Ca2l9100

ENT1^−/− mice were scanned following each time point of behavioral testing (2.5, 4.5, and 6.5 months of age) in order to correlate behavioral changes to radiographic features of spine calcification. µCT imaging was performed using a cone-beam imaging system (eXplore SpeCZT scanner, GE Healthcare Biosciences: London, CAN), as previously reported [23 (link)]. Mice were anesthetized for µCT scanning using two to three percent inhaled isoflurane (CA2L9100, Baxter: Mississauga, CAN) infused with oxygen at a flow rate of 1.0 mL/min. To maintain sedation, a nose cone apparatus was used to administer 1.75% inhaled isoflurane for 15 min while scanning was performed.
Quantitative analysis of vertebral cortical bone (three mice each time point) created a radiodensity range for mineralized tissue (289 to 1278 HU). The cervical-thoracic spine and sternocostal regions were manually segmented for analysis of hyperdense calcifications, defined as volumes exceeding the radiodensity of cortical bone (≥ 1278 HU) within these anatomical structures. Imaging data were analyzed using MicroView (Version 2.5.0–4118, Parallax Innovations Inc.: Ilderton, CAN) and VGStudio MAX (Version 2.0.4, Volume Graphics GmbH: Heidelberg, DEU).

Forty-eight hours before sacrifice, μCT imaging was performed using a cone-beam imaging system (eXplore SpeCZT scanner, GE Healthcare Biosciences: London, CAN). For imaging, mice were anesthetized using 2–3% inhaled isoflurane (CA2L9100, Baxter: Mississauga, CAN) infused with oxygen at a flow rate of 1.0 mL/min. To maintain sedation, a nose cone apparatus was used to administer 1.75% inhaled isoflurane for 20 min while scanning was performed. During a single 5 min rotation of the gantry, 900 X-ray projections were acquired (peak voltage of 90 kVp, peak tube current of 40 mA, and integration time of 16 ms). A calibrating phantom composed of air, water, and cortical bone-mimicking epoxy (SB3; Gammex, Middleton WI, USA) was included in each scan. Data were reconstructed into 3D volumes with an isotropic voxel spacing of 50 μm and scaled into Hounsfield units (HU). Using MicroView software (GE Healthcare Biosciences) three signal-intensity thresholds (− 200, − 30, and 190 HU) were used to classify each voxel as adipose, lean, or skeletal tissue, respectively. Custom software was used to calculate tissue masses from assumed densities of 0.95 (adipose), 1.05 (lean), and 1.92 (skeletal) g/cm³, as previously reported [42 ].