Vevo 770

At the end of 24-hour reperfusion, cardiac function was evaluated by using a high-frequency ultrasound system Vevo770 (Visual Sonics Inc., Canada) with a 35 MHz central frequency scan head. Mice (n = 5, each group) were anesthetized with pentobarbital sodium (50 mg/kg) and positioned on the table. Two-dimensional image was obtained by using the conductive adhesive to connect the mouse limbs to the four electrodes and fixed with the adhesive tape. The scan head was placed on the left chest of the mice and the two-dimensional image was obtained at the short axis level. M-type echocardiography was obtained at the level of the papillary axis perpendicular to the interventricular septum and the posterior wall of the left ventricle. The scanning velocity was 800 mm/s. We measured the left ventricular end-systolic diameter (LVESd), left ventricular end-diastolic diameter (LVEDd), left ventricular posterior wall end-systolic thickness (LVPW), interventricular septum end-systolic thickness (IVSs), and interventricular septum end-diastolic thickness (IVSd). The left ventricular ejection fraction (LVEF%), left ventricular fractional shortening (LVFS%), and stroke volume (SV) were calculated by Visual sonics Vevo770 software. Each of the mice underwent an average of 3–5 consecutive cardiac cycles of ultrasound process.

Transthoracic echocardiography was performed using a Vevo 770 high-resolution in vivo imaging system (Vevo770, Visual Sonics Inc., Toronto, ON, Canada) equipped with a RMV 707B scan head. Mice were anesthetized with isoflurane in oxygen. M-mode images were obtained when the heart rates of mice were stably maintained at 450–500 bpm. The left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured. All records were averaged for 5 consecutive cardiac cycles.

Firstly, hemodynamic measurements were performed with echocardiography. Echocardiography was evaluated with the high-resolution Micro-Ultrasound system (Vevo 770, VisualSonics, Toronto, Ontario, Canada). Mice were anesthetized with 1.5% isoflurane. Right ventricle wall thickness during diastole (RVWTD) were obtained from parasternal short axis view at papillary muscle level using M-mode. Pulmonary arterial (PA) peak flow velocity and PA acceleration time/ejection time (AT/ET) were obtained from parasternal short axis view at aortic valve level using pulsed Doppler mode [46 (link)–51 (link)]. The data were measured using VisualSonics Vevo 770 analysis software with a cardiac measurement package based on the average of at least 3 cardiac cycles. Secondly, a 22-gauge needle connected to the PowerLab system (AD Instruments, Sydney, Australia) was inserted into the right ventricles of the mice to record RVSP using Chart program in PowerLab system. Lastly, mice were sacrificed for assessing right ventricular hypertrophy (RV/LV + S).

On Day 1, Day 2, Day 4, and every 3-5 days thereafter for a total of 10 time points in 28 days post-implantation, high-frequency small animal ultrasound (US) examination of the liver was performed in five of the animals with a dedicated small-animal high-resolution imaging unit (Vevo 770; VisualSonics, Toronto, Canada). During imaging, rats remained anesthetized with 2% isoflurane in oxygen at 2 L/min on a heated stage. Real-time imaging was performed with a 40-MHz high-frequency linear transducer. Two-dimensional B-mode images were acquired in two directions (transversal and sagittal) with manual optimization of the gain, and the maximum tumor diameter measured in millimeters was measured in millimeters using electronic calipers. Images were recorded digitally and analyzed offline using micro-US imaging software (Vevo 770; VisualSonics, Toronto, Canada).

Cardiac function of MI/R mice models was first evaluated by transthoracic echocardiography (Visual Sonics, Vevo 770) 1 day before and 4 weeks after treatment. Mice were anesthetized with low-dose isoflurane, left ventricular ejection fraction (LVEF) and fraction shortening (LVFS) were calculated in six consecutive cardiac cycles, following two-dimensional targeted M-mode traces at the papillary muscle level. Then, mouse hearts were harvested and cut into 5-μm sections, fibrosis extension and infarct size were visualized and measured by a common histochemical procedure including Masson.

Mice were anesthetized in a chamber containing 2-4% isoflurane mixed with 0.2 l/min 100% O₂, and during measurements, narcosis was retained using 1-2% isoflurane with 0.2 l/min 100% O₂. Mice were fixed on a heated table on a rail system (VisualSonics, Canada). Ultrasound measurement was conducted using the Vevo 770 or 3100 System (VisualSonics) with a 400 MHz mouse transducer.