Medicine
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Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice
Chapters
Summary February 1st, 2022
There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.
Transcript
Right ventricular is more resistant to ischemia than the left one. We introduced a technique to generate a right ventricular infection model, which can clarify the pathophysiological mechanism during the development of a right ventricular infarction. We located the right coronary artery through vascular costing and defined the ligation area which is critical for successfully generating a mouse right ventricular infarction model.
Demonstrating the procedure will be Linhai Ruo, a poster doctor from my laboratory. Place the mouse supine on a pad and ensure the depth of anesthesia by the absence of a toe withdrawal response. Fix its incisors with a suture and immobilize the limbs with adhesive tape.
Remove hair from the neck to xiphoid with a depilatory cream. Pull out the tongue slightly with tweezers, lift the mandible with a tongue depressor to expose the glottis. And perform intra tracheal intubation by inserting a 22 gauge cannula into the glottis.
Connect the electrocardiography electrodes to the mouse limbs correctly, and record the ECG. To open the chest, make a one centimeter long incision in the skin, parallel to the third right rib with ophthalmic scissors. Determine the third intercostal space again, and ensure adequate space according to the sternum angle.
Separate and cut the pectoralis major and pectoralis minor muscles with scissors and micro forceps above the third intercostal space. After that, bluntly separate the intercostal muscle with elbow forceps to expose the surgical field, then incise the pericardium. Lift the right atrium with sterile cotton and ligate the right coronary artery with a sterile 8-0 nylon thread, with a ligation range of three to five millimeters.
After ligating the right coronary artery, note the monitoring ECG lead 3, showing ST-segment elevation. Remove the cotton and suture muscles and skin with a sterile 5-0 nylon thread. Then place the mouse on the heating pad to promote recovery.
Place the mouse in the supine position on an ultrasonic platform for animal fixation and ultrasonic operation. Tape its claws to the electrode to obtain an ECG recording, through a system attached to the ultrasonic machine. Remove the hair from the mouse's chest with a depilatory cream and apply ultrasound gel to the skin of the chest.
Set the platform to the horizontal position. Orient the transducer parallel to the left leg and obtain the left ventricular long axis image. Rotate the probe 90 degrees clockwise to obtain the left ventricle short access view.
Press the cine store button to save the images. Tilt the upper left of the platform at the lowest point. Move down the transducer vertically, maintaining its position over the upper abdomen and below the mouse's diaphragm under B-Mode.
Adjust the platform position slightly by rotating its X and Y axis until the right ventricle, right atrium, left atrium and left ventricle are seen on the screen. And save the image by pressing cine store or frame button. Press M-Mode.
After the two X indicator line appears locate the indicator line at the tricuspid valve orifice, to obtain the movement of the tricuspid annual plane. Press M-Mode again. Press the cine store or frame store button to save data and images.
Tilt the left side of the platform at the lowest point. Place the probe at a 30 degrees angle to the horizontal axis along the right anterior axillary line. Rotate the X and Y axis of the platform to display the right ventricle.
Press the M-Mode button and locate the indicator line at the septum hyperechoic point, to obtain the M-Mode image of the right ventricle interface. Press the M-Mode button again. Press the cine store button to save the picture.
Perform tracheal intubation and set the parameter of the animal ventilator. Make a one centimeter bilateral incision on the skin above the xiphoid process and transect the diaphragm and rib with ophthalmic scissors to expose the heart. Puncture the right ventricular free wall with a 32 gauge needle.
Remove the needle and press the wound with cotton to staunch bleeding. Insert the tip of the catheter into the right ventricle through the puncture site, and push the catheter forward, slowly. Adjust the position of the tip to obtain a typical RV pressure wave form, shown on a monitor and recording system.
After 10 minutes of stabilization, record the data of RV systolic blood pressure, RV and diastolic pressure, and RV maximum right ventricular pressure rising decreasing rate. Click on the select button to select cardiac cycles for calculation. And then click on analyzed button to calculate the mean values of the selected cycles.
Place the animal supine on the pad and intubate for artificial ventilation. Open the chest with surgical scissors and expose the heart. Make a three millimeter notch with ophthalmic scissors on the right atria and perfuse the heart with 5mL of normal saline through the cardiac apex with an injector.
Block the blood from the aorta with an aortic clamp. And perfuse 0.1mL of 1 mg/mL nitroglycerin, through the cardiac apex with an injector to dilate the coronary artery. Perfuse the heart with a 1mL cast reagent through the cardiac apex and wait two to three hours.
Erode the heart with 50%sodium hydroxide for two to three days. And remove the muscle tissue or connective tissue by rinsing with normal saline. After RCA ligation, ST-segment elevation was seen in lead 3 of the ECG.
2, 3, 5-Triphenyltetrazolium chlorid staining showed that the infarct area accounts for 45%of the RV free wall, 24 hours postoperatively. Echocardiography showed that the RV internal dimension at the end of diastole, increased in the RV1. And it was more than two times in the Sham group.
RV fraction shortening, RV ejection fraction, and tricuspid annular plane systolic excursion were significantly smaller in the RVI. The RV by LV area ratio increased by approximately 50%RV hemodynamic measurement showed that in the RVI group, right ventricular systolic blood pressure, the maximum and minimum rising rate of RV pressure, RV contractility, were significantly smaller. Right ventricular and diastolic pressure and the exponential time constant of RV relaxation index, were considerably more significant than those in the Sham group.
An RV aneurysm was visible in the infarcted area. The heart weight to body weight ratio and heart weight to tibia length ratio in the RVI group were slightly larger. Masson staining indicated significant fibrosis in the RV free wall.
And seldomly, fibrosis occurred in the septum of RVI group. In contrast, a few surviving cardiomyocytes were in the infarct area. It is crucial expose the ligation area and grasp the inserted needle's depth and the range to increase the success and the survival rates.
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