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

描述（由申请人提供）：神经肌肉失神经支配是周围神经损伤和神经系统疾病的常见后果。由于目前使用的方法通常导致较差的功能恢复，因此迫切需要寻求新的神经移植方法来修复瘫痪的肌肉。推动该项目的全球假设是，通过使用丰富的完整神经末梢和运动终板来源对瘫痪肌肉进行神经再支配可以获得更好的结果。这个想法促使我们发展神经-肌肉-终板带移植术（NMEG）的方法来重新神经麻痹的肌肉。选择颈带状肌肉在大鼠模型中进行可行性研究。从肩胛舌骨肌（OH）上采集NMEG，然后移植到实验瘫痪的胸锁乳突肌（SM）上。同时进行神经-肌肉蒂（NMP）和端到端吻合（EEA）再神经移植手术进行比较。我们使用即时神经再生模型进行的初步研究表明，与NMP和EEA相比，NMEG能够成功地实现神经再生和功能恢复。本研究旨在确定NMEG与经典EEA技术相比，对麻痹颈带肌即刻和延迟神经再支配的疗效。本提案中设计的研究将记录功能恢复的基本神经基础和影响结果的其他主要因素。我们假设再神经支配肌肉的功能恢复程度在很大程度上取决于重建的神经-肌肉接触的数量和去神经支配引起的肌肉改变，并且NMEG将是肌肉再神经支配的更好选择，而不是常用的EEA和其他方法。这些假设将通过以下3个具体目标进行检验。具体目的1是通过分析肌电图（EMG）记录、肌肉力量和运动测量以及糖原耗尽肌纤维的分布来评估再神经支配肌肉的功能恢复。具体目的2是通过量化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来证明再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）组成，探索再神经支配肌肉的手术相关和时间依赖性形态学、免疫细胞化学和生化变化。结果将允许NMEG在肌肉麻痹康复的功效可靠的文件。提出的工作的意义远远超出了目前的理解。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将是巨大的，因为从这项研究中获得的数据将有助于在不久的将来最终临床应用于麻痹性神经肌肉疾病患者的治疗。由于目前还没有找到完全令人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）来重建大鼠模型的瘫痪颈带肌。我们的初步工作表明，这种技术比目前使用的方法效果更好。本研究所得的数据对将来临床应用于肌肉麻痹的治疗有帮助。

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.