Prolonged high force high repetition pulling induces osteocyte apoptosis and trabecular bone loss in distal radius, while low force high repetition pulling induces bone anabolism

Abstract

We have an operant rat model of upper extremity reaching and grasping in which we examined the impact of performing a high force high repetition (High-ForceHR) versus a low force low repetition (Low-ForceHR) task for 18weeks on the radius and ulna, compared to age-matched controls. High-ForceHR rats performed at 4 reaches/min and 50% of their maximum voluntary pulling force for 2h/day, 3days/week. Low-ForceHR rats performed at 6% maximum voluntary pulling force. High-ForceHR rats showed decreased trabecular bone volume in the distal metaphyseal radius, decreased anabolic indices in this same bone region (e.g., decreased osteoblasts and bone formation rate), and increased catabolic indices (e.g., microcracks, increased osteocyte apoptosis, secreted sclerostin, RANKL, and osteoclast numbers), compared to controls. Distal metaphyseal trabeculae in the ulna of High-ForceHR rats showed a non-significant decrease in bone volume, some catabolic indices (e.g., decreased trabecular numbers) yet also some anabolic indices (e.g., increased osteoblasts and trabecular thickness). In contrast, the mid-diaphyseal region of High-ForceHR rats' radial and ulnar bones showed few to no microarchitecture differences and no changes in apoptosis, sclerostin or RANKL levels, compared to controls. In further contrast, Low-ForceHR rats showed increased trabecular bone volume in the radius in the distal metaphysis and increased cortical bone area its mid-diaphysis. These changes were accompanied by increased anabolic indices, no microcracks or osteocyte apoptosis, and decreased RANKL in each region, compared to controls. Ulnar bones of Low-ForceHR rats also showed increased anabolic indices, although fewer than in the adjacent radius. Thus, prolonged performance of an upper extremity reaching and grasping task is loading-, region-, and bone-dependent, with high force loads at high repetition rates inducing region-specific increases in bone degradative changes that were most prominent in distal radial trabeculae, while low force task loads at high repetition rates induced adaptive bone responses.

Keywords: Mechanical loading; Osteoactivin; Osteoprotegerin; Overuse injury; RANK; RANKL; Sclerostin.

Figure 1

Design and microCT images. (A) Young adult (2.5 months at onset) Sprague-Dawley rats were randomly assigned as food-restricted control (C) rats, rats that performed a high force high repetition task (High-ForceHR) for 18 weeks, or rats that performed a low force high repetition task (Low-ForceHR) for 18 weeks. Numbers per group shown at right. (B) Regions of interest (ROI) in radius (R) and ulnar (U) analyzed using micro-computerized tomography. (C) Upper row: Representative transaxial 2D image slices of distal ulnar and radial metaphyses. Lower row: Representative longitudinal 3D images of the distal radius. The ulna is also shown for a HFHR rat.

Figure 2

Reach parameters in High-ForceHR (HFHR) and Low-ForceHR (LFLR) rats across weeks of task performance (n=12 and 15/gp, respectively). (A) Number of reaches per minute; the target was 4 reaches/minute (dashed line). (B) Mean reach force on the lever bar (grams force, gf). Target for HFHR rats was 130 grams of force (gf); target for LFLR rats was 17 gf. (C) Mean duration of grasping the lever bar (msec) per reach. The minimum duration required for a food reward was 90 msec. (D) Mean Reach Impulse (Nmsec), defined as mean reach force (N) multiplied by grasp duration (msec). (E) Percent Duty Cycle, defined as reaches/cycle multiplied by grasp duration (in seconds (s)) on the lever bar per reach, divided the cycle time (60 s), and then multiplied by 100. (F) Representative graph of force time history of one 30 minute session (1800 seconds) by a High-ForceHR rat, extracted from Force Lever program files using a custom-written MATLAB program. The vertical blue lines indicate a pull on the force lever bar; the horizontal red line indicates the target of 130 gf; the green lines indicate the minimum (110 gf) and maximum (150 gf) thresholds of force. Baseline as indicated. Mean ± SEM is shown; ** p<0.01, compared to LFLR rats.

