Skyscan 1176

Mouse hind limbs and skulls were harvested, soft tissues were removed, and the remaining tissues were stored in 70% ethanol. The femurs of 4-week-old Osx^Cre;Stat3^fl/fl mice and control littermates were scanned with a SkyScan1176 (Bruker, Kartuizersweg, Belgium) at a 8.96 μm resolution for quantitative analysis. The turn on voltage was 65 kV and current density was 300 μA. The region from −40 to −240 slices below the growth plate was analyzed for BV/TV, Tb.Th., Tb.N., and Tb.Sp. The region from −400 to −456 slices below the growth plate was analyzed for Ct.Th. Skulls of 4-week-old Osx^Cre;Stat3^fl/fl mice and control littermates were used for Micro-CT analysis (SkyScan 1176, Bruker, Kartuizersweg, Belgium) with 9 μm voxel size. The turn on voltage was 60 kV and current density was 280 μA Micro-CT analysis of the femurs of 6-week-old Col1^{cre ERT2};Stat3^fl/fl and 9-week-old mice that had been injected with AG490 for 5 weeks, 5-week-old tail-suspended mice injected with colivelin or PBS and their corresponding WT littermates were scanned with a Quantum GX2 (PerkinElmer, Waltham, USA) instrument. The turn on voltage was 80 kV and current density was 88 μA, and 101 slices (9 μm each) immediately below the growth plate in the distal metaphysis of the femur were used for quantification of the bone parameters.

BBB permeability assay: after being anesthetized by intraperitoneal injection of urethane (1.8 g/kg), rats were intravenously injected with Cy5-Dextran (10 kDa, Stargraydye, China). One hour later, after transcardial perfusion with 300 ml of PBS, the whole brain tissue was removed for observation under a 646 nm excitation wavelength exposed by a small animal live imaging system (SkyScan 1176, Bruker, Belgium) and photographed.
Detection of supplemental acetate in the brain: after being anesthetized by intraperitoneal injection of urethane (1.8 g/kg), rats were given an intravenous injection of Sulfo-Cyanine5-labelled acetate (Cy5-acetate, provided by Xi’an Rui Xi Biotechnology Co., Ltd., China). Thirty minutes later, the rats were transcardially perfused with 300 ml of PBS to flush out the Cy5-acetate from the blood vessels. Then the rats were exposed to a 646 nm excitation wavelength using a small animal live imaging system (SkyScan 1176, Bruker) and photographed.

After removing the intramedullary pin, whole right tibias with callus were dissected from the attached muscle and stored in 10% neutral formalin for 48 h. Samples of 28 d post fracture were scanned using a high-resolution micro-CT (SkyScan1176 Software: Version1.1 (build 6), Bruker, Kontich, Belgium) [17 (link)]. The region of interest of each callus was the area 2 mm above and below the fracture line [18 (link)]. To better describe our findings, we used the coronal plane through the intramedullary pin to divide the callus into anterior and posterior parts (Fig. 3) and calculated architectural parameters in the anterior site, posterior site, and total callus respectively, as previously described [16 (link)].Fig. 3

Position of anterior and posterior callus. a Cross plane. b Sagittal plane

The right proximal tibia was repeatedly imaged (in the control and at 17 and 28 days) using in vivo micro-CT (SkyScan 1176, Bruker, Kontich, Belgium) with a voxel size of 17.5 μm. The imaging was conducted under ketamine-xylazine anesthesia, following previously established protocols (average anesthesia duration: 30–40 min, scan duration: 7–10 min). Bone structural changes were assessed using CT Analyser^® software (CT Analyzer 5.30 Software, OMICRON, Vienna, Austria), employing regions of interest (ROIs) of identical sizes around the tibia. ROIs of standard dimensions were delineated around the respective regions to calculate bone volume.