Identification and Imaging of Skeletal Muscle Response to Graded Nerve Crush

项目摘要 神经肌肉创伤后骨骼肌的萎缩和纤维化是神经肌肉损伤的重要障碍。 严重神经肌肉创伤后的功能恢复。尽管如此，肌肉萎缩的动态评估工具 和功能障碍是有限的，留下了一个关键的差距，整形外科医生的能力，以评估程度的神经源性 肌肉损伤及其最终预后。这一差距部分源于对联合国作用的不完全理解， 与萎缩相关的转录因子和蛋白酶表达增加， 他们临床上。钙蛋白酶是神经性萎缩肌原纤维破坏的核心蛋白酶之一， 因此有可能作为肌肉萎缩的标志物。然而，将这种关系转化为 诊断工具受到缺乏用于钙蛋白酶活性的真实的时间评估的技术的限制。拟议的工作旨在 探索使用光学探针通过检查神经与肌肉萎缩之间的关系来识别肌肉萎缩的可能性。 损伤和肌肉收缩性以及钙蛋白酶活性。目的1a将确定钙蛋白酶的表达和活性是否会增加 与神经损伤和肌肉功能障碍成比例。将对相同数量的雄性和雌性小鼠进行 单侧坐骨神经不同程度挤压伤。在随后的手术中，分阶段进行，功能恢复 将通过步行轨迹分析和握力测试进行评估。后肢肌肉将经历离体收缩 检测以及组织形态学分析和相关转录因子。钙蛋白酶活性将 使用ELISA试剂盒和临床前成像系统检测近红外荧光（NIR）进行定量 在注射钙蛋白酶敏感探针后后肢肌肉内。在Aim 1b中，单侧坐骨神经 将在小鼠中进行神经切断和修复，并进行相同系列的功能测试，转录， 将采用光学探针进行分析和近红外成像。同样，目标1c将采用相同的方法 在去除一段坐骨神经后，以相同的时间间隔进行评估。神经增大程度 损伤和建议的评估将有助于描绘神经损伤后肌肉萎缩的典型途径， 并且所提出的光学探针将提供一种强大的新诊断工具。 作为一名骨科医生，我的工作是护理受损的肢体，我理解这对临床的影响。 但是需要保护的时间和资源来发展在分子水平上研究这些的技能。在 除了上述调查外，我还将参加研究生课程，以提高我对 分子生物学以及光学和生物成像。我会定期参加学术活动，如 通过肌肉骨骼研究中心的期刊俱乐部和赠款研讨会，以提高我的赠款写作能力， 提高我对实验方法的理解。除了我的主要和次要导师，我还有 组建了一个指导委员会，对结果提供反馈，并协助进行实验设计。这个星座 计划的活动，沿着上述建议的研究方法将为我提供必要的培训， 经验，以发展为临床科学家与骨骼肌萎缩的光学成像的兴趣。

Project Summary Atrophy and fibrosis of skeletal muscle after neuromuscular trauma is a significant impediment to the restoration of function after severe neuromuscular trauma. Despite this, dynamic assessment tools for muscle wasting and dysfunction are limited, leaving a critical gap in the orthopedic surgeon’s ability to assess the degree of neurogenic muscle injury and its ultimate prognosis. This gap stems in part from an incomplete understanding of the role of increased expression of transcriptional factors and proteases related to atrophy, and inability to dynamically assess them clinically. Calpain is one of these proteases central to the myofibril destruction of neurogenic atrophy, and therefore has potential to serve as a marker of muscle atrophy. However, translation of this relationship into a diagnostic tool is limited by a lack of techniques for real time assessment of calpain activity. The proposed work seeks to explore the potential for use of optical probes to identify muscle atrophy by examining the relationship between nerve injury and muscle contractility and calpain activity. Aim 1a will determine if calpain expression and activity will increase proportionally with nerve injury and muscle dysfunction. Equal numbers of male and female mice will be subjected to a varying degree of unilateral sciatic nerve crush injury. At a subsequent surgery, at staged intervals, functional recovery will be assessed with walking track analysis and grip strength testing. Hindlimb muscles will undergo ex-vivo contractility testing, as well as histomorphometric analysis and relevant transcriptional factors will be assayed. Calpain activity will be quantified with ELISA kits and with use of a pre-clinical imaging system to detect near-infrared fluorescence (NIR) within the hindlimb muscles after administration of an injectable calpain sensitive probe. In Aim 1b, a unilateral sciatic nerve transection and repair will be performed in the mice, and the same series of functional tests, transcriptional assays and NIR imaging with the optical probe will be undertaken. Similarly, Aim 1c will utilize the same methodologic assessments, at the same time intervals, after removal of a segment of sciatic nerve. The increasing degree of nerve injuries and proposed assessments will help to delineate the canonical pathways of muscle atrophy after nerve injury, and the proposed optical probe will provide a powerful new diagnostic tool. As an orthopedic surgeon with a practice devoted to the care of mangled limbs, I understand the clinical impact of such injuries, but need protected time and resources to develop the skills to study these at a molecular level. In addition to the investigations described above, I will participate in graduate coursework to improve my understanding of molecular biology, as well as optical and biological imaging. I will regularly participate in scholarly activities such as journal clubs and grant seminars through the Musculoskeletal Research Center to enhance my grant writing abilities and improve my understanding of experimental methodologies. In addition to my primary and secondary mentor, I have assembled a mentoring committee to give feedback on results and assist with experimental design. This constellation of planned activities, along with the proposed research methods above will provide me the requisite training and experience to develop as a clinician scientist with an interest in optical imaging of skeletal muscle atrophy.