Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

It is possible to take CT images of blood vessels in the lower limbs and the abdominal organs using step protocol. The method enables the evaluation of the entire blood vessels comprehensively. In addition, it is possible to evaluate only arteries or at the same time, arteries and veins.

It is very important to prevent blood clots, leaked or blood well, and avoid air bubbles. Before the operation, inject 1 unit/gram heparin in PBS into the intraperitoneal cavity of the mouse. After 30 minutes, make a midline incision in the sternum of the euthanized mouse and fix the open thorax with pins.

Cut the ascending aorta before removing the heart, and then remove the lung to expose the descending aorta. Cut the descending aorta diagonally to reveal the cross-section of the vessel. Next, insert a 22-gauge catheter into the descending aorta while running the vasodilation buffer.

Pin the root of the catheter and make a knot to prevent leakage due to the backflow. Once done, start the perfusion with a warmed vasodilating solution at a fixed pressure between 13 to 15 kilopascals. After three minutes, perfuse a solution of 4%paraformaldehyde in PBS for 3 min.

Stop the perfusion, and fill the extension tube connected to the catheter with 2 mL of the freshly-prepared contrast medium diluted in the ratio 1:1 to perfuse at a fixed between 13 to 15 kilopascals. To visualize arteries, check the toenail of the mouth to confirm that the contrast medium has reached the artery. In the same way, check the inferior vena cava of the diaphragm to confirm complete circulation of the contrast medium and visualize all vessels.

If the circulation of the contrast medium is complete, close the three-way stopcock and remove the tube. Incubate the sample overnight at 4 degrees Celsius. In this protocol, all the vessels in the lower extremities of the mouse were visualized.

The results of a hind-limb ischemia model confirmed that the non-ligated femoral artery was running parallel to the femoral vein. Also, the interruption in the contrast media made it easy to view a ligated femoral artery and the development of collateral vessels. A few vessels filled with the contrast medium could be viewed.

On the other hand, the disruption in the contrast media due to mixing of noncontrast media, or insufficient perfusion, was also noticed. It was impossible to view the arteries on the surface of the body by microscopic of stereomicroscopic observation of the contrast medium, which made it easier to assess defects in the vasculature. It is very important to avoid air bubbles when inserting the catheter.

Since their abdominal organs are also relaxed, it is possible to take out of the organs and take a CT scan. By using this method, we were able to identify a new quarter of saturation.