Pulmonary Capillary Hemorrhage Induced by Super Sonic Shear Wave Elastography in Rats

Abstract

The occurrence of the pulmonary capillary hemorrhage (PCH) bioeffect of diagnostic ultrasound in rats was investigated for a SuperSonic Imagine (SSI) shear wave elastography system (Aixplorer, Supersonic Imagine, Aix en Province FR). The elastography imaging repeated at 1 Hz and consisted of widely spaced B mode image pulses, supersonic push (SSP) pulses and shear wave imaging (SWI) pulses. Groups of rats anesthetized with ketamine and xylazine, or with ketamine only, were imaged on their right side in a warm water bath for 1 frame, 30 s, and 300 s. The image focus and region-of-interest were adjusted to give exposure only with the background B mode imaging, or primarily with the SSP and SWI pulses. A sham group had only low power aiming scans. The lungs were removed 5 min after exposure and evaluated for PCH area and volume. The B mode was notably ineffective, and produced significant PCH only at the maximum 0 dB output. The SSP pulses together with the SWI pulses produced significant PCH for 300 s, 30 s and even single image exposures. Peak rarefactional pressure amplitude PCH thresholds for 300 s exposure were the same with or without the B mode pulses at 1.5 MPa (in situ Mechanical Index, MI_IS=0.67). A 30 s duration resulted in a slightly increased threshold of 1.7 MPa (MI_IS=0.76). The omission of xylazine from the anesthetic, which reduces the sensitivity of rat lung to PCH occurrence, resulted in an increased threshold of 2.1 MPa (MI_IS=0.94). The unique SSP pulses were much more effective than the B mode, but thresholds were comparable to previous results with other diagnostic ultrasound modes on other systems.

Introduction

Diagnostic ultrasound (DUS) imaging can induce pulmonary capillary hemorrhage (PCH) in mammalian lung (Child et al. 1990; Miller, 2012). This phenomenon has received attention in research designed to gauge the potential safety issue for patients who have pulmonary ultrasound examinations (AIUM 2000; Church et al. 2008). However, uncertainty remains with regard to the physical mechanism of PCH, the influence of diagnostic ultrasound exposure parameters and to the vulnerability of patients with different conditions. Direct pulmonary DUS examination is performed in various clinical settings for diagnosis of pneumonia, pulmonary edema, pulmonary embolism, pneumothorax, and other patient conditions (Sartori et al 2010; Volpicelli 2013; Lichtenstein 2014). The use of portable ultrasound machines allows DUS to be performed by the physician at the point of care for routine monitoring (Ahmad and Eisen 2015; Irwin and Cook 2016; Sekiguchi 2016). Research is needed to understand the etiology of DUS PCH and to clarify the potential risks for induction of PCH in patients.

Research on diagnostic ultrasound exposure of rats has shown that DUS machines can cause PCH, and display its occurrence in the B mode images as growing comet-tail artifacts (also known as B-lines) (Miller 2012). The PCH increased with ultrasonic output above an exposure response threshold. Different modes of ultrasound including B mode, M mode, color Angio Doppler and pulsed Doppler were found to have different PCH exposure-response characteristics (Miller et al. 2018). The new DUS mode of acoustical radiation force impulse (ARFI) elastography involves high energy “push-pulses” that move tissue and generate shear waves (Palmeri et al 2005; Palmeri and Nightingale 2011). Scanning the push-pulse and tissue motion allow formation of an image of tissue elastic responses.

PCH induction was examined for ARFI elastography frames with 5.7 MHz push-pulses (Acuson S3000, Siemens Medical Solutions USA, Mountain View, CA, USA)(Miller et al. 2019). Only a maximal exposure was available for the manually triggered ARFI elastography exposure for one image. The influence of positive pressure ventilation was tested because ventilation was previously found to greatly reduce the PHC effect (Miller et al. 2018b). 20 ARFI elastography exposures produced substantial PCH over the imaging scan plane, and even a single ARFI elastography exposure produced significant PCH relative to shams. Ventilation with positive end expiratory pressure of 8 cm H₂O produced about half the PCH area induced by elastography without PEEP. The PCH results for 20 ARFI elastography exposures were comparable to those from B mode or color Doppler mode exposure for 300 s; however, these modes delivered many more pulses for real-time imaging. The results suggested that the relatively long duration push-pulses were more effective for PCH induction than the pulses of other DUS modes.

