The Mechanism Of Spinal Cord Stimulation For The Treatment Of Neuropathic Pain By Affecting CSF1 In CCI Rats

Introduction:Neuropathic pain(NP)is an important factor that threaten the health and quality of us.However,traditional drugs have limitations in the treatment of neuropathic pain.The emergence of Spinal cord stimulation(SCS)provides a new treatment strategy for NP.However,not all patients have well efficacy to SCS.Therefore,it is very important to explore an optimal and most suitable SCS mode for different patients,and the premise of realizing this dream is to understand the mechanism of SCS therapy.However,the precise mechanisms underlying pain relief by SCS remain poorly understood in the present.Previous studies have suggested that SCS alleviates pain by activating axons to produce numbness that masks the pain or by activating intermediate neurons in the spinal horn to induce brain chemical release including serotonin,epinephrine,gamma aminobutyric acid(GABA),acetylcholine,and adenosine.Microglial are now considered to play an important role in the development and maintenance of NP.And Some researches showed that SCS reduces chronic pain partially by inhibiting spinal microglial activation now.However,the critical pathway leading to the reduced activity of microglia by SCS is unclear.Recent evidence reveals that injured sensory neuron-derived colony-stimulating factor 1(CSF1)induces microglial activation in the spinal cord,contributing to the development of neuropathic pain(NP).Objective:To observe whether SCS could improve the mechanical withdrawal threshold of paws(PWT)in Chronic constriction injury(CCI)rats.And the level of CSF1 expression and microglial activation in the dorsal root ganglion(DRG),dorsal root(DR)and dorsal horn(DH),respectively.To investigate whether SCS alleviates pain via inhibiting microglia activation by affecting the level of CSF1 in CCI rats.Methods:Part I: The CCI rat model was used to explore the level of CSF1 expression,microglial activity and the Mechanical allodynia after nerve injury.1.Mechanical allodynia were assessed by measuring the mechanical withdrawal threshold of paws(PWT)on day 1,3,7,14,and 21 after CCI.2.Immunofluorescence double-label CD11 b and CSF1 were used to evaluate CSF1 expression level and microglia activation in dorsal horn.(CD11b,also known as OX42,is a specific marker of microglia)3.Western blot was used to evaluate the protein expression of CSF1 in dorsal root ganglion and dorsal horn after CCI.4.After intrathecal injection of different concentrations of recombinant CSF1 protein,PWT and immunofluorescence single label CD11 b were done to further evaluate the effect of CSF1 on microglia activity and mechanical allodynia.Part II: The effect of SCS on CSF1 expression,microglial activity and mechanical allodynia were explored in CCI rat model with or without SCS.1.The mechanical withdrawal threshold of paws of CCI rats with SCS were measured to evaluate the analgesic effect of SCS;2.Immunofluorescence double-label CD11 b and CSF1 were used to evaluate the effect of SCS on CSF1 expression level and microglia activation in the dorsal horn(DH)of CCI rats.3.Western blot was used to evaluate the effect of SCS on CSF1 protein expression in dorsal horn(DH),dorsal root ganglion(DRG)and dorsal root nerve(DR)of CCI rats.4.Quantitative real time PCR(q RT-PCR)was used to evaluate the effect of SCS on proinflammatory cytokines in the dorsal horn of CCI rats.5.Immunofluorescence single label CSF1 was used to evaluate the effect of SCS on CSF1 protein level in dorsal root ganglion(DRG)and dorsal root nerve(DR)of CCI rats.Part III: Some rats received a once-daily intrathecal injection of CSF1 for 3 days during SCS significantly reduced PWT and enhanced CD11 b fluorescence intensity in dorsal horn.1.Mechanical allodynia were evaluated by observing the PWT in rats;2.Immunofluorescence single label CD11 b was used to assess changes in microglia activity in the dorsal horn.Results:Part I:1.Mechanical withdrawal threshold of the paws(PWT)was significantly decreased in CCI rats;2.CSF1 expression levels in dorsal horn and dorsal root ganglion were significantly increased in CCI rats;3.The fluorescence intensity of CD11 b were significantly enhanced in CCI rats;Part II:1.SCS could significantly improve PWT in CCI rats;2.SCS significantly decreased CSF1 expression in the dorsal horn and dorsal root nerve(DR)of CCI rats,but no significant change in CSF1 expression in the dorsal root ganglion(DRG).3.SCS significantly reduced the fluorescence intensity of CD11 b in the spinal dorsal horn of CCI rats;Part III:Intrathecal injection of recombinant CSF1 protein in CCI rats with SCS significantly reduced PWT and significantly enhanced CD11 b fluorescence intensity in dorsal horn.Conclusion:1.In CCI model rats,CSF1 is an important factor on the activation of microglia in spinal dorsal horn,and can directly induce neuropathic pain in normal rats.2.SCS down-regulated CSF1 expression levels in the spinal dorsal horn(DH)and dorsal root nerve(DR)of CCI rats,but did not affect CSF1 expression levels in the dorsal root ganglion(DRG).3.The effects of SCS on inhibiting microglial activation in CCI rats were mediated by downregulation of CSF1 in the dorsal horn(DH).