| ObjectivePain is an unpleasant sensation that affects the health of more than 1/6 of the world’s population.The classification of pain can be divided into visceral,muscle,superficial,and neuropathic(NP)according to its location and cause.Among them,neuropathic pain is one of the most common pain caused by damage or disease of the somatosensory nervous system.Although its complex pathophysiological development mechanism is still unclear,it is currently believed that the central sensitization of the spinal dorsal horn is one of the mechanisms of chronic neuropathic pain.Microglia and astrocytes are the most glial cells in the spinal cord,and they jointly regulate the central sensitization of dorsal horn neurons in neuropathic pain.Therefore,exploring the crosstalk between these two cells in neuropathic pain will help to understand the central sensitization process in the spinal dorsal horn.In our previous studies,it was found that the complement levels in the dorsal horn were elevated in a variety of chronic pain conditions.Studies have reported that in other diseases of the nervous system,astrocytes and microglia in the brain communicate through the C3/C3aR axis to regulate the development of the disease.Therefore,we hypothesized that the C3/C3aR axis could transmit signals between astrocytes and microglia in neuropathic pain,affecting the sensitization of the spinal cord dorsal horn to regulate chronic pain.At present,the first-line treatment of neuropathic pain drugs mainly targets neuronal cells.Additional use of antiinflammation drugs improves the effectiveness of treatment.But the current treatment effect for neuropathic pain is not satisfactory.Therefore,this study intends to provide new ideas for the treatment of neuropathic pain by studying the regulation of complement C3 and its receptor C3aR on chronic pain in the spinal dorsal horn.MethodsThis study established chronic constriction injury of the sciatic nerve(CCI)models.Then,the threshold levels of mechanical and thermal pain were detected through behavioral tests to confirm that the neuropathic pain model was successfully established.After establishing the neuropathic pain model,the spinal dorsal horn was collected.Real-time polymerase chain reaction(qRT-PCR)technology was used to detect C3 and C3aR mRNA expression levels in the spinal dorsal horn in neuropathic pain.To clarify which cells expresses C3 and C3aR,immunofluorescence co-staining was used to co-label C3 and Neu,GFAP,and Iba-1,which are separately the marker of neurons,astrocytes,and microglia.So did for C3aR.Then,intrathecal injection of the C3 antibody was used to neutralize the increased expression of C3 in the spinal dorsal horn on CCI rats.And the change in pain threshold after neutralizing C3 was detected by pain behavior tests.We also observed the activation of astrocytes and microglia in the spinal dorsal horn and the polarization of microglia after this intervention.Finally,we inhibited the upregulated C3aR in neuropathic pain by intrathecal injection of the C3aR-specific inhibitor Trifluoroacetate.The changes in the mechanical pain threshold and thermal pain threshold of CCI rats after this intervention were detected again.We also detected whether the activation of astrocytes and the activation and polarization of microglia in the spinal dorsal horn after the inhibition of C3aR.We upregulated and downregulated the expression of nuclear factor kappa-B(NF-κB)in astrocytes to verify the changes in the expression of C3 and chronic neuropathic pain after inhibiting NF-κB.ResultsWe successfully established the rat CCI model.Mechanical and thermal pain thresholds started to decrease at postoperative day 3 and became stable at postoperative day 7-14.qRTPCR showed that the mRNA expression levels of C3 and its receptor C3aR in the spinal dorsal horn were increased in neuropathic pain.Immunofluorescence co-staining showed that C3 mainly co-localized with GFAP,and C3aR mainly co-localized with Iba-1 and Neu.This showed that C3 was mainly expressed on astrocytes in neuropathic pain,while C3aR was primarily expressed on microglia and neurons.Intrathecal injection of C3 antibody and C3aR inhibitor could relieve neuropathic pain.After neutralizing C3 and inhibiting C3aR,GFAP,and Iba-1 expression were reduced.And the mRNA levels of M1 markers CD86 and INOS in microglial cells decreased,while the mRNA levels of M2 markers CD206 and Argl increased.Afterward,the upregulation of NF-κB directly regulated the expression of C3 in astrocytes while inhibiting NF-κB could alleviate chronic neuropathic pain induced by CCI.In vivo experiments showed that the mechanical and thermal responses of CCI rats were reduced after inhibiting NF-κB,and downstream C3 of NF-κB was involved in this process.ConclusionsInhibition of the C3/C3aR axis can alleviate pain in the CCI neuropathic pain model in rats,providing relevant theoretical support for the anti-inflammatory treatment of this pain and offering a novel therapeutic target for the management of chronic neuropathic pain. |
