The overall goal of this procedure is to fill the coronary vasculature with a radiopaque dye in preparation for micro computed tomography or micro CT imaging. First, the mouse's abdominal cavity is opened and the abdominal aorta is cannulated. Then the vasculature is perfused with a vasodilating buffer, followed by perfusion with AFF fixative.
The vessels are then ligated both to direct the PERFUSE eight, as well as to block potential leakage points. Finally, the coronaries are filled with radiopaque dye. The complete filling of the coronary vasculature can be evaluated by visual analysis of the heart or via micro CT imaging and analysis.
Though this method can provide insight into coronary vasculature, branching patterns, as well as vessel blockages and other coronary vessel diseases. It can also be applied to other vascular networks in other organ systems such as the brain, kidney, or hind limb. Visual demonstration of this method is critical as the vessel ligation steps are difficult to learn.
If you are unfamiliar with the vessel anatomy, finding and tying off the correct vessels in the surrounding tissue can also be challenging. Before starting the procedure, fill one side of a pressure perfusion apparatus with vasodilator buffer and the other with 4%paraform aldehyde in PBS. Next, fill a half CC insulin syringe with a 29 gauge half inch needle with 0.1 milliliters of one to 100 heparin.
Then bend the needle to approximately a 120 degree angle with the bevel up. Finally, fill a one milliliter syringe with a 26 gauge half inch needle with 0.4 milliliters of saturated potassium chloride solution, and then bend the needle to 120 degrees as just demonstrated. After anesthetizing the mouse and pinning the animal ventral side up to the dissecting tray, spray the animal with ethanol until their hair is wet.
Then open the abdominal cavity with a midline incision. Retract the skin to expose the organs and move the intestines to one side so the posterior vena CVA or PVC is accessible. Clean away any fat or connective tissue as necessary, and inject the prepared heparin solution into the PVC.
As you extract the needle. Cover the needle hole with a cotton tipped applicator to prevent leakage and hold the applicator in place for a few seconds until the PVC wall clots and seals. After waiting two to three minutes for the heparin to disperse throughout the mouse circulation, dissect the diaphragm and rib cage so the beating hard can be visualized.
Then slowly inject the prepared potassium chloride solution into the PVC until the heart arrests. Next, cut the PVC near the level of the diaphragm so the portion proximal to the heart is easy to locate in subsequent steps. Then remove all the organs below the diaphragm and excise the posterior portion of the mouse.
Now locate the cut end of aorta and place one long length of six dash zero braided silk suture doubled back on itself underneath the aorta, a few millimeters anterior from the cut end. Cut this long piece of suture so there are two under the aorta. Finally, insert the catheter into the cut end of the aorta and tie each suture with a double knot to hold the catheter in place and to prevent any back pressure within the aorta from leaking out.
Begin by connecting the pressure perfusion apparatus to the catheter. Then pump the perfusion apparatus to a driving pressure of 100 to 110 millimeters of mercury to perfuse the vessels with vasodilator buffer. Ensure the buffer is perfusing through the coronaries by confirming that liquid is exiting from the PVC while waiting for the liquid to run clear.
Dissect the ribs and pin back the rib cage to expose the heart. Once exposed, squeeze drops the buffer onto the heart from a buffer soaked piece of gauze to keep it from drying out. Clear away the thymus.
To expose the aortic arch Ligation of the vessels is the single most difficult aspect of this procedure. Locating the vessels attached to the aortic arch can be very frustrating for the beginner, but once the anatomy of the vessels is understood, one learns to trust that the vessels are there even if they cannot be seen clearly through the fat and other connective tissue. Placing the sutures requires patience and a steady hand, but it is a skill that becomes easier with practice.
Then using six dash zero braided silk suture lige, the three major aortic branches to ensure that the fluid is diverted through the coronaries rather than through these larger low resistance vessels. Next, perfuse the heart with fixative for 15 minutes. Then rinse with vasodilation buffer for at least two minutes.
Meanwhile, to prevent the microfill from leaking out of the heart after injection ligate the first anterior vena cava and then ligate the second anterior vena cva. Place sutures around the PVC and the aorta, but do not tighten them until after filling. After preparing the microfill and loading it into a one milliliter syringe, fill the dissection tray with an F water to cover the catheter.
Now, disconnect the perfusion apparatus from the catheter and connect the microfill syringe. Inject the microfill into the aorta. It will first fill the arteries, then spill into the capillaries causing the tissue to flush with the color of the microfill.
Note how the hydrophobic nature of the microfill causes it to initially appear as independent spheres as it emerges from the smaller vessels. Complete filling is evident when the microfill is continuous within the vessels and is exiting through the PVC. After the filling is complete, quickly tighten the sutures around the PVC and aorta to prevent the elastic nature of the cardiac tissue from squeezing the microfill out of the vessels.
Avoid any external pressures on the heart during the polymerization process, such as lifting or turning the heart in an attempt to get an early view of the filled vessels in the back of the heart. Then soak some gauze in water from the dissecting tray and cover the heart with the gauze to prevent its drying out. Let the heart sit for approximately one hour at room temperature until the microfill has polymerized.
Finally, remove the heart and post fixx it in 4%PFA overnight at four degrees Celsius. Then move the heart into 70%ethanol and store it at four degrees Celsius. Vessels that are effectively perfused by microfill will have continuous unbroken microfill throughout the vessels.
As demonstrated in this first figure, the extent of the filling of the coronary vessels can be judged by the eye. The veins are epicly located and are easily observed as indicated by the arrowhead. The arteries which are more intra myocardial are also visible through the surface of the heart and are indicated here by the arrow.
Capillary filling is also evident. Observe a capillary dense area of the heart in this figure as denoted by the star. Occasionally, blockages may prevent the filling of a portion of the vascular bed.
Thus, any vascular networks that fail to fill will be noticeable due to the lack of microfill as indicated here with an arrow. Further breaks in the microfill can occur, examples of which are denoted with asterisk. Incomplete filling can also result if too little pressure is used during microfill administration, as the microfill will not be forced into all the capillary networks.
Notice how the tissue has not taken on the yellow color of the microfill indicating that the microfill did not penetrate into the capillaries. Conversely, too much pressure can cause the capillaries to burst and extravasate microfill into the surrounding tissue as highlighted by the arrow. While attempting this procedure, it's important to remember not to inject the microfill too fast or with too much pressure as the vessels will burst and ruin the vascular cast.
Following the procedure in micro CT scanning. Other methods like immunohistochemical analysis can be performed in order to answer additional questions and to better characterize the vessels and surrounding tissue.
