Bone loss from high repetitive high force loading is prevented by ibuprofen treatment

Abstract

We examined roles of loading and inflammation on forearm bones in a rat model of upper extremity overuse. Trabecular structure in distal radius and ulna was examined in three groups of young adult rats: 1) 5% food-restricted that underwent an initial training period of 10 min/day for 5 weeks to learn the repetitive task (TRHF); 2) rats that underwent the same training before performing a high repetition high force task, 2 hours/day for 12 weeks (HRHF); and 3) food-restricted only (FRC). Subsets were treated with oral ibuprofen (IBU). TRHF rats had increased trabecular bone volume and numbers, osteoblasts, and serum osteocalcin, indicative of bone adaptation. HRHF rats had constant muscle pulling forces, showed limited signs of bone adaptation, but many signs of bone resorption, including decreased trabecular bone volume and bone mineral density, increased osteoclasts and bone inflammatory cytokines, and reduced median nerve conduction velocity (15%). HRHF+IBU rats showed no trabecular resorptive changes, no increased osteoclasts or bone inflammatory cytokines, no nerve inflammation, preserved nerve conduction, and increased muscle voluntary pulling forces. Ibuprofen treatment preserved trabecular bone quality by reducing osteoclasts and bone inflammatory cytokines, and improving muscle pulling forces on bones as a result of reduced nerve inflammation.

Figure 1

Experimental design and rat body weights over time. (A). Experimental design showing onset of food restriction following a 1 week period of daily handling. All rats were food restricted to 5% less than weights of age-matched normal controls rats. Food-restricted control (FRC) rats rested until euthanasia at matched time points as HRHF rats. Trained and task rats underwent a 5 week training period (rats reached the HRHF level by last week of training). Trained only rats (TRHF) were euthanized after training. Task rats performed a high repetition high force (HRHF) task for 12 weeks. FRC+IBU rats received daily ibuprofen (IBU) treatment in final 8 weeks, as did HRHF+IBU rats (arrow indicates onset of ibuprofen treatment). TRHF+IBU rats received ibuprofen treatment prophylactically during training. Number/group shown at far right. (B) Rats were weighed weekly from the naïve time point to euthanasia, and showed an increase in weight across weeks of experiment. No differences in weight were observed between groups at matched time points (n.s. = not significant).

Figure 2

MicroCT images of distal radius and ulna. (A) A representative 3D model of the distal radius and ulna, cut sagittally, showing the site of the epiphyseal (epi) and metaphyseal (meta) analyses (encircled by black lines), and the sites of the images shown in panels B-D, as indicated by the letters B, C and D. (B) Representative transaxial microCT slices of the epiphysis of the radius and ulna (at 27 slices, 243 micrometers from the proximal edge of their respective growth plates) from a food restricted control (FRC), a 12-week high repetition high force (HRHF), and 12-week HRHF rat treated with ibuprofen for 8 weeks (HRHF+IBU). (C) Representative transaxial microCT slices of the metaphysis of the radius and ulna (at 166 slices, 1.5 millimeters from the distal edge of their respective growth plate) from a FRC, HRHF, and HRHF+IBU rat. (D) Representative 3D models of transaxial microCT slices through the metaphyseal region of the left radius (R) of a FRC, HRHF, and HRHF+IBU rat, with the surrounding cortical bone segmented away from the cortical bone in order to show the trabecular architecture. The transaxial reconstructions are located from 1.0 to 2.0 mm proximal to the respective growth plates, and are viewed from the bottom looking towards the growth plate. Increased separation and loss of trabeculae are seen in the HRHF rats, changes not present in FRC or HRHF+IBU rats (panel D).

Figure 3

MicroCT analysis of trabeculae of distal radial (A-D) and ulnar epiphyses (E-H). Results for trabecular bone volume (BV/TV), trabecular number (Tb.N.), and trabecular separation (Tb.Sp.) are shown. Significant 2-way ANOVA findings shown in individual panels and hereafter. Symbols: *:p<0.05 and **:p<0.01, compared to FRC rats; &: p<0.05, compared to untreated HRHF rats.

Figure 4

MicroCT analysis of trabeculae of the distal radial (A-D) and ulnar metaphysis (E-H). Results for trabecular bone volume (BV/TV), trabecular number (Tb.N.), trabecular separation (Tb.Sp.) and trabecular thickness (Tb.Th.) are shown. Symbols: *:p<0.05 and **:p<0.01, compared to FRC rats; &: p<0.05, compared to untreated HRHF rats.

Figure 5

Bone cell histomorphometry and bone mineral density in distal radial trabeculae, and serum bone turnover markers. (A-B) Density of osteoclasts (N.Oc.) and osteoblasts (N.Ob.), normalized to bone surface (BS), of distal radial metaphyseal trabeculae. (C) Bone mineral density (BMD) of radial metaphyseal trabeculae. (D-F) Serum levels of Trap 5b, CTX1 and osteocalcin, assayed using ELISA. Symbols: *:p<0.05 and **:p<0.01, compared to FRC rats; &&:p<0.01, compared to untreated HRHF rats.

Fig 6

Mean voluntary pulling force, muscle cross sectional area, and bone cytokine levels (serum cytokine levels are provided in the text). (A) Mean voluntary pulling force on the lever bar used for the reaching and grasping task, in grams. Arrow indicates onset of ibuprofen treatment. (B) Mean cross-sectional area of myofibers in flexor digitorum muscle, quantified at mid-muscle level. (C&D) IL-1beta and TNF-alpha in forelimb bones, tested using ELISA. Symbols: *:p<0.05 and **:p<0.01, compared to FRC rats; &:p<0.05, compared to untreated HRHF rats.

Fig 7

Changes in the median nerve in the carpal tunnel. (A) Nerve conduction velocity (NCV) in meters/second (m/sec), measured electrophysiologically. (B) Mean number of ED1+ in media nerve at the level of the wrist. (C-E) Representative microscopic photos showing width of epineurial connective tissues (CT) located around median nerve (N) at wrist level (double arrows). Arrowheads indicate ED1+ macrophages (detected immunohistochemically). Eosin counterstain. Each photo in C-E was taken at same magnification; thus, the scale bar is the same for panels C through E. Symbols: *:p<0.05 and **:p<0.01, compared to FRC rats; &:p<0.05 and &&:p<0.01, compared to untreated HRHF rats.