Joint inflammation and early degeneration induced by high-force reaching are attenuated by ibuprofen in an animal model of work-related musculoskeletal disorder

Abstract

We used our voluntary rat model of reaching and grasping to study the effect of performing a high-repetition and high-force (HRHF) task for 12 weeks on wrist joints. We also studied the effectiveness of ibuprofen, administered in the last 8 weeks, in attenuating HRHF-induced changes in these joints. With HRHF task performance, ED1+ and COX2+ cells were present in subchondral radius, carpal bones and synovium; IL-1alpha and TNF-alpha increased in distal radius/ulna/carpal bones; chondrocytes stained with Terminal deoxynucleotidyl Transferase- (TDT-) mediated dUTP-biotin nick end-labeling (TUNEL) increased in wrist articular cartilages; superficial structural changes (e.g., pannus) and reduced proteoglycan staining were observed in wrist articular cartilages. These changes were not present in normal controls or ibuprofen treated rats, although IL-1alpha was increased in reach limbs of trained controls. HRHF-induced increases in serum C1,2C (a biomarker of collagen I and II degradation), and the ratio of collagen degradation to synthesis (C1,2C/CPII; the latter a biomarker of collage type II synthesis) were also attenuated by ibuprofen. Thus, ibuprofen treatment was effective in attenuating HRHF-induced inflammation and early articular cartilage degeneration.

Figure 1

Immunohistological staining cyclooxygenase 2 (COX 2) and ED1 in HRHF distal radiocarpal articular cartilage, synovium and ligaments of longitudinally sections of paraffin embedded bones. ED1 detects a 90 kDa lysosomal membrane glycoprotein in osteoclasts, phagocytic macrophages and their progenitors. (a)–(f) COX 2 staining was visualized using DAB intensified with cobalt (black signal); light hematoxylin and eosin ((h) and (e)) staining was used as a counterstain. (a) No COX 2 immunostaining was observed in distal radial articular cartilage and synovium of a normal control (NC). (b) Higher-power image of same section as shown in (a). (c) COX2+ cells are present in the articular cartilage and synovium of a 12-week high-repetition high-force (HRHF) rat's distal radius. (d) Higher-power image of same section as shown in (c). (e) COX2+ cells are present in the distal radial articular cartilage and radiocarpal ligament of a 12 week HRHF rat. (f) Higher-power image of same section as shown in (e). (g)–(i) Several COX2+ cells are present in the radiocarpal synovium of an HRHF 12 rat (red cells). (g) Same section double labeled for ED1 (green cells, (h)). (i) Merged photo. Arrow indicates a group of COX2+/ED1+ double labeled cells. (j) ED1+ cells (brown DAB signal) were absent in distal radius articular cartilage of an NC rat. Section is lightly counterstained with hematoxylin. (k) Mononucleated ED1+ cells (arrows, presumably osteoclast progenitor cells) were observed in the subchondral bone of the distal radius of an HRHF rat. (l) ED1+ cells were also present in HRHF carpal bones. Scale bars = 50 μm. b: bone, c: articular cartilage, lig: ligament, s: synovium.

Figure 2

Cytokine concentrations in reach and support wrist joint (distal most radius, distal ulna, first row of carpal bones, and associated joint regions), tested using ELISA. Levels of (a) Interleukin (IL)-1α, (b) tumor necrosis factor (TNF)-α, (c) IL-10, (d) IL-1β, and (e) IL-6 are shown for NC (normal control), NC + IBU (normal controls receiving ibuprofen for 8 weeks), TR (trained controls), TR + IBU (trained controls receiving ibuprofen treatment in final 8 weeks), HRHF (rats performing high-repetition high-force task for 12 weeks), and HRHF + IBU (HRHF rats receiving ibuprofen treatment in final 8 weeks). Abbreviations: 1 = P < .05 compared to NC, 2 = P < .05 compared to NC + IBU, 3 = P < .05 compared to TR; 4 = P < .05 compared to TR + IBU; 5 = P < .05 compared to HRHF + IBU; a = support limb is significantly different from reach limb, P < .05.

Figure 3

Distal radii and carpal bones stained with safranin O and fast green. (a)–(g) are longitudinal sections of paraffin embedded radius, ulna and carpal bones. (a)–(c) Low-power micrographs showing distal radius, scaphoid bones and articular cartilage in (a) NC (normal control), (b) TR (trained control), and (c) HRHF rat (performed the high-repetition high-force task for 12 weeks). (a) also indicates the 4 zones of the articular cartilage assessed in the radius. (d)–(g) Higher-power images showing the distal radii articular cartilage of (d) untreated TR, (e) TR + IBU (trained controls receiving ibuprofen treatment in final 8 weeks), (f) HRHF, and (g) HRHF + IBU rats. (f) demonstrates an HRHF animal with structural changes (surface pannus, arrows), cellular changes (hypocellularity in areas indicated with ∗), and dramatically reduced proteoglycan staining in the articular cartilage (red-pink safranin O staining). The inset in (f) is a higher-power micrograph from another HRHF rat showing chondrocytes that appear to be proliferating. The inset in (g) shows a higher-power micrograph from area indicated with arrow that also contains proliferating chondrocytes. (h)-(i) Loss of safranin O staining in HRHF articular cartilage was verified in plastic embedded bones that were also cut longitudinally. Radii and scaphoid articular cartilages of (h) HRHF and (i) HRHF + IBU are shown. Scale bars = 50 μm. B: bone, S: scaphoid bone.

