Vevo 2100 imaging system

At 6 weeks, we performed high‐resolution ultrasonography measurements of PWV in rat abdominal aorta as previously described (Lee et al., 2016). We used transit time method validated for abdominal aorta using the Vevo2100 imaging system (VisualSonics). PWV was calculated as Δd/Δt, where d is the distance between two points divided by the difference in transit time (t) of the pressure wave arrival at two said points and was measured reproducibly via noninvasive ultrasound micro‐imaging and Doppler ultrasound transit time approach. The transit time was defined as the peak of the ECG R‐wave to the foot of the velocity upstroke with the foot defined as the point at the end of the diastole when the steep rise of the wavefront begins. All measurements were obtained in triplicate; the mean value was used for data analysis. The data were analyzed with VevoLab 1.7.1 software (VisualSonics). The Doppler ultrasound studies were done with the animals sedated with 0.8% isoflurane anesthesia, which is not expected to induce nonphysiological artifacts since it is less than the 1% isoflurane anesthesia level defined as ‘‘baseline level’’ for coronary blood flow with heart rates similar to sleeping rats and documented to not alter aortic impedance (Hartley et al., 2008; Herrera, Decano, Giordano, Moran, & Ruiz‐Opazo, 2014).

After skin preparation of the chest and upper abdomen, mice were fixed on the operating table in supine position and anaesthetized with 1.5% isoflurane. Non‐invasive transthoracic echocardiography was carried out to assess heart morphology and function using the Vevo2100 Imaging system (VisualSonics, Toronto, Ontario, Canada) equipped with a 30 MHz phased‐array transducer. Left atrium (LA) diameter was determined from M‐mode at end‐systole. All these images were processed using Vevolab 3.1 software (VisualSonics).

Left ventricular function was evaluated on nonanesthetized conscious mice by transthoracic echocardiography using a Vevo 2100 Imaging System (Visual Sonics) with a 30 MHz probe. Left ventricular parasternal short- and long-axis views were obtained in B-mode imaging, and left ventricular parasternal short-axis views were obtained in M-mode imaging at the papillary-muscle level. The short-axis M-mode images were used to measure left ventricular end-diastolic internal diameter (LVIDd), left ventricular end-systolic internal diameter (LVIDs), diastolic and systolic septal wall thickness (IVS), and left ventricular diastolic and systolic posterior wall (LVPW) thickness in 3 consecutive beats according to the American Society of Echocardiography leading edge method [76 (link)]. Some additional parameters such as fractional shortening, corrected left ventricular mass, stroke volume, and heart and respiratory rate were calculated from the above measured parameters [77 (link)].
For the first cohort of B6 mice, only n = 7 animals were examined.

Animals were imaged at 5, 12, 20, and 25 weeks of age. Twelve weeks was chosen to represent a central timepoint between 5 weeks, where initial onset of disease occurs, and 25 weeks, where the diabetic condition is at its most severe. Twenty weeks was chosen as a secondary endpoint due to the potential of animals perishing prior to 25 weeks of age due to the severity of untreated diabetes mellitus and the deterioration caused by the diabetic condition. Therefore, animals were imaged at both 20 and 25 weeks to ensure that all animals received an echo during the most severe stage of cardiovascular dysfunction. A single trained individual in the WVU Animal Models and Imaging Facility acquired ultrasound images in a blinded fashion in conscious mice to maintain normal left ventricle (LV) function and heart rate [46 (link)–49 (link)]. Images were acquired using a 32–55 MHz linear array transducer on the Vevo2100 Imaging System (Visual Sonics, Toronto, Canada) as previously described [9 (link),50 (link)–52 (link)], and were acquired at the highest frame rate (233–401 frames/second) as determined by image resolution. M-mode images were acquired by placing a gate through the center of the short-axis B-mode images to obtain recordings of the internal features of the myocardium. Long-axis B-mode images and short-axis B-mode images were acquired for STE analysis.