This method provides a useful technique for studying the mechanisms of right ventricular failure. The main advantages of this technique are saving time and increasing the success rate for surgery. This technique can help us imitate right heart hypertrophy and failure in humans by constriction of the pulmonary artery to create a pulmonate pressure overload.
This method can be applied to the transverse aortic constriction technique and can provide insight into post heart transplantation complications, and congenital heart disease studies. The procedure will be demonstrated by Xiaoxia Huang, a technician from my laboratory. Before beginning the procedure, pass a piece of 6-0 braided silk suture through a 25 gauge needle disassembled from a one milliliter syringe.
Use hemostatic forceps to bend the needle 90 degrees. Then bend a 22 gauge needle 120 degrees. After confirming a lack of response to toe pinch, place an anesthetized mouse in the supine position on a 37 degrees Celsius heating pad, and secure the incisors with a suture.
Fix the limbs with tape, and use depilatory cream to remove the hair from the neck to the xiphoid process. Disinfect the exposed skin with sequential iodine and 75%ethanol wipes, and use pointed pliers to extract the tongue slightly. Elevate the mandible with a custom made spatula to expose the glottis, and use a cold light source to direct the careful insertion of a custom made trachea cannula through the glottis.
Adjust the mini ventilator parameters and set the respiratory rate to 150 breaths per minute, and a tidal volume of 300 microliters. Then connect the tube and the ventilator to confirm that the cannula has been correctly inserted before fixing the cannula with tape. To open the chest, make an approximately 10 millimeter skin incision parallel to the second rib.
Use scissors and elbow tweezers to separate the pectoralis major and minor muscles above the second intercostal space. Count the ribs from the sternal angle to identify the second intercostal space and use scissors to open this space. Then bluntly separate the parenchyma and thymus until the pulmonary trunk is visible.
To constrict the pulmonary artery, use the elbow tweezers to bluntly separate the pulmonary trunk and the ascending aorta. Use the 90 degree latch needle to pass the suture through the connective tissue between the pulmonary trunk and the ascending aorta. Place the 120 degree padding needle on the pulmonary trunk.
Use the 6-0 braided silk suture to ligate the padding needle to the pulmonary trunk, removing the padding needle immediately after filling of the pulmonary conus is observed. Cut both ends of the sutures and continue to observe the filling of the pulmonary conus to evaluate whether a constriction is present. Then evaluate the animal's pedal reflex again to ensure the success of the ligation and close the chest and skin with a 5-0 nylon suture.
At least three days after the surgery, apply ultrasound gel to the chest of the pulmonary artery constricted animal. Place a 30 megahertz probe on the chest at a 30 degree counter clockwise position relative the the left parasternal line, while orienting the notch in the caudal direction. Regulate the Y and X axes under the B-mode until the mitral valve, aorta, and left ventricle chamber are clearly visible.
Rotate the probe 30 to 40 degrees on its Y axis toward the chest. Regulate the Y and X axes until the pulmonary conus is clearly visible and place the cursor at the tip of the pulmonary valve leaflets to measure the peak flow velocity. Select color and PW and place the pulsed wave dashed line parallel to the direction of the blood flow.
Adjust the left side of the pad so that it is higher than the right side, keeping the probe oriented 30 degrees to the horizon along the right interior axillary line, with the notch pointed in the caudal direction. Regulate the X and Y axes until the right ventricle is clearly visible. Then click M-mode two times to show the indicator line.
Eight weeks after the surgery, intubate the mouse as demonstrated, and make a 1.5 centimeter bilateral incision below the xiphoid process through the diaphragm to open the chest cavity. Cut through the ribs to expose the heart, and use a 23 gauge needle to penetrate the right ventricle free wall. Press the puncture point gently with a cotton swab to stop any bleeding, and use the catheter tip to puncture the ventricle through the wound.
When the curve is stable, regulate the tip of the catheter toward the right ventricle outflow tract. Click select and analyze to record the right ventricle's systolic blood pressure and diastolic pressure, maximal rate of rise, exponential time constant of relaxation, and heart rate for 10 minutes. Then transfer the catheter from the heart into a container of saline.
Compared to the Sham group, a higher peak velocity, greater pressure gradient, and greater right ventricle wall thickness are obtained from the parasternal long axis view at eight weeks after surgery in the Pulmonary Artery Constriction group. Additionally, the systolic function of the right ventricle is significantly reduced in the Pulmonary Artery Constriction group, compared to the Sham group, eight weeks after the surgery. The Pulmonary Artery Constriction group has a higher right ventricle pressure in the systole and diastole, and the contractility index is reduced compared to that of the Sham group.
The right ventricle Tau and the maximal rate of rise are also greater in the Pulmonary Artery Constriction group than that in the Sham group. Demonstrating that right ventricle dysfunction is induced in mice after eight weeks of pulmonary artery banding. Invasive hemodynamic testing in the right ventricle, reveals that the heart rate remains stable before and after the catheter monitoring.
Compared to the Sham group, the right ventricle dimensions are significantly enlarged, and the right ventricle weight is higher. Moreover, histological examination shows that cardiac fibrosis in the area covered by cardiomyocytes are greater in the Pulmonary Artery Constriction group than in the Sham group. The mostly important part of the procedure is ensuring that the pulmonary artery is separated correctly and completely.
To avoid an incorrect separation, the separation point should be near the branch of the pulmonary artery, where the connective tissue between the arteries will be looser. With this method, we have provided a more convenient and efficient approach to inducing right ventricular hypertrophy. Generally, the surgical procedure and evaluation approaches are very safe.
