Reinnervation of Paralyzed Muscle by Nerve-Muscle-Endplate Band Grafting

描述(申请人提供)：神经肌肉失神经是周围神经损伤和神经疾病的常见后果。由于目前使用的方法普遍导致功能恢复不良，迫切需要寻找新的神经再支配方法来恢复瘫痪肌肉。推动这一项目的全球假说是，通过用丰富的完整神经末梢和运动终板来源重新支配瘫痪的肌肉，可以获得更好的结果。这个想法促使我们开发了一种神经-肌肉-终板带移植(NMEG)方法来重新支配瘫痪的肌肉。选择颈带肌肉在大鼠模型上进行可行性研究。NMEG取自大舌骨(OH)肌，移植到实验性瘫痪的胸锁乳突肌(SM)。同时采用神经肌蒂(NMP)和端端吻合(EEA)两种术式进行比较。我们采用即刻神经再支配模型进行的初步研究表明，NMEG组神经再生成功，功能恢复优于NMP组和EEA组。本研究旨在与经典的EEA技术比较，确定NMEG对瘫痪颈带肌肉的即刻和延迟性神经支配的疗效。本建议中设计的研究将记录功能恢复的基本神经基础和其他影响结果的主要因素。我们假设，神经肌肉功能恢复的程度在很大程度上取决于重建的神经-肌肉接触的数量和失神经引起的肌肉改变，NMEG将是肌肉重新神经支配的更好的选择，而不是通常使用的EEA和其他方法。这些假设将通过以下三个具体目标进行检验。具体目标1是通过分析肌电(EMG)记录、肌力和运动测量以及糖原耗竭的肌肉纤维的分布来评估重新神经支配的肌肉的功能恢复。具体目的2是通过对逆行辣根过氧化物酶(HRP)标记的运动神经元、再生的轴突和芽以及新形成的运动终板进行定量，来说明神经支配肌肉功能恢复的神经基础。具体目标3是通过分析肌肉质量、纤维大小、纤维类型分组、纤维类型和肌球蛋白重链(MHC)的组成，探索神经再支配肌肉中与程序相关和时间相关的形态、免疫细胞化学和生化变化。这一结果将使NMEG在肌肉瘫痪康复方面的疗效得到可靠的记录。拟议工作的意义远远超出了目前所理解的范围。一旦NMEG的优势被广泛的动物研究充分证明，这项研究对科学和医疗保健的影响可能是巨大的，因为从这项研究获得的数据有助于在不久的将来最终临床应用于瘫痪神经肌肉疾病患者的治疗。由于尚未找到完全满意的修复瘫痪骨骼肌的方法，我们开发了一种新的技术(神经-肌肉-终板带移植)来重新支配瘫痪的大鼠颈带肌。我们的初步工作表明，这项技术的结果比目前使用的方法更好。本研究获得的数据对今后肌肉麻痹的临床应用有一定的参考价值。

DESCRIPTION (provided by applicant): Neuromuscular denervation is a common consequence of peripheral nerve injuries and neurological diseases. There is a pressing need to seek novel approaches of reinnervation for restoration of paralyzed muscles as the presently used methods generally result in poor functional recovery. The global hypothesis driving this project is that better outcomes could be achieved by reinnervating a paralyzed muscle with an abundant source of intact nerve terminals and motor endplates. This idea promoted us to develop a nerve- muscle-endplate band grafting (NMEG) method to reinnervate paralyzed muscles. Cervical strap muscles were selected to perform feasibility studies in a rat model. The NMEG was harvested from the omohyoid (OH) muscle and then transplanted to the experimentally paralyzed sternomastoid (SM) muscle. Meanwhile, nerve- muscle pedicle (NMP) and end-to-end anastomosis (EEA) reinnervation procedures were also carried out for comparison. Our pilot studies using immediate reinnervation model showed that NMEG resulted in successful neuroregeneration and better functional recovery than the NMP and EEA. This research is to determine the efficacy of the NMEG for the immediate and delayed reinnervation of paralyzed cervical strap muscles as compared with the classic EEA technique. Studies designed in this proposal will document the fundamental neural basis of the functional recovery and other major factors influencing outcomes. We hypothesized that the extent of functional recovery of a reinnervated muscle is largely dependent on both the quantity of the reestablished nerve-muscle contacts and the denervation induced muscular alterations and that the NMEG would be a better option for muscle reinnervation than the commonly used EEA and other methods. These hypotheses will be tested with the following 3 specific aims. Specific Aim 1 is to evaluate functional recovery of the reinnervated muscles by analyzing electromyographic (EMG) recordings, muscle force and movement measurements, and distribution of the glycogen depleted muscle fibers. Specific Aim 2 is to demonstrate the neural basis of the functional restoration of the reinnervated muscles by quantifying the retrograde horseradish peroxidase (HRP) labeled motoneurons, regenerating axons and sprouts, and newly formed motor endplates. Specific Aim 3 is to explore procedure-related and time-dependent morphological, immunocytochemical, and biochemical changes in the reinnervated muscles by analyzing muscle mass, fiber size, fiber type grouping, and fiber type and myosin heavy chain (MHC) composition. The results will allow the reliable documentation of the efficacy of the NMEG in rehabilitation of muscle paralysis. The significance of the proposed work extends far beyond what is currently understood. Once the advantages of the NMEG are fully documented by extensive animal studies, the impact of this research on science and health care could be substantial as the data obtained from this research are useful for ultimate clinical application in the near future to the treatment of patients with paralytic neuromuscular disorders. As an entirely satisfactory solution to restoration of the paralyzed skeletal muscles has not yet been found, we developed a new technique (nerve-muscle-endplate band grafting) to reinnervate paralyzed cervical strap muscles in a rat model. Our preliminary work showed that this technique results in better outcomes than currently used methods. The data obtained from this research is useful for future clinical application to treat muscle paralysis.