|Mice are euthanized
||Left femurs are harvested and fixed
||Femurs are analyzed via µCT
||Parameters determined in the metaphyseal trabecular bone
- µCT System (µCT 50, Scanco Medical AG, Switzerland)
- 5% isoflurane
- 4% paraformaldehyde
- 70% ethanol
Procedure: Femur preparation and µCT evaluation
- Mice are intraperitoneally euthanized with cervical dislocation performed approximately one min after breathing stops owing to 5% isoflurane inhalation.
- Left femurs are harvested and fixed for 24h in 4% paraformaldehyde solution, and then stored in 70% ethanol.
- Whole femurs are examined by a µCT system as previously described (Hiram-Bab et al., 2015).
- Scans are performed at a 10 µm resolution in all three spatial dimensions.
- Mineralized tissues are differentially segmented by a global thresholding procedure as described by Ruegsegger et al, 1996.
- All morphometric parameters are determined by a direct 3D approach as described by Hildebrand et al., 1999.
- Parameters analyzed are determined in the metaphyseal trabecular bone.
Hildebrand T, Laib A, Muller R, Dequeker J, Ruegsegger P. Direct three-dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus. J Bone Miner Res. 1999. 14:1167-74.
Hiram-Bab S, Liron T, Deshet-Unger N, Mittleman M, Gassmann M, Rauner M, et al., Erythropoietin directly stimulates osteoclast precursors and induces bone loss. FASEB J. 2015. 29:1890-900.
Ruegsegger P, Koller B, Muller R. A microtomographic system for the nondestructive evaluation of bone architecture. Calcif Tissue Int. 1996. 58:24-9.
- Femur length
- Trabecular bone volume fraction
- Trabecular thickness
- Trabecular number
- Trabecular connectivity density