The Mechanism Of Anterior Cingulate Cortical Microglia-Mediated Diabetic-associated Pain

Pain is one of the complications of diabetes,manifested as burning,tingling,knife cut-like or electric shock-like pain,accounting for 25～50%of diabetic patients.Because the pathophysiological mechanisms underlying diabetic pain remain elusive,it has always been a difficult point in clinical treatment.Microglial cells are the resident immune cells in the brain.Under pathological conditions,microglia drive the abnormal activities of neurons and produce different disease phenotypes,which are essential for the formation of neuropathic pain.How the diabetic environment changes the plasticity of brain microglia,resulting in abnormal neural activity and neuropathic pain,is not yet clear.Therefore,an in-depth analysis of the neurosensitization mechanism of pain in the process of diabetes from the perspective of microglia will provide new insights for understanding the occurrence and development of diabetic neuralgia,and provide new ideas for finding analgesic targets related to inflammatory factors.Anterior cingulate cortex(ACC)is located in the forebrain,and is an important brain area for processing and regulating pain and emotional information.In the current study,a mouse model of diabetic neuropathic pain(DNP)was established using streptozotocin(STZ,intraperitoneal injection).Combined with immunofluorescence staining and three-dimensional reconstruction analysis,we found that the microglia in the ACC of DNP mice were activated.Using the microglia inhibitor minocycline to inhibit the activation of microglia rescued pain sensitization in DNP model mice and reduced the activity of glutamatergic neurons in the ACC.Using the whole-cell patch clamp recordings,optical fiber recordings and in vivo multi-channel recordings,we found that compared with control mice,the excitability of glutamatergic neurons in the ACC of DNP model mice was increased.Chemogenetic inhibition of the glutamatergic neurons in the ACC could relieve the mechanical hyperalgesia of the model mice.These results indicated that,ACC microglia activation participates in the formation of hyperalgesia by regulating the avtivity of glutamate neurons in the diabetic environment.In order to explore the molecular basis of the interaction between microglia and glutamatergic neurons in the ACC,using mass spectrometry and immunofluorescence staining,we found that,in the ACC of DNP model mice,the expression of the chemokine CXCL12 on microglia was up-regulated,and the degradation of CXCR4 receptors expressed on the glutamatergic neurons was accelerated resulting from the CXCL12 excessive combined with the CXCR4 receptor,accompanied by the hyperactivity of glutamatergic neurons,which subsequently cause the development of pain sensitization in diabetic mice;Inhibition of microglia decreased STZ-induced upregulation of microglial CXCL12.In addition,ACC infusion of CXCL12 protein induced significant pain sensitization in normal mice and increased the activity of glutamatergic neurons.Blockade of CXCR4 in the ACC alleviated painsensitization,and reduced the ACCGlu neuronal hyperactivity in STZ mice.These results suggest that neural plasticity produced by the CXCL12/CXCR4 signaling pathway after ACC microglial activation is an important basis for the formation of diabetic neuralgia.Interestingly,after ACC was injected with inhibitory chemical genetic viruses,some mice showed relief of pain symptoms.Our anatomical analysis of these mice found that hM4Di virus was expressed in the ACC;and a few mice showed excessive grooming or spinning,which was similar to the obsessive-compulsive behavior of mice in previous studies.Our anatomical analysis of these mice found that the hM4Di virus was mainly expressed in the medial prefrontal cortex(mPFC)rather than the ACC(the location of the ACC and mPFC are adjacent).Thus then,is mPFC related to obsessivecompulsive disorder or compulsive behavior?Based on this problem,we established a mouse model of obsessive-compulsive checking behavior by subcutaneously injecting quinpirole into the neck.Using immunofluorescence staining,we found that the c-Fos expressions in the medial prefrontal cortex(mPFC)and basolateral amygdala(BLA)neurons of the model mice were both increased.Virus traces showed that BLA glutamatergic(BLAGlu)neurons innervate mPFC glutamatergic(mPFCGlu)neurons,and at the same time they also innervate mPFC GABAergic(mPFCGABA)neurons that have a direct inhibitory effect on mPFCGlu neurons.Using brain slice whole-cell patchclamp recordings,compared with the control mice,the activity of BLAGlu neurons and mPFCGABA neurons in model mice were significantly increased,while the activity of mPFCGlu neurons was significantly decreased.These results suggest that it is likely that the over-excitability of BLAGlu neurons may lead to the increase of feedforward inhibition of mPFCGlu neurons.We used optogenetical and chemogenetical methods to manipulate the activities of different neurons in the BLA-mPFC neural circuit,and found that BLAGlu→mPFCGABA→Glu neural circuit participates in the formation of obsessive-compulsive checking behaviors in mice.This study provides a theoretical basis for in-depth understanding of the neural mechanism of obsessive-compulsive disorder.