The overall goal of echocardiography and right heart catheretization in rodents is to determine the function of the right heart in various cardiac and pulmonary disease models. This method can help answer key questions in cardiovascular physiology research, such as, what are the effects of genetic manipulations or drug effects on the development of pulmonary hypertension? The main advantage of this technique is that it provides a comprehensive analysis of right heart function in these models of cardiac and pulmonary disease.
To view the parasternal long axis of a rodent heart, begin by placing an anesthetized rodent in the supine position. Confirm the appropriate level of sedation by toe pinch and apply veterinary ointment to the animal's eyes. Next, shave the chest and apply ultrasound gel.
Select B-Mode to project a 2D live image. Then, align an ultrasound transducer with the appropriate frequency to the left parasternal line. Rotate the transducer counterclockwise 30 degrees, with the probe indicator pointing in the caudal direction.
Then, angle the transducer slightly, rocking the instrument along the short axis of the transducer in the same tomographic plane to obtain a full left ventricle chamber view in the center of the screen. Locate and view the lumen of the left ventricle, the interventricular septum, the lumen of the right ventricle, the ascending aorta, and the left atrium. To view the parasternal short axis at the aortic level, in B-Mode, rotate the transducer 90 degrees and angle the transducer toward the cranium to identify the aortic valve cross section view.
Next, holding the transducer steady at the same position, switch to the Pulse Wave Doppler mode. Then, place the sample volume proximal to the level of the pulmonary valve in the center of the right ventricular outflow tract and position the cursor parallel to the direction of blood flow through the vessel. Adjust the scale along the direction of the blood flow as necessary to obtain a good doppler envelope, that is, an image with white borders and a dark hollow inside, indicative of laminar blood flow, and record the doppler tracing.
To perform a closed chest right heart catheterization, place the anesthetized rodent under a dissection microscope and incise the skin from the mandible to the sternum. Place a pair of retractors on each side of the incision to fully expose the cervical area. Then, separate the salivary glands by blunt dissection, followed by use of fine, blunt tip forceps to expose the right external jugular vein.
Carefully isolate the right external jugular vein from the surrounding connective tissue, and place two pieces of silk suture beneath the right external jugular vein. Distally ligate the vein as close to the mandible as possible and tie a loose knot proximally. Using iris scissors, make a small incision in the external jugular vein proximal to the distal tied knot.
Next, mark the catheter at a length roughly corresponding to that from the external jugular vein to the heart. Then, using forceps, insert the catheter into the incision and tighten the proximal knot. Gently push the catheter into the right heart, monitoring the depth of advancement according to the mark on the catheter and verifying the location of the catheter in situ by the pressure tracing on the computer monitor.
Once a right ventricular pressure tracing has been identified, stabilize the catheter for data collection. To obtain right ventricular pressure volume loops, next intubate the rodent. Then, make an incision below the xiphoid process and bilaterally dissect the skin with scissors towards the flank.
To open the abdominal cavity through the abdominal wall, make a bilateral dissection through the abdominal wall under the diaphragm. Then, open the diaphragm to expose the apex of the heart and bilaterally cut the rib cage. Carefully isolate the inferior vena cava from the surrounding connective tissue and place sterile gauze soaked in normal saline over the abdominal cavity and intubation sites.
Then, using a small piece of PE 60 tubing, guide the puncture of the conductance catheter into the right ventricle apex and insert the catheter tip through the stab wound until all of the electrodes are inside the ventricle. Now monitor the pressure volume loop in the software, adjusting the position of the catheter to obtain consistently shaped loops that do not demonstrate significant respiratory variation as necessary. To obtain a family of pressure volume loops, gently apply pressure to the inferior vena cava to decrease the right ventricular filling.
A number of hemodynamic parameters can then be calculated from these data. To perform inflation perfusion of the lung, first, connect inflation tubing to a ring stand. Next, bluntly dissect the trachea from the surrounding muscle and connective tissue, and place a piece of silk suture around the trachea.
Then, tie a loose knot in the suture, press the head to gently stretch the trachea, and cut 70%of the circumference of the trachea close to the mandible. Keeping the trachea stretched, insert a tracheal cannula and secure the cannula with a suture. Connect the cannula to the inflation tubing, then stab the right ventricle free wall with a 10 milliliter syringe filled with PBS, and flush the lungs, injecting toward the pulmonary artery.
When the lungs begin to blanch, nick the left atrium once. Then, cut the root of the aorta to harvest the heart. Finally, inflate the lung with 10%buffered neutral formalin for five minutes and remove the tracheal cannula.
Then, ligate the trachea, excise the lung from the thorax, and fix the lung tissue with fresh 10%buffered neutral formalin. A parasternal short axis view at the aortic level can be easily visualized in rodents, enabling the recording and measurement of pulmonary arterial doppler tracings, the shapes of which have been associated with the degree of pulmonary hypertension. In a closed chest right heart catheterization of a mouse exposed to chronic hypoxia, the right ventricle systolic pressure is elevated to 45 millimeters of mercury, consistent with significant pulmonary hypertension.
In an open chest right heart catheterization of a normal rat, the right ventricle systolic pressure is observed at a significantly lower level of 27 millimeters of mercury. The procedure can be just completely in few minutes if the technique can be mastered and performed properly. While attempting this procedure, it is important to remember to make sure that the rodent is adequately anesthetized and that there are no significant sources of bleeding or tissue drying that could result in hypervolemia.
After watching this video, you should have a good understanding of how to perform a comprehensive hemodynamic characterization of the right heart. Practice is critical to mastering these techniques.
