Extracellular Microvesicles Aid Peripheral Nerve Regeneration

描述（由申请人提供）： 这一系列研究的总体目标是更好地了解决定周围神经系统成功神经再生的潜在机制;这是VA医疗保健使命的高优先级研究领域。周围神经损伤后，失神经支配的远端神经节段经历显著的变化，包括血-神经屏障的丧失、雪旺细胞增殖、巨噬细胞侵袭以及许多细胞因子和神经营养因子的产生。这些变化的总体结果是，失神经支配的神经成为从近端神经节段再生轴突的允许的甚至优选的靶。然而，人们经常感到惊讶的是，尽管周围神经显示出强大的轴突再生，但只有大约10%的成年人会使用最先进的修复技术恢复正常的神经功能。自从几十年前利用现代手术显微镜引入增强的显微外科技术以来，这种成功率并没有显著提高。显然，需要对这一问题提出新的想法和办法，以改进这些成果统计数据。 包括我们自己在内的几个实验室以前的工作表明，早期对终末器官靶点（如肌肉）进行神经再支配会导致更高水平的整体功能恢复。因此，改善功能恢复的一个主要关键将是减少远端终末器官目标（如肌肉）保持失神经支配的时间。另一个将改善神经修复后功能恢复的因素是增加原始终末器官靶的精确神经再支配的程度。失神经终末器官靶本身（例如肌肉）在再生期间可能在这些现象中发挥的可能作用在很大程度上尚未探索。本申请中提出的工作将开始使用小鼠中的模型系统来解决这个问题，以评估在终末神经分支水平上运动神经元再生的程度和准确性。 外泌体（纳米大小的细胞外囊泡）几乎由已检查的每种细胞类型分泌，最近发现它们成为各种形式的RNA和蛋白质的保护环境，因此可以作为新型长距离信使发挥作用。我们的工作假设是，肌肉作为一个终末器官，阐述了在去神经支配的神经内传播的信号，这些信号影响再生轴突的速度和准确性。我们进一步认为，肌肉产生的外泌体可能是这样一种机制，通过这种机制，这些信号在失神经支配的神经内传播。为了支持这一假设，我们提出了在体内的数据表明，再生运动神经元的准确性是依赖于失神经支配的远端神经节段保持不间断的连续性与肌肉。我们还提供了初步数据，即从肌肉细胞系中收获的外泌体并应用于体内失神经支配的神经，在两种不同的再生模型中导致显著更大和更快的轴突再生。本申请中提出的实验可以通过关注靶终末器官产生的外泌体的作用，为我们的外周神经修复方法提供范式转变。这种创新的想法是对直接针对运动神经元本身的现有干预措施的重要补充，并试图通过突出远端神经元来影响临床和研究的关注。 去神经支配的神经作为新疗法的一般递送装置。

DESCRIPTION (provided by applicant): The overall goal of this line of research is to gain a better understanding of the underlying mechanisms that determine successful nerve regeneration in the peripheral nervous system; this is a high priority research area for the VA healthcare mission. Following injury to a peripheral nerve the denervated distal nerve segment undergoes remarkable changes including loss of the blood-nerve barrier, Schwann cell proliferation, macrophage invasion, and the production of numerous cytokines and neurotrophic factors. The aggregate consequence of these changes is that the denervated nerve becomes a permissive and even preferred target for regenerating axons from the proximal nerve segment. However, it often surprises people that although peripheral nerves display robust axonal regeneration, only approximately 10% of adults will recover normal nerve function using state-of-the-art repair techniques. This success rate has not markedly improved since the introduction of enhanced microsurgical techniques utilizing modern surgical microscopes several decades ago. Clearly there is a need for new ideas and approaches to this problem in order to improve these outcome statistics. Previous work from several laboratories, including our own, has shown that earlier reinnervation of an end-organ target such as muscle results in a greater overall level of functional recovery. Thus a major key to improving functional recovery will be to decrease the amount of time that distal end-organ targets such as muscle remain denervated. Another factor that will improve functional recovery following nerve repair will be to increase the extent of accurate reinnervation of origina end-organ targets. The possible role that the denervated end-organ target itself (e.g. muscle) may play in these phenomena during the regeneration period is largely unexplored. The work proposed in this application will begin to address this question using model systems in the mouse to assess both the extent and accuracy of motor neuron regeneration at the level of the terminal nerve branch. Exosomes (nanometer sized extracellular vesicles) are secreted by just about every cell type that has been examined, and it has recently been discovered that they become a protected environment for various forms of RNA and proteins and thus can function as novel long-distance messengers. Our working hypothesis is that muscle, as an end-organ, elaborates signals that travel within the denervated nerve and that these signals influence the speed and accuracy of regenerating axons. We further suggest that exosomes produced by muscle may be one such mechanism by which such signals travel within the denervated nerve. In support of this hypothesis we present in vivo data that demonstrates that the accuracy of regenerating motor neurons is dependent upon the denervated distal nerve segment remaining in uninterrupted continuity with muscle. We also present preliminary data that exosomes harvested from a muscle cell line and applied to a denervated nerve in vivo results in significantly greater and faster axonal regeneration in two different regeneration models. The experiments proposed in this application could provide a paradigm-shift in our approach to peripheral nerve repair by focusing on the role of exosomes produced by target end-organs. This innovative idea is a significant addition to existing interventions that directly target the motor neuron itself, and seeks to impact clinical and research attention by highlighting the distal denervated nerve as a general delivery device for novel therapeutics.