Mechanism Of Microglia In The Anterior Cingulate Cortex Regulating Neuropathic Pain

Chronic pain affects up to 30%of world population,and its pathogenesis is complicated and heterogeneous.Chronic pain involves the maladaptation of multiple neural networks related to sensation,emotion,and cognition.However,the molecular and cellular mechanisms of chronic pain remain unclear.The current treatments cannot meet the needs of clinical patients,and cause a huge economic burden to the society.Therefore,understanding the molecular and cellular mechanisms underlying chronic pain will provide new targets and ideas for the development of analgesic drugs,which has crucial basic and clinical research significance.The anterior cingulate cortex(ACC)is known to participate in transmitting internal and external sensory stimuli and encoding pain information.The long-term maladaptive of ACC glutamatergic(ACCGlu)neurons are involved in chronic pain perception and emotion,but the detailed mechanisms remain largely unknown.In addition,chronic inflammation is the basis of many nervous system diseases,and microglia are the most important resident immune cells in the brain.However,the involvement of microglia in regulating ACCGlu neuronal plasticity in chronic pain is unknown.Here,the molecular and cellular mechanisms of microglia regulating ACC neuronal plasticity in neuropathic pain formation are explored by electrophysiology,in vivo multi-channel and fiber photometric recording,in vivo two-photon calcium imaging,western blot,photogenetic and chemogenetic.Based on the mouse model of diabetic neuropathic pain established by streptozotocin(STZ,180 mg/kg),we found that the mice 7 weeks after STZ injection(STZ 7W)showed significant spontaneous pain and pain sensitization using von Frey and gait tests,and ACCGlu neuronal hyperactivity using electrophysiology,in vivo multichannel,and fiber photometric recording.Chemogenetic inhibition of ACCGlu neuronal activity alleviated DNP.These results suggest that ACCGlu neuronal overexcitation is involved in the occurrence of diabetic neuropathic pain.In order to explore whether microglia contribute to the change in ACCGlu neuronal activity and thus affect pain behavior in mice,we found that microglia in the ACC of STZ 7W mice were significantly activated,and that ACC or intraperitoneal injection of microglia inhibitor(minocycline)could significantly alleviate the pain sensitivity of STZ 7W mice.In addition,microglial chemokine CXCL12 expression was upregulated in the ACC of STZ 7W mice,while its receptor expression,CXCR4,in neurons was decreased.Pharmacological inhibition of microglia or blockade of CXCR4 in the ACC alleviated DNP by preventing ACCGlu neuronal hyperactivity in STZ 7W mice.Furthermore,inhibition of microglia decreased STZ-induced upregulation of microglial CXCL12.Administering recombinant CXCL12 protein in the ACC increased ACCGlu neuronal activity and induced pain sensitization in na(?)ve mice.These results suggest that microglia-mediated ACCGlu neuronal hyperactivity via the CXCL12/CXCR4 signaling drives the development of diabetic neuropathic pain.The formation of chronic pain requires the synergy of multiple genes or proteins.The expression of CXCL12 and its receptor is regulated by multiple factors.The upregulation of lipocalin-2(LCN2)expression can induce the activation of microglia and the production of chemokines under inflammatory or injury conditions.Therefore,we further explored the function of LCN2 in chronic pain.We found that LCN2 expression was upregulated in the ACC of mice at 14 days after spared nerve injury(SNI 2W),resulting in ACCGlu neuronal hyperactivity and pain sensitization.Suppressing LCN2 protein levels in the ACC with viral constructs or exogenous application of neutralizing antibodies lead to significant attenuation of neuropathic pain by preventing ACCGlu neuronal hyperactivity in SNI 2W mice.In addition,administering recombinant LCN2 protein in the ACC could increase ACCGlu neuronal activity and induce pain sensitization in naive mice.These results suggest that LCN2 may participate in the process of neuropathic pain by exciting ACCGlu neurons,but whether it is involved in this process in synergy with CXCL12/CXCR4 signal pathway mediated by microglia activation needs further investigation.In summary,this study reveals that ACCGlu neuronal plasticity mediated by microglial activation is involved in the development of neuropathic pain,and CXCL12 and LCN2 could participate in this process.These research results not only provide a new theoretical basis for the occurrence of neuropathic pain,but also provide potential targets for the development of new analgesic drugs.