Ms550d transducer

A high-resolution ultrasound (US) imaging system designed for small experimental animals (Visual Sonics Vevo 2100, Canada) with an MS-550D transducer with a center operating frequency of 22–55 MHz was used for ultrasound measurements of 4T1 tumors. During imaging, body temperature was controlled by the heating pad and maintained at 37°C. Anesthesia was induced by 3 vol% isoflurane (Aerrane, Baxter Polska Sp. z o. o., Poland) and then maintained at 1.5–2.0 vol% isoflurane in air, delivered at 1.2 l/min via a nose cone. Ultrasonographic imaging of tumors was conducted on the same day as EPR oximetry. For the initial confirmation of the tumor, a B-mode ultrasound (greyscale) was performed with a central frequency of 40 MHz. Doppler imaging is used in ultrasound to detect the presence of blood flow and to evaluate the direction and speed of flow in vessels. Power Doppler (PD) measurements were performed with a central frequency of 32 MHz and pulse repetition frequency (PRF) 3–4 kHz. 3D images of tumors, each containing about 40 scans per tumor, were obtained with a 0.2 mm step using a steady-arm-held transducer. The PD measurements were performed only on one of the most promising ITPP protocols (4T1 tumor model with three ITPP/saline doses at day 8,12,16), due to prolong anesthesia during the multi-imaging session.

Cardiac ultrasonography was performed by a trained technician during the fourth week of exposure, using a Vevo 2100 imaging system from Visual Sonics with a 22–55 MHz MS550D transducer. Mice were anaesthetized with isofluorane (1%) supplemented with O₂. Body temperature, respiratory rate and ECG were monitored and heart rate was maintained at 350–400 beats/min during the measurements of cardiac parameters. Systolic function parameters, including the ejection fraction (EF) and fractional shortening (FS) were measured by two‐dimensional parasternal short‐axis imaging plane of M‐mode traces close to the papillary muscle level.

Animals were maintained under steady-state isofluorane anaesthesia and placed on a heated platform with ECG and respiration monitoring. Core temperature was measured using a rectal probe (Indus Instruments) and maintained at 37.5 °C throughout recording. Echocardiography was performed using a Vevo2100 high resolution, pre-clinical in vivo ultrasound system (VisualSonics) with the MS-550D transducer at 40 MHz frequency and 100% power. Imaging was performed on a layer of aquasonic gel after the pre-cordial skin had been clipped and de-epliated with cream (Veet). Parasternal long-axis view (PLAX) images were obtained in EKV mode (set at 1000 Hz for recording) over the entire cardiac cycle. The left ventricular area was traced in end-distole (LVAd) and end-systole (LVAs) and used to derive the ejection fraction (EF) with the Vevo LAB cardiac package software. The investigator performing sonography was blinded to the genotype of the animals. Transverse EKV recordings were also obtained over the abdominal aorta just below the diaphragm using the same settings as described for the heart. These images were evaluated in the VevoVasc software package to determine vessel distensibility. Maximal anteroposterior aortic diameter (from inner wall to inner wall) was measured in the same images in systole and diastole using Vevo LAB general imaging package software.