Microfil mv 122

Blood vessels in EP sites were imaged by angiography of microphil-perfused bones. In detail, the thoracic cavity of mice was opened after anesthesia, and the inferior vena cava was severed. The vascular system was flushed with PBS containing heparin sodium (100 U/ml) through a needle inserted into the left ventricle. The specimens were then pressure fixed with 10% neutral buffered formalin which was washed off from the vessels by heparinized saline solution. A radio paque silicone rubber compound containing lead chromate (Microfil MV-122, Flow Tech) was injected to label the vasculature. Samples were stored at 4 °C overnight for contrast agent polymerization. Mouse C_8–9 along with EPs were dissected and decalcified in 10% EDTA after fixation in 10% neutral buffered formalin for 24 h. Images were obtained using a μCT imaging system (Skyscan 1172).

Mice were sacrificed at 2–3 weeks of age to evaluate the coronary artery and vein morphology. Mice were euthanized by CO₂ inhalation, and the thoracic cavity was opened surgically. The large branches from the aorta were ligated. The vasculature was flushed with normal saline containing heparin (200 U ml⁻¹) via a needle inserted into the descending aorta until heart became visibly blanched. The heart was then pressure-fixed with 2% paraformaldehyde. Paraformaldehyde was flushed from the heart in heparinized saline, and coronary vasculature was injected with a radiopaque silicone rubber compound (Microfil MV-122; Flow Tech, Carver, MA) solution prepared in a volume ratio of 1:1 of Microfil diluent with 5% curing agent. Once filling is complete, to prevent the Microfil leakage from the coronary vessels, the accessible major vascular exit points were ligated immediately after filling. Heart was stored at 4 °C for contrast agent polymerization.

The vasculature of SD rats was injected with 20 mL of silicone rubber compound (Microfil MV-122, Flow Tech, Carver, MA) after they were euthanized at 12 weeks post-operation 34 (link). Briefly, the animals were anesthetized and the rib cage was opened. The descending aorta was clamped and the auricula dextra was incised. Heparinized saline and Microfil were successively perfused into the left ventricle with an angiocatheter. Successful perfusion was defined as a yellow color change in the eyes and tongue. Finally, the rats were stored at 4 °C overnight to ensure plasticization of the contrast medium, after which the crania were dissected and fixed in 4% paraformaldehyde for another 48 h. The fixed crania were decalcified in 10% ethylenediaminetetraacetic acid (EDTA; Sigma, US) for four weeks. Images were obtained with a high-resolution micro-CT imaging system at 9 µm resolution, and the number and volume of vessels within the 5 mm diameter region surrounding the bone defect were evaluated.

Mice were killed and perfused with Microfil (Microfil MV-122; Flow Tech, Carver, MA, USA). Then, the samples were analyzed by micro-CT (Skycan 1176; Burker), and three-dimensional images were reconstructed with the CTVol program (Bruker). The number of blood vessels was determined with the ImageJ software.

Directly after euthanasia the abdominal cavity of the animal was opened to expose the abdominal aorta and the junction into the arteria communis of the right leg. This vessel was cannulated and was flushed first with 40 mL heparinized saline solution (100 U/mL in 0.9% NaCl) and then with the silicone rubber polymer containing lead chromate contrast agent (Microfil MV 122, Flow Tech, Massachusetts, USA). The mixing ratio of the contrast agent compounds was 5 mL diluent, 5 mL compound and 1 mL curing agent. The contrast agent was allowed to polymerize for 3 h at 4°C inside the animal, followed by fixation of the specimens in 4% Paraformaldehyde (PFA, Science Services, Munich, Germany) for 24 h at 4°C. The specimens were then stored at 4°C in neutral buffered PBS.

After the mice were euthanized, right atrium was broken with microscissors. Then the blood circulation system was lavaged with 0.9% warm normal saline solution containing heparin sodium (100U ml⁻¹) by a needle inserted into the left ventricle. Thereafter, we pressure 10% neutral buffered formalin to fixed the specimen and washed with heparinized saline solution. In the same channel, radiocontrast agent (Microfil MV-122, Flow Tech) was then injected and the specimens were stored at 4 °C overnight. The knee joint of the mice was harvested and soaked in 10% neutral buffered formalin for 5 days. After that, decalcification was performed with a formic acid-based solution (Cal-Ex II) for 72 h to minimize the influence of surrounding tissues. The specimens were also scanned using high-resolution micro-CT (SkyScan 1176) with the resolution of 9 μm isotropic voxel size.