Identification and Imaging of Skeletal Muscle Response to Graded Nerve Crush

项目摘要 神经肌肉损伤后骨骼肌萎缩和纤维化是严重的障碍。 严重神经肌肉损伤后的功能恢复。尽管如此，肌肉损耗的动态评估工具 和功能障碍是有限的，在骨科医生评估神经源性疾病程度的能力上留下了一个关键的缺口 肌肉损伤及其最终预后。造成这一差距的部分原因是对 与萎缩相关的转录因子和蛋白水解酶的表达增加，无法动态评估 临床上是这样的。钙蛋白酶是神经源性萎缩中肌原纤维破坏的核心酶之一，并且 因此有可能作为肌肉萎缩的标志。然而，将这种关系转化为 诊断工具受到缺乏实时评估钙蛋白酶活性的技术的限制。拟议的工作旨在 通过检查神经与肌肉萎缩之间的关系，探索使用光学探头识别肌肉萎缩的可能性 损伤与肌肉收缩和钙蛋白酶活性的关系。目标1a将确定是否会增加calain的表达和活性 与神经损伤和肌肉功能障碍成比例。同等数量的雄性和雌性小鼠将受到 不同程度的单侧坐骨神经挤压伤。在随后的手术中，每隔一段时间进行功能恢复 将通过步道分析和握力测试进行评估。后肢肌肉将进行体外收缩 测试，以及组织形态计量学分析和相关转录因子将被检测。钙蛋白酶活性将 用酶联免疫吸附试验试剂盒和临床前成像系统检测近红外荧光(NIR)进行定量 在注射了可注射的钙蛋白酶敏感探针后，在后肢肌肉内。在Aim 1b中，单侧坐骨神经痛 将在小鼠身上进行神经切断和修复，并进行相同的一系列功能测试，转录 将使用光学探头进行分析和近红外成像。同样，目标1c将使用相同的方法 在同一时间间隔内，在切除一段坐骨神经后进行评估。神经递增的程度 损伤和拟议的评估将有助于描绘神经损伤后肌肉萎缩的典型途径， 而提出的光学探头将提供一种强大的新诊断工具。 作为一名致力于四肢损伤护理的整形外科医生，我理解这对临床的影响。 但需要受保护的时间和资源来发展在分子水平上研究这些损伤的技能。在……里面 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，比如 通过肌肉骨骼研究中心的杂志俱乐部和赠款研讨会来提高我的赠款撰写能力和 提高我对实验方法论的理解。除了我的主要和次要导师，我还有 组织了一个指导委员会，对结果进行反馈，并协助实验设计。这一星座 有计划的活动，加上上述拟议的研究方法，将为我提供必要的培训和 作为一名对骨骼肌萎缩的光学成像感兴趣的临床科学家的经验。

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.