Vevo 2100 high resolution small animal ultrasound system

Echocardiography was performed as previously described [34 (link)]. Briefly, transthoracic echocardiography was performed using a high-resolution echo machine with a 30 MHz probe. Animals were anesthetized with 3% isoflurane, their chests were shaved, and temperature-controlled anesthesia was maintained with 1.5% isoflurane. The probe was situated perpendicular to the heart to determine the position, and then, the probe was rotated clockwise 30-45 degrees to determine the left ventricle of the heart. The following parameters were measured from the M-mode images and two-dimensional images obtained in the short-axis views by the corresponding matching software (Vevo 2100 high-resolution small animal ultrasound system, VisualSonics, Canada): the percentage of fractional shortening (FS, %), ejection fraction (EF, %), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV). Stroke volume (SV) was calculated using the formula: LVEDV − LVESV (Figure S1).

A Visual Sonics Vevo 2100 high-resolution small animal ultrasound system (probe frequency: 40 MHz) was used for cardiac ultrasonography. The mice were placed in an airtight anesthesia induction chamber, filled with an oxygen gas mixture containing 3% isoflurane to rapidly induce anesthesia, and were removed when the mice were unable to turn freely, and fixed on the ultrasound table. At this point the concentration of isoflurane in the gas mixture is reduced to 1.5–2% to maintain mouse anesthesia. The mice were debrided on the chest, coated with an appropriate amount of ultrasound coupling agent, and two-dimensional ultrasound images were acquired at the long-axis and short-axis levels of the left ventricle, respectively, while satisfactory M-mode ultrasound images were obtained for preservation. The left ventricular ejection fraction (EF%) was measured on the M-mode ultrasound images using the analysis software provided with the ultrasound imaging system [35 (link)].