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, 37 (9), 2004-2018

Blocking CCN2 Reduces Progression of Sensorimotor Declines and Fibrosis in a Rat Model of Chronic Repetitive Overuse

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Blocking CCN2 Reduces Progression of Sensorimotor Declines and Fibrosis in a Rat Model of Chronic Repetitive Overuse

Mary F Barbe et al. J Orthop Res.

Abstract

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.

Keywords: muscle; nerve; tendinopathy; tendon; work-related musculoskeletal disorders (WMSDs).

Figures

Figure 1
Figure 1
Operant performance behaviors assayed in task week 3 in rats performing a high repetition high force (HRHF) reaching and grasping a lever bar task. (A) Target reach rate was 4 reaches/min. (B) Mean voluntary grasp force on the lever bar. *p < 0.05, and **p < 0.01 compared with groups as shown. FRC, food‐restricted control; IgG, immunoglobulin G [Color figure can be viewed at wileyonlinelibrary.com]
Figure 2
Figure 2
Reflexive grip strength and forepaw mechanical sensitivity. (A) and (B) Reflexive grip strength in food‐restricted control (FRC) and HRHF task groups. HRHF rats were tested at a naïve timepoint immediately prior to the 5‐week shaping period, immediately post‐shaping (task week 0) and after performing the HRHF task for 3 weeks, with subsets receiving treatments in the final 2 weeks. FRC rats were tested at similar times. (C) Forepaw mechanical sensitivity assayed in task week 3 using nylon monofilaments with milliNewton (mN) sizes shown. a: p < 0.05, compared with matched FRC group; b: p < 0.05, compared with untreated HRHF rats; c: p < 0.05, compared with HRHF + hIgG rats; n.s., not significant. Number of limbs tested per group: HRHF, n = 12; HRHF + FG‐3019, n = 10–12; HRHF + hIgG, n = 10; FRC, n = 10–25; FRC + Saline, n = 10; FRC + hIgG, n = 10, and FRC + FG‐3019, n = 10, bilateral results included for latter two groups [Color figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3
Collagen type 1 in flexor digitorum muscle and serum. (A) Percent area with collagen type 1 immunoreactivity (green) and 4′,6‐diamidino‐2‐phenylindole (DAPI) nuclear stain (blue) in cross‐sectionally cut muscles of food‐restricted control (FRC), 3‐week untreated high repetition high force (HRHF) and HRHF + FG‐3019‐treated rats. (B) Enzyme‐linked immunosorbent assay (ELISA) detected levels in muscles. (C) Collagen type 1 serum levels, tested using ELISA. aa: p < 0.01, compared with matched FRC group; bb: p < 0.01, compared with HRHF rats; c: p < 0.05, and cc: p < 0.01, compared with HRHF + hIgG rats. Scale bars = 50 μm. The 0‐week HRHF rat results from Supplementary Table 1 are indicated with dotted lines [Color figure can be viewed at wileyonlinelibrary.com]
Figure 4
Figure 4
Cellular communication network factor 2 (CCN2) in flexor digitorum muscles. (A–C) Immunoexpression and enzyme‐linked immunosorbent assay (ELISA) detected levels in muscles and serum. (D) An untreated 3‐week high repetition high force (HRHF) muscle probed for both SMA and CCN2 immunoexpression. Only the arteriole shown in the insets shows co‐localization. (E) Location of CCN2 immunoexpression in muscle cross‐sections. (F) Anti‐human IgG detection of the FG‐3019 agent in muscles of HRHF + FG‐3019 rats; staining absent in untreated HRHF rats. a: p < 0.05 and aa: p < 0.01, compared with matched FRC group; b: p < 0.05 and bb: p < 0.01, compared with HRHF rats; cc: p < 0.01, compared with HRHF + hIgG rats. Scale bars = 50 μm. The 0‐week HRHF rat results from Supplementary Table 1 are indicated with dotted lines [Color figure can be viewed at wileyonlinelibrary.com]
Figure 5
Figure 5
Anti‐CCN3/NOV probed Western blots showing truncated forms of CCN3 with approximate molecular weights of 32 and 28 kDa (arrows). (A) Western blot showing flexor digitorum muscle samples from food‐restricted control rats (F; FRC), untreated high repetition high force rats (H; HRHF), and HRHF rats treated with the FG‐3019 agent (H + F; HRHF + FG‐3019). (B) Ponceau‐S red staining of same membrane shown in panel A. (C) Densitometry results in which CCN3 bands were compared with the total protein loaded per lane, determined from Ponceau‐S red stained membranes. Gels were repeated until three different samples per group were assayed. a: p < 0.05, compared with FRC levels [Color figure can be viewed at wileyonlinelibrary.com]
Figure 6
Figure 6
Transforming growth factor β1 (TGFβ1) in flexor digitorum muscles. (A) and (B) Immunoexpression and enzyme‐linked immunosorbent assay (ELISA) detected levels. a: p < 0.05 and aa: p < 0.01, compared with matched food‐restricted control (FRC) group; b: p < 0.05, compared with high repetition high force (HRHF) rats; c: p < 0.05, compared with HRHF + hIgG rats. (C) Location of TGFβ1 immunoexpression in muscle cross‐sections. (D) Presence and location of αSMA+/tcf4+/PDGFR+and αSMA+/tcf4+/PDGFR cells in HRHF + FG‐3019 and HRHF rat muscles. A few triple‐labeled cells were found on the perimeter of myofibers in 3‐week HRHF rat muscles (arrows). These cells are shown at higher magnification in (E), right side. Left side of (E) shows examples of αSMA+/tcf4+/PDGFR cells (i.e., double labeled only) in the endomysium. Scale bars = 50 μm [Color figure can be viewed at wileyonlinelibrary.com]
Figure 7
Figure 7
Task‐ and treatment‐induced changes in flexor digitorum tendons at wrist level. (A) Location of cellular communication network factor 2 (CCN2) staining in tendons showing increases in tenocytes and epitendon cells of high repetition high force (HRHF) rats (arrows). (B) Anti‐human immunoglobulin G (IgG) detection of FG‐3019 agent in tendons of HRHF + FG‐3019 rats; staining absent in untreated HRHF rats. (C) and (D) Quantification of epitendon cellularity and thickness. (E) Representative images of hematoxylin and eosin stained tendons. Arrows indicate epitendon region. a: p < 0.05 and aa: p < 0.01, compared with matched food‐restricted control (FRC) group; b: p < 0.05, compared with untreated HRHF rats; cc: p < 0.01, compared with HRHF + hIgG rats. Scale bars = 50 μm [Color figure can be viewed at wileyonlinelibrary.com]
Figure 8
Figure 8
Masson's Trichrome staining around the median nerve branches and in upper dermis of forepaw digits. (A) Masson's trichrome staining with collagen stained blue around the median nerve (N) at wrist level in longitudinal sections. (B) Masson's trichrome staining in the upper dermis. Arrows indicate regions containing nerves within dermal papillae. (C) Quantification of blue‐stained collagen in paraneural regions (i.e., areas surrounding nerve) at wrist level. (D) Quantification of blue‐stained collagen in the upper dermis. aa: p < 0.01, compared with matched food‐restricted control (FRC) group; b: p < 0.05 and bb: p < 0.01, compared with untreated high repetition high force (HRHF) rats [Color figure can be viewed at wileyonlinelibrary.com]

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