DUS elastography is also performed using an improved method with novel supersonic push-pulses (Bercoff et al. 2004a). The push-pulses are delivered along an axis at a rate exceeding the shear wave velocity (hence supersonic). This perturbation generates a spreading shear wave in adjacent tissue that is followed in real time by very high frame rate (e. g. 3500 fps) B-mode imaging, and the method is termed shear wave elastography (SWE) (Bercoff et al. 2004b). The shear wave velocity is measured and mapped to form an image of shear elasticity. The two methods produce comparable results in elasticity phantoms (Dillman et al. 2017), but may be somewhat different in clinical practice (Woo et al. 2015).

In this study, an Aixplorer (Supersonic Imagine, Aix-en-Provence, FR) was used to examine the induction of PCH with the SWE method. The SWE method produces elasticity images at 1 fps without manual triggering, and allows for adjustment of ultrasonic output. This featured allowed determination of exposure-response thresholds for comparison to thresholds for other DUS modes. The pulses delivered for creation of an elasticity image included normal B mode background image pulses, the supersonic push (SS Push) pulses, and the fast shear wave imaging pulses (SW image). These different pulse sequences could be separated to some extent to identify the pulse type responsible for the PCH.

Materials and Methods

Animal preparation

All in vivo animal procedures were conducted with the approval and guidance of the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan. Female rats (Sprague Dawley, Charles River, Wilmington, MA, USA) were anesthetized by intraperitoneal injection of 91 mg/kg ketamine (Zetamine™ ketamine hydrochloride injection, MWI, Boise, ID, USA) plus 9 mg/kg IP xylazine (XylaMed™ xylazine injection, MWI, Boise, ID, USA). For some rats, the xylazine was omitted from the anesthetic mix to give a light anesthesia with reduced susceptibility to PCH. For ultrasound transmission, the right thorax was shaved and depilated. Each rat was mounted on a holding board in dorsal recumbent position, and the board was mounted vertically in a 38 °C degassed water bath for ultrasound imaging exposure. This allowed for accurate ultrasound imaging of the right lung by the ultrasound probe mounted in the water bath on a mechanical positioning gantry. The rats were all weighed and respiration was manually counted. Heart rate and SpO2 were measured with a pulse oximeter probe (SurgiVet V3395 TPR, Smiths Medical Inc. St Paul, MN USA) placed on a front paw. The physiological parameters measured at the time of exposure (mean ± standard deviation) were: weight 241 ± 14 gm, heart rate 288 ± 26 bpm, SpO₂ 75 ± 4.5 and respiration 67 ± 14.

Ultrasound

An Aixplorer diagnostic ultrasound machine (Aixplorer, Supersonic Imagine, Aix en Province FR) was used for aiming and imaging exposure using the SL15–4 probe. B mode imaging at 5% power was used with a 3 cm by 5 cm image area to aim through an intercostal space at the right cranial or medial lobe of the rat lung and obtain a pre-SWE image. For SWE imaging, the elastography image box was reduced to 1 cm by 1 cm at a 1–2 cm depth and 3 cm to 4 cm horizontal position. The box was normally placed over the lung image for exposure with the bright lung surface image at a 1.25 cm depth. This normal setup exposed the lung to pulse sequences associated with the B mode background image pulses, the SSP pulses and the SWI pulses. This setup is illustrated in Figure 1. This complex exposure setup presumably could induce PCH from all three pulse sequences. For this research, an effort was made to separately identify the PCH induction potential for each of the pulse sequences. For this purpose, the elastography image box was moved to the opposite side of the image area at a 1 cm to 2 cm horizontal position, avoiding lung exposure to the SSP and SWI pulses, and leaving exposure by only the B mode imaging pulses. Alternatively, the B mode focal zone, normally set to 1.2–1.7 cm, could be moved to 0.2–0.7 cm to greatly reduce the B mode exposure pulse amplitudes (by −8 dB) and thereby give exposure primarily from the SSP and SWI pulses. Furthermore, the distinct positioning of the SSP pulses to four lines spaced 5 mm apart allowed some separation of the effect from the SW image pulses for the region between the SSP pulse axes.

