149. Distal Forepaw Muscle Fibrosis and Entheseal Damage in a Repetitive Overuse Injury Model

PURPOSE: Overuse injury is a frequent complaint and diagnosis in workplace medicine and athletics. Research to date shows adaptive and low inflammatory responses in forearm and forepaw musculoskeletal tissues of young adult (5-9 mo of age) rats performing a high-repetition low-force (HRLF) task. In contrast, very mature female rats (14-18 mo of age) performing the HRLF task showed elevated inflammatory responses in upper extremity tissues and systemically, and marked bone degradation, concomitant with sensorimotor declines. We sought here to determine if mature rats performing the HRLF task for 12 weeks also develop fibrotic and degradative changes in their wrist and forepaw regions that might contribute to motor declines. METHODS: 57 female rats underwent a 4-week training period, before performing a high-repetition low-force (HRLF) task for 9-12 wks, with results compared to age-matched controls. Forepaws and forelimbs of 37 rats were fixed, sectioned, and stained with Masson’s Trichrome, Safranin Orange-Fast Green, or CD68+ stains to detect histopathological changes in muscle fibrosis, entheses, radiocarpal articular cartilage, and osteoclast/macrophage presence, respectively. Forepaw/forelimb tissues, and serum, were collected flash frozen from 49 rats and analyzed using ELISA for inflammatory cytokines. Reach success and reflexive grip strength was assessed and correlated with these results. RESULTS: HRLF animals showed significant increases in unsuccessful reaches and declines in grip strength, compared to baseline (p<0.01 each), indicating loss of function. Biochemical analyses revealed elevated IL1-α (p<0.05) and TNF-α (p<0.01) in distal bones and entheses, and elevated TNF-α, MIP1-α/CCL3, and CINC-2/CXCL-3 (p<0.05) systemically (i.e., in serum), yet no elevation of cytokines in distal forelimb muscles (although we recently found elevated inflammatory cytokines in 6-week HRLF rats), in these 9-12 week HRLF rats versus Control rats. Since elevated inflammatory cytokines can lead to inflammation-induced fibrosis or tissue degradative changes, we next examined wrist and forepaw regions for fibrotic or degradative changes. Forepaw muscle fibrosis, entheseal microdamage, and radiocarpal articular degradation were evident in HRLF rats (p<0.01 each, compared to Control rats). CD68+ multinucleated osteoclasts were elevated in radial and ulnar metaphases of HRLF animals (N.Oc/BS and % Oc/BS, p<0.01 and p<0.05, respectively, compared to Control rats), as were CD68+ single nucleated cells (pre-osteoclasts or macrophages, p<0.05). Grip strength declines correlated inversely with muscle fibrosis, entheseal microdamage, and articular cartilage damage (r = -0.40, -0.57, -0.53, respectively), suggestive of histopathology contributions to the functional declines. CONCLUSION: We show here for the first time that moderate demand repetitive tasks contribute to pathological changes in forepaw musculoskeletal tissues of mature female animals, including palmar muscle fibrosis, forepaw and wrist entheses, and radiocarpal articular cartilage, and serum cytokines. These findings correlated with grip strength declines. Although inflammatory responses observed previously in muscles of 6-week HRLF mature rats was resolved at this 9-12 week timepoint, the elevated inflammatory cytokines in distal bones/entheses and serum indicate a potential biochemical basis for the observed structural changes. These findings contribute to the understanding of effects of repetitive overuse injuries in upper extremity tissues and have significant clinical relevance for work-related overuse injury in older human females.