Mouse Echocardiography: A Non-invasive Technique to Assess Cardiac Morphology

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Echocardiography

1. Determine the body weight of the mouse using a standard laboratory balance while holding it lightly by the tail to ensure proper positioning.
2. Anesthetize the animal by the intraperitoneal (i.p.) injection of 50 mg/kg pentobarbital.
   NOTE: Any other kind of anesthesia can be used if the same protocol is used throughout the study. Advantages and disadvantages will be discussed below.
3. Put the mouse back in its own cage and wait until it is unresponsive, it shows steady breathing, and rear foot reflexes are absent. To test this, squeeze a foot slightly and observe whether the leg still retracts.
4. Shave the left side of the thorax and the left armpit using a commercial rodent shaver.
   NOTE: The use of a dedicated rodent shaver allows for the complete removal of fine mouse hair to avoid interference in the echocardiographic measurement. Commercial hair removal creams or solutions should be avoided, as they are usually perfumed, which will disturb the animal after it awakens. Avoid excessive shaving, as it increases heat loss.
5. Put the sleeping animal on a warm pad set to 40–42 °C in a shallow left-sided position, with the head at 12 o'clock and the tail at 6 o'clock. Fix the left arm, left leg, and tail with tape.
6. Apply pre-warmed echocardiography gel onto the shaved chest and the head of the transducer.
7. Place the transducer parasternal-left, directing it to the right side of the neck to obtain a two-dimensional (2D) parasternal long-axis view on the level of the papillary muscle. Turn the transducer 90° clockwise to obtain a short-axis view at papillary muscle level. Use a minimal depth setting and a zoom to maximize image quality and frame rate. Set the sweep speed to the maximum.
   1. To obtain these settings, different zoom and depth setting options may be used, depending on the machine and software. Record 2D-guided M-mode images in short-axis view. Refer to Figure 1A and B.
      NOTE: Take care to avoid applying excessive pressure to the chest, as this may cause bradycardia.
8. Record at least 3 series of 3 heartbeat cine loops for each animal.
   NOTE: For the echocardiograph software used in this study, press the "Acquire/Save" button only once. This methodology is specific to the echocardiograph with this particular software. Other software packages may be used with different machines.
9. After successful recordings, wipe off the echocardiography gel from the mouse thorax, heating pad, and transducer. Remove the tape from the limbs and tail.
10. Leave the mouse under observation on the heating pad, covered with tissue to avoid unnecessary light exposure and heat loss, until it wakes up.
11. Put the animal back in its cage.
12. Analyze recorded M-mode images from parasternal short-axis view to determine left ventricular (LV) dimensions and function. Measure the thickness of the LV anterior wall in systole and diastole (LVAWs and LVAWd), the LV internal end-systolic and end-diastolic diameters (LVIDs and LVIDd), and the LV posterior wall thickness (LVPW) in systole and diastole (LVPWs and LVPWd) using the identification of the tissue-blood interface on the stored images.
13. Measure the diastolic dimensions at the time of the apparent maximal LV diastolic dimensions and LV end-systolic dimensions at the time of the most anterior systolic excursion of the LV posterior wall. Tap the touchscreen on the "Analyze" icon and then on the "LVAWd" icon. Position the electronic caliper on the interface between the right ventricular cavity and the LV anterior wall in diastole.
14. Position the electronic caliper on the interface between the LV anterior wall and the LV cavity to obtain the LV diastolic anterior wall thickness; the software will directly switch to the LV internal end-diastolic measurement.
15. Position the caliper on the interface between the LV cavity and the LV posterior wall to obtain the LV internal end-diastolic diameter; the software will switch to the LV posterior wall thickness measurement.
16. Position the caliper on the interface between the LV posterior wall and the pericardium to obtain the LV diastolic posterior wall thickness. For LV systolic dimensions, tap the touch screen on the "LVAWs" icon and position the electronic caliper on the interface between the right ventricular cavity and the LV anterior wall in systole.
17. Position the electronic caliper on the interface between the LV anterior wall and the LV cavity to obtain the LV systolic anterior wall thickness. Repeat the process as described above for the LV internal end-systolic diameter and the LV systolic posterior wall thickness. Use the leading-edge convention adopted by the American Society of Echocardiography to trace the endocardial and epicardial borders.
    NOTE: LV contractile function parameters will be automatically calculated using the previous measurements. The LV fractional shortening (FS) is defined as FS (%) = [(LVIDd – LVIDs)/LVIDd] x 100. The LV ejection fraction (EF) is calculated with the modified Teicholz formula, where EF (%) = [(LVIDd³ - LVIDs³)/LVIDd³] x 100¹². Refer to Figure 1A, B.
18. Store the data on compact discs or USB memory sticks and make backup copies.
19. Import, analyze, and export the data using the appropriate software.