Evaluation of Right Ventricular Function in Experimental Models of Pulmonary Arterial Hypertension

We explore regenerative therapies, particularly mesenchymal stem cells, and mitochondria, to reverse vascular remodeling and improve cardiopulmonary function in experimental pulmonary arterial hypertension. Invasive procedures, such as right ventricular puncture and heart dissection, are both terminal, limiting longitudinal studies. Additionally, variability in echocardiographic measurements can affect reproducibility across different operators and time points. Our protocol combines non-invasive echocardiography with invasive hemodynamic measurements and postmortem analysis, providing a comprehensive assessment of pulmonary hypertension progression, and right ventricular remodeling in rats. This integrated approach enhances data reliability and reduces the amount of animals needed. In the future, we aim to better understand the brain-heart-lung interaction and cognitive deficits in patients with pulmonary arterial hypertension to understand the clinical relevance and potential underlying mechanisms.

[Instructor] To begin, use gauze dressing soaked in detergent and water to wet the fur on the chest of the anesthetized animal. With a razor blade attached to Kelly forceps, shave the fur from the animal's chest. Apply a generous amount of conductive gel to the animal's chest. Press the 2D button on the control panel. Place the transducer between the third and fifth intercostal space to acquire a short axis view. then direct the transducer toward the animal's left shoulder at a 90 degree angle relative to the sternum to capture a cross-sectional image of the base of the heart, showing the right ventricle, aortic valve, pulmonary valve, and left atrium. Press the freeze button on the control panel to freeze the image, then press the save button on the control panel to save the image. When the correct angle is located, hold that position and press the PW pulse wave button to activate pulse wave spectral doppler mode. Use the trackball on the control panel to move the yellow cursor in any direction on the screen and place it over the pulmonary valve. Press the five button on the control panel to acquire the blood flow curve across the pulmonary valve. Now, position the transducer between the third and fifth intercostal space to acquire a long axis view. Direct the transducer toward the animal's right shoulder at a 45 to 60 degree angle relative to the sternum, capturing a longitudinal section showing the left ventricle, right ventricle, left atrium, mitral valve, and aortic valve. Press the freeze button on the control panel to freeze the image, then press the save button on the control panel to store the image. Press the end exam button on the control panel to conclude the imaging session and store the images in the patient database. For echocardiography, select search on the screen and choose the appropriate animal ID from the database to begin analysis. Press the SonoView button on the control panel. Click once inside the image area on screen to start the analysis. Use the trackball to select the short axis image in pulsed wave spectral doppler mode. Now, press the calculator button and select heart rate HR on the screen. Use the trackball to draw cursor lines peak to peak of two curves. Repeat this three times to obtain the average. Press the calculator button again and choose pulmonary valve, PV, and then PV acceleration time/ejection time on the screen. Use the trackball to place a cursor from the start to the peak, and then from the start to the end of the same curve. Repeat three times to obtain the average. Press the save button on the control panel to save the results. Using the trackball, select the long axis image from the upper right section of the screen. Press the calculator button on the control panel and select the right ventricular outflow tracked, RVOT, parameter and then the RVOT diameter on the screen. Using the trackball, position the cursor from one wall of the right ventricle to the opposite wall. Perform this three times to obtain the average. For right ventricular puncture, first, tracheostomize an anesthetized animal. Ensure the computer is powered on, the LabVIEW software is open, and the baseline is configured for signal acquisition. After locating the right ventricle in the software, verify that signal acquisition has begun. Click the save button on the screen and enter the animal's ID. Use a heparinized saline-filled 19 gauge scalp vein set to puncture slightly above the anatomical reference point, taking care not to insert the needle too deeply. Verify accuracy of the puncture by observing the pressure values. Record at least 10 stable pressure wave peaks to ensure data reliability. At the end of signal acquisition, administer heparin using a heparinized syringe for blood collection via puncture of the left ventricle or abdominal vena cava. For data analysis, open the MATLAB program, click the browse for paste button, and select the file on the computer's internal hard drive that contains the correct code. On the left side of the MATLAB screen, observe the list of files that include codes for each channel recognized by the transducer. Choose the code corresponding to the selected channel configuration used earlier in the experiment. Press the run button and choose the .bin file on the external hard drive for analysis. Select the most uniform section of the curve by clicking once at the beginning and once at the end, identifying the portion with visually equal peaks and troughs. Then select a ten-second segment of the curve by clicking once at zero seconds and once at 10 seconds. Adjust the baseline by subtracting the lowest trough value in the command window and pressing enter on the keyboard. The software will automatically generate all relevant data. Observe the data in two columns that are time of peak and pressure value. Copy and paste the systolic pressure values into a .txt file and remove the time values. Replace all periods with commas in the file to enable calculation of the average. Select 10 peak pressure values and calculate their average. For right ventricular hypertrophy index, measure and record the animal's body weight at the start of the experiment. Measure the right ventricular systolic pressure, then euthanize and remove the thoracic organs. Dissect the aorta, pulmonary artery, vena cava, and pulmonary veins from the base of the heart, then dry the entire heart using gauze and record the dry weight using a precision balance. After dissecting and removing the atriums, separate the right ventricle from the left ventricle along with the septum. Record the weights of the dried right ventricle and the left ventricle plus septum separately using the precision balance. The PAT to PET ratio was significantly reduced in pulmonary arterial hypertension animals compared to controls, indicating increased pulmonary vascular resistance and altered blood flow pattern in the pulmonary artery. The right ventricular outflow diameter was increased in pulmonary arterial hypertension animals compared to controls, reflecting the degree of ventricular hypertrophy as an adaptive response of the right ventricle, surrogating the development of the disease. Representative echocardiography images showed visibly enlarged right ventricular outflow diameters and altered flow patterns in pulmonary arterial hypertension animals relative to controls. Right ventricular systolic pressure was significantly elevated in pulmonary arterial hypertension animals compared to controls, reflecting the increased pulmonary vascular resistance. The pressure waveform recordings showed higher peaks in pulmonary arterial hypertension animals, consistent with elevated RVSP. Right ventricular hypertrophy index was significantly elevated in pulmonary arterial hypertension animals compared to controls.