O-Ring Aortic Banding Versus Traditional Transverse Aortic Constriction for Modeling Pressure Overload-Induced Cardiac Hypertrophy

This novel technique of aortic banding allows induction of pressure overload cardiac hypertrophy in mice with less variance and higher reproducibility. Placing a rubber ring with a fixed diameter around the aorta induces a consistent increased flow velocity in the transverse aorta, thus generating reproducible cardiac hypertrophy. This procedure should be performed by someone who has already experience with anesthesia and microsurgery techniques in rodents, or has someone with experience who can guide.

Begin by performing one cut of the O-ring under the microscope using scissors, or a scalpel to enable the placement around the aorta. Pierce each ring side close to the cut with a needle connected with an 8-0 non-absorbable suture, and pull the thread. Cut, and leave two to three centimeters on one side, and two centimeters on the other side to fasten the O-ring around the aorta in the final step.

Before surgery, take the ligation aid, and pull the end of the thread of one ring side through the hole of the ligation aid. Put the ligation aid with the attached O-ring aside for placement. Shave the fur on the left thorax side of the anesthetized mouse.

Then place the mouse back into the isoflurane-filled chamber for sufficient sedation before intubation. Turn the heating pad on before the surgery to maintain the animal's body temperature at 37 degrees Celsius. Connect the heating pad to a rectal probe to maintain the mouse's body temperature automatically.

Stretch a rubber band around the heating pad to fix the mouse with the front teeth on the plate. Place the anesthetized mouse on the heating pad in a supine position. Place the rubber band over the animal's front teeth to extend the neck on the plate.

Focus a light source on the throat for good visibility of the opening of the trachea for endotracheal intubation. Open the mouth gently with one hand, positioning an intubation aid with the other hand and small forceps. Gently move the tongue to clear the opening of the trachea.

Introduce the endotracheal tube into the trachea and on the other hand, continue holding the intubation aid. Connect the endotracheal tube position via a three-way stopcock to a ventilator for mice to confirm the correct endotracheal tube position. Monitor the right ventilation according to the manufacturer's instructions.

Confirm sufficient anesthetic depth by a toe pinch reflex check. Apply ophthalmic ointment on the eyes to avoid dryness during the surgery. Disinfect the surgery area using a commercially available disinfectant solution.

Using scissors, make a 0.5 to one centimeter long skin incision in the middle of a line between the xiphoid process and left axilla. Use forceps to separate the muscle layer from the underlying ribs, and place two five-millimeter retractors into the incision to expose the ribcage. To start the left thoracotomy, perform a one to two millimeter incision in the intercostal muscles between the second and third rib using micro-spring scissors.

Open the thoracic cavity, and spread the incision with 45-degree angled forceps. Place three chest retractors into the incision for opening the thoracic cavity to improve visualization. To expose the aortic arch, try to lift and gently separate the thymus and fat tissue from the arch with fine-tip 45-degree angled forceps.

Expose the aortic arch with 45-degree angled forceps in one hand. On the other hand, position the O-ring connected with the ligation aid via the threads of one side attached earlier. Pass the threads using the ligation aid under the aortic arch from caudal to the cranial side of the transverse aorta between the brachiocephalic and left common carotid arteries.

Take both threads between the ligation aid and the aortic arch with the forceps carefully. Remove the ligation aid and gently position the O-ring around the arch by pulling the threads on each side. After successful positioning, fix the O-ring with the threads and a surgical knot.

Make an additional knot to avoid opening it on each side. After ligation of the O-ring, remove the three chest retractors from the incision. If needed, eliminate the residual air from the thorax by filling it with warm 0.9%isotonic salt solution.

To expose the thorax incision for suture, take two five-millimeter retractors again to hold the skin on the side. Close the thorax with two, or three 6-0 non-absorbable sutures. Remove the two retractors and close the skin with three to five 4-0 absorbable sutures.

Then turn off the isoflurane and monitor. Pinch off the outflow of the ventilator for two seconds to reinflate the lungs. One day after surgery, verify the stenosis using ultrasound by measuring the maximal flow velocity over the stenosis.

For measurements, use echocardiography with an ultrasound system and a transducer probe with a frequency of 30 megahertz. Place the anesthetized animal on the heating pad in a supine position and connect the heating pad with a rectal probe to maintain the body temperature at 37 degrees Celsius, and monitor the heart rate with an ECG using four mouse paw sensors. A successfully performed O-ring aortic banding results in an increased flow velocity over the stenosis as measured by ultrasound.

For this measurement, position the transducer head parasternally on the right thorax side to localize the aortic arch by two-dimensional imaging. Use the color doppler to visualize the blood flow in the aorta, and measure with the pulsed wave doppler blood flow velocity over the stenosis. Visualize the right and left common carotid arteries by two-dimensional imaging.

Position the transducer head horizontal on the left and right sides of the neck at a 45-degree angle, and use the pulsed wave doppler to determine the blood flow velocity. Successful induction of aortic stenosis was seen by increased blood flow velocity over the stenosis in the aorta measured by pulsed wave doppler ultrasound. Additionally, the blood flow velocity ratio between the right and left common carotid arteries was determined as a functional marker of O-ring aortic banding.

Two weeks post-surgery, increased left ventricular posterior wall and intraventricular septum diameters in diastole were observed. Additionally, a progressive worsening of cardiac function was documented by measuring decreased left ventricular ejection fraction. While securing the O-ring around the aortic arch, it's important to close the ring on both sides with a knot to get an optimal result.