Treating Spinal Cord Injury By Olfactory Ensheathing Cells And The Mechianisms

Spinal cord injury refers to the damage to the spinal cord from various causes,resulting in temporary or permanent dysfunction of motor,sensory,and autonomic nerves below the level of injury.Self-repair after spinal cord injury is extremely limited,and various clinical methods,including surgery,drugs,rehabilitation training,etc,have limited efficacy,have limited efficacy.In recent years,cell transplantation has been considered as one of the most promising treatments for spinal cord injury.Neural stem cells(NSCs)are the most important cells in cell replacement therapy.They can self-renew and differentiate into neurons,astrocytes or oligodendrocytes/oligodendrocyte precursor cells,thereby replacing or supplementing the dead cells after spinal cord injury to play a role in nerve repair.Although several studies have shown the effectiveness of neural stem cell transplantation in the treatment of spinal cord injury,the ethical,source,immune rejection,and tumorigenicity issues of exogenous cells have limited their clinical application prospects.Therefore,activation of endogenous neural stem cells involved in nerve repair after spinal cord injury has become a research hotspot.The central nervous system of adult mammals is thought to be difficult to regenerate,but the olfactory system is an exception.Olfactory neurons can continuously regenerate throughout life,and this special ability may be related to the special glial cells in the olfactory system-olfactory ensheathing cells(OEC).OEC is a kind of glial cell that can contribute to the axons growth and regeneration of olfactory neuronal throughout life.Olfactory ensheathing cells have the functions of proliferation,ensheathing,and secretion of cytokines,which make olfactory ensheathing cells the most promising candidate cells for cell transplantation in spinal cord injury.Previous studies have shown that olfactory ensheathing cells can promote the proliferation of neural stem cells in vitro,and the efficacy of combined transplantation of olfactory ensheathe cells and neural stem cells in the treatment of spinal cord injury is better than that of single cell transplantation.It may be related to the secretion of cytokines by OECs to support the survival and proliferation of NSCs.However,it remains to be shown whether olfactory ensheathing cells can activate endogenous neural stem cells in the spinal cord.Our previous study observed that co-culture of olfactory ensheathing cells and neural stem cells in vitro could significantly promote the proliferation and differentiation of neural stem cells,which suggested that olfactory ensheathing cells transplanted into the myeloid might activate neural stem cells in situ to participate in nerve repair after SCI.However,the mechanism by which olfactory ensheathing cells promote the proliferation and differentiation of neural stem cells remains unclear.In our previous experiments,we found that compared with the supernatant of olfactory ensheathing cells,olfactory ensheathing cells themselves could more effectively activate the proliferation of neural stem cells,indicating that this effect also occurred in the form of intercellular communication,rather than just the role of cytokines secreted by OECs.Studies have shown that multiple signaling pathways,such as Notch,WNT and TGF,are involved in the proliferation regulation of neural stem cells.Notch signaling pathway plays a fateful role in the proliferation and differentiation of NSCs.Unlike other signaling pathways,Notch signaling occurs through cell-to-cell communication,in which transmembrane ligand on one cell activates transmembrane receptors on neighboring cells.During normal development of the nervous system,neighboring cells expressing Notch receptors and Delta ligands interact to mediate the lateral inhibition mechanism that determines the distinct differentiation fates of cells.It was shown that olfactory ensheathing cells express a high level of Notch3 receptor,which binds to the Dleta ligand of Müler cells,the major glial cell in the retinal membrane,and medilates a side inhibitory effect on the proliferation of Müller cells.Neural stem cells express Delta ligand on the surface.When olfactory ensheathing cells are transplanted into the medulla,can they also regulate the proliferation of neural stem cells through the Notch-Delta signaling system?We propose that OECs transplantation can inhibit Notch signaling activation through the Notch-Delta signaling system,thereby promoting endogenous neural stem cell proliferation and participating in nerve regeneration and repair after spinal cord injury.Therefore,this study will be verified from the following three aspects:Part Ⅰ:The therapeutic effect of olfactory ensheathing cell transplantation on spinal cord injuryObjectiveTo observe the effects of OEC transplantation on the motor and autonomic functions,spinal cord structure,endogenous NSC activation,newborn neurons and synapse changes in rats,and to clarify the therapeutic effect of OEC transplantation on SCI..MethodsEighty rats were divided into four groups:the sham group,SCI group,SCI+OEC-CM group,and SCI+OEC group.Sham group was given only T10 laminectomy,and the other three groups were given SCI modeling.After successful modeling,the cells were transplanted The SCI group:DMEM/F12 medium was injected;The SCI+OEC group:OECs suspension,concentration 1.0×105 cells/μL;The SCI+OEC-CM group:OEC conditioned medium(OEC-CM)was injected.The BBB score and sympathetic nervous system skin response(SSR)were measured 1-4 weeks after surgery to assess motor and