Research Of Neural Stem Cells Transplantation Following Traumatic Brain Injury

Background and Objective:In daily life, due to traffic accidents, productive accidents, natural disasters and other common causes, the incidence of traumatic brain injury(TBI)has been high so far. However, just because of its high disability rate and morbidity rate, TBI has become one of the most common diseases threatening people’s health and life. Due to the lack of regeneration of neurons, most survivors’ nerve functions are uncomplete or missing after traumatic brain injury, which results in the loss of part or all of the labor ability and self-care ability, meanwhile it also bings huge burden to society and family. Protection and restoration of neurological function as the key to clinical therapy after traumatic brain injury, has been faced with serious challenges. As for the nerve dysfunction after TBI, there is no specific effective treatment method. That’s the reason why we need to explore new effective treatment methods to improve the situation.Due to the limitation of their self-repair after injury of the central nervous system, the discovery of neural stem cells( NSCs) appeared to bring the light for the treatment of traumatic brain injury. At present, the research of NSCs in vitro has become increasingly mature, and the transplantation of NSCs in the treatment of traumatic brain injury has got more and more attention.In this research, one hand we studied the isolation,culture and identification of NSCs in vitro,on the other hand we marked NSCs and studied their proliferation and differentiation of phenotypic characteristics. Based on this, the NSCs marked in vitro were transplanted into the brain of the model rats, and the distribution, migration and differentiation of NSCs in the host brain were observed. To explore new ways and provide theoretical support for the treatment of TBI, we evaluated the effects of NSCs’ transplantation on the repair of TBI from the following aspects: morphology, structure and function recovery.Contents and methods:1.Neural stem cells: in vitro culture, identification and differentiated phenotypeTaking 1 to 2 days’ newborn kunming mice, mechanically extracting NSCs fromhippocampus and olfactory bulb. After 3 weeks of screening culture, the suspended cells were labeled with 5-bromo-2-deoxyuridine(Brd U) and identified by immunofluorescence staining of anti-Brd U, Nestin and Hochest33258.Attachment and differentiation of NSCs was induced in vitro, then the daughter cells were identified by composite immunofluorescent staining of anti-Brd U, β-tubulinⅢ, glial fibrillary acidic protein(GFAP) and Hochest33258 to confirm differentiated phenotype.2.Transplantation of NSCs after Traumatic Brain InjuryThe improved Feeney’s method of free-fall impact was applied to build closed traumatic brain injury model.One day later, NSCs were transplanted into the brain of model.Neurological Severity Scores(NSS) and time of walking on the rotarod were used to test the function at the 0d,3d, 7d, 14 d, 21 d post transplantation. Carrying on immunofluorescence staining anti-Brd U,Nestin,GFAP, β-TubulinⅢ at 7d, 21 d post transplantation.Results:1.Hundreds of cells from hippocampus and olfactory bulb of newborn mice were aggregated to form neurospheres which showed suspended growth and different sizes after long-term culture.Immunofluorescence staining showed both nestin and Brd U were positive in neurospheres.Under induced differentiation in vitro, NSCs could turn into daughter cells which positively expressed β-tubulin Ⅲor GFAP.2.There was significant difference in NSS between transplanting group( 10.22±1.42、7.44±1.17、6.22±1.06) and control group( 12.44±2.01、9.44±1.91、7.33±1.63) at 7d,14 d, 21 d post transplantation( P<0.05). There was significant difference in rotarod performance between transplanting group( 274.21±4.34s、292.88±4.53s) and control group( 244.32±9.55、250.48± 10.87) at 14 d,21d post transplantation( P <0.05). Immunofluorescence staining of brain tissue slices was performed at 7d after transplantation, we found that Brd U/nestin double positive cells were confined to the "tunnel" of needle. 21 d after transplantation, Brd U/GFAP double positive cells and Brd U/β-tubulinⅢ double positive cells were found distributing in the brain injury area and the surrounding.Conclusion:1.Newborn mice’ hippocampus and olfactory bulb contained abundant source of NSCs.NSCs owned the ability of proliferation, self-renewal and poly-potential for differentiation. They had the tendency to form stablely suspended neurospheres during culture in vitro.They could differentiate into neurons and astrocytes under induction in vitro,which laid the foundation of NSCs transplantation in living body.2.A Part Part of NSCs transplanted into the host brain could survive, migrate and differentiateinto astrocytes and neurons in damaged areas,which could improve nerve function after TBI within a certain period.In a word, the transplantation of exogenous NSCs to repair TBI had a certain effect, but the long-term effect and the specific treatment mechanism remained to be further studied.