Identification and Imaging of Skeletal Muscle Response to Graded Nerve Crush

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.

Grant Number: 5K08AR080260-04
NIH Institute/Center: NIH
Principal Investigator: David Brogan