Minimally Invasive Transverse Aortic Constriction in Mice

The overall goal of this procedure is to induce left ventricular hypertrophy by constricting the transverse aortic arch with a simple surgical procedure that doesn't require artificial ventilation. This method can help answer key questions in the cardiology field about myocardial remodeling, cardiac hypertrophy, apoptosis and fibrosis, and the left ventricular pressure overload conditions. The main advantage of this technique is that it conserves the essentials of regular transverse aortic constriction while eliminating the need of a ventilator and tracheal intubation.

Before beginning the procedure, use a needle holder to curve the tips of two to four 30-gauge needles, and mount the needles onto individual cotton applicators. Next, blunt a 27-gauge needle and use the needle holder to bend the tip 90 degrees. Then, smooth the tip on a hard surface to make a 0.4 millimeter spacer.

After confirming the appropriate level of sedation by a lack of response to toe pinch, apply ointment to the animal's eyes and place the mouse on a 37 degrees Celsius warmed heating pad in the supine position. Secure the limbs to the heating pad with surgical tape. Then, disinfect the skin with three consecutive alcohol and povidone-iodine solution wipes and place a sterile drape with an exposed operation field over the animal.

To begin the ligation procedure, use a sterile scalpel to open the skin at the midline position of the neck and chest. Next, under a dissecting microscope, use blunt scissors to gently separate the connective tissues and pull the thyroid gland towards the head. Using the curved needles, separate the muscle layer on the trachea at the midline, towards both sides of the animal and use the blunt scissors to cut the sternum approximately five millimeters to the second rib.

Open the incision with curved forceps and use a curved needle to separate the thymus lobes and the lower chest wall. The transverse aortic arch and two carotid arteries will be clearly visible at this point. Place the curved needle under the arch and use the tip to make a perforation between the vessel wall and the connective tissue on the other side.

Use the needle to place a 6-0 monofilament suture under the aortic arch, and place the previously prepared spacer into the loop. Fix the suture in place with a double knot and gently remove the spacer. After confirming a successful knot constriction, cut the ends of the suture and use a 6-0 silk suture to close the chest wall with a simple interrupted suture pattern, followed by closure of the skin with a 6-0 monofilament suture in a continuous suture pattern.

Then, apply povidone-iodine solution to the suture site and place the animal in a pre-warmed cage for recovery and post-operative monitoring. After one week, apply electrode gel to the experimental animal's paws and tape them to a 37 degrees Celsius warmed heating pad with electrodes. Next, apply ultrasound gel to the mouse chest and select the B-mode on a high-frequency ultrasound system.

Using a 40 megahertz MS550D probe, locate the left ventricle. Then, in M-mode, measure the systolic and diastolic wall thickness. Return to B-mode to locate the left common carotid artery before the branching point, and use pulsed-wave and color doppler to evaluate the flow, storing the images for later analysis as appropriate.

Tilt the animal handling platform to the far left so that the mouse is in the left decubitus position and place the probe at a 30 degree offset from a position parallel to the head in the parasternal position. Manipulate the x and y torques to find the aortic arch and locate the constriction using the color doppler mode to make the site of the constriction more visible. Using pulsed-wave and color doppler, measure the peak flow velocity at the point where the blood is flowing between the two carotid arteries branching out from the aortic arch.

Then, allow the animal to recover on a heating pad in the prone position with monitoring until fully recovered. Minimally invasive transverse aortic constriction, or MTAC reliably produces left ventricular pressure overload in ligated mice with a clearly visible constricted transverse aorta and an increased peak-flow velocity through the constriction site. In comparison, the sham-operated animal demonstrates no apparent constriction with a normal peak-flow velocity.

As a consequence of the ligation, the flow velocity is significantly increased in the right carotid artery, and pressure can build up in the ascending aorta and the heart, to produce left ventricular hypertrophy, significantly reducing the flow in the left common carotid artery. Near identical blood flow is observed in the right and left carotid arteries in the sham-operated mouse, however. The resulting hypertrophy-related parameters are readily observed in the significantly enlarged and deformed hearts of MTAC ligated mice, and their reduced overall survival.

These changes are further observed at the cellular level, including an increased cardiac muscle cell size and fibrosis in the MTAC mice, compared to non-ligated animals. Once mastered, this technique can be completed in five to ten minutes, if it is performed properly. While attempting this procedure, it is important to remember to be careful not to damage the vessels.

After watching this video, you should have a good understanding of how to induce left ventricular hypertrophy via transverse aortic arch constriction, using a simple surgical procedure that does not require artificial ventilation.