Fig. 4

Everolimus (EV) protects ovariectomized mice from bone loss. Female C57BL/6 mice were divided into sham (SHAM) and ovariectomized (OVX) groups and subdivided into control or 1 mg/kg/day EV treatment groups (eight to ten mice per group). Four weeks post-OVX, treatment with EV commenced for 4 weeks. Bone parameters of the femur were assessed by micro-computed tomography (μCT) (a), and bone parameters of the tibia were assessed by bone histomorphometry (b). Parameters assessed included bone mineral density (BMD), bone volume over total volume (BV/TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp). The number of osteoclasts per unit of bone surface (Oc.N/BS) was assessed by tartrate-resistant acid phosphatase (TRAP) staining (femur), and assessment of the double calcein labels (tibia) was used to determine the bone formation rate per unit of bone surface (BFR/BS). Representative μCT images are shown of the trabecular bone of the femur for the control OVX and EV OVX groups. Representative TRAP staining (with red arrowheads indicating osteoclasts) (original magnification × 40, scale bar 20 μm) and double calcein labels for these two groups are also provided (original magnification × 20, scale bar 100 μm). Data represent mean ± SD. Statistical analysis was performed by two-way analysis of variance for the effect of surgery, treatment, and the interaction of the two (surgery × treatment). Statistical significance of multiple comparisons are denoted (* p < 0.05, ** p < 0.01, *** p < 0.001). Equal volumes of dimethyl sulfoxide used to prepare and administer EV concentrations were used in all control conditions. HA Hydroxyapatite