Overuse injury is a frequent diagnosis in occupational medicine and athletics. Using an established model of upper extremity overuse, we sought to characterize changes occurring in the forepaws and forelimbs of mature female rats (14–18 months of age). Thirty-three rats underwent a 4-week shaping period, before performing a high-repetition low-force (HRLF) task for 12 weeks, with the results being compared to 32 mature controls. HRLF animals showed a reduced grip strength versus controls. ELISAs carried out in the HRLF rats, versus controls, showed elevated levels of IL1-α in tendons, IL1-α and TNF-α in distal bones/entheses, and TNF-α, MIP1-α/CCL3, and CINC-2/CXCL-3 in serum, as well as IL-6 in forelimb muscles and tendons, and IL-10 in serum. HRLF rats had elevated collagen deposition in the forepaw intrinsic muscles (i.e., fibrosis), entheseal microdamage, and articular cartilage degradation versus the control rats. CD68/ED1+ osteoclasts and single-nucleated cells were elevated in distal forelimb metaphyses of the HRLF animals, versus controls. Declines in grip strength correlated with muscle fibrosis, entheseal microdamage, articular cartilage damage, distal bone/enthesis IL1-α, and serum IL-6. These data demonstrate inflammatory and persistent degradative changes in the forearm/forepaw tissues of mature female animals exposed to prolonged repetitive tasks, changes with clinical relevance to work-related overuse injuries in mature human females.

The matricellular protein cell communication factor 2/connective tissue growth factor (CCN2/CTGF) is critical to development of neuromuscular fibrosis. Here, we tested whether anti-CCN2 antibody treatment will reduce established forepaw fibro-degenerative changes and improve function in a rat model of overuse injury. Adult female rats performed a high repetition high force (HRHF) task for 18 weeks. Tissues were collected from one subset after 18 wks (HRHF-Untreated). Two subsets were provided 6 wks of rest with concurrent treatment with anti-CCN2 (HRHF-Rest/anti-CCN2) or IgG (HRHF-Rest/IgG). Results were compared to IgG-treated Controls. Forepaw muscle fibrosis, neural fibrosis and entheseal damage were increased in HRHF-Untreated rats, compared to Controls, and changes were ameliorated in HRHF-Rest/anti-CCN2 rats. Anti-CCN2 treatment also reduced phosphorylated-β-catenin (pro-fibrotic protein) in muscles and distal bone/entheses complex, and increased CCN3 (anti-fibrotic) in the same tissues, compared to HRHF-Untreated rats. Grip strength declines and mechanical sensitivity observed in HRHF-Untreated improved with rest; grip strength improved further in HRHF-Rest/anti-CCN2. Grip strength declines correlated with muscle fibrosis, entheseal damage, extraneural fibrosis, and decreased nerve conduction velocity, while enhanced mechanical sensitivity (a pain-related behavior) correlated with extraneural fibrosis. These studies demonstrate that blocking CCN2 signaling reduces established forepaw neuromuscular fibrosis and entheseal damage, which improves forepaw function, following overuse injury.

A role for substance P has been proposed in musculoskeletal fibrosis, with effects mediated through transforming growth factor beta (TGFβ). We examined the in vitro effects of substance P on proliferation, collagen secretion, and collagen deposition in rat primary dermal fibroblasts cultured in medium containing 10% fetal bovine serum, with or without TGFβ. In six-day cultures, substance P increased cell proliferation at concentrations from 0.0002 to 100 nM. TGFβ increased proliferation at concentrations from 0.0002 to 2 pg/mL, although higher concentrations inhibited proliferation. Substance P treatment alone at concentrations of 100, 0.2, and 0.00002 nM did not increase collagen deposition per cell, yet when combined with TGFβ (5 ng/mL), increased collagen deposition compared to TGFβ treatment alone. Substance P treatment (100 nM) also increased smooth muscle actin (SMA) expression at 72 h of culture at a level similar to 5 ng/mL of TGFβ; only TGFβ increased SMA at 48 h of culture. Thus, substance P may play a role in potentiating matrix deposition in vivo when combined with TGFβ, although this potentiation may be dependent on the concentration of each factor. Treatments targeting substance P may be a viable strategy for treating fibrosis where both substance P and TGFβ play roles.

Neurotrophic factors and reactive oxygen species (ROS) modulate neuronal plasticity. In a model of a lower motor neuron lesioned bladder, somatic nerve transfer was used as a reinnervation strategy. Levels of neurotrophins, ROS, and TNF-α in bladder mucosa and muscle layers collected from three groups of adult female dogs: (1) Decentralized, via bilateral transection of coccygeal and sacral spinal roots, lumbar 7 dorsal roots, and hypogastric nerves, then 6–21 mo recovery; (2) reinnervated (ObNT-Reinn), after similar decentralization for 12 mo, then bilateral obturator-to-vesical nerve transfer and 8–12 mo recovery; and (3) Controls. In mucosa, BDNF and ROS levels were highest in ObNT-Reinn bladders, GDNF and TNF-α levels were restored to Control levels in ObNT-Reinn bladders (lowest in Decentralized). NT-3 and ARTN were lower in ObNT-Reinn and Decentralized bladders versus Controls. In muscle, ROS was lower in ObNT-Reinn muscle versus Controls. BDNF mucosa levels correlated with bladder axonal density and detrusor layer thickness; and GDNF mucosal correlated with bladder contraction after vesical or transferred obturator nerve electrical stimulation, as did BDNF and GDNF muscle levels. The increased BDNF and GDNF in bladders that underwent somatic nerve transfer with subsequent recovery suggest that BDNF and GDNF may help promote the reestablishment of bladder innervation.

Chronic sleep disturbance is postulated to enhance pain and inflammatory responses, although systemic inflammation has been primarily investigated to date. We sought to examine the effects of chronic intermittent sleep disturbance on pain-related behaviors and peripheral forelimb neural- and myofascial tissues in the context of aging. Uninjured young adult and mature female rats (3 mo. and 9–10 mo. of age) were exposed to either normal or disturbed sleep for 9 h/day on 4 d/week (using in-cage environmental stimulation), for 6 weeks. Compared to rats with normal sleep, sleep disturbed rats of both ages displayed: (1) mild declines in grip strength; (2) low-grade elevations in immune cells and collagen in forepaw neural- and myofascial tissues; and (3) higher protein expression of collagen type I, CTGF, and phosphorylated AKT in forearm flexor muscles. Young adult sleep disturbed also showed increased TGF-beta. Mature sleep disturbed rats also displayed low-grade yet greatest forepaw mechanical sensitivity, muscle and serum TNF-α, and peripheral immune cell numbers. These data suggest that chronic intermittent sleep disruption can enhance pain-related behaviors, inflammation, and fibrosis-related changes in peripheral neural- and myofascial tissues in uninjured rats. These effects occurred in both age groups, yet were more pronounced in the older rats.