形態学/組織学的変化: in in vivo tests: bone mineral density (every 2 weeks during exposure; DXA); trabecular bone volume, thickness, spacing and number in femur (micro computed tomography); histology in femur slices (cell densities of osteoblasts and tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts; hematoxylin-eosin stain, TRAP stain, light microscopy); histomorphometry in tibia slices (calcein green and tetracycline hydrochloride injections prior to sacrifice, van Gilson stain; light microscopy and fluorescence microscopy); in in vitro tests: length of primary cilium (immunofluorescence staining of acetylated α-tubulin and cell nuclei, confocal laser scanning microscopy)
in in vivo tests: bone turnover (concentrations of osteocalcin (bone formation marker) and C-terminal crosslinked telopeptides of type I collagen (bone resorption marker) in blood (commercial kits))
分子生合成: in in vivo tests: gene expression of 89 osteoporosis-related genes in femur (PCR Array Rat Osteoporosis kit); in in vitro tests: activation of Wnt signal pathway (TCF/LEF1 luciferase reporter assay; commercial kit); gene expression and protein expression of signal pathway genes Wnt3a, Wnt10b, Dickkopf1 (DKK1), Disheveled (Dsh), Frizzled and LRP6 (only protein expression), β-catenin, GSK3β, COL1α1, BMP-2, RUNX2 and OSX (quantitative real-time RT-PCR and Western Blot as well as immunofluorescence, confocal laser scanning microscopy, respectively)
細胞生存力／細胞分裂/増殖: in in vitro tests: osteogenic differentiation of rat calvarial osteoblasts (enzyme activity of alkaline phosphatase (method not explained) and histochemical stain of alkaline phosphatase (fast blue RR and 1-naphthyl phosphate sodium stain; automated image analyser)); mineralization of rat calvarial osteoblasts (Alizarin Red S stain; automated image analyzer)
