Ralationship Between Complement 3 And Neuropthic Pain: An Experimental Study

Background: Mechanism of neuropathic pain is complex variety.Central theory of the neurons can not fully explain some Pathological phenomena, Such as phantom limb pain, specular image pain.There is growing recognition that glia in spinal cord could contribute to neuropathic pain powerfully。Astrocyte is immunologically competent, it not only secrete a range of inflammatory cytokines, but also secrete complement proteins. These complement proteins may constitute a complete complement system of the central nervous system. The expression of complement 3 was the highest in the complement system, and it play a central role in three complement activation pathways. Wangjinbao's study found that the expression of complement 3 in rats cerebrospinal fluid with neuropathic pain is consistent with the formation time of hyperalgesia.Nevertheless,they did not further investigate whether Complement 3 is involved in the immune regulation on signal transmission of NPP. And Complement 3 is to promote, maintain and amplify the pain state ?Objective: In the study, the production of the CCI model induced NPP by C3-gene knock out mice, And complement 3 by exogenous interventions in this model.The aim of this study was to observe behavioral changes in NPP mice, oxidative inflammation damage of spinal cord, activation effect on astrocytes.Method: This study was composed of three parts as follows.1.behavioral assessment of C3-gene knock out miceEighteen C3-gene knock out mice were divided into three groups randomly:Sham group,(S group n=6); Model control group(M group,n=6); Experimental protein intervention group(E group, n=6).The threshold in nerve injured hind paw to thermal stimulus (pain threshold) and behavioral changes of mice were recorded at pre-operation and1,3,5,7,10,14days after operation.2. Effect of complement 3 on astrocytes activation in the neuropathic pain model miceEight-one C3-gene knock out mice were divided randomly into nine groups as sham 1,3,7d, Model control 1,3,7d, Experimental protein intervention1,3,7d. the mRNA and protein of GFAP were determined by methods of RT-PCR, immunohistochemistry in different time according to the groups. astrocyte Morphologic changes in spinal dorsal horn neurons were observed under Laser scanning confocal microscope by Immunofluorescence staining.3. Effect of complement 3 on oxidative inflammation injury response in the neuropathic pain model mice One hundred forty-four C3-gene knock out mice were divided randomly into three groups as sham, Model control, Experimental protein intervention. The L4-6 segments of the spinal cord were isolated in 1,3 and 7 days after surgery. SOD activity and MDA content in the spinal cord homogenate were determined in different time according to the groups, while the mRNA and protein of TNFαand IL-1βwere determined by methods of RT-PCR,ELISA.Results:1. mice in M group and E group exhibited flexion, Close together, valgus of the affected foot toe, and it were not weight-bearing ,limping when they were walking. the pain threshold in both groups decreased significantly. Compared to M group, the pain threshold in E group showed more magnitude of changing range. However, there is not significant difference in both groups. 2. The expressions of mRNA and protein of GFAP in spinal dorsal horn increased at 1, 3, 7days after CCI , but those changes did not show in sham operation group. The expressions of mRNA and protein of GFAP in spinal cord astrocytes of E group was significantly higher than M group at the same time point. 3. Compared with the mice in S group,those in M group decreased in SOD activity and increased in MDA level significantly in spine tissues.And the change magnitude in E group was obviously more than those in M group. SOD activity increased and MDA level decreased on 7th day than those on 3rd day in E group. Compared with the mice in S group,TNFa, IL-1βprotein and their mRNA expression began to increased 1d after operation. TNFa, IL-1βprotein and mRNA in E group was obviously more than those in M group.Conclusion:1.The sensitivity of pain was significantly increased in the CCI models of C3 gene knockout mice by exogenous C3 protein lateral ventricle injection, The activation state of Complement 3 may be in intimate relationship with pain and analgesia in NPP model mice.2. The existence of complement C3 was significantly related to astrocyte activation in the neuropathic pain model mice, thus it might affect the occurrence and maintaining of neuropathic pain.3. C3 promoted oxidation inflammation injury in spinal cord of C3 gene knockout mice, The mechanisms might be that C3 induced Complement cascade in spinal dorsal horn.