Identification and Imaging of Skeletal Muscle Response to Graded Nerve Crush

骨骼肌对分级神经挤压反应的识别和成像

• 批准号: 10351778
• 负责人: David Micah Brogan
• 金额: $ 15.64万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10351778
• 关键词: AKT Signaling Pathway; Affect; Animal Model; Assessment tool; Atrophic; Biological; Biological Assay; Calpain; Caring; Cells; Clinical; Crush Injury; Custom; Data; Denervation; Desmin; Early Intervention; Enzyme-Linked Immunosorbent Assay; Evaluation; Event; Excision; Experimental Designs; Feedback; Female; Fibrosis; Filament; Fluorescence; Fluorescent Probes; Functional Imaging; Functional disorder; Future; Genetic Transcription; Goals; Grant; Hamsters; Hand Strength; Health; Hindlimb; Hippocampus (Brain); Image; In Vitro; Injectable; Injections; Injury; Investigation; Journals; Lesion; Limb structure; Literature; Mediating; Mediator of activation protein; Mentors; Methodology; Methods; Modeling; Molecular; Molecular Biology; Molecular Probes; Molecular Target; Mus; Muscle; Muscle denervation procedure; Muscular Atrophy; Musculoskeletal; Myofibrils; Myogenin; Nature; Near-infrared optical imaging; Nerve; Nerve Crush; Neuregulins; Operative Surgical Procedures; Optics; Orthopedics; Outcome; PI3K/AKT; Pathway interactions; Patients; Penetration; Peptide Hydrolases; Peripheral Nerves; Process; Prognosis; Proteins; Recovery; Recovery of Function; Reporting; Research; Research Methodology; Resected; Resources; Role; Scientist; Secondary to; Series; Severities; Signal Transduction; Skeletal Muscle; Surgeon; System; Techniques; Technology; Testing; Time; Training; Translations; Trauma; Traumatic injury; Up-Regulation; Walking; Wallerian Degeneration; Work; Writing; axon injury; base; biomarker identification; bone imaging; clinical translation; depolymerization; design; diagnostic tool; differential expression; disability; ex vivo imaging; experience; functional outcomes; iatrogenic injury; imaging probe; imaging system; improved; improved outcome; in vivo; in vivo imaging; insight; interest; male; molecular marker; mouse model; nerve injury; nerve repair; nerve transection; neuromuscular; novel; novel diagnostics; optical imaging; preclinical imaging; predictive marker; protein aminoacid sequence; repaired; response; restoration; sciatic nerve; skeletal muscle wasting; skills; stem; time interval; tool; transcription factor

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.

项目概要 神经肌肉创伤后骨骼肌萎缩和纤维化是严重阻碍神经肌肉损伤的因素。 严重神经肌肉创伤后功能的恢复。尽管如此，肌肉萎缩的动态评估工具 且功能障碍有限，这使得骨科医生评估神经源性程度的能力存在严重差距 肌肉损伤及其最终预后。这种差距部分源于对角色的不完全理解。 与萎缩相关的转录因子和蛋白酶的表达增加，并且无法动态评估 他们在临床上。钙蛋白酶是这些蛋白酶之一，对神经源性萎缩的肌原纤维破坏至关重要，并且 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 诊断工具因缺乏实时评估钙蛋白酶活性的技术而受到限制。拟议的工作旨在 探索使用光学探针通过检查神经之间的关系来识别肌肉萎缩的潜力 损伤和肌肉收缩力和钙蛋白酶活性。目标 1a 将确定钙蛋白酶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。相同数量的雄性和雌性小鼠将接受 单侧坐骨神经不同程度挤压损伤。在随后的分阶段手术中，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 将进行测试、组织形态分析和相关转录因子。钙蛋白酶活性将 使用 ELISA 试剂盒进行定量，并使用临床前成像系统检测近红外荧光 (NIR) 注射钙蛋白酶敏感探针后，在后肢肌肉内。在目标 1b 中，单侧坐骨神经痛 将在小鼠中进行神经横断和修复，并进行同一系列的功能测试、转录 将使用光学探针进行测定和近红外成像。同样，目标 1c 将采用相同的方法 在切除一段坐骨神经后，以相同的时间间隔进行评估。神经程度的增加 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， 所提出的光学探针将提供一种强大的新诊断工具。 作为一名致力于护理残肢的骨科医生，我了解其临床影响 此类伤害，但需要受保护的时间和资源来培养在分子水平上研究这些伤害的技能。在 除了上述调查之外，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，例如 通过肌肉骨骼研究中心参加期刊俱乐部和资助研讨会，以提高我的资助写作能力和 提高我对实验方法的理解。除了我的小学和中学导师之外，我还有 组建了一个指导委员会来提供结果反馈并协助实验设计。这个星座 计划的活动以及上述建议的研究方法将为我提供必要的培训和 培养对骨骼肌萎缩光学成像感兴趣的临床科学家的经验。