小胶质细胞-单核吞噬细胞协同调控急性疼痛慢性化的机制研究

Chronic pain is debilitating diseases and dramatically impact the life quality of patients. Mechanisms underlying the transformation of acute neural lesions to neuropathic pain is one of the most important and challenging problems in the field of pain research. Neuro-Immune inflammation is an indispensable factor in the regulation of the transformation, but the specific mechanism is still unclear. Microglia and peripheral monocytes have the same embryonic origin, sharing many molecular markers. They have been reported contributed to hypersensitivity in rodent models of neuropathic pain. However, the precise respective function of microglia and peripheral monocytes has not been investigated in these models. To address this question, we combined transgenic mice and pharmacological tools to specifically and temporally control the depletion of microglia and monocytes in a mouse model of spinal nerve transection (SNT). Our previous studies found that either resident microglia or peripheral monocytes are sufficient in gating neuropathic pain after SNT. Resident microglia and peripheral monocytes act synergistically to initiate hypersensitivity and promote the transition from acute to chronic pain after peripheral nerve injury. We further found that spinal microgliosis predominantly results from local proliferation of resident microglia but not from infiltrating monocytes after SNT by using two genetic mouse models (CCR2RFP/+:CX3CR1GFP/+ and CX3CR1creER/+:R26tdTomato/+ mice) as well as specific staining of microglia and macrophages. Those results have been published in Nat Commun and Cell Rep. But it is still unclear how these two kinds of cells co-mediated the chronicity of acute pain. Our preliminary experiments showed that microglia and peripheral monocytes had different activation peak time, and the late depletion of mononuclear phagocyte could only relieve the thermal hyperalgesia, but not mechanical allodynia. Therefore, we hypothesized that spatial distribution and temporal onset of microglia/monocytes activation orchestrate the transformation of acute neural injury to neuropathic pain, while mononuclear phagocytes were mainly involved in the thermal hyperalgesia. In this study, we develop multiple transgenic mice as a platform. The current study is to explore the role of microglia/monocytes in different time windows and locations by genetic and pharmacological tools. We look forward this project will provide new insights to the transformation mechanism underlying neuropathic pain.

小胶质细胞和单核吞噬细胞参与的神经免疫炎症在急性疼痛慢性化中发挥重要作用。然而，既往研究未能准确区分二者、明晰它们的特定作用。申请者前期发现只有同时敲除二者才能遏制神经病理性疼痛的发生；深入研究发现SNT术后外周单核吞噬细胞未能进入脊髓背角，二者活化部位不同。成果先后发表于Nat Commun和Cell Rep。但两种细胞如何协同介导急性疼痛慢性化呢？我们新近研究发现二者有不同的活化高峰时间；晚期耗竭单核吞噬细胞仅可缓解热痛敏，却对机械性异常痛觉无效。提示：小胶质细胞和单核吞噬细胞通过不同时间和空间活化协同介导热痛敏和机械性痛觉的急性疼痛慢性转化，单核吞噬细胞主要参与了热痛敏的转归。本课题以多品系转基因小鼠为平台，联合药理学工具，证实两种免疫细胞在不同时空的活化是不同急性疼痛慢性转化的关键，根据病程先后靶向抑制两种免疫细胞将为开发阻止急性疼痛慢性化的临床治疗策略提供理论依据。

项目背景：小胶质细胞和单核吞噬细胞参与的神经免疫炎症在急性疼痛慢性化中发挥重要作用。然而，既往研究未能准确区分二者、明晰它们的特定作用。课题负责人前期发现：只有同时敲除二者才能遏制神经病理性疼痛的发生；深入研究还发现SNT术后外周单核吞噬细胞并不能侵润进入脊髓背角，二者活化部位不同，有不同的活化高峰时间。为此，我们提出了“小胶质细胞-单核吞噬细胞通过不同时间和空间活化协同介导急性疼痛慢性化”这一假说，拟初探其可能机制。.主要研究内容及结果：一，系统探索了周围神经损伤后小胶质细胞在痛觉传递通路（尤其是脊髓上水平）的活化、增殖情况。明确神经病理性痛状态下，小胶质细胞活化、增殖主要发生在DRG和脊髓背角，低位脑干PAG、RVM等痛觉传递通路上的部位也可观察到少量但明确的小胶质细胞的活化。聚焦DRG，深入研究了周围神经损伤后在损伤直接相关的L4、术侧相邻L3节段DRG固有单核细胞/卫星细胞和外周血液来源单核/巨噬细胞的活化时程变化。二，利用L4 SNL模型，研究了不同时间点单独敲除单核/吞噬细胞、小胶质细胞（中枢神经系统CX3CR1+细胞）对神经病理性痛急慢性转化的影响。三，利用福尔马林诱导炎性痛模型，研究了敲除CX3CR1+细胞或外周单核/吞噬细胞对福尔马林诱导炎性痛的影响。发现特异性敲除CNS CX3CR1+细胞可显著缓解福尔马林诱导的具有神经病理性特性的II相炎性疼痛；在福尔马林注射诱导的II相持续炎症疼痛期间，脊髓背角小胶质细胞突起动力指数显著增加；CNS小胶质细胞敲除显著抑制福尔马林诱导炎性疼痛相关脊髓背角神经元的过度激活，提示小胶质细胞-神经元交互作用很可能增强了福尔马林诱导炎性疼痛期间脊髓痛觉传递相关神经元的激活；P2Y12受体在小胶质细胞突起动力学、神经元兴奋性和福尔马林II相持续炎性疼痛机制中的发挥重要作用。.科学意义：上述结果为单核/吞噬细胞、小胶质细胞参与神经病理性痛急慢性转化的机制提供新的学术见解，为开发阻止急性疼痛慢性化的临床治疗策略提供理论依据。