Figure 1.

A diagram illustrating the layout of the SWE image. The image area was 3 cm by 5 cm. The supersonic push pulses were directed along 4 axes spaced 5 mm apart. The square region of interest could be move and was set to 1 cm by 1 cm with the lung surface at 1.25 cm depth. The B mode focal zone was normally set to 1.2–1.7 cm depth but was moved up to reduce the amplitude of the B mode pulses at the lung. The shear wave imaging pulses spanned 1.5 cm across the scan plane.

For animal exposure, after aiming with a 5 % power setting, an ultrasound shield (cut from a thin foam plastic tray) was manually inserted between the probe and the rat to avoid any accidental exposure. After switching to SWE mode and setting the desired output power for exposure, the scan was “frozen”, the ultrasound shield was removed, and the scan was “unfrozen” for the desired exposure duration. At the end of the desired duration, the scan was “frozen” and the ultrasound shield again was inserted for readjustment to the 5% power B mode imaging. B mode images were recorded to assess any development of comet tail artifacts (CTA) in the image. The elastography image was color mapped for elasticity with a range of 100 kPa. The elasticity display was presumably valid for muscle layers, ribs and spine but were not meaningful when displayed inside the air-filled lung regions (these artifactual displays were due to multiple reflections).

The ultrasound pulse parameters were measured in the water bath using a calibrated hydrophone with a 0.2 mm sensitive spot (model HGL-0200, Onda Corp., Sunnyvale, CA), which was mounted in the water bath. The ultrasound probe was mounted on a micrometer positioning gantry and was moved within the scan plane to maximize the received signal. The hydrophone tip was visible in the B mode image and was located at the 1.25 cm depth corresponding to the surface of the lung. Pulse waveforms were digitized with an oscilloscope and transferred to a computer for applying the calibration factor and determining pulse characterization parameters. Figure 2 shows the signal received for an entire 1 s elastography sequence with the hydrophone at a SSP pulse axis. This sequence was expanded to shorter time spans to observe the details. Figure 3 details the four SWI scans which accompany the four SSP pulse axes. Each set had 42 groups of 3 pulses spaced 285 μs intervals (3509 frames per second) as shown in Fig. 4. The SSP pulses are detailed in Fig. 5. The four 154 μs duration pulses were closely spaced in time and focused at different depths to induce a push axis moving inward at a speed faster than the shear wave velocity (hence, supersonic), and generating an approximately cylindrical shear wave spreading from the push line (Bercoff et al. 2004a). The SWI at the rate of 3509 frames per s then follow the shear wave as it moves away.

Figure 2.

A plot of the hydrophone output in peak detect mode over the entire 1 s pulse sequence. The time interval marked by the red diamond line (lower left) is expanded in Fig. 3.

Figure 3.

A plot of the hydrophone output in peak detect mode over the time interval with the supersonic push pulses and shear wave imaging pulses. The time interval marked by the red diamond line (lower left) is expanded in Fig. 4.

Figure 4.

A plot of the hydrophone output in peak detect mode over the shear wave imaging (SWI) pulse accompanying the supersonic push pulses. The entire SWI pulses were in sets of 3 for each frame, with a frame rate of 3509 fps. The time interval marked by the red diamond line (lower left) is expanded in Fig. 5.

Figure 5.

A plot of the hydrophone output in peak detect mode showing the four 154 μs supersonic push (SSP) pulses. The SSP pulses are focused at different depths and yield different amplitudes at the position of the hydrophone.

