Antegrade Perfusion Model: An Ex Vivo Technique to Perfuse Isolated Mouse Heart

Begin by placing an anesthetized mouse in the supine position. Dissect the thoracic cavity to visualize the heart. Use a pipettor fitted with a wide bore tip to apply suction pressure and aspirate the heart from the mouse’s body into the tip. Excise the heart at the aorta to harvest it from the mouse's body.

Immerse the isolated heart into a beaker containing a nutrient-rich buffer supplemented with an anticoagulant agent. The anticoagulant in the buffer forms a complex with calcium present in the cardiomyocytes - the heart muscle cells, ceasing contraction of cardiac muscles and preventing blood coagulation.

Next, place the heart apex-side down on a heart stand kept in a Petri dish containing suitable buffer. Clamp the aorta above the atria and transfer the clamped heart to a perfusion plate.

Subsequently, take a syringe-needle assembly containing an appropriate perfusion buffer. Insert the needle into the left ventricle piercing the ventricular chamber.

With the aorta clamped, intraventricular injection pressurizes aortic valves to open. Consequently, the buffer flushes blood antegradely through the coronary arteries, thoroughly perfusing heart muscles. Finally, the buffer effuses through the pulmonary veins. This provides a successfully perfused heart for downstream applications.

For mouse heart harvest, after confirming euthanasia and shaving the abdomen, open the thoracic cavity quickly to expose the heart, and use a plastic transfer pipette with the tip cut to approximately the size of the heart, to suck the heart into the pipette. Raise the pipette to create enough space to insert curved scissors, and use the scissors to excise the heart from the dorsal side, taking care to avoid damaging the atria.

Immediately, place the heart into a 30 milliliter glass beaker containing ice-cold CIB EGTA for about 1 minute. When the contractions have stopped, place the heart in a 35 milliliter culture dish containing ice-cold CIB EGTA, and remove the lung and other visible tissue.

Place the roughly cleaned heart onto a heart stand filled with chilled CIB EGTA, apex side down, and place the stand under a stereoscopic microscope. Remove the fat and connective tissues from around the aorta. If the length of the cut aorta is too long, trim the aorta just under the brachiocephalic artery, and orient the heart so that the anterior surface is facing forward. Use tweezers to lift the end of the aorta, and use a small vascular clamp to clamp the aorta near the atria, while gently pushing down on the atria.

Then, place the clamped heart on a perfusion plate, with the anterior side facing up, and hydrate the heart with a few drops of CIB EGTA. For antegrade perfusion, load a 20 milliliters syringe containing pre-warmed CIB EGTA connected to a flexible extension tube and a marked injection needle, onto the infusion pump, and start the pump at a 0.5 milliliter per minute flow rate. When the needle and pump have been filled, place the injection needle on the perfusion plate, with the shorter side of the diagonal shape in front, and slide the needle until it is just touching the apex of the heart.

Carefully, insert the needle near the apex of the left ventricle, into the ventricular chamber without twisting or detaching the needle from the plate, watching the mark to estimate the depth of the needle insertion. When the needle insertion is complete, blood should begin to flow from the coronary artery.

Use tape to fix the injection needle to the plate and increase the pump speed to 1 milliliter per minute. If the heart is perfused successfully, the flow of the buffer in the capillary should be visible just under the epicardium.