The involvement of microglia and peripheral macrophages in the permanent deletion of proprioceptive IA afferents from spinal motoneurons following peripheral nerve injury

 描述（由申请人提供）：周围神经损伤（PNI）每年影响近100万美国人，给他们留下永久性运动缺陷，如伸展反射丧失、肢体协调缺陷和过度肌肉共同收缩。即使周围神经再生成功，这些不可避免的结果仍然存在，这表明中枢机制可能是导致运动恢复不良的原因。PNI后发生的一种已知现象是本体感受IA传入的中枢投射消失，这可能是造成这些缺陷的部分原因。同时，在PNI后，中央小胶质细胞被激活，并发出信号，外周巨噬细胞浸润到脊髓中。这两种细胞都是吞噬细胞，并包围受损的IA传入突触，但它们在突触重塑中的确切作用仍然是一个争论的话题。该建议的假设是，小胶质细胞和外周巨噬细胞特异性地识别在外周神经中受损的IA传入的中枢轴突和突触，并且直接参与其降解。我们将使用新的转基因小鼠模型，首次从遗传上区分中央小胶质细胞（CX 3CR 1-EGFP）和浸润性巨噬细胞（CCR 2-RFP），并研究它们与IA传入神经中枢末端的关系。所获得的知识将产生对新的治疗方法的见解，通过防止运动回路的重组和改善神经损伤后再生后的运动功能恢复来改善患者的生活质量。目标1：降低中枢小胶质细胞活性和阻断外周巨噬细胞浸润可保护PNI后IA传入突触，并改善运动功能恢复。为了测试小胶质细胞和外周巨噬细胞是否参与IA传入突触的清除，我们将通过鞘内给予 米诺环素或针对MCP-1的中和抗体，其是外周巨噬细胞募集所必需的。此外，米诺环素治疗的动物将用于确定减少神经炎症是否改善神经再生后的运动结果。通过记录跑步机运动期间踝关节屈肌和伸肌的肌电图（EMG）活动来测试运动功能。目标二：使用活细胞成像来表征中央小胶质细胞和外周巨噬细胞与受损的IA传入中枢轴突和突触相互作用的过程。受伤和未受伤的IA传入神经都将用荧光示踪剂标记，用于活体检测。 将研究成人脊髓切片的成像实验及其与遗传标记的小胶质细胞或外周巨噬细胞的相互作用。我们将使用双光子显微镜和延时成像来观察它们之间的关系。这些相互作用的性质将使我们能够推断IA突触是如何被识别为退化的以及它们被去除的机制。

 DESCRIPTION (provided by applicant): Peripheral nerve injuries (PNI) affects nearly one million Americans every year leaving them with permanent motor deficits such as a loss in the stretch-reflex, deficiencies in limb coordination, and excessive muscle co- contraction. These inevitable outcomes persist even when peripheral nerve regeneration is successful, suggesting that central mechanisms might be responsible for poor motor recovery. One known phenomenon that occurs after PNI, that may be in part responsible for these deficiencies, is the disappearance of the central projections of proprioceptive IA afferents. Concurrently, after PNI central microglia become activated and signal the infiltration of peripheral macrophages into the spinal cord. Both of these cells are phagocytic and surround injured IA afferent synapses, but their exact roles in synaptic remodeling remain a topic of debate. The hypothesis of this proposal is that microglia and peripheral macrophages specifically recognize the central axons and synapses of IA afferents that are injured in the peripheral nerve and are directly involved in their degradation. We will use novel transgenic mouse models to genetically distinguish, for the first time, central microglia (CX3CR1-EGFP) from infiltrating macrophages (CCR2-RFP) and study their relationships with the central terminations of IA afferents. The knowledge gained will generate insights into novel therapeutic approaches for improving patient's quality of life by preventing the reorganization of motor circuits and improving motor function recovery following regeneration after nerve injuries. Aim 1: Decreasing central microglia activity and blocking peripheral macrophage infiltration preserves IA afferent synapses following PNI and results in improved motor function recovery. To test if microglia and peripheral macrophages are involved in the removal of IA afferent synapses, we will block their activity by intrathecally administering minocycline or a neutralizing antibody against MCP-1, which is necessary for peripheral macrophage recruitment. Furthermore, minocycline-treated animals will be used to determine if reducing neuroinflammation improves motor outcomes after nerve regeneration. Motor function will be tested by recording electromyographic (EMG) activity from ankle flexor and extensors during treadmill locomotion. Aim 2: Use live cell imaging to characterize the process by which central microglia and peripheral macrophages interact with injured IA afferent central axons and synapses. Both injured and uninjured IA afferents will be labeled with fluorescent tracers for live imaging experiments in adult spinal cord slices and their interactions with genetically labelled microglia or peripheral macrophages will be investigated. We will use two-photon microscopy and time-lapse imaging to observe their relationships. The nature of these interactions will allow us to infer how IA synapses are recognized for degradation and the mechanism(s) of their removal.