Spinal cord neuroinflammation and synaptic plasticity after peripheral nerve injury

项目摘要/摘要 每年有近100万美国人在神经损伤后接受神经再植手术，但 尽管显微外科技术不断改进，但这些患者中的大多数仍 永久性运动缺陷。通常认为这是由于周围神经再生不良所致。 然而，当实验性神经损伤在动物模型中设计为快速、 特定而有效的神经再生和肌肉再神经支配。在过去，我们报告了结构性的 神经损伤后脊髓回路的重塑是部分原因。损伤后，中央突触 周围损伤的感觉本体感觉轴突和运动神经轴突的分支从 脊髓腹角导致几个关键的运动控制回路功能障碍。其作用机制 因此，突触和轴突的去除显然很重要，但尚不清楚。我们的初步数据显示 周围损伤后脊髓内神经炎性反应的发生 运动神经元和周围损伤感觉神经轴突的中央投射。小胶质细胞在 尽管它们吞噬突触的能力经常被提出，但它们的确切 神经损伤后的功能仍然存在争议。此外，单核细胞渗入脊髓并转化为 变成巨噬细胞。这些细胞在以前的研究中被遗漏了，因为它们变得无法区分 小胶质细胞。因此，它们与驻留的小胶质细胞的相互作用以及在突触可塑性中的可能作用尚不清楚。 最近，我们发现，在神经损伤触发大的突触回路重塑后，有额外的 T细胞的浸润。T细胞在突触重构中扮演的角色是完全未知的。为了调查 小胶质细胞和不同免疫细胞浸润与突触可塑性的关系我们将使用小鼠 其中小胶质细胞被GFP标记，免疫细胞被RFP渗透，并执行许多 干扰一个或另一个细胞的功能并测试可能的信号机制的遗传操作 导致小胶质细胞活化、免疫细胞渗入和突触移除。在目标1中，我们将调查 外周来源的单核细胞、巨噬细胞和T细胞在肌肉传入突触移除中的作用 感官传入。在目标2中，我们将调查小胶质细胞的激活是否是招募 从血液中提取免疫细胞，并研究促进这些入侵的信号。在目标3中，我们将使用两个- 光子时间推移成像直接观察和分析感觉神经突触和神经细胞的移除过程 促进周围神经损伤的轴突的特定识别的机制。最后，在目标4中，我们 将阻断中枢神经炎性反应以保留本体感觉突触输入和测试模式 肌电图仪检测神经再生后跑步机运动时肌肉的激活情况。结果是 将提供有关神经炎症的作用和涉及移除特定的 来自脊髓的输入，并将提供对干预这一过程后的运动结果的第一次洞察。

Project Summary / Abstract Every year nearly one million Americans undergo surgery for nerve reattachment after nerve injuries, but despite continued improvements in microsurgical techniques, a majority of these patients are left with permanent motor deficits. Usually these are believed to result from poor regeneration of the peripheral nerve. However, deficits are still present when experimental nerve injuries are designed in animal models for rapid, specific and efficient nerve regeneration and muscle re-innervation. In the past we reported that structural remodeling of spinal cord circuitry after nerve lesions is in part responsible. After injury, the central synaptic branches of sensory proprioceptive axons and motor axons injured in the periphery are removed from the ventral horn of the spinal cord resulting in dysfunction of several critical motor control circuits. The mechanisms of synapse and axon removal are therefore clearly important, but unknown. Our preliminary data implicate the neuroinflammatory response that occurs inside the intact spinal cord around cell bodies of peripherally-injured motoneurons and along the central projections of peripherally-injured sensory axons. Microglia is activated in these regions and although their capacity for synapse phagocytosis has been frequently proposed, their exact function after nerve injury remains controversial. In addition, monocytes infiltrate the spinal cord and transform into macrophages. These cells were missed in previous studies because they become indistinguishable from microglia. Thus, their interactions with resident microglia and possible roles in synaptic plasticity are unknown. More recently we found that after nerve injuries triggering large synaptic circuit remodeling there is additional infiltration by T-cells. The roles T-cells play in synaptic remodeling are completely unknown. To investigate the relation between microglia and different immune cell infiltrates in relation to synapse plasticity we will use mice in which microglial cells are labeled with GFP and infiltrating immune cells by RFP and perform a number of genetic manipulations to interfere with the function of one or other cell and test possible signaling mechanisms leading to microglia activation, immune cell infiltration and synapse removal. In Aim 1 we will investigate the role of peripherally derived monocytes, macrophages and T-cells in the synaptic removal of inputs from muscle sensory afferents. In Aim 2 we will investigate whether microglia activation is necessary for recruitment of blood-derived immune cells and investigate the signals promoting these invasion. In Aim 3 we will use two- photon time-lapse imaging to directly observe and analyze the process of removal of sensory synapses and the mechanisms that facilitate specific recognition of axons injured in the peripheral nerve. Finally, in Aim 4 we will block the central neuroinflammatory response to preserve proprioceptive synaptic inputs and test patterns of muscle activation during treadmill locomotion after nerve regeneration with electromyography. The results will inform about the role of neuroinflammation and the cellular mechanisms involved in removing specific inputs from the spinal cord and will provide first insights into motor outcomes after interfering with this process.