Figure 3

Dynamic histomorphometry in distal radial metaphyseal trabeculae (n=6–12/gp) and serum biomarkers of bone formation and turnover (n=8–12/gp). (A) Mineral apposition rate (MAR). (B) Bone formation rate (% BFR/BS). (C) Calcein incorporation in C and LFLR rats was observed as double-labeled lines (arrowheads) in distal radial trabeculae, while calcein incorporation in HFHR rats was observed only as woven bone or single-labeled lines. (D) Serum levels of osteocalcin (a biomarker of bone formation). (E) Serum levels of CTX1 (a biomarker of bone degradation). Magnification 400X. Mean + SEM shown. * and **: p<0.05 and p<0.01, compared to control rats; † and ††: p<0.05 and p<0.01, compared to High-ForceHR rats.

Figure 4

Increased empty lacunae and TUNEL stained cells in distal radial metaphyseal trabeculae (n=4–7/gp). (A–C) Representative distal radial trabeculae stained with H&E showing disrupted organization and increased empty lacunae (arrows in B and C) in High-ForceHR (HFHR) rats, relative to a Control rat (C). (D) Percentage of empty lacunae to all lacunae. (E and F) Percentage of TUNEL positive cells as a ratio of all DAPI stained cells in radial metaphyseal trabeculae and mid-diaphyseal cortical bone. (G–L) TUNEL stained cells (green), DAPI stained cells (blue) and merged representative images in C and HFHR rats. Asterisks indicate an area in panels J–L that is enlarged in the insets, showing examples of cells double-labeled with both TUNEL and DAPI. Mean ± SEM shown. *: p<0.05, compared to C rats.

Figure 5

Microcracks in radius bones. (A) Small linear microcracks in a trabeculae adjacent to a resorptive space (RsSp). (B–D) Examples of microcracks in interstitial bone of the metaphyseal radius cortical shell. Panel D shows same crack as in panel C using orange fluorescence. (E) Crack density (Cr.Dn) quantification, n=3/gp. 400× magnification. Mean ± SEM shown. * and **: p<0.05 and 0.01, compared to same region in control rats.

Figure 6

Sclerostin expression in forelimb bones. (A) Sclerostin protein levels in forelimb bones assayed using ELISA (n=4–6/gp). Distal region included radial and ulnar metaphyses and epiphyses; mid-diaphyseal region included the radial and ulnar diaphyses. (B) Quantification of sclerostin immunoexpression in distal metaphyseal trabeculae of the radius and ulna (n=4–7/gp). (C and D) Representative low power images of sclerostin immunostaining (brown) in osteocytes and matrix (asterisks) of a control (C) and a High-ForceHR (HFHR) rat, respectively. (E and F) Representative higher power images showing sclerostin immunostaining in a C rat’s trabecular and mid-diaphyseal cortical bone, respectively. (G and H) Representative higher power images showing sclerostin immunostaining in a HFHR rat’s trabecular and mid-diaphyseal cortical bone, respectively. Small arrows indicate empty lacunae observed in HFHR 18W rats’ trabecular bone. Asterisks indicate an area in panel G enlarged in the inset. Bones in panels C–H were counterstained lightly with H&E. Mean + SEM shown. * p<0.05, compared to C rats.

Figure 7

RANKL, RANK and OPG analyses. (A–C) qPCR quantification of RANKL, RANK and OPG mRNA in forelimb bones (bones and regions as described in Figure 7 legend). Fold changes compared to housekeeping gene beta-actin are reported. (D) The ratio of RANKL mRNA levels to OPG mRNA levels. (E and F) RANKL protein levels in forelimb bones, assayed using ELISA and immunohistochemical methods (n=4–7/gp). (G and H) Representative images showing RANKL immunostaining (brown staining) in distal radial trabeculae of control (C) and High-ForceHR (HFHR) rats, respectively. Arrowheads indicate RANKL immunostained cells; asterisk indicates RANKL immunoreactive matrix. Sections were counterstained lightly with hematoxylin. Mean + SEM shown. Symbols as in Figure 3’s legend; n.s. = not significant.