The overall goal of the following experiment is to study the structure and hemodynamic parameters of the fetal circulation, using a high-frequency ultrasound system. This method provides comprehensive phenotyping of cardiac morphology and flow indices during development in real time, and allows studying of congenital heart defects in experimental settings. Compared to low frequency ultrasound systems, this technique provides higher sensitivity and resolution by generating two-dimensional dynamic images at high-frequency frame rates.
To begin, turn on the ultrasound imaging system and the physiology monitoring unit. Then connect the 30-45 megahertz transducer, and place the corresponding scan head on its holder near the imaging platform. Next select the cardiac measurement program option.
Place the ultrasound gel upside down in its pre-warming container, set to 37 degrees Celsius. Then confirm the appropriate tubing system for anesthesia, and verify the levels of oxygen and isoflurane. Disinfect the imaging platform and the working area.
Finally, set the heat level of the imaging platform to maintain a constant body temperature of 37 degrees Celsius for the dam. Transfer the sedated dam from anesthesia induction chamber to the imaging platform, and place the animal in a supine position. Provide steady state sedation with isoflurane, using a face mask connected to the anesthesia tubing system.
Apply electrode gel to the embedded electrocardiographic electrodes. Then gently tape the limbs to the electrodes for constant monitoring of maternal cardiac and respiratory rates. Monitor the dam's temperature and heart rate, displayed on the physiology controller unit.
Adjust the level of isoflurane to maintain an average heart rate close to 450 beats per minute. Confirm the appropriate level of anesthesia by evaluating the mouse's posture, heart rate, and response to toe pinches. Then use a depilatory cream to remove the fur from the mid-chest level to the lower limbs, to minimize ultrasound attenuation.
Palpate the abdominal wall gently to locate the fetuses and spread them out. Avoid spreading the fetuses forcefully. Using the cervix as a landmark, annotate each embryo on the dam's abdomen, and define their orientations.
Exclude those fetuses where the orientation is unreliable. Label the fetuses on the left uterine horn with the prefix L, and those on the right uterine horns with an R prefix, as shown here. Apply pre-warmed ultrasound gel on the abdomen, and spread it carefully, to avoid bubble formation.
Add additional amount of gel to the area of scanning. Place the ultrasound probe on its mechanical holder, and move it gradually towards the abdomen to contact the thick gel layer, while looking for the beating heart, using B mode scanning. Confirm the right and left orientation of the individual fetus in real time, by moving the imaging platform in the horizontal plane.
Scan the fetus from head to tail to annotate the snout, the limbs, and the spine, as landmarks. Visualize the beating heart and annotate the left ventricle and the right ventricle. Switch to color Doppler mode to optimize the heart visualization.
Click the B-Mode button to obtain a peristernal short access view. Ensure that the left and right ventricles are displayed in their maximum diameter at the center of the data acquisition frame, and start live-imaging. Change the scanning planes to obtain a longitudinal four chamber view.
First identify the remaining structures of the heart, such as the atria, interventricular septum, and left and right outflow tracts. Next have the ventricular and atrial chambers displayed in their maximum length. Start image acquisition by clicking the Cine button, to obtain continuous recording cine-loops for a minimum of 10 seconds.
Next click the scanning M-Mode button to obtain cardiac images from four chamber planes to evaluate fetal heart rate and ventricular function. Exclude any non-optimal images. Then click the Analysis button to measure the ventricular wall thickness and internal diameter at diastoli and systoli.
First adjust the sector to an angle of acquisition less than 60 degrees, to visualize the bifurcation of the pulmonary artery and identify the right outflow tract. Next click Pulsed Wave Doppler button to obtain the flow pattern through the pulmonary and the aortic valves. Next place the pulse wave Doppler sample volume to record aortic mitral flow Doppler patterns, and measure early diastolic velocity and atrial contraction velocity.
Use the color Doppler scan to visualize the uterine artery and fetal placental vascular tree, using the 45 megahertz transducer. Place the pulsed wave Doppler sampler volume to obtain the umbilical artery flow pattern. Use the pulsed wave Doppler scanning record to measure vascular peak flow parameters, including acceleration time, ejection time, and peak flow velocity at end systoli.
Turn off the isoflurane container, following the completion of the imaging process. Continue monitoring body temperature, respiratory rate, and heart rate during the recovery phase. Document the time to full resumption of normal activity.
The data in this table demonstrates the feasibility of using high-frequency ultrasound, as outlined in this protocol, to perform a comprehensive phenotyping of fetal heart morphology and function, and fetal placenta circulatory parameters, and their alterations in fetal mice exposed to chronic hypoxia in utero. Following this procedure, other methods like speckle tracing and stain analysis can be employed in order to answer additional questions, like measuring regional myocardial function of the developing myocardium in future studies. After watching this video, you should have a good understanding of how to operate high-frequency ultrasound effectively and to evaluate fetal cardiac morphology and circulatory parameters systematically in real time.