Passage of the ultrasound through the 5 mm thick rat chest wall attenuates the pulse amplitudes. The water values of ultrasound pressure amplitudes were derated by a 1.2 dB/cm/MHz attenuation coefficient to estimate in situ values (Miller et al. 2015a). The pulses shapes observed in water showed substantial non-linear deviation from a simple sinusoidal shape, and therefore contain higher frequency harmonics. The derating method can involve simple linear reduction of the pulse, or complex Fourier analysis of the pulses with frequency-dependent reduction. Frequency-dependent derating is performed using the “wideband technique” of Schafer (1990). The recorded signal was decomposed into its frequency components via a fast Fourier transform and was then derated in the frequency domain. Finally, a derated time-domain signal was reconstructed by applying an inverse fast Fourier transform to the derated, frequency-domain signal. An attenuation coefficient of 1.2 dB/cm/MHz (reference frequency = 1 MHz), and a depth of 0.5 cm were used in the derating process. Calculations were performed using Matlab (Mathworks Inc., Natwick, MA). The modifications of the water-pulse shapes are presented in the supplemental material as Figure S1, S2 and S3 for B mode, SWI and SSP pulses for both methods. The use of frequency-dependent derating tends to preferentially reduce the sharp positive peaks due to their high frequency content; however, given the mixed water and tissue path, the most accurate method is not clear. The peak rarefactional pressure amplitude (PRPA) was the primary exposure parameter, and the PRPA values were about the same for the two derating methods. Therefore, linear derating was used for characterizing the exposures here, to allow straightforward comparison to results reported for previous studies. Pulse parameters are listed in table 1 for the three different pulse sequences. The derating reduction was −4.6 dB for the 7.6 MHz pulses and −3.0 dB for the 5.0 MHz pulses. In addition, the ultrasonic radiation surface pressure (P_RSa) was calculated from the derated I_SPPA intensity, as described in Miller (2016), and was 45 cm H₂O (4.4 kPa) during the maximal push-pulses.

Table 1.

Ultrasound exposure parameters associated with each of the three different pulse sequences included in the SSI elastography image generation. The B mode imaging provides the 3 cm by 5 cm background tissue image. The rapid shear wave imaging (SWI) pulses follow the shear waves emanating from the supersonic push (SSP) pulses. The ultrasound pulse frequency is the center frequency for a single ultrasound cycle. The pulse duration is the value from the determination of the spatial peak, pulse average intensity (I_SPPA). The repetition frequency is essentially the frames-per-second for each sequence. Linearly derated maximal (0 dB) pulse parameters, see the text, are given for each of the sequences.

Pulse Sequence	Frequency MHz	Durationμs	Repetition Hz	p+ MPa	p− MPa	ISPPAW cm−2	MIISMPa MHz−½
B Mode	7.6	0.18	12	4.5	2.8	338	1.0
SWI	7.6	0.73	3,509	2.8	1.9	118	0.69
SSP	5.0	154	1	6.2	3.3	512	1.5

Measured endpoints

The ultrasound images were used to assess PCH-related comet tail artifacts (CTAs), which approximate the actual size of PCH seen on the lungs (Miller, 2012). This was expressed as the percentage of the bright-line image of the lung surface, which was involved with comet-tail artifacts (CTAs) at 5 min post exposure, both measured using the simple distance measurement tool of the ultrasound machine. After the rat was euthanized, the trachea was tied off, and the lungs were removed. The exposed right lungs were then examined and photographed using a stereomicroscope with digital camera (Spot Flex, Diagnostic Instruments Inc., Sterling Heights, MI USA). The photographs of the lungs were used to measure the approximate PCH areas. PCH areas on the lung surface were measured by manual outline of the PCH region on the photographs, using image analysis software (Spot v. 5.1, Diagnostic Instruments, Inc., Sterling Heights, MI USA). The lungs were fixed in neutral buffered formalin for a minimum of one week. As described previously (Miller et al. 2016), the fixed lungs were used to estimate the depth of the larger PCH regions and, together with the surface area measurements, the volume of the hemorrhage region. Depths were measured in 2 mm intervals by slicing 2 mm-thick sections and measuring under the stereomicroscope. The mean depth of a PCH region was then multiplied by the PCH area measured in fresh tissue to obtain an estimate of the PCH volume.

