Our protocol describes a surgical procedure to remove ascending aortic banding in a rat model of pulmonary hypertension due to left heart disease, which leads to the reversal of pulmonary hypertension. This technique presents a valuable tool to study mechanisms of physiological reverse remodeling in the pulmonary circulation and the right ventricle. It can help in developing strategies for treating pulmonary hypertension.
After the surgical instruments are prepared, place an anesthetized rat in a supine position on a sterilized surgical table. Then, fix the animal's abdomen and limbs with an adhesive tape, and disinfect animal skin with povidone-iodine/iodophor solution. Note scars and sutures from the primary surgical aortic banding, or AoB, surgery.
After ensuring adequate depth of anesthesia with toe pinching, remove the sutures from the primary AoB surgery. Then, perform a tracheotomy by making a 7-to 10-millimeters-long cervical midline incision with fine scissors. With the help of a pair of blunt forceps, dissect the cervical soft tissue to expose the infrahyoid muscles.
Then, by splitting the muscles in the midline, visualize the trachea. When done, make a two-millimeter trachea incision between two cartilaginous rings using angled Noyes spring scissors to insert the tracheal cannula of outer diameter two millimeters into the trachea. Then, secure the cannula with a 4-0 silk suture.
Connect the other end of the tracheal cannula to a mechanical ventilator while keeping the dead space minimum. Keep perioperative lung ventilation at a respiratory rate of 90 breaths per minute at a tidal volume of 8.5 milliliters per kilogram of body weight. For the aortic debanding, make a 20-millimeter-long skin incision between the second and third ribs using fine scissors.
With the help of smaller surgical scissors, spread and cut the muscles layer by layer, followed by making a 10-millimeter lateral incision along the intercostal space between the second and third rib. Use a rib spreader to expand the intercostal space between the second and third rib, to create a surgical window. With the help of blunt forceps, separate the thymus from the heart and conduit arteries to visualize the aorta with the clip.
Hold the clip with the help of the forceps, followed by removing the connective tissue around the clip to expose the aorta. Open the clip with a needle holder, and remove the clip from the thoracic cavity. Before closing the chest, open up lung atelectasis and ensure adequate lung recruitment without over-distention with mechanical ventilation, with a tidal volume of 9.5 milliliters per kilogram body weight for 10 minutes, and return to a tidal volume of 8.5 milliliters per kilogram body weight.
Later, close the deep muscles by a simple interrupted suture using 4-0 silk, and connect the upper muscles with a simple continuous suture. Then, close the skin with a simple continuous suture. Disconnect the tracheal cannula from the ventilation machine while observing the rat for spontaneous breathing.
If the animal fails to breathe spontaneously upon disconnection, reconnect the ventilator and continue ventilating for an additional five minutes before repeating the procedure. After spontaneous breathing is reestablished, remove the cannula from the trachea and clean the liquid around the trachea with sponge points. Close the trachea with a simple suture using 6-0 Prolene, and close the infrahyoid muscles in a simple interrupted suture using 4-0 silk.
Connect the skin in a simple continuous suture, followed by cleaning and disinfecting the muscles and the skin in the process with povidone-iodine/iodophor solution. After completing the surgical procedure, move a single animal to a recovery cage with supplemental oxygen and an infrared lamp, and place the oxygen mask close to the rat's snout. In the study, the position of the clip on the ascending aorta in the AoB animals and its absence after the debanding, or Deb, surgery was visualized.
Also, aortic blood flow was evaluated by pulsed-wave Doppler imaging before and after the clip in AoB animals prior to the debanding and in corresponding aortic segments after the Deb surgery. The results showed a marked attenuation of the blood flow gradient in line with the functional debanding. At week five, the Deb rats expressed brain natriuretic peptide, or BNP, at levels comparable to the sham animals, indicating the reversal of left ventricle, or LV, failure by the aortic debanding.
The evaluation of LV function by transthoracic echocardiography revealed an increased LV ejection fraction and LV volume in the Deb animals compared to the AoB rats. The debanding surgery performed at week three after AoB resulted in a significant reduction of left ventricular systolic pressure and LV hypertrophy in comparison to the AoB animals. Compared to the AoB rats at week three and week five, the Deb animals also showed a significant reduction in right ventricular systolic pressure and right ventricular hypertrophy, demonstrating a successful reversal of pulmonary hypertension due to left heart disease.
The most critical steps in this procedure are removal of the clip and the recruitment of the lung after the aortic debanding. These steps, when correctly performed, increase animal survival.
