Fig. 2

Parkin deficiency enhances the bone-resorption capacity leading to bone loss in vivo. A Femurs from WT (n = 5) and Parkin^−/− (n = 5) male mice were isolated at 8 weeks of age, fixed in 4% paraformaldehyde, and then examined by micro-CT imaging. Representative micro-CT images of the trabecular bone of the mouse femurs are shown. The histograms represent the three-dimensional structural parameters of the femurs. B–H Three-dimensional morphometric analysis of bone parameters (i.e., B bone mineral density; BMD); C bone volume per tissue volume (BV/TV), D trabecular thickness (Tb. Th), E trabecular number (Tb. N), F trabecular separation (Tb. Sp), G total porosity (%), and H structure model index (SMI). I Representative TRAP staining images from the femurs of 8-week-old WT and Parkin^−/− mice are shown (left), and the size of the TRAP⁺ OCs was measured using ImageJ software (right). Scale bar, 200 μm (left) and 100 μm (right). J CTX-1 protein levels in the plasma were determined by ELISA. K, L BMMs from WT or Parkin^−/− mice were K (upper) treated with RANKL for 3 days, fixed, and TRAP-stained, and also K (lower) plated onto bone slices and incubated with M-CSF and RANKL for 7 days. Scale bar, 100 μm. L (left) The numbers of TRAP-positive multinucleated cells (≥ 3 nuclei) were quantified under a light microscope. L (middle) The area of TRAP-positive OCs was evaluated by ImageJ software. Resorption pits were visualized by K (lower) staining with toluene blue, and L (right) resorption pit areas were then estimated. *P < 0.05, **P < 0.005, and ***P < 0.001 between the indicated groups. Data are represented as means ± SD from three independent experiments; P-values were calculated by Kruskal–Wallis or Tukey post hoc comparison tests