Increased CCN2, substance P and tissue fibrosis are associated with sensorimotor declines in a rat model of repetitive overuse injury

Abstract

Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Here, we characterized the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFalpha in 6-week HRHF rats. Serum had increased IL-1beta, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNγ increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNγ, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFB1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression.

Fig. 1

Sensorimotor behavioral changes in food restricted control rats (FRC) and in high repetition high force task (HRHF) rats. a Incidence of spontaneous pain behaviors in HRHF rats during task performance, from week 0 through week 18: bilateral pulling of lever bar (rather than with preferred reach limb), switching limb used to pull, supinated pulls (rather than typical pronated pull), and sitting in corner (rather than participating). *: p < 0.05 and **: p < 0.01, compared to week 0. b Temperature aversion compared to room temperature of 22 °C, assayed using cold and hot temperature place preference instrument in FRC rats, and HRHF rats at naïve and after 18 weeks of task. c Palmar mechanical sensitivity in age-matched FRC and 18-week HRHF rats, assayed using von Frey filaments of sizes indicated on x-axis. Number of responses out of 10 are reported. d Maximum grip strength in grams (g) in FRC and HRHF rats at naïve time point and across weeks of experiment. Symbols of panels b–d: *: p < 0.05 and **: p < 0.01, compared to naïve timepoint of HRHF rats; &:p < 0.05 and &&: p < 0.01, compared to age-matched FRC rats. Two-way ANOVA values are reported in individual panels

Fig. 2

Cytokines in flexor digitorum muscles, analyzed using ELISA. Data is shown for a MCP-1, b IL-18, c TNFα, d CCN2, e TGFB1, f Leptin, and g RANTES in normal control (NC), food-restricted controls (FRC), trained only (TRHF), and 6-, 12-, and 18-week high repetition high force (HRHF) rats. ANOVA values shown in individual panels. *:p < 0.05 compared to NC; &:p < 0.05 compared to FRC; #: p < 0.05 compared to TRHF rats; ϕ: p < 0.05 compared to 6-week HRHF rats

Fig. 3

Western Blot analysis of CCN2 and collagen type I protein in flexor digitorum muscles. a A representative Western blot of muscle homogenates from trained only (TRHF), 12- and 18-week HRHF rats, probed with anti-CCN2 (at 37 kDa). GAPDH used as a loading control (at 40.2 kDa). b Densitometric analysis of three replicate Western Blots, showing ratio of CCN2 bands normalized GAPDH levels. c A representative Western blot of muscle homogenates from TRHF, 12-, and 18-week HRHF rats, probed with anti-collagen type I, showing increased procollagen band (top procollagen band is approximately 150 kDa) in 12 week HRHF rats, and increased mature collage collagen type I band (75 kDa) in 12- and 18-week HRHF rats. d Densitometric analysis of three replicate Western blots, showing the ratio of mature collagen type I (75 kDa), normalized to GAPDH protein. # p < 0.05 and ## p < 0.01, compared to TRHF rats

Fig. 4

(a–h) CCN2 (red) and collagen type I (green), and (i–j) CCN2 (red) and smooth muscle actin (SMA; green) immunostaining in mid-region of flexor digitorum muscles (epimysium, myofibers, endomysium, and surrounding extracellular matrix regions) of FRC and 18 week HRHF rats. Muscle cross-sections are from mid-muscle region. (a,b) CCN2 and collagen type I immunostaining in FRC and 18 week HRHF rats, with double-headed arrows indicating in epimysium (epim) thickness. Asterisks indicate myofibers shown magnified in insets. (c) Quantification of percent area with CCN2 immunostaining in epimysium. (d–f) CCN2 and collagen type I immunostaining in myofibers, perimysium (perim) and endomysium region of FRC (d) and 18-week HRHF rats (e,f). Asterisks indicate myofibers shown enlarged in insets. (f) A fibrous scar-type region in the mid-muscle is outlined. (g,h) CCN2 and collagen type I immunostaining in extracellular matrix (ECM) around flexor digitorum muscle of FRC and 18 week HRHF rats. (i,j) CCN2 and SMA immunostaining in myofibers of FRC and 18-week HRHF rats, showing double-labeling of small cells surrounding subsets of myofibers, indicating subsets of small CCN2 stained cells are myofibroblats. Art = artery, cap = capillary, ct = connective tissue, M = muscle, N = nerve. *:p < 0.05, compared to NC. Scale bar = 50 μm

Fig. 5

IFNγ, CCN2, and TGFB1 immunostaining in flexor digitorum muscles and tendons of FRC and 18-week task rats. DAPI (blue) used as nuclear stain. (a,b) IFNγ (red) and CCN2 (green) immunostaining in muscle of FRC and 18 week HRHF rats. Inset in b: IFNγ immunostaining in small cells on myofiber perimeter. Arrows indicate examples of IFNγ stained cells; arrowheads indicate CCN2 deposition around myofiber perimeter. (c,d) TGFB1 (green) immunostaining in muscle of FRC and 18 week HRHF rats. (e,f) TGFB1 in tenocytes of FRC and 18 week HRHF rat tendons (inset shows enlargement of area indicated with arrow). The Western blots for IFNγ and TGFB1 show the bands recognized by the antibodies used for IHC. Scale bar = 50 μm

Fig. 6

Substance P (SubP; red) and either collagen type 1 (COL1) or pgp9.5 (a pan neuronal marker) green immunostaining in FRC and 18 week HRHF tissues. DAPI (blue) used as nuclear stain in panels a,b and d. (a,b) SubP and COL1 immunostaining in extracellular matrix surrounding mid-forearm region of flexor digitorum muscle showing increased SubP axon profiles and COL1 in extracellular matrix of 18 week HRHF rat, compared to FRC rat. (c) SubP and pgp9.5 double-labeled axons (arrows) in extracellular matrix (ECM) and nerve bundle (N). (d,e) SubP (red) and COL1 (green) immunostaining in flexor digitorum muscle of FRC and 18 week HRHF rats. Arrow in panel E indicates longitudinal profile of a SubP immunostained axon between myofibers. (f) Arrows indicate SubP immunostained axons around myofibers at myofiber tendinous (T) junction. (g) SubP and pgp9.5 double-labeled axon in muscle. (h,i) SubP and COL1 immunostaining in epidermis and dermis of mid-palmar forepaw of FRC and 18 week HRHF rat. Arrows indicate SubP immunostained axon profiles. Profiles indicated by arrow and asterisk are enlarged in inset in I. (j,k) SubP and pgp9.5 double-labeled axons in dermis (arrows indicate same axon regions that are double-labeled or pgp9.5 labeled). Scale bar = 50 μm