The Effects Of Co-transplant Olfactory Ensheathing Cells And Neural Stem Cells On The Ethology Of Rats Subjected To Transected Spinal Cord In Acute Stage

objective We investigated the effects of co-transplanted neural stem cells(NSCs) and olfactory ensheathing cells(OECs)on the rats'hind limb motor and sensory function recovery,which were subjected to transected spinal cord.And the survival, immigration and differentiation of the transplanted NSCs were observed as well as the regenerating nerve in injured spinal cord.It would provide the theoretical and experimental evidence for treating injured spinal cord.Methods NSCs were dissociated and cloned from the hippocampal tissue of GFP transgenic pregnant mice.OECs were also dissociated and purified from the olfactory bulbs of 1-2day GFP transgenic mice.These cells were identificated by immunostaing.SD female rats were randomly divided into 4 groups:operated group, NSCs transplanted group,OECs transplanted group,and co-transplanted group.The operated group was completely transected at T9-10.In cells grafted groups,a small piece of gelfoam,which was preinjected with cell suspension containing NSCs,or OECs,or both,was placed into the transected cord.The recovery of hind limb motor or sensory function of all rats was evaluated by BBB scoring every week and cortical somatosensory evoked potentials(CSEP) at 4 and 8 weeks post operation(wpo) respectively.At 8 wpo,the response of the rats'hind limbs to stimulate the brain cortical somatomotor area was observed.Then the injured spinal cords were harvested and done frozen section,survival and immigration of transplanted NSCs in vivo were investigated under a fluorescence microscope.Histomorphology was observed by HE staining.The differentiation of transplanted NSCs and regenerating nerve were detected by immunohistoehemistry.BDA tracing were performed to observe the corticospinal tract.The SPSS 11.5 statistical analysis software package was used to analyze the data. Results 1.The primary culture of NSCs presented nestin positive in vitro,while OECs were p75NGFR positive before transplantation.2.Behavioral evaluation showed an improvement in animals with all rats.The results of repeated measures showed that cell-transplantation groups had more scors compared with operation control group(p<0.05).The repair of rat's hind limb motor function in co-transplantation group was better than OECs transplanted rats(p<0.05).There was no statistical difference between in each other group(p>0.05).The results by one-way ANOVA showed that,compared with the operated control group,the BBB scores of NSCs transplanted group were higher in each week after operation,the scores of OECs transplanted group were better at 5,7 and 8 wpo,the scores in co-graft group were better from 4 to 8 wpo.However,at 6 to 8 wpo,hindlimb function of co-transplanted group showed much better recovery than OECs transplanted group(p<0.05).3.No CSEP could be recorded throughout the experimental period.However,cells transplantation reversed this trend,as demonstrated in a significant improvement in sensory function during 8 wpo (p<0.05),the amplitude of P1-N1 wave was higher in grafted groups(p<0.05). Moreover,at 8 wpo,the amplitude of P1-N1 wave in co-graft group was better than single transplanted groups(p<0.05).4.At 8 wpo,the response to stimulate the brain cortical somatomotor area in rats'hind limbs was not observed in all groups.5.After transplantation,both of NSCs and OECs were survived in host,migrated to the rostral and caudal spinal cord.And more NeuN and APC positive cells derived from grafted NSCs were detected in co-graft group than NSCs transplanted group.6.After spinal cord transection,there were many different cavities in host injuried cords of all rats,with forming scar tissue.7.BDA tracing showed that little brown positive fibers were observed in rostral to transected side of operated group,some fibers were in cell grafted rats,and a few were disperse sparsely at scar tissue of co-transplant group.8.Detection of immunohistochemistry:Some the 5-HT,CGRP,GAP-43 and SY positive fibers were observed in rostral to transected side of operated group, more fibers were in cell grafted rats,and several positive fibers were disperse sparsely at scar tissue of co-transplant group.In addition,there were several CGRP, GAP-43 and SY positive fibers in caudal sides of injured cords in cotransplanted group than single cell transplantation.There was no any positive fiber in scar tissue of all rats. Conclusions 1.The transplanted of NSCs and OECs,derived from the hippocampus of GFP transgenic mouse,could be survive and migrate rostrally and caudally.NSCs of grafted groups were differentiated into neurons,astroeytes and oligodentrocytes. Moreover,co-transplantation could increase the percentage of neurons, oligodendraeytes,which derived from grafted NSCs.2.Cells transplantation could improve hindlimb motor and sensory function.However,co-transplant rats showed better in repairing sensory ruction than single transplanted rats,not in improving motor function.3.Co-graft could promote nerve fibres regeneration,but not through the scar tissue.4.No response of rats'hind limbs to stimulate the brain cortical somatomotor area was observed in each group,it could be no synapse forming in injuried spinal cords.