In this study, we used fresh bovine skeletal muscle samples from the same animal sourced from a local butcher shop, so this work was not sent to an Ethics committee or institutional review board.
Supersonic Shear Imaging
The elastographic equipment used in our experiments is based on the SSI technique and is commercially called ShearWave™ Elastography (version 9, Aixplore, Aix-en-Provence, France). It operates sequentially in three modes: imaging, pushing, and imaging. When the device is activated, it initially operates in the imaging mode to acquire a reference image of the medium, then begins to operate in the pushing mode. In this mode, the equipment applies acoustic radiation force by means of beam ultrasonication that is highly focused at three or more different successive depths for a period of approximately 100 µs. In this manner, three or more sources of spherical waves are created, which interact in the form of a Mach cone, creating a quasi-plane wavefront in a 3D conical shape. In the next step, the system returns to operation in the imaging mode and excites the same transducer in bursts with a 5–30-kHz (5,000 to 30,000 images per second) pulse repetition rate to detect the medium vibrations caused by propagation of the shear waves. Initially, the group shear wave speed (cs) is estimated and used to calculate the shear modulus (μ) based on the product of cs squared and the density (predefined as 1000 kg·m-3). Finally, the device presents a map of the E (E ≈ 3μ) of the tissue, which is reconstructed by estimating the shear wave speed between two points in the displacement image by means of a time-delay estimation algorithm. The displacement images are obtained by using the cross-correlation technique between the reference image and images acquired after pushing is activated. ShearWave™ Elastography was described in detail by Lima et al. (2018).
In our first experiment, E images were recorded from a fresh bovine skeletal muscle sample (13 × 7 × 19 cm), which was heated via physiotherapeutic ultrasound. The transducer was aligned such that its length was parallel to the muscle fibers. Initially, an E image was collected with the physiotherapeutic ultrasound equipment (AVATAR III; KLD Biosisstemas Equipamentos Eletrônicos Ltda, Amparo, SP, Brazil) deactivated. The equipment was then configured to generate a continuous signal with a nominal frequency of 3 MHz, nominal intensity of 2 W·cm-2, and application time of 2 min. Immediately following the cessation of physiotherapeutic ultrasound irradiation (in the time of 2 min), ShearWave™ Elastography was used to record an E image, which was saved on the device. Over the first five minutes, an E image was saved every minute. Next, from 7 – 32 min, an elastographic image was saved every five minutes. Thereafter, an image was saved 60 min after the completion of irradiation using the physiotherapy equipment. The temperature of a muscle sample at a distance of 7 cm from the center of the area irradiated by the physiotherapeutic ultrasound equipment ranged from 17.6 – 18.8 °C throughout the experiment and the ambient temperature was 18.8 °C. The transducer with a 3-MHz frequency, which was responsible for irradiating the upper face of the sample, was fixed in a certain position. When irradiation was completed, the therapeutic transducer was removed and the ShearWave™ Elastography transducer was positioned in the same region to evaluate mechanical properties. In addition, the both transducers were coupled to the bovine muscle sample using clinical water-based gel and an acoustic absorber (rubber plate) was placed underneath the muscle sample to avoid unwanted reflections of the ultrasonic wave emitted by the transducer and, thus, prevent the formation of hot spots inside the sample.
A region of interest (ROI) with dimensions of 3.0 × 3.0 cm was selected in all images. In the center of the ROI relative to the x-axis, circular Q-Boxes with a diameter of 5 mm were positioned at the following depths: 0.4, 0.9, 1.4, 1.9, 2.4, and 2.9 cm (Figure 1). ShearWave™ Elastography calculates and displays the mean and standard deviation of the E within each Q-Box, as shown in Figure 1.
In the following experiment, the same fresh bovine skeletal muscle sample was used 60 minutes after the end of the first experiment, but the transducer axis of the physiotherapeutic ultrasound equipment was displaced by 2 cm compared to its previous position along the depth axis. Additionally, the application time for the AVATAR III device was changed to 10 min. Immediately following the cessation of physiotherapeutic ultrasound irradiation, ShearWave™ Elastography was used to record the first E image. E images were then recorded at 10, 13, 18, 20, 25, 30, 35, 40 and 70 min. In this experimental setup, three circular Q-boxes were positioned in the center of the ROI at the following depths: 0.4, 0.9, and 1.4 cm.
