The Mechanism Of CCL2 And KCC2 Between Peripheral Nociceptors And Lower Central Nervous System In Brachial Plexus Induced Neuropathic Pain Sensitization

BackgroundBrachial plexus avulsion(BPA)is the most severe trauma of peripheral nerve injury of the upper extremity.It is a combined injury of central nervous system(CNS)and peripheral nervous system(PNS),which usually involves the spinal cord and dorsal root ganglion(DRG)of corresponding segments simultaneously.BPA is often accompanied by severe pain complications called neuropathic pain(NPP),which is characterized by spontaneous hyperalgesia and hypersensitivity to stimuli.It will be resistant to a variety of analgesics once formed.BPA induced NPP will not only seriously affect the quality of patients’ life,but also bring heavy burden to patients and society,which also leads to it gradually becoming a thorny issue faced by clinicians and researchers.The unclear mechanism of NPP after BPA is one of the important reasons for its poor therapeutic effect.In recent years,with the deepening of NPP related studies,it has been confirmed that pain sensitization plays an important role in the occurrence and maintenance of NPP,which is called central sensitization when it occurs at the CNS level,and called peripheral sensitization at the PNS level.The causation of pain sensitization can be divided into two categories which include enhancement of excitatory input and impairment of inhibitory input.Pain information is received at the level of nociceptors,integrated into the spinal cord,and travelled upward to the brain.Sensitization at any part on the sensory pathway will finally lead to NPP.Therefore,at the level of the peripheral nociceptors-DRG and lower central nervous system-spinal cord circuit,the study of NPP after BPA not only can uncover its deep mechanisms,but also may supply new ideas and novel targets for the treatment of this kind of pain syndrome,in order to achieve the final purpose of pain management,which has important clinical significance.Part 1 Study on the mechanism of CCL2 in pain sensitization after BPAObjective To investigate the mechanism of spinal CCL2 and its receptor CCR2 contributed to neuropathic pain sensitization following BPA via modulating glutamate N-methyl-D-aspartate receptor(NMDAR).Methods A rat model of BPA on lower trunk(C8-T1)was established and pain behavioral tests were performed.Seven days after BPA modeling,the sham-and BPA-operated animals were intrathecally injected with saline,CCR2 inhibitor INCB3344 and NMDAR antagonist DL-AP5.The behavioral performance was used to evaluate the analgesic effect of the treated animals,and expressions of CCL2,CCR2,and NMDAR subunit NR2 B in spinal cord sections of each group were examined through immunofluorescence,western blot and RT-PCR.Results It was shown that BPA injury significantly reduced mechanic withdrawal thresholds the next day after surgery until the end of the observation(21 d).Both CCL2 and CCR2 expressions increased in BPA rats compared to those in sham rats,which is significant in deference compare with sham group(P < 0.05).CCL2 was mainly localized in astrocytes,and CCR2 was preferably expressed on astrocytes and neurons.Besides,NMDAR subunit NR2 B increased in BPA-operated rats(P < 0.05),which was reversed in response to CCR2 and NR2 B inhibition.However,these inhibitors didn’t change the spinal NMDAR level in sham rats.CCR2 and NMDAR inhibition efficiently alleviated mechanical allodynia caused by BPA either at early or late phase of neuropathic pain.Conclusion Spinal CCL2 and its receptor CCR2 potentiate the occurrence and maintenance of pain sensitization after BPA by elevating NMDAR signaling.Part 2 Study on the mechanism of KCC2 in pain sensitization after BPAObjective To investigate the mechanism of nociceptor localized KCC2 contributed to neuropathic pain sensitization following BPA.Methods A novel mouse model of BPA on middle trunk(C7)was established and pain behavioral tests were performed.Seven days after BPA modeling,expressions of BDNF and its receptor TrκB,KCC2 and GABAR in spinal dorsal horn(SDH)and DRG were examined through immunofluorescence,western blot and fluorescence in situ hybridization(FISH).Five days before BPA modeling,KCC2 overexpression virus was injected into DRGs both in sham and BPA group,the behavioral performance was used to evaluate the analgesic effect of the treated animals 7 d and 14 d after BPA modeling.Single cell patch clamp technique was used to evaluate excitability in DRG neurons,and spinal fiber optic calcium imaging was used to evaluate whether DRG overexpression of KCC2 affects the excitability of SDH neurons.Results It was shown that BPA injury significantly reduced mechanic withdrawal thresholds the next day after surgery until the end of the observation(28 d).In SDH,BDNF and TrκB expressions increased in BPA mice compared to those in sham mice,and KCC2 and GABAR-α1 subunit expressions decreased,which is significant in deference compare with sham group(P < 0.05).BDNF in SDH was mainly localized in neurons,and the distribution of KCC2 in DRG showed no cell specific according to immunofluorescence and FISH results.Overexpression of KCC2 in DRG could downregulate neuronal excitability,which could alleviate the mechanical allodynia of BPA mice(P < 0.05).The overexpression of KCC2 in DRG can significantly reduce the calcium response signal and excitability of neurons in SDH,with statistical difference compared with the control group(P < 0.05).Conclusion BDNF and its receptor participate in the occurrence and maintenance of pain sensitization after BPA by regulating its downstream KCC2,and peripheral sensitization can be regulated to reverse central sensitization by targeting KCC2 in DRG at the peripheral level.