Effects of strength training and raloxifene on femoral neck metabolism and microarchitecture of aging female Wistar rats

Abstract

The aim of this study was to prevent female osteoporosis using strength training (ST), raloxifene (Ral) or a combination of ST plus Ral during the natural female aging process, specifically in the periestropause period. For a total of 120 days, aging female Wistar rats at 18-21 months of age performed ST on a ladder three times per week, and Ral was administered daily by gavage (1 mg/kg/day). Bone microarchitecture, areal bone mineral density, bone strength of the femoral neck, immunohistochemistry, osteoclast and osteoblast surface were assessed. We found that the treatments modulate the bone remodeling cycle in different ways. Both ST and Ral treatment resulted in improved bone microarchitecture in the femoral neck of rats in late periestropause. However, only ST improved cortical microarchitecture and bone strength in the femoral neck. Thus, we suggest that performing ST during the late period of periestropause is a valid intervention to prevent age-associated osteoporosis in females.

Figure 1

Plasma bone biomarkers in aged female Wistar rats after ST realization, Ral treatment or association of ST and Ral. (A) TRAP, (B) ALP and (C) summary of p values in two way ANOVA analysis, n = 8–10 animal/group. Abbreviations and symbols: +main effect of Ral, *interaction of ST plus Ral. TRAP = tartrate-resistant acid phosphatase and, ALP = alkaline phosphates, ST = strength training, Ral = raloxifene, NT-Veh = non-trained and treated with vehicle; NT-Ral = non-trained and treated with raloxifene; ST-Veh = strength training and treated with vehicle; ST-Ral = strength training and treated with raloxifene.

Figure 2

Characterization of 3D morphometric analysis. (A) Scanned image of proximal epiphysis, with dimensions of the region of interest (femoral neck) indicated. The region of interest in the trabecular femoral neck is marked by crosshatched and the cortical femoral neck is marked by*. The 3D images show a typical example of trabecular and cortical bone in the femoral neck of animals that did not undergo strength training (NT-Veh – B and F), animals who did not undergo strength training but received raloxifene (NT-Ral – C and G), animals who underwent strength training (ST-Veh – D and H) and animals who underwent strength training and received raloxifene (ST-Ral – E and I).

Figure 3

Ex vivo trabecular bone microarchitecture. (A) BV/TV, (B) Tb.Th, (C) Tb.N, (D) Conn.Dn, (E) Tb.Sp, (F) SMI, (G) summary of p values in two way ANOVA analysis, n = 7 animals/group. Data of the femoral neck assessed by microCT. Each column represents mean ± standard error of the mean (SEM). Statistical analysis was performed with two-way ANOVA, followed by Tukey post-hoc testing (p < 0.05) to analyze the effect of strength training (ST) and raloxifene (Ral) treatment, and any interactions (ST*Ral). Abbreviations and symbols: +main effect of Ral, #main effect of ST, *interaction of ST plus Ral^a, vs NT-Veh; ^bvs NT-Ral; ^cvs ST-Veh. NT-Veh = non-trained and treated with vehicle; NT-Ral = non-trained and treated with raloxifene; ST-Veh = strength training and treated with vehicle; ST-Ral = strength training and treated with raloxifene; BV/TV = bone volume fraction; Tb.Th = trabecular thickness; Tb.N = trabecular number; Conn.Dn = connectivity density; Tb.Sp = trabecular separation; SMI = structure model index.

Figure 4

Ex vivo cortical bone microarchitecture. Data of the femoral neck assessed by microCT. (A) Ct.Ar, (B) Ct.Th, (C) Imax, (D) Imin, (E) J, (F) summary of p values in two way ANOVA analysis, n = 7 animals/group. Each column represents mean ± standard error of the mean (SEM). Statistical analysis was performed with two-way ANOVA, followed by Tukey post-hoc testing (p < 0.05) to analyze the effect of strength training (ST) and raloxifene (Ral) treatment, and any interactions (ST*Ral). Abbreviations and symbols: +main effect of Ral, #main effect of ST, *interaction of ST plus Ral^a, vs NT-Veh; ^bvs NT-Ral; ^cvs ST-Veh. NT-Ral = non-trained and treated with raloxifene; ST-Veh = strength training and treated with vehicle; ST-Ral = strength training and treated with raloxifene; Ct.Ar = cortical bone area; Ct.Th = average cortical thickness; Imax = maximum moment of inertia; Imin = minimum moment of inertia; J = polar moment of inertia.

Figure 5

Ex vivo areal bone mineral density (aBMD) and maximum load. (A) Data of the femoral neck assessed by DXA. (B) Ex vivo maximum load data of the femoral neck, data assessed by biomechanical compression bending testing. (C) Summary of p values in two way ANOVA analysis, n = 7 animals/group. Each column represents mean ± standard error of the mean (SEM). Statistical analysis was performed with two-way ANOVA, followed by Tukey post-hoc testing (p < 0.05) to analyze the effect of strength training (ST) and raloxifene (Ral) treatment, and any interactions (ST*Ral). Abbreviations and symbols: + main effect of Ral, # main effect of ST, *interaction of ST plus Ral^a, vs NT-Veh; ^bvs NT-Ral; ^cvs ST-Veh. NT-Veh = non-trained and treated with vehicle; NT-Ral = non-trained and treated with raloxifene; ST-Veh = strength training and treated with vehicle; ST-Ral = strength training and treated with raloxifene.

Figure 6

Immunolabeling for OCN, TRAP and SOST assessed by immunohistochemistry. (A–D) Photomicrographs showing osteoblasts OCN-positive (A), osteoclast TRAP-positive (B), osteocytes SOST-positive (C) and osteoclast SOST-positive. (E–J): Graphs showing the distribution of scores for OCN (E and H), TRAP (F and I) and SOST (G and J) in cortical and trabecular bone in the different experimental groups. (K) summary of p values in two way ANOVA analysis, n = 7 animals/group. Statistical analysis was performed with two-way ANOVA, followed by Tukey post-hoc testing (p < 0.05) to analyze the effect of strength training (ST) and raloxifene (Ral) treatment, and any interactions (ST*Ral). Abbreviations and symbols: +main effect of Ral, #main effect of ST, *interaction of ST plus Ral^a, vs NT-Veh; ^bvs NT-Ral; ^cvs ST-Veh. OCN = osteocalcin, TRAP = tartrate-resistant acid phosphatase and SOST = sclerostin. NT-Veh = non-trained and treated with vehicle; NT-Ral = non-trained and treated with raloxifene; ST-Veh = strength training and treated with vehicle; ST-Ral = strength training and treated with raloxifene.

Figure 7

Osteoclast perimeter/Trabecular perimeter (Oc.Pm/Tb.Pm) and Osteoblast perimeter/Trabecular perimeter (Ob.Pm/Tb.Pm) assessed in TRAP positive cells by immunohistochemistry counterstaining with hematoxilin and eosin, analyzed by Image J. (A) Oc.Pm/Tb.Pm (%), (B) Ob.Pm/Tb.Pm (%), (C) summary of p values in two way ANOVA analysis, n = 7 animals/group. Abbreviations and symbols +main effect of Ral, *interaction of ST plus Ral. NT-Veh = non-trained and treated with vehicle; NT-Ral = non-trained and treated with raloxifene; ST-Veh = strength training and treated with vehicle; ST-Ral = strength training and treated with raloxifene.