autonomic function.At 4 weeks after operation,the spinal cord of SCI rats was harvested and the spinal cord was stained with HE,Nissl and LFB to observe the changes of spinal cord structure.The expression levels of Nestin,DCX,NF200,GFAP and SYP were detected by immunohistochemistry,immunofluorescence and Western blot,respectively.ResultsBBB scores of rats in each group after SCI were significantly lower than those in sham group.The BBB scores of SCI+OEC group were significantly higher than those of SCI Group and SCI+OEC-CM Group at all time points.SSR detection:after SCI,the extraction rate of SSR decreased,the latencies of SSR were prolonged and the amplitudes were decreased.The changes of latency and amplitude in SCI+OEC group were significantly less than those in SCI Group and SCI+OEC-CM Group.HE staining showed that the structure of medulla spinalis in SCI+OEC group was intact.The number of neurons stained with Nissl staining and the relative staining area of LFB were higher than those of SCI and SCI+OEC-CM groups.The protein expressions of Nestin,NF200,DCX and SYP in SCI+OEC group were increased,and the expression of GFAP was decreased compared with the other three groups.ConclusionOEC promoted the proliferation of NSCS in spinal cord,generated new neurons and synapses,and improved the motor and autonomic functions of SCI rats.Part Ⅱ:The regulatory role of olfactory ensheathing cell on the proliferation and differentiation of endogenous neural stem cells in the spinal cordObjectiveOECs and NSCs were cultured in vitro,and SCI-like micro-environment was prepared with injured spinal cord supernatants to investigate the effects of OECs on proliferation and differentiation of NSCs in normal and SCI microenvironment.Methods1.Cell groups in the normal microenvironment:NSC+OEC Group,NSC+OEC-CM Group,NSC Group.SCI microenvironment cells were divided into naive-h(normal spinal cord supernatant),SCI-h(injured spinal cord supernatant)and SCI-h+OEC groups.The proliferation of NSC was measured by CCK-8 and Cytometry.2.NSC differentiation was induced by serum and OEC,respectively,after 5 days of culture,GFAP and β-Ⅲ tubulin were used for immunofluorescence double staining,and the ratio of GFAP+ and β-Ⅲ tubulin+cells was determined by Image J to evaluate NSC differentiation.Results1.The number of cells and OD value in NSC+OEC group were higher than those in NSC Group and NSC+OEC-CM group(p<0.05).There was no significant difference between NSC+OEC-CM Group and NSC Group.2.In SCI microenvironment,the number of cells and OD value of SCI-h+OEC group were significantly higher than those of the other two groups at each time point(p<0.05).There was no significant difference between naive-h group and SCI-h group.3.Compared with the NSC +serum group,the NSC+OEC group differentiated more neurons.ConclusionIn both normal and SCI microenvironments,OEC could promote NSC proliferation and neuronal differentiation.Part Ⅲ Role of the Notch signal pathway in the activation of endogenous neural stem cell proliferation and differentiation by olfactory ensheathing cellObjectiveTo investigate whether OEC promotes endogenous NSC proliferation through Notch signaling pathway and the mechanism by which OEC regulates Notch signaling pathway.Methods1.The rat model of SCI was established,and the mRNA and protein of spinal cord were extracted 1-7 days after SCI.The expression of Notch signaling pathway was detected by qPCR and Western blot.2.OECs and NSCs were co-cultured in normal and SCI microenvironments,and SCI rat models were established for OEC transplantation.The expression of Notch signaling pathway was detected by qPCR and Western blot.3.Interfering with NOTCH3 expression of OECs and DLL 1(Delta 1)expression of NSCs by siRNA,respectively.The proliferation of NSCs was determined by CCK-8 and Cytometry,and the expression of Hesl was detected by Western blot.Results:1.The mRNA and protein expressions of Notch2,Hesl and RBP were the highest on the 3rd day after SCI(p<0.05),and decreased to normal on the 7th day after SCI.The expressions of β-Ⅲ tubulin and CD133 were significantly decreased(p<0.05).2.The mRNA and protein expressions of Notch2 and Hesl in NSC+OEC group were significantly lower than those in NSC group after co-culture in normal microenvironment for 3 days.3.The mRNA and protein expressions of Notch2,RBP and Hesl in SCI-h group were significantly higher than those in naive-h group.Compared with SCI-h group,NOTCH2 receptor and RBP,HES1 protein expression in SCI-h+ OEC group were significantly decreased.4.Compared with sham group,the mRNA and protein expressions of Notch2,RBP and Hes1 were significantly increased after SCI.Compared with SCI Group and SCI+OEC-CM Group,the expression of Notchl,Notch2,RBP,HES1 mRNA and protein decreased and the expression of Mash1 increased in SCI+OEC Group.5.The results of Cytometry and CCK-8 test showed that the proliferation of NSC was decreased after siRNA interference,and the difference was statistically significant compared with NC Group.Western blot analysis showed that the expression of Hes1 was significantly higher in the siRNA-treated group than that in the NC group after interfering with Notce3 and DLL1,respectively.ConclusionNotch signaling pathway is activated after SCI.Both coculture of OECs with NSCs and transplantation of intramedullary OECs can inhibit Notch signaling pathway activation through NOTCH3-DLL1.SummaryOEC can inhibit the activation of Notch signaling pathway through NOTCH3-DLL1 signal,thus promote the proliferation of endogenous nscs,the generation of new neurons and synapses,and improve the motor and autonomic functions of SCI rats.