The Effects Of Rats' Behavior After Co-transplantation Of Neural Stem Cells And Olfactory Ensheathing Cells Into Transected Spinal Cord In Chronic Phase

【Objective】To investigate the therapy of neural stem cells(NSCs) and olfactory ensheathing cells(OECs) were isolated from the ICR(Institute of Cancer Researcch) embryo mouse' hippocamp and neonatal mouse' olfactory bulb,co-transplantation on adult rats' transected spinal cord.We explored the effect on the hind limb motor as well as sensory function recovery.And the cell survival as well as migration after transplantation was also observed.So as to provide the experiment evidences for the usage of stem cells transplantation for transected spinal cord injury(SCI) therapy.【Methods】72 female SD(Sprague Dawley) rats were divided into six groups,sham group,simple transected spinal cord group(control group),control + CsA(Ciclos porin A)group,NSCs group,OECs group and NSCs+OECs group(n=12 in each group).NSCs(labeled by Hoechst33342) isolated from the hippocampus and OECs(OECs simple grafted group labeled by Hoechst33342;co-grafted group OECs labeled by BrdU) isolated from the olfactory bulb of ICR mouse in vitro,were transplanted into the rostral,scar and caudal site of transected spinal cord of rats in chronic phase(postoperative 14 day) under stereotaxic apparatus.All rats were estimated the motor functional recovery of hind limb by system of the Basso-Beattie-Bresnahan (BBB) locomotion score at each weekend in 12 weeks after injury or cells transplantation respectively.The sensory functional recovery of hind limb was evaluated using Von-Frey Filament and Plantar Test at 12 week post-operation.After the spinal cord was obtained and done frozen section,the survival and migration of transplanted cells were observed under inversion fluorescence microscope and used immunohistochemistry technique to analysis the differentiation of NSCs,which grafed into spinal cord.The SPSS 15.0 statistical analysis software package was used to analyze the data.【Results】1.At 1-2 days after primary culture,cell aggregation and the neurosphere was observed;at 5-6 days after primary culture,neurosphere growth floating and arrange tight among cells,Immunocytochemistry showed that the Nestin positive staining. The OECs display a very characteristic morphological appearance.Most of OECs are bipolar or tripolar with long and thin processes.Positive staining of P75 was observed in OECs by immunocytochemistry,2.The BBB scoring of control group and control + CsA group,increased slowly as the time prolonged,no statistically significant differences were observed between two groups.The BBB scoring of all transplantion groups were improved significantly contrasting to that of control group,from 2 weeks to 12 weeks,especially the co-grafted group(P＜0.05).After the transected spinal cord injury of rats,the hind limb sensory function of cells transplantation groups improve more than that of operated group,especially the co-grafted group,were improved significantly contrasting to the control group at 12 week post-operation(P＜0.05).3.Transplanted NSCs and OECs can survive for at least twelve weeks in the rostral, scar and caudal site of transected spinal cord of rats.The number of surviving neural stem cells and differentiated neuron-like cells and asrrocyte-like cells were significantly increased in the co-grafted(NSCs and OECs) group compared with the NSCs group(P＜0.05).【Conclusion】1.Neural stem cells were in neurosphere growth in vitro The result of immunocytochemistry demonstrated that the cultured cells were neural stem cells. Olfactory ensheathing cells were in bipolar or tripolar growth in vitro,Positive staining of P75 was observed in OECs by immunocytochemistry.2.Transplantion of NSCs and OECs can enhance recovery of locomotion and sensory function for adult rats with spinal cord injury.Especially Co-grafted group was more effective on rats' behavior. 3.Transplanted NSCs and OECs can survive for at least twelve weeks in spinal cord. OECs can promote the survival and differentiation of transplanted neural stem cells in the injured spinal cord.