Fibrosis may be a key factor in sensorimotor dysfunction in patients with chronic overuse‐induced musculoskeletal disorders. Using a clinically relevant rodent model, in which performance of a high demand handle‐pulling task induces tissue fibrosis and sensorimotor declines, we pharmacologically blocked cellular communication network factor 2 (CCN2; connective tissue growth factor) with the goal of reducing the progression of these changes. Young adult, female Sprague–Dawley rats were shaped to learn to pull at high force levels (10 min/day, 5 weeks), before performing a high repetition high force (HRHF) task for 3 weeks (2 h/day, 3 days/week). HRHF rats were untreated, or treated in task weeks 2 and 3 with a monoclonal antibody that blocks CCN2 (FG‐3019), or a control immunoglobulin G (IgG). Control rats were untreated or received FG‐3019, IgG, or vehicle (saline) injections. Mean task reach rate and grasp force were higher in 3‐week HRHF + FG‐3019 rats, compared with untreated HRHF rats. Grip strength declined while forepaw mechanical sensitivity increased in untreated HRHF rats, compared with controls; changes improved by FG‐3019 treatment. The HRHF task increased collagen in multiple tissues (flexor digitorum muscles, nerves, and forepaw dermis), which was reduced with FG‐3019 treatment. FG‐3019 treatment also reduced HRHF‐induced increases in CCN2 and transforming growth factor β in muscles. In tendons, FG‐3019 reduced HRHF‐induced increases in CCN2, epitendon thickening, and cell proliferation. Our findings indicate that CCN2 is critical to the progression of chronic overuse‐induced multi‐tissue fibrosis and functional declines. FG‐3019 treatment may be a novel therapeutic strategy for overuse‐induced musculoskeletal disorders. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2004–2018, 2019

Fibrillar collagen type 1 is the most abundant type of collagen within the body and is a critical component of extracellular infrastructure. In order to assess collagen synthesis and extracellular accumulation in fibrotic disorders, improved methods are needed to detect changes in procollagen versus mature collagen at the protein level. Using Western blot methodology, we systematically examined: (1) gel composition (Tris-glycine vs. bis-Tris, gradient vs. non-gradient, sodium dodecyl sulfate (SDS) vs. no SDS); (2) sample preparation (SDS vs. no SDS, β-mercaptoethanol (BME) vs. no BME, boiling vs. no boiling); and (3) running buffer composition (SDS vs. no SDS). Our results indicate full native gel conditions prevent resolution of all collagen type 1 bands. The best resolution of type 1 procollagens is achieved using 4%–12% Tris-glycine gels without the presence of SDS in the gel itself, although SDS in the running and sample buffers are needed. Also, BME must not be added to the sample buffer and samples should not be boiled. For characterization of mature collagen 1(I), both 8% and gradients type gels are appropriate, although still without SDS, yet with SDS included in both running and sample buffers, BME must be added to the sample buffer, and samples should not be boiled. Boiling is to be avoided as the antigenic site recognized by the monoclonal antibody used is sensitive to thermal denaturation, as is the case with many monoclonal antibodies available on the market. Thus, the exact parameters employed are dependent upon the collagen protein product that the scientist desires to identify.

Purpose/Aim: Substance P-NK-1R signaling has been implicated in fibrotic tendinopathies and myositis. Blocking this signaling with a neurokinin 1 receptor antagonist (NK1RA) has been proposed as a therapeutic target for their treatment. Materials and Methods: Using a rodent model of overuse injury, we pharmacologically blocked Substance P using a specific NK1RA with the hopes of reducing forelimb tendon, muscle and dermal fibrogenic changes and associated pain-related behaviors. Young adult rats learned to pull at high force levels across a 5-week period, before performing a high repetition high force (HRHF) task for 3 weeks (2 h/day, 3 days/week). HRHF rats were untreated or treated in task weeks 2 and 3 with the NK1RA, i.p. Control rats received vehicle or NK1RA treatments. Results: Grip strength declined in untreated HRHF rats, and mechanical sensitivity and temperature aversion increased compared to controls; these changes were improved by NK1RA treatment (L-732,138). NK1RA treatment also reduced HRHF-induced thickening in flexor digitorum epitendons, and HRHF-induced increases of TGFbeta1, CCN2/CTGF, and collagen type 1 in flexor digitorum muscles. In the forepaw upper dermis, task-induced increases in collagen deposition were reduced by NK1RA treatment. Conclusions: Our findings indicate that Substance P plays a role in the development of fibrogenic responses and subsequent discomfort in forelimb tissues involved in performing a high demand repetitive forceful task.