This protocol can be used to study the role of genetic alterations or pharmacological interventions in cardiac function and coronary flow at early postnatal ages. The main advantage of this technique is the early recognition of cardiopathologies and longitudinal follow-up studies in left ventricular hemodynamics and coronary flow parameters in different mice models. Echocardiographic studies in neonatal mice are technically challenging.
This protocol is designed to study the early onset of cardiovascular disease in neonatal mice that may allow researchers to design the appropriate therapeutic measures. Start with placing the ECG gel on the warmed platform electrode pads. Place aluminum foil strips on top of the electrode pads to extend the electrode range and secure them with tape.
Subsequently, place the ECG gel on top of the aluminum strips. Ensure that the gel underneath the aluminum foil strips does not dry out during the procedure. If that occurs, add more gel to maintain the conductivity.
Place the anesthetized pup in a supine position on the imaging platform and secure the cut-out glove finger around the pup's nose. Place the paws on top of the aluminum foil pads and secure them with tape. Ensure that the electrical circuit is complete and that the ECG is recording.
Use two gauze rolls to keep the ultrasound gel in place. Place a thick layer of pre-warmed ultrasound gel on top of the pup's upper body. Use a heating lamp to maintain the pup's normal body temperature.
Perform transthoracic echocardiography using an echocardiography instrument equipped with a linear array transducer at 40 megahertz for B-Mode and at 32 megahertz for Doppler following adult mice echocardiography protocols. Place the transducer in the holder with the index mark toward the right shoulder of the pup. Lower the transducer until it is in contact with the gel and visualize the left ventricular outflow tract in B-Mode.
Avoid placing excessive pressure on the pup's chest cavity when placing the echotransducer during echocardiographic image acquisition. Capture the parasternal long axis view of the left ventricle, left ventricular outflow tract, and the left atrium. Use M-Mode at the aortic leaflets to measure the left atrium maximum diameter at N-systole.
Record the data by pressing the Cine Store button. Rotate the transducer around 90 degrees clockwise of the parasternal long axis to obtain the parasternal short axis view. Capture the parasternal short axis view of the left ventricle to measure the chamber dimensions, wall thickness, aortic flow, and pulmonary flow.
Place the probe at the level of the papillary muscles and use M-Mode to measure the left ventricular internal diameters and wall thickness during systole and diastole. Record the data by pressing the Cine Store button. Move the transducer toward the base of the heart and use the Color Doppler to visualize the pulmonary artery.
Press Pulse Wave Doppler to quantify the pulmonary peak flow velocity, pulmonary flow profiles, pulmonary ejection time, and pulmonary acceleration time. Record the data by pressing the Cine Store button. Move the transducer further toward the base and use Color Doppler to visualize the aortic flow.
Use Pulse Wave Doppler to visualize the blood flow and measure the aortic ejection time. Record the data by pressing the Cine Store button. To capture the apical four-chamber view, place the platform in the Trendelenburg position, tilt it to the left, and adjust the probe to visualize the four chambers.
Use Color Doppler to visualize the blood flow and Pulse Wave Doppler at the tip of the mitral valve leaflets in the center of the mitral valve orifice to record the mitral flow. Record the data by pressing the Cine Store button. Use Tissue Doppler at the septal side of the mitral valve annulus in a four-chamber view to measure the peak myocardial relaxation velocity in the early diastolic filling and late diastolic filling, as well as the peak systolic myocardial contraction velocity.
Record the data by pressing the Cine Store button. Capture the modified parasternal long axis view to examine the left anterior descending coronary artery. Use a modified parasternal long axis view, moving the transducer laterally.
Move the probe and use Color Doppler to visualize the origin of the left main coronary artery that generates from the aorta. Identify the left anterior descending artery that generates from the left main coronary artery and runs between the left ventricular anterior wall and the right ventricular outflow tract. In this position, measure the left anterior descending coronary artery flow by pressing Pulse Wave Doppler.
Record the data by pressing the Cine Store button. Increase the isoflurane concentration to 2.5%and wait for five minutes to achieve maximal flow. Record the data by pressing the Cine Store button.
In the parasternal long axis view, M-Mode was used to measure the left atrium diameter. The parasternal short axis view was used to measure the left ventricular chamber dimensions and wall thickness, pulmonary flow, and aortic flow. The apical four-chamber view was used to examine the blood flow velocities across the mitral valve as well as the myocardial relaxation and contraction velocities at the mitral valve annulus.
The modified parasternal long axis view was used to examine the left anterior descending coronary artery flow parameters. The increased values of peak coronary flow velocity, mean coronary flow velocity, and velocity/time integral five minutes after isoflurane increment confirmed the expected response to hyperemia in the neonatal mice. Due to the small size of the pups, it is important to maintain their normal body temperature and to attach aluminum foil strips to their limbs to reach the electropads and to create an ECG circuit.
