional brain tissues and the excitation,assembly,distribution of microglia in TBI insults with seawater immersion rats.The treatment work of cells transplantation for the model of TBI immerged with seawater was started when completed the previous preparation.There were total three groups:combined therapy group(NSCs combined GABAergic neurons),simple therapy group(only NSCs),simple control group(just injured).In the combined therapy group,the NSCs combined with GABAergic neurons were transplanted into the model rats on day 4 after the model rats were made.In the simple therapy group,only NSCs were transplanted in the same way.The rats in simple control group were injured only without any therapy.The rats' neurobehavioral manifestation would be recorded;the survival rate and the distribution of the transplanted cells would be observed in the different time.Result:1.90%-95%cells were adherent and differentiated on day 14.The result of anti-GABA immunofluorescence stain showed the positive cell rate was 64.0%in anti-Hes1 differentiated group and 16.6%in control group(P<0.01).2.The survival rates of the model rats were all 100%in 30min group and 45min group while was only 20%in 60min group.The rate above 3 grade of the neural function defect was 80%in 45min group which was distinctly suitable for the further investigation compared with other two groups.3.The neuro-inflammation was most severe after injured 48h-72h,the primary characteristics included:blood cells increased,brain edema became more and more severe,microglia actives constantly.The number of activated microglia got the maximum value in 48h-72h after injury and the activation of phagocytosis was also found.The blood cells were decreased,brain edema was alleviative and the number of microglia was decrease in 72h-96h.4.Two therapy groups' results of behavioral tests were superior to the simple control group.Compared with the simple therapy group,the combined therapy group's score of modified neurological severity scores(mNSS)was better on 14-31days after transplantation(P<0.05)while the result of Limb use asymmetry test(LUA)was better only on day 21(P<0.05).Conclusion:1.Blocking Hesl can significant increase the differentiation rate of GABAergic neurons in human NSCs.2.The modified model of TBI immerged with seawater can simulate the characteristic of TBI immerged with seawater veritably while satisfy the necessary for scientific research.3.The neuro-inflammation was most severe and the number of activated microglia got the maximum value in the initial 72 hours in TBI model immerged with seawater.Then the changes were starting to level off in 72-96 hours.4.Cell therapy is an effective approach.The therapeutic effect of transplanting NSCs combined with GABAergic neurons is better than that of transplanting NSCs merely on motor function defect,but the effect on memory is remain to explore.