The overall goal of this protocol is to establish minimally invasive transverse aortic constriction in mice. This method can help answer key questions about implementing transverse aortic constriction in mice, such as how to create a passage under the aortic arch. The main advantages of this technique are that it is simple, user-friendly, and that it eliminates the need for intubating and ventilating the mice.
I will be demonstrating the procedure with our postdoc Simona Nemska who will be performing the presurgical animal preparation and postoperative surveillance steps. On the day of the procedure, shave the neck and chest of an anesthetized mouse and disinfect the exposed skin with 70%ethanol. After confirming the appropriate level of sedation by toe pinch, secure the animal in the supine position onto a clean cork working pad.
Next, use a number 11 knife blade to make a 10 millimeter longitudinal midline cervical incision from the suprasternal notch to the mid chest to expose the sternum. Using a Crile-Wood needle holder, pass a 4-0 monofilament polypropylene stay suture to retract the thyroid. And tape the suture to the working pad.
Then move the working pad under a surgical microscope and use microsurgical forceps to bluntly separate the pretracheal muscles to uncover the trachea. Gently slide the closed tips of a pair of curved microsurgical forceps over the trachea and behind the sternum and carefully open and close the curved forceps to bluntly dissect the pleura. Use the straight microsurgical forceps to grasp the right supraclavicular muscles and gently pull up on the chest.
Sliding the inferior tip of the bone nipper under the sternum, perform a three to four millimeter upper partial sternotomy, directing the lower part of the mini sternotomy slightly toward the left. Using a microsurgical needle holder, pass a 7-0 monofilament polypropylene stay suture from the inside to the outside of the second intercostal space on each side of the mini sternotomy. Use 7-0 monofilament polypropylene stay sutures to spread the sternal edges on each side.
And use the curved forceps to gently move the pretracheal muscles, mediastinal fat, and thymus aside to visualize the aortic arch under low power magnification. To expose the soft tissue under the aortic arch, insert tying forceps under the arch with the tips closed and gently open the forceps. Use a second tying forceps to make a tunnel in the soft tissue under the aortic arch by gently opening and closing the forceps in the soft tissue.
Then pass a piece of 6-0 silk ligature threaded through the eye of a ligation aid under the aortic arch, using a tying forceps to retrieve the suture from between the origin of the right innominate and left common carotid arteries. The singular most difficult step of the procedure is inserting the suture under the aortic arch. Taking adequate care while creating the tunnel under the arch will make the subsequent suture placement easier.
Now use the straight forceps to position a blunted 27 gauge needle next to the aortic arch and use the two tying forceps to tie the suture snugly around the needle and the aorta between the right innominate and left common carotid arteries. When the suture has been secured, quickly but gently remove the needle to achieve a 0.4 millimeter diameter narrowing and a reproducible transverse 65 to 70%aortic constriction. After confirming hemostasis, remove the 7-0 monofilament polypropylene stay sutures used for spreading the sternal edges.
Use a microsurgical needle holder to pass a simple 6-0 monofilament polypropylene suture from the outside to the inside of the left second intercostal space and from the inside to the outside of the right second intercostal space. Use the Crile-Wood needle holder to bring the sternal edges together with a 6-0 monofilament polypropylene suture. And close the skin with a 5-0 monofilament polypropylene running suture in one layer with a Crile-Wood needle holder.
Then place the animal back in its cage in the prone position under warming with monitoring until full recovery. The efficacy of the left ventricular hypertrophy induction can be validated by comparing the heart weight to body weight ratios of the sham animals to that of the experimental animals at days three, seven, 14, and 28 postsurgery. The ratio significantly increases in the banded animals compared to the sham groups from postoperative day seven on, remaining significantly higher in the constricted animals up through day 28 postsurgery.
The observed increase in the heart to body weight ratios is due solely to a rise in the left ventricle body weight ratio as the right ventricle body weight ratio remains comparable between the transverse aortic constriction and the sham operated animals over the entire observation period. In addition, the mRNA expression of the brain natriuretic peptide, a positive control for cardiac hypertrophy in the left ventricles, is significantly higher in the aortic banded animals compared to the control sham operated animals at day 14, further validating the efficiency of the technique. Once mastered, this technique can be completed in 20 minutes if it is performed properly.
While attempting this procedure, it is important to use a ligation aid and tying forceps similar to those used in our protocol. After its development, this technique provided a reliable and simplified model for exploring the mechanisms involved in the genesis of pressure overload induced left ventricular hypertrophy. After watching this video, you should have a good understanding of how to perform minimally invasive transverse aortic constriction in mice with a special emphasis on the passage under the aortic arch.
Don't forget that creating the passage under the aortic arch is the most critical step of the procedure, and using a ligation aid and tying forceps considerably reduces the risk of injury to the arch.