Figure 4

Histopathological Mankin scores shown for distal radius articular cartilage of the reach limb in NC (normal control), NC + IBU (normal controls receiving ibuprofen for 8 weeks), TR (trained controls), TR + IBU (trained controls receiving ibuprofen treatment in final 8 weeks), HRHF (rats performing high-repetition high-force task for 12 weeks), and HRHF + IBU (HRHF rats receiving ibuprofen treatment in final 8 weeks). (a) Total scores for zones 2 and 3 are presented separately by group. (b) Mean of total scores for both zones 2 and 3. (c) Mankin score for structural changes in zone 2. (d) Mankin score for structural changes in zone 3. (e) Mankin score for cellular changes in zone 2. (f) Mankin score for cellular changes in zone 3. (g) Mankin score for staining changes in zone 2. (h) Mankin score for staining changes in zone 3. Abbreviations: 1 and 2 = P < .05 and P < .01, respectively, compared to NC. 3 and 4 = P < .05 and P < .01, respectively, compared to TR; 5 and 6 = P < .05 and P < .01, respectively, compared to TR + IBU; 7 and 8 = P < .05 and P < .01, respectively, compared to HRHF + IBU. 2* = P < .001 when comparing zones 2 and 3 (zone 2 is higher). Statistical results compared to NC + IBU are not presented as they were comparable to NC results.

Figure 5

Distal radii and carpal bones stained with TUNEL (longitudinal sections of paraffin embedded radius and carpal bones). (a)–(f) Micrographs showing distal radius articular cartilage (indicated as c, for cartilage) in (a) NC (normal control), (b) TR (trained control), and (c) HRHF + IBU (performed the high-repetition high-force task for 12 weeks and received ibuprofen treatment daily in last 8 weeks), (d)-(e) HRHF rat (12 weeks HRHF rat with no treatment). (d) is a lower-power photo showing medial edge of radius and increased TUNEL stained cells at that edge as well as stained cells in adjacent articular cartilage. Inset shows higher-power image of indicated cell. (e) is a higher-power photo of same section shown in (d). (f) shows the radial articular cartilage from a different rat than shown in (d) and (e). (g) Image showing the lateral edge of the radius of an NC rat at the site of the attachment of the radiocarpal ligament. (h) Image showing the lateral edge of the radius of an HRHF rat at the site of the attachment of the radiocarpal ligament. (i) Higher-power photo of same cell as indicated in (f) with an arrow. (j) Higher-power photo of same cell as indicated in (h) with an arrow. Scale bars = 50 μm. b: bone, c: articular cartilage, lig: radiocarpal ligament, s: synovial ligament.

Figure 6

Carpal bone articular cartilages (intracarpal joints) and epiphyseal plates from longitudinal sections of paraffin embedded bone that were stained with safranin O and fast green. (a)–(c) Carpal bone articular cartilages in (a) TR (trained controls), (b) HRHF (rats performing high-repetition high-force task for 12 weeks), and (c) HRHF + IBU (HRHF rats receiving ibuprofen treatment in final 8 weeks). Reduced safranin O staining and a paucity of cells in lower cartilage layers are present in an HRHF rat, compared qualitatively to TR and HRHF + IBU. Chondrocytes that appear to be proliferating are present in the HRHF carpal articular cartilage (arrows). (d)-(e) Distal epiphyseal plate of the radius showing (d) an HRHF rat with a near absence of safranin O staining (arrowheads) and an irregularly shaped epiphyseal plate, as compared to (e) HRHF + IBU rat with an intensely stained and thicker epiphyseal plate. Scale bars = 50 μm. epi: epiphyseal plate.

Figure 7

Serum concentrations of collagen degradation and synthesis markers, and the ratio of collagen degradation to synthesis in NC (normal control), NC + IBU (normal controls receiving ibuprofen for 8 weeks), TR (trained controls), TR + IBU (trained controls receiving ibuprofen treatment in final 8 weeks), HRHF (rats performing high-repetition high-force task for 12 weeks), and HRHF + IBU (HRHF rats receiving ibuprofen treatment in final 8 weeks). (a) C1, 2C, a marker of collagen Type I and II cleavage (degradation). (b) CPII, a marker of procollagen II C-propeptide synthesis. (c) Ratio of C1, C2 (degradation) to CPII (synthesis). 1 = P < .05 and 2 = P < .01 compared to NC; 3 = P < .01 compared to NC + IBU; 4 = P < .05 compared to TR; 5 = P < .05 compared to TR + IBU; 6 = P < .01 compared to HRHF + IBU.