Experimental plan and statistics

The SSI machine allowed adjustment of the output power, including that of the SSP pulses. The experimental plan was set to determine the thresholds for PCH using groups of rats exposed to output power steps −2 dB apart (i. e. 0 dB, −2 dB, −4 dB, etc.) until a low step had no significant effect. This process was performed for the normal, B mode only and reduced B mode exposures described above (Table 1) for 300 s duration. In addition, thresholds were determined for the normal condition for 30 s duration, and for one single elastography sequence. An additional test was performed to determine the influence of anesthesia without xylazine (using ketamine only) on the PCH induction, which had been shown previously to lead to reduced PCH impact (Miller et al. 2015b). Each test condition was performed in 5 rats except, 8 rats were tested using the 0 dB B mode only condition, and 6 rats were tested at the −4 dB output for normal elastography and 30 s duration. A sham condition was performed in 7 rats at the 0 dB setting for 300 s but with the B mode focus moved to 0.2–0.7 cm and the elastography box moved to 1–2 cm. Statistical significance of the results was determined using the simple t-test against shams (p<0.05), which gave the same result as an occurrence determination using the Z test with Yates correction. Linear regression on the means was utilized to estimate the threshold value as the zero-crossing point as described previously (Miller et al. 2018a).

Results

The SSI elastography was tested in the normal setup and proved to be highly effective for PCH induction. Figure 6 shows the ultrasound image before and after 300 s of elastography (0 dB) with the bright lung surface reflection showing CTAs after scanning. The fresh lung image in Fig. 6 reveals substantial PCH that is concentrated in spots spaced 5 mm apart (the spacing of the SSP pulse lines (Fig. 1), but connected with somewhat less severe PCH areas. The SSP pulse axes were spaced 5 mm apart and explain the 5 mm spacing of the high impact PCH spots. The hemorrhages extended to the posterior of the medial lobe as shown in Fig. 7. Results for the bioeffect endpoints are listed in Table 2, and the PCH areas are plotted in Figure 8. The thresholds, determined by linear regression as the PRPA intercept for the PCH areas, are listed in Table 3. For the SWE plus B mode the threshold was 1.5 MPa. B mode images and the resulting PCH are shown in Fig. 9 for one of the positive results for the −8 dB setting. Small spots of PCH appeared in the lung image between the two larger spots, which were spaced 5 mm apart and likely were due to the SSP pulses. The PCH occurrence between the SSP axes even at −8 dB seems to indicate an important role for the SWI pulses in the overall PCH impact.

Figure 6.

The low power aiming B mode images before (top) and after (middle) the 300 s of elastography imaging in the normal configuration at 0 dB. The middle image shows the comet tail artifacts arising from the pulmonary capillary hemorrhage (PCH) induced by the imaging pulses. The bottom image is a photograph of the cranial and medial lobes with the PCH visible as the very red blood filled regions (scale 5 mm).

Figure 7.

Photographs of the lung shown in Fig. 6 with the cranial lobe moved to reveal the medial lobe PCH (left) (scale 5 mm) and with the medial lobe moved to show the anterior side, which reveals the penetration of the PCH to the back of the lobe.

Table 2.

Pulmonary capillary hemorrhage results for diagnostic ultrasound scanning in the SSI elastography mode: CTA %, the percentage of CTAs in the lung surface image. The volume of PCH was estimated for the higher impact conditions. Stats MWRST

