The overall goal of the following experiment is to study cardiac structure and function in utero using high frequency ultrasonography. This is achieved by properly sedating and preparing the dam. As a second step, identify the fetuses with the uterine horns.
Next, obtain B mode, M mode, and pulse stoper data. In order to identify and quantify structural or functional abnormalities, results are obtained that show the in vivo cardiac structure and function of fetal mice with genetic alterations. The main advantage of this technique over existing methods like histopathology is that fetal echocardiography provides real-time insight into cardiac function and may identify structural heart defects in vivo.
Prepare the mouse for imaging by first anesthetizing it with two to 3%isof fluorine in an induction chamber. Place the anesthetized mouse supine on a heating pad with embedded ECG leads to maintain body temperature, secure the paws with tape. Remove the mouse from the chamber and use hair clippers to shave its fur from the mid chest level to the lower limbs.
Use depilatory cream to remove the remaining body hair. Then place the snout in a nose cone connected to the anesthesia system to maintain a steady sedation level throughout the procedure. Adjust the level of anesthesia to maintain a target heart rate of 450 plus or minus 50 beats per minute.
Then apply electrode gel to the four paws and tape them to the ECG electrodes. Next, lubricate and gently insert a rectal probe in the mouse in order to monitor body temperature via the heating pad. Maintain the body temperature within 37 degrees Celsius, plus or minus 0.5 degrees.
Controlled anesthesia and constant body temperature are essential for the hemodynamic stability of both mother and fetuses. Begin imaging using the mother's bladder as a landmark first, adjust the Z manipulators, then adjust the XY manipulators. Fetuses on the left uterine horn are labeled as L one, two and three.
While those on the right uterine horn are labeled R one, two and three. Noting embryo location is useful when retrieving embryos. After imaging, scan the embryos that are situated deep within the abdomen to document their presence, but exclude them from the data analysis because of poor resolution.
Modify scan planes by changing the orientation of the mouse with respect to the scan plane. Obtain images in two orthogonal planes for each embryo. Try to obtain views approximating the transverse frontal and sagittal planes, although sometimes views are limited to oblique planes by the position of the uterus in the abdomen.
Use scanning B mode images to identify basic cardiac structures such as the atria, intraventricular septum, ventricular chambers, and left and right ventricular outflow tracts. Then obtain M mode images from the short axis view and use them to measure ventricular wall thickness and chamber dimensions. Next, visualize the left and right ventricles.
The echogenic embryonic blood creates visible flow streams, which facilitates the accurate placement of the Doppler sample volume in the mitral orifice. Determine the left ventricular inflow velocity from the mitral valves in the apical four chamber LV long axis views. Measure systolic ejection time using aortic outflow measurements.
Calculate heart rate from the measurements of one flow cycle to the following flow cycle. Use scanning B mode images to identify common structural congenital abnormalities such as ventricular septal defects. Identify flow across the ventricular septum using Doppler sample volume within the ventricles.
Representative assessments of right ventricular functions are shown here. Here are representative images of 2D echocardiography of the long axis view of the heart at embryonic day, 14.5 and a four chambered view here. M mode tracing is shown with lines clearly indicating left and right ventricular internal diameter at diastole marked with the lowercase D and systole marked with the lowercase S.The interventricular septum or IVS is also visualized here.
Representative images of 2D echocardiography of embryonic day 14.5 heart in an apical four chamber view are seen. The left atrium and left ventricular cavity are outlined in blue. Shown here is a representative placement of pulse wave Doppler sample volume for recording mitral inflow shown here are mitral inflow doppler patterns from which early diastolic velocity denoted E and atrial contraction denoted a velocities can be measured.
Shown here is the representative aortic doppler waveform. The aortic doppler jet can be used to measure ejection time. Heart rate can also be calculated from the measurement of one flow cycle to the next.
Here we see representative detection of a ventricular septal defect or VSDB mode, images of embryonic day 14.5 heart in an apical four chamber view are seen here. The right and left ventricular cavities are outlined here in blue and red respectively. Note the presence of the interventricular septum shown here is a transverse section of this imaged heart stained with hemat, toin, and eosin.
This is B mode imaging of an embryonic day, 14.5 heart with A VSD as indicated by the white arrow. Here we see right and left ventricular cavities outlined in blue and red respectively. Effectively shown here is a transverse section of this imaged heart stained with hematin and eoin.
Here is a B mode image of an embryonic day, 14.5 heart with a ventricular septal defect indicated by the arrow with superimposed placement of pulse wave Doppler sample volume for recording of flow across the interventricular septum. Here is the representative doppler tracing from the previous figure demonstrating flow from the left to right ventricle After its development. This technique paved the way for researchers in the field of cardiac development to explore heart function prior to field of the mines.