In the final experiment, during ultrasonic irradiation, the transducer coupled to the physiotherapeutic equipment was applied for 10 min in an area corresponding to 5.5-ERA (ratio between the irradiated area and the ERA, effective radiating area) in a fresh bovine skeletal muscle sample in a circular motion and with an angular velocity of 3.54 ± 0.30 rad·s-1 (33.8 ± 2.82 rpm), which was measured 10 times by counting the number of revolutions per minute of the transducer inside an acrylic ring with internal radius of 2.68 ± 0.01 cm. When the physiotherapeutic equipment operates at 3 MHz, the ERA is 4.09 cm2. Immediately following the cessation of ultrasound irradiation (in 10 minutes), ShearWave™ Elastography was employed to record an E image. Over the first five minutes, an E image was captured every minute. From 15–40 min, an E image was saved every five minutes. Thereafter, an image was saved at 70, 100, and 130 min after the completion of irradiation with the physiotherapy equipment. Six circular Q-boxes with a diameter of 5 mm were positioned at the following depths: 0.4, 0.9, 1.4, 1.9, 2.4 and 2.9 cm.
Figure 2 summarizes the parameters (nominal frequency, nominal intensity, application time and transducer movement) used in the therapeutic ultrasound device, which was used to heat the bovine muscle. In addition, the instant of time that the elastographic images were acquired in each experiment were presented.
Two bovine muscle samples (7.0 × 3.0 × 12.0 cm) connected at one end by a thin layer of fat (< 0.3 mm) were used to study the maximum temperature and thermal field generated by physiotherapeutic ultrasound inside the samples with the same experimental configuration as that used for the SSI experiments, for tests 1 (application time of 2 min) and 2 (application time of 10 min and with transducer in a fixed position). These samples were placed parallel and in contact to each other, forming a unique sample with dimensions of 7.0 × 6.0 × 12.0 cm. Additionally, the physiotherapeutic transducer was positioned in the same position (top surface of the sample) as the transducer coupled to the ShearWave™ Elastography device that captured E images. An infrared camera lens (E6, FLIR® Systems Inc, Boston, MA, USA) was positioned parallel to the upper surface of the fresh bovine skeletal muscle sample, at a distance of 50 cm and the ambient temperature was 23.3 °C. Initially, the physiotherapeutic transducer was coupled to the upper face of the bovine muscle sample using clinical water-based gel and the muscle sample was placed on an acoustic absorber (rubber plate). So, the physiotherapeutic device was configured in a similar way to the first test performed with SSI, then the tissue irradiation was started. Immediately after the physiotherapeutic equipment completed ultrasonic irradiation, the bottom faces of the two samples (connected at one end by a thin layer of fat) were placed in contact and a thermal image was recorded by the infrared camera. This process took approximately 10 s (see Figure 3). In the static situation, the axis of symmetry of the ultrasonic transducer was positioned at the top between the two samples. In the dynamic situation, the center of the treatment area was positioned at the top between the two samples. Finally, the same procedure described above was performed with the physiotherapeutic ultrasound equipment configured in a similar way to test 2.
The thermal images also showed the thermal field inside the tissue, which can be related to information regarding E as a function of depth. These thermal images were obtained considering the configurations of the physiotherapeutic device used in the two tests performed with ShearWave™ Elastography device. In order to confirm the temperature value obtained with the infrared camera, the values obtained with this device were compared with the temperature values recorded by a digital thermometer (Model 52, Fluke, Everett, WA, USA). An ultrathermostat bath (524-2D, Nova Ética, São Paulo, SP, Brazil) was used to facilitate comparison of the temperature values obtained by the two devices. For this purpose, the ultrathermostat bath was set to maintain the temperature of its reservoir at values of 25, 30, 35, 40, 45, 50, 55, and 70 °C, and the water temperature was recorded five times for each thermal device. The temperature values measured with the infrared camera (TIC) and the digital thermometer (TDT) were applied in erro = [(TIC - TDT)/TDT] · 100 to estimate the relative error of each measurement. Finally, the mean and standard deviation of the relative error will be calculated.