Condition	n	Durations	PRPA MPa	CTA %	PCH Proport	Area mm2	Signif. p	Volume μL
Norm 0 dB	5	300	3.2	95 ± 4	5/5	63 ± 26	0.003	164 ± 79
Norm −2 dB	5	300	2.7	84 ± 3	5/5	32 ± 8	0.003	66 ± 30
Norm −4 dB	5	300	2.2	60 ± 12	5/5	13 ± 11	0.003	23 ± 22
Norm −6 dB	5	300	1.7	38 ± 37	5/5	8.4 ± 6.4	0.003	20 ± 17
Norm −8 dB	5	300	1.4	6.7 ± 8.9	3/5	1.2 ± 1.5	0.11	1.3 ± 1.7
Norm 0 dB	5	30	3.2	54 ± 20	5/5	11.7 ± 7.1	0.003	22 ± 24
Norm −2 dB	5	30	2.7	38 ± 26	5/5	11.1 ± 9.7	0.003	13 ± 21
Norm −4 dB	5	30	2.2	18 ± 17	4/5	2.1 ± 1.6	0.018	0.8 ± 1.9
Norm −6 dB	6	30	1.7	8 ± 15	2/6	0.4 ± 0.9	0.37	0
Norm 0 dB	5	1	3.2	5 ± 5	4/5	1.1 ± 1.1	0.018	0
Low B 0 dB	5	300	3.2	78 ± 29	5/5	66 ± 21	0.003	139 ± 51
Low B −2 dB	5	300	2.7	91 ± 14	5/5	34 ± 14	0.003	61 ± 37
Low B −4 dB	5	300	2.2	70 ± 20	5/5	24 ± 10	0.003	44 ± 17
Low B −6 dB	5	300	1.7	30 ± 16	5/5	3.8 ± 3.5	0.003	11 ± 14
Low B −8 dB	5	300	1.4	5 ± 7	2/5	0.4 ± 0.7	0.27	0
B only 0 dB	6	300	2.8	10 ± 10	4/6	1.8 ± 3.1	0.051	0
B only −2 dB	5	300	2.6	3 ± 7	2/5	0.9 ± 1.7	0.27	0
KO 0 dB	5	300	3.2	48 ± 30	5/5	38 ± 14	0.003	69 ± 22
KO −2 dB	5	300	2.7	45 ± 29	4/5	18 ± 12	0.018	40 ± 31
KO −4 dB	5	300	2.2	9 ± 13	3/5	2.7 ± 3.5	0.11	4.3 ± 5.8
Sham	7	300	1.1	0	0/7	0	-	0

Figure 8.

A plot of the PCH areas measured on the fresh lung surfaces after exposure to the elastography imaging pulses. The lines represent linear regressions on the means which approximate the PCH thresholds at the zero crossing point (Table 3).

Table 3.

Results for the linear regressions plotted in Fig. 8. The zero crossing values of the peak rarefactional pressure amplitude approximate the threshold for each condition. The thresholds for SWE with or without the B mode imaging were the same, and about the same for 300 s or 30 s of SWE imaging. The use of ketamine alone for anesthesia resulted in a higher threshold. The value of MIIS was calculated based of the 5 MHz frequency for the supersonic push pulses.

Pulse Sequence	Durations	Intercept mm2	Slope mm2/MPa	r2	Threshold MPa	MIISMPa MHz−½
SWE+B	300	−51.6	33.4	0.72	1.5	0.67
SWE	300	−56.3	36.4	0.80	1.5	0.67
SWE+B	30	−14.6	8.5	0.42	1.7	0.76
SWE (KO)	300	−71.6	33.9	0.67	2.1	0.94

KO, ketamine only anesthesia

Figure 9.

The low power aiming B mode images before (top) and after (middle) the 300 s of elastography imaging in the normal configuration at −8 dB. The middle image shows two small comet tail artifacts arising from the pulmonary capillary hemorrhage (PCH) induced by the supersonic push pulses. The bottom image is a photograph of the cranial lobe with the PCH visible as the small red spots. (scale 5 mm).

The B mode imaging was notably ineffective at causing PCH. Figure 10 shows a lung after exposure with only the B mode at the maximum output of 2.8 MPa (B only 0 dB). The PCH occurrence was insignificant for −2 dB. When the SWE was set up with greatly reduced B mode (focal zone at the top of the image), the results were about the same as for the SWE plus normal B mode, which shows that the B mode pulses were not important in the overall PCH impact. Figure 11 shows a lung for exposure with SWE and reduced B mode, which is very similar to the SWE plus normal B mode shown in Fig. 6. The thresholds (Table 3) were identical for the SWE exposure with normal or reduced B mode imaging. Final SWE elasticity images after 5 min for the three conditions of SWE plus B mode, B mode only and SWE plus reduced B mode are shown in Fig. 12. These illustrate the different configuration of the SWE window and the B mode focal zone. A low elasticity value (blue color) was displayed in the upper parts of the window, due to the presence of some intercostal tissue in the SWE box.

