2.3. Histomorphometry

The animals were killed by exsanguination under anesthesia. The left femur was excised, and the surrounding soft tissue was cleaned off. After fixation in 70% ethanol at 4 °C, femurs were trimmed and the bone distal halves were post-fixed in 70% ethanol, dehydrated in xylene at 4 °C, and embedded without demineralization in methyl methacrylate. Histomorphometric parameters were measured as recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (Dempster et al., 2013; Parfitt et al., 1987) on 5-mm thick sections using a Nikon microscope interfaced with a software package from Microvision Instruments. For tartrate-resistant acid phosphatase (TRAP) detection, sections were stained with 50 mM sodium tartrate and naphthol AS-TR phosphate (Sigma-Aldrich). Measurements of the trabecular bone were performed in a region of the secondary spongiosa. OC number and bone surface were measured according to Parfitt's nomenclature. We also counted the number of nuclei per OC.