Lunar piximus

Changes in body composition were determined by dual-energy X-ray absorptiometry (DEXA) using a densitometer (Lunar PIXImus; GE Medical Systems, Madison, WI, USA). This system employs a cone beam X-ray source generating energies of 35 and 80 keV and a flat 100 × 80 mm detector with individual pixel dimensions of 0.18 × 0.18 mm. The ratio of energy attenuation in the luminescent panel separates bone, lean tissue, and fat mass. A quality control procedure was routinely performed with a calibration phantom before imaging. Mice were anesthetized by intraperitoneal (IP) injection of 100 mg/kg ketamine and 10 mg/kg xylazine (Cristália, SP, Brazil) to ensure good immobilization and positioning during a five-minute acquisition [27 (link)]. Analyses were performed at baseline and at the end of the 24th week of diet, and the results were expressed as a percentage of body fat.

Mice were subjected to Dual Energy X-ray Absorptiometry (DEXA; Lunar PIXImus; General Electric, Boston, MA, USA) and images were analyzed to assess lean body mass (LM) [34 (link)].

Dual-energy x-ray absorptiometry (DXA, GE Lunar PixiMus, Madison, WI) was performed on excised right femora and L4 vertebrae for measures of areal bone mineral density (aBMD g/cm²), bone mineral content (BMC, g) and area (cm²). Peripheral quantitative computed tomography (pQCT, XCT Research SA+, Stratec Medizintechnik GmbH, Pforzheim, Germany) was performed on the right femur midshaft for cortical bone morphometric properties (volumetric BMD (vBMD), BMC, cortical area and thickness, periostal and endosteal circumferences and x-axis cross-sectional moment of inertia). Micro-computed tomography (µCT, Brucker Skyscan 1172, Kontich, Belgium) was performed on L4 vertebral bodies and the right distal femur for cancellous bone morphometric properties. Scans were done at 8 µm resolution, 65 kV and 120 µA using a 0.7° rotation step. Reconstructed µCT images (NRecon software) were analyzed using CT Analyzer software (Skyscan, Kontich, Belgium). The same parameters/thresholds were used for each site for reconstruction and analysis. Outcome measurements included whole vertebral body bone volume (BV, mm³), trabecular bone volume fraction (BV/TV,% [where TV is tissue volume]), trabecular number (Tb.N, mm⁻¹), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm), connectivity density (Conn.D, mm⁻³), and structural model index (SMI).

This study was approved by the Institutional Animal Care and Use Committee at the University of Wisconsin, Madison. Male B6C3F1 hybrid mice were housed under controlled pathogen‐free conditions. Mice were randomized into control or restricted groups at 2 months of age and fed 87 kcal week⁻¹ (Bio‐Serv diet #F05312), which is ~95% of ad libitum intake, or 73 kcal week⁻¹, which is a 23% reduction in calorie intake from ad libitum levels and 16% reduction from controls (Bio‐Serv diet #F05314). Total daily intake for CR mice was proportionately lower in carbohydrates than controls (20% lower) with the difference made up in equivalent proportional increases in fat and protein contents (Table S6, Supporting information). Fatty acid composition was identical for both diets. Mice were individually housed to ensure consumption of all food and so that precise caloric intake could be known. Body composition was determined on anesthetized mice using dual‐energy X‐ray absorptiometry (GE Lunar Piximus) 2 weeks prior tissue harvest. A list of all measured parameters for each mouse is provided in Table S7 (Supporting information).

Mice were anesthetized with ketamine/xylazine, and a dual-energy X-ray absorptiometry (DXA) scan was performed as described previously [41 (link)] before (mice aged 8 weeks) and after the administration of the diet for 3 months (mice aged 20 weeks) using a Lunar PIXImus mouse bone densitometer (General Electric). BMD was analyzed manually with PIXImus software as described previously [41 (link)].