Figure 10.

The low power aiming B mode images before (top) and after (middle) the 300 s of elastography imaging by the B mode only at 0 dB. The middle image shows the comet tail artifacts arising from the pulmonary capillary hemorrhage (PCH) induced by the imaging pulses. The bottom image is a photograph of the cranial lobe with the PCH visible as a few small red spots (scale 5 mm).

Figure 11.

The low power aiming B mode images before (top) and after (middle) the 300 s of elastography imaging with the B mode focal zone moved to the top of the elastography image. The middle image shows the comet tail artifacts arising from the pulmonary capillary hemorrhage (PCH) induced by the imaging pulses. The bottom image is a photograph of the cranial and medial lobes with the PCH visible as the very red blood filled regions (scale 5 mm).

Figure 12.

The elasticity images for the normal configuration (top) for the PCH shown in Fig. 6, for the region of interest (and the SSP and SWI pulses moved to the left) to give exposure from the B mode pulses only (middle) for the PCH shown in Fig. 10, and the reduced B mode configuration with the B mode focal zone raised to the minimum depth for the PCH shown in Fig. 11. Low elasticity results were shown in blue.

Reduction of the normal SWE plus B mode exposure duration of 300 s to 30 s reduced the overall PCH impact. An example of the B mode images and PCH effect is shown in Fig. 13. Although the impact was much less for this reduced duration exposure, the threshold of 1.7 MPa was about the same. Exposure from a single SWE image produced at least one small spot of PCH each for each test (n=5). One result is shown in Fig. 14: a minimal CTA effect was evident in the B mode image for one of the two small PCH spots, which were spaced 5 mm apart (the SSP pulse axis spacing).

Figure 13.

The low power aiming B mode images before (top) and after (middle) the 30 s of elastography imaging with the normal configuration. The middle image shows the comet tail artifacts arising from the pulmonary capillary hemorrhage (PCH) induced by the imaging pulses. The bottom image is a photograph of the cranial and medial lobes with the PCH visible as the red blood filled regions, with other PCH regions on the medial lobe hidden by the cranial lobe (scale 5 mm).

Figure 14.

The low power aiming B mode images before (top) and after (middle) one elastography image in the normal configuration. The middle image shows small perturbations of the lung surface image (which would lengthen into comet tail artifacts for longer exposures). The bottom image is a photograph of the cranial lobe with two small red spots 5 mm apart, which were produced by the SSP pulses (scale 5 mm).

Finally, the normal SWE plus B mode condition was tested for anesthesia with only ketamine. The omission of xylazine reduced the PCH substantially, but not as much as the reduction for the B mode exposure in a previous study (Miller et al. 2015b). The B mode and lung images for this condition at 0 dB are shown in Fig. 15. The SSP pulse axes appeared more prominently for this condition. The lung image in Fig. 15 includes the typical miss-alignment of the scan plane during photography. Figure 16 is another photograph of the same lung held vertically to approximately re-align the lobes as they were positioned in vivo, which clearly shows the four spots at the four positions of the SSP axes (illustrated in Fig. 1).

Figure 15.

The low power aiming B mode images before (top) and after (middle) the 300 s of elastography imaging with the normal configuration for a rat having anesthesia by ketamine only. The middle image shows four comet tail artifacts spaced 5 mm apart, arising from the pulmonary capillary hemorrhage (PCH) induced by the SSP pulses. The bottom image is a photograph of the cranial and medial lobes with the PCH visible as the very red blood filled regions spaced 5 mm apart (scale 5 mm).

Figure 16.

A photograph of the fresh lung presented in Fig. 15 but suspended by the trachea to show the alignment of the scan plane and PCH approximately as they were in vivo.

