Prior to performing echocardiography measurements, the mouse is placed to top a heated EKG board with its pause taped to ECG electrodes that are embedded in the stage. Appropriate anesthesia level and body temperature are maintained throughout the experiment. Using the short axis mode, also known as B mode.
A proper image is obtained when both interlateral and post medial papillary muscles, as well as all myocardial walls are easily viewable. The viewing mode can be changed to M mode to evaluate and quantify measurements of systolic cardiac function. After targeting the left ventricular posterior wall, the tissue doppler imaging mode can be used to quantify measurements of diastolic function.
In the four chamber view mode, a proper image of the mitral valve and septal wall can be obtained. Finally, by changing to pulse wave Doppler mode, the mitral valve blood flow pattern can be visualized. Hi, my name is Jonathan Repress from the laboratory of Dr.Zander Wars in the Department of Molecular Physiology and biophysics at Baylor College of Medicine.
Today we're gonna show you a procedure for transthoracic echo cardiography in mice. We use this technique in our lab to study cardiac function and dimensions over time. Let's get started.
One day prior to the imaging studies, anesthetize the mouse and use hair clippers to shave the fur from the neck line to mid chest level. Then remove the remaining body hair with hair removal cream and apply dura loop gel to both eyes to prevent drying of the sclera. Place the anesthetized mouse in a supine position atop a heating pad with embedded ECG leads.
In order to maintain body temperature, place the snout within a nose cone connected to the anesthesia system to maintain a steady state sedation level throughout the procedure. If necessary, the level of anesthesia can be adjusted to obtain a target heart rate of 450 plus or minus 50 beats per minute. Or BPM gently insert a rectal probe to continuously monitor and adjust body temperature via the heating pad.
It is important to maintain the body temperature within a narrow range as even moderate changes in temperature and heart rate, affect cardiac function. In mice. Apply electro gel to the four paws and tape them to the ECG electrodes.
To begin this procedure, apply a layer of ultrasound gel to the chest, primarily the area overlying the heart. Avoid air bubbles in the gel which can interfere with ultrasonic imaging. Immobilize the mouse in a slight upward position, head up under a slight angle using a micro manipulator.
Immobilize the ultrasound probe with a 90 degree angle between the probe and the heart. To begin making short axis echocardiographic measurements, lower the probe onto the thorax sternly. Avoid placing the probe directly over the sternum as this will distort the signal.
First, we will perform two dimensional imaging or B mode to obtain a view along the per sternal short axis, adjust the viewing area of the probe to obtain a complete image. Depending on the size of the animal's heart, a proper image in this orientation will include the left ventricle and a slight portion of the right ventricular wall. The arrow indicates the anterolateral and posterior medial papillary muscles, the posterior and anterior walls of the left ventricle, the intraventricular septal wall, and a slight portion of the right ventricular wall.
Next, we'll use mode echocardiography, which provides a one dimensional view to obtain fine measurements of cardiac dimensions and contractility store acquired images for later evaluation of parameters of systolic left ventricular function. While still viewing the parasternal short axis, we can perform tissue doppler imaging or TDI to measure velocity of myocardial motion. The region of interest is marked to include a posterior left ventricular wall for radial axis evaluation.
The waveform obtained will have four peaks to evaluate diastolic function, IVRT ePrime AP prime and I-V-C-T-S prime represents systolic velocity. These parameters will be described later in the representative results section. Pulse wave doppler or PWD can be used to measure blood flow velocity within a small area at a specific depth in the myocardial tissue.
To image the trans mitral flow pattern, tilt the animal backwards in the trendelenberg position, tilt the probe upward such that the probe is orthogonal with the apex of the heart. Now increase the ISO fluorine level to lower the heart rate to 300 to 350 BPM, which will slow the movement of the mitral valve. For comparison studies, it is important to maintain similar heart rates between animals.
Visualize the movements of the mitral valve as a reference point to measure the flow across the valve. Measure the flow patterns across the mitral valve. Using PWD two waves are characteristically seen, one representing passive filling of the ventricle early EWA, and one consistent with active filling with atrial systole.
Atrial a wave in a healthy heart, the EWA velocity is slightly greater than that of the A wave. Once all measurements are completed, allow the animal to recover atop the heated ECG pad and turn off the anesthesia. When the mouse awakens return it to its cage.
We show here a 2D echocardiography image or B mode obtained along the parasternal short axis, which shows the left ventricular anterior and posterior walls, the intraventricular septal wall and the lateral wall. The diameter of the left ventricular lumen can be measured as the left ventricular internal diameter. The asterisk indicates the postal medial papillary muscle.
This next 2D echocardiography image or B mode shows an apical four chamber view with a viewing area over the mitral valve for determination of E over a peak velocities. MVAL indicates the mitral valve anterior leaflet. MVPL indicates the mitral valve.
Posterior leaflet RV indicates the right ventricle and LV indicates the left ventricle. In this representative M mode tracing the large arrows indicate the end systolic diameter ESD and the end diastolic diameter. EDD of the left ventricle.
LV A WDS indicates left ventricular anterior wall thickness and L-V-P-W-D-S indicates left ventricular posterior wall thickness during diastole and systole respectively. A representative tracing for tissue doppler imaging of the LV posterior wall is shown here where IVRT represents the ISO volumetric relaxation time and IVCT represents the ISO volumetric contraction time. The ePrime wave corresponds to the motion of the mitral annulus during early diastolic filling of the LV and the APR wave originates from atrial systole during late filling of the LV S prime represents systolic velocity.
Pulse wave doppler recording of mitral valve leaflet tips provides mitral inflow velocity patterns from which early diastolic velocity, late diastolic velocity with diastolic contraction and the E over a ratio can be derived. The IVRT is also a useful variable to characterize diastolic function and filling pressures. ET in this tracing indicates ejection time in a healthy heart.
The EWA velocity is slightly greater than that of the a wave as observed here. Hi, we've just shown you how to perform transthoracic echocardio in mice. Remember, when doing this procedure, please remember to handle the animal properly and also adjust the levels of anesthesia accordingly.
So that's it. Thanks for watching and good luck with your experiments.
