The protocol for non-invasively loading the

mouse tibia has been reported previously [5], [8] and [12]. In brief, the flexed knee and ankle joints are positioned in concave cups; the upper cup, containing the knee, is attached to an actuator arm and the lower cup to a dynamic load cell. The tibia is held in place Ruxolitinib datasheet by a 0.5 N continuous static pre-load. In this study, 40 cycles of dynamic load were superimposed with 10 s rest intervals between each cycle. The protocol for one cycle consisted of loading to the target peak load, hold for 0.05 s at the peak load, and unloading back to the 0.5 N pre-load. From the strain gage data (see “ex vivo strain measurements”), peak loads of 13.3 N for males and 13.0 N for females were required to engender 2200 με on the medial surface of the tibia. Strain rate at this site was normalized to a maximum of 30,000 μεs− 1 by applying the load at rates of 460 N/s in males and 450 N/s in females. Following sacrifice, lower legs were stored in 70% ethanol and whole tibiae imaged using the SkyScan 1172 (SkyScan, Kontich, Belgium) with a voxel size of 4.8 μm (110 μm3). The scanning, reconstruction and method of analysis has been previously reported [8] and [14]. We evaluated the effect of housing and sex on both tibiae and changes [(right–left)/left] due to loading in bone volume fraction (BV/TV), trabecular selleck kinase inhibitor thickness (Tb.Th), trabecular

separation (Tb.Sp) and trabecular number (Tb.N) in the trabecular region (0.25–0.75 mm distal to the proximal physis) and cortical bone area (Ct.Ar), total cross-sectional area inside the periosteal envelope (Tt.Ar), medullary area (Ma.Ar), cortical area fraction (Ct.Ar/Tt.Ar),

cortical thickness (Ct.Th) and polar moment of inertia (J), a parameter of structural bone strength, at the cortical site (37% from the proximal end), according to ASBMR guidelines Cobimetinib molecular weight [15]. Three days after the final anesthesia/loading session, animals were euthanized by asphyxiation with carbon dioxide prior to cardiac puncture to minimize changes in corticosterone. Serum was separated by centrifugation and stored at − 80 °C until the time of analysis. Serum testosterone was measured using a competitive binding assay kit (R&D systems, MN) following manufacturers’ instructions. Serum corticosterone was assayed using a competitive radioimmunoassay (Cort RIA, Izoto, Hungary) as previously described [16]. The effect of housing, sex and their interaction on each bone parameter was assessed using a two-way ANOVA with interaction. When interactions were found to be significant, post-hoc t-tests were used for pair-wise comparisons to further examine the effect of housing within each sex. The effect of loading was assessed using paired samples t-tests. Differences in fighting and serum hormones were assessed using independent samples t-tests. Significance was set at p < 0.05. Analyses were performed using SPSS (version 18.0; SPSS Inc., Chicago, USA).