Discussion and Conclusion

The occurrence of the PCH bioeffect was investigated for the SSI shear wave elastography system. The SWE image-exposures, repeated at 1 fps, consisted of widely spaced B mode image pulses, SSP pulses and SWI pulses. The B mode was notably ineffective, and gave significant PCH only at the maximum 0 dB output. The SSP pulses together with the SWI pulses produced significant PCH for 300 s, 30 s and 1 image exposures (Table 2). Thresholds for 300 s exposure were the same with or without the B mode pulses at 1.5 MPa PRPA (MI_IS=0.67). A 30 s duration resulting in a slightly increased threshold of 1.7 MPa (MI_IS=0.76). The separate effects of the SWI and SSP pulses could be gauged at least qualitatively in the space in the scan plane between the four SSP pulse axes (Fig. 9), and appeared to occur with about the same threshold. The SWI pulses had a much shorter duration (0.73 μs compared to 154 μs) but there were many more of them in an image sequence due to the high frame rate of 3,509 fps. The omission of xylazine from the anesthetic, which reduced the sensitivity of rat lung to PCH occurrence, resulted in an increased threshold of 2.1 MPa (MI_IS=0.94) for 300s exposures.

The exposures were different from the previous study of an ARFI elastography system (Miller et al. 2019) in two ways: the SSI imaging exposure repeated at 1 fps and generated 5 MHz SSP pulses along 4 axes (5 mm apart), while the ARFI system repeated at a manually triggered interval of about 15 s and 94 ARFI push pulses at 5.7 MHz were scanned. The SSI exposure was more effective with a maximum 66 ± 21 mm² PCH area compared to 36.9 ± 9.2 mm² for the ARFI system. This difference was likely due to the greater number of SWE image-exposures in 300 s: 300 for the SSI system and 20 for the ARFI system. The ARFI system output was fixed and no threshold was determined.

The SSP pulses appeared to represent a unique and high level diagnostic ultrasound exposure. Four 154 μs duration pulses of 3.3 MPa PRPA were delivered along 4 separate axes every second. The SSP pulses can generate fountains and atomization at an air-water interface (Patterson and Miller, 2019), and the SWE mode was much more effective than the B mode on this system. However, the PRPA threshold for PCH was comparable to previous results with other diagnostic ultrasound modes on other systems. In a previous study of different DUS modes (Miller et al. 2018), the 6.6 MHz B mode used 0.27 μs pulses with a 3.3 MPa maximum PRPA for 60 fps imaging. The PCH area for 300 s of exposure was 45 ± 17 mm² and the threshold was 1.5 MPa (MI_IS = 0.58), values remarkably similar to the SSI results. Furthermore, the pulsed Doppler mode with 1.13 μs pulses and 6.8 kHz PRF aimed along one axis was significantly more effective with a threshold of 0.6 MPa (MI_IS = 0.24). The PCH results were greater for anesthesia with ketamine only compared to previous results with a different system (Miller et al. 2015b), but nevertheless were reduced from the results for ketamine and xylazine anesthesia (Table 3). Overall, the elastography systems, designed to comply with the present DUS regulation by the Food and Drug Administration, do not appear to present a clearly increased risk in terms of the PRPA thresholds, compared to other DUS modes on other systems, but appear to be more effective. The SSI SWE system operates at 1 fps and produced an impact equal or greater than a B mode running at 60 fps (Miller et al. 2018). Even one SSI elastography frame produced small PCH areas (Fig.14).

For most patients having an exam with an elastography system, the risk of PCH seems low because the air-filled lungs would not be directly exposed (and does not seem to give a useful elastography image, see Fig. 12). For some patients, however, pulmonary assessment for elasticity changes using ARFI or SSI elastography in consolidated regions of the lung at the level of the visceral pleura (e. g. tuberculosis or cancer) may be of diagnostic value (Wei et al. 2018), and may present some risk of PCH. Other methods of elasticity assessment may be prudent for these patients. Pulmonary elasticity can be tested by stress/strain methods not involving high energy push-pulses for impulsive displacement generation (Lim et al. 2017; Zhang et al. 2016; Sperandeo et al. 2015; He et al. 2017), which may involve a reduced risk of PCH induction. The PCH induced by SSI shear wave elastography reported herein may be avoided simply by utilizing different assessment methods for pulmonary elastography examination with lower ultrasound exposure.