Bone Marrow Mesenchymal Stem Cells Alleviate Neuropathic Pain After Spinal Cord Injury Through TLR4/MyD88

Background and purpose Spinal cord injury(SCI)is a serious central nervous system(CNS)trauma characterized with high morbidity?disability rate and expenditure but low mortality.And it has been considered as one of the stubborn diseases which is a burden to the patients and their families,affecting their psychological,physical and social well-being.Epidemiological researches show that annually 100,000 individuals live with the disease of SCI in china.Pain often happens following SCI.International regulations divided pain following spinal cord injury into sensory pain,neuropathic pain and other pain.Amang them,the incidence of sensory pain is 50% and the incidence of neuropathic pain is 59%.Neuropathic pain is thought to be the pain that the most common and most difficult to treat.Medicines are available for relieving neuropathic pain with a large side effect and only about a third of the patients can relieve half of the pain.In the present treatment of SCI,mesenchymal stem cells(MSCs)as the representative of adult stem cell transplantation has been given high hopes and has been preliminarily applied in clinic.Thus,it is essential to study the effect of MSCs transplantation on SCI-NP and the mechanism.In recent years,stem cell transplantation on sensory recovery and pain relief after SCI show a great effect.Some studies domestic and overseas have indicated that transplantation of MSCs after spinal cord injury can relieve mechanical pain and heat pain,and prevent the development of chronic pain.However,the effect of stem cell transplantation on pain relief has been controversial.Hofstetter found that NSCs transplantion into SCI rats can promote the motor function recover but the forelimb of the rats suffered allodynia.Therefore,to explore the effect of MSCs transplantation on SCI-NP and the molecular mechanism is the main purpose of this study.Toll-like receptor 4(TLR4)is a kind of the pattern recognition receptor which is closely related to immune and inflammatory diseases,its activation an promote the release of proinflammatory cytokine through NF-?B signaling pathway.In the CNS,TLR4 is mainly expressed in microglia cells and plays an important role in the activation of microglia and inflammatory process.Sorge RE's study shows that TLR4 is related to inflammatory pain and neuropathic pain.MSCs is known as “guardian”against the excessive inflammatory reaction.But it is not clearly that whether MSCs transplation can relieve hyperalgesia induced by chronic inflammation and injury through down-regulate the expression of TLR4.In order to evaluate the effect of MSCs transplantation on SCI-NP and the mechanism,we transplant MSCs into SCI-NP rat and observed the histological and behavioral changes before and after transplantation.Our study will provide theoretical basis and experimental basis for transplantation therapy on SCI-NP.Method1.In order to analysis the feasibility of the study,we search the literature with the key words "MSCs","Spinal cord injury" and "Neuropathic pain " and make a meta-analysis to evaluate the effect of MSCs transplantation on SCI-NP.2.In order to determind the best impact force,we set four groups with different impact force such as 100 kdynes,150kdynes,180 kdynes and 200 kdynes.Detect the changes of mechanical pain threshold at 1d before operation and 7d,14 d,21d and 28 d after operation and the changes of BBB score and inclined plane test at 1d before operation and 1d,3d,5d,7d,14 d,21d and 28 d after operation.3.Establish the SCI-NP model with the best impact force.Detect the the changes of mechanical pain threshold at 1d before operation and 7d,14 d,21d and 28 d after operation and the changes of BBB score and inclined plane test at 1d before operation and 1d,3d,5d,7d,14 d,21d and 28 d after operation.Immunofluorescence(IF)and Western blot are use to test the activation of glia and the expression of TLR4,My D88,p-p38 and p-NF-?B.The expression of IL-6 and TNF-? are test through ELISA.Pathological changes are detected by HE staining and myelin staining.4.Cultured the primary MSCs in vitro derived from the SD rat,Then obtain the second generation and test the expression of CD34,CD45,CD44 and CD90 by flow cytometry to identified MSCs.5.Establishment the SCI-NP model.Then transplant MSCs at 7d after the operation and test the mechanical pain threshold motor function of rat before and after MSCs transplantation.Immunofluorescence(IF)and Western blot are use to test the activation of glia and the expression of TLR4,My D88,p-p38 and p-NF-?B.The expression of IL-6 and TNF-? are test through ELISA.Pathological changes are detected by HE staining and myelin staining.Results1.the results of meta-analysis showed that MSCs transplantation could relieve the mechanical hyperalgesia after SCI,but had no effect on thermal hyperalgesia,and the best time of MSCs transplantation was 7d after SCI.2.The explorarion of impact force showed that the 100 kdneys and 150 kdynes group,there was no neuropathic pain,while the 180 kdynes and 200 kdynes groups had neuropathic pain,but the pain threshold of 200 kdynes group decreased early than180 kdynes group but the motor function between the two groups did not have significant differences,so 200 kdynes is the best impact force.3.The mechanical pain threshold decreased can be observed at 7d after SCI and was gradually stabilized at 21 d after SCI with the impact of 200 kdynes and can last more than 28 d.The BBB score showed that at 1d after SCI,the BBB score was 0,with the injury process,motor function recovered gradually and reached stable at 14 days after SCI,but still can not reach the normal value.The results of inclined plane test showed that the support angle was decreased after SCI,with the injury process,The angle were increased,but still can not reach the normal value.4.The congestion occurs at anticipation after SCI in the local damage.Damage deformation,inflammatory cell infiltration,demyelination and syringomyelia was occured accompanied with the damage process.At the same time the glial was activated at the injured spinal cord and the expression of TLR4,My D88,p-p38,p-NF-?B was increased in microglia,The expression of IL-6/TNF-? was also increased in serum at 24 h after SCI and can last more than 28 d.5.Cultured the primary MSCs in vitro and the expression of CD34,CD45,CD44 and CD90 was detect by flow cytometry.The results showed that CD90 and CD44 was positived and the positive rate was 99.7% and 86.5%,respectively.CD34 and CD45 was negetived.These result showed that the cells that we obtain was MSCs.6.The result showed that MSCs transplantation can significant relief the mechanical pain compared with vehicle group.There was no statistical difference between the two groups on motor function.Molecular biology experiment results showed that the expression of TLR4,My D88,p-p38,p-NF-?B was down regulated,and the expression of IL-6/TNF-was also down regulated in serum.ConclusionMSCs transplantation can relieve the neuropathic pain after SCI by inhibiting the TLR4/My D88 signaling pathway and down regulating the expression of IL-6/TNF-?.