前扣带皮层（ACC）在痛分辨、痛情绪反应中扮演重要角色，但其免疫机制尚不十分清楚。本项目预实验将携带有人M3/M4毒蕈碱受体（hM3Dq或hM4Di）基因的AAV病毒转染ACC，在给予配体氯氮平-N-氧化物后可通过Gq或Gi通路兴奋或抑制ACC锥体神经元，行为学结果表明ACC参与痛调制；分子生物学实验显示神经损伤后ACC内趋化因子CX3CL1、致炎细胞因子TNF-α上调，并与神经元兴奋标志物c-Fos共表达；ACC内预处理TNF-α中和抗体抑制神经损伤诱导的c-Fos表达、缓解触诱发痛和痛情绪。本项目拟用多种神经科学手段进一步揭示不同表型神经病理性痛模型ACC的痛调制差异；ACC神经元↔胶质细胞交互激活网络中趋化因子-致炎细胞因子级联反应在脊髓下行易化中的作用；NF-κB、p38 MAPK、JNK通路介导ACC兴奋、细胞因子微环境改变的分子机制。本项目旨在为缓解慢性痛和痛情绪提供新靶点。

Anterior cingulate cortex (ACC) has been regarded as necessary in the encoding of the sensory discriminative and affective consequences of nociceptor stimulation. However, the immune mechanisms underlying the pain-related regulation of ACC is still unknown. In our preliminary experiment, we used the stimulatory designer receptors exclusively activated by designer drugs (DREADD-hM3Dq or hM4Di, where hM3Dq indicates Gq-coupled human M3 muscarinic receptor and hM4Di indicates Gi-coupled human M4 muscarinic receptor), which couples through a Gq or Gi pathway to depolarize or hyperpolarize neuronal membrane potential upon the application of synthetic ligand clozapine-N-oxide (CNO).We found that inhibition of ACC pyramidal neurons with use of Gi-DREADD reversed the mechanical allodynia induced by spared sciatic nerve injury (SNI). On the contrary, administration of Gq-DREADD into the unilateral ACC layer Ⅴ/Ⅵ without any nerve injury decreased the bilateral paw withdrawal thresholds to von Frey hairs through activating ACC. Furthermore, the protein levels of chemokine CX3CL1 and tumor necrosis factor-alpha (TNF-α) and interleukin-6 were increased in ACC following SNI surgery. The increased TNF-α was co-localized with c-Fos (neuronal excited marker) and GFAP (astrocyte marker). Pre-treatment with anti-TNF-α antibody into ACC significantly blocked c-Fos expression, increased the animal paw withdrawal threshold and attenuated the pain-related negative emotion, suggesting the cascade of chemokines-proinflammatory cytokines in ACC might be responsible for the development of chronic pain. Based on our present works, we will further investigate the ACC mechanisms underlying different models of neuropathic pain, reveal the role of chemokine-proinflammatory cytokine cascade in the glial-neuronal interactions as well as the regulations of NF-κB, p38 MAPK and JNK signals on the cytokines secretions in ACC following nerve injury. The present project aims to reveal the immune mechanism of ACC in neuropathic pain, and to provide a new target for clinical therapy.