Radiology and serum biochemistry on Sam68+/+ and Sam68−/− mice.
Radiography, BMD, and micro-CT were performed essentially as described previously [63]. Mice were administered a lethal dose of anesthetic at the indicated times, exsanguinated, and imaged using a Faxitron MX20 equipped with an FPX-2 Imaging system (Dalsa Medoptics, Waterloo, Ontario, Canada). Body fat, BMC, and BMD were evaluated using a Lunar PixiMUS 1.46 (GE-Lunar, Madison, Wisconsin, United States). Morphometric parameters were determined on anesthetized mice at the time of sacrifice by direct measurement or from the Faxitron radiograph.
Micro-CT was performed on the left femur and fourth lumbar vertebra after removal of soft tissues and overnight fixation in 4% paraformaldehyde. The distal metaphysis was scanned with a Skyscan 1072 micro-CT instrument (Skyscan, Antwerp, Belgium). Image acquisition was performed at 100 kV and 98 μA, with a 0.9° rotation between frames. The two-dimensional images were used to generate three-dimensional reconstructions to obtain quantitative data with the 3D Creator software supplied with the instrument.
Serum biochemistry (ALP, Ca, PO4, Mg) was determined at the Rodent Diagnostics Lab (McGill University, Montréal, Quebec, Canada) using routine automated techniques. Commercial assays were used to determine serum levels of CTX (RatLaps ELISA, Nordic Bioscience), 17β estradiol (IBL Immuno-Biological, Hamburg, Germany), leptin (R & D Systems, Minneapolis, Minnesota, United States), and interleukin-6 (R & D Systems).