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. Author manuscript; available in PMC: 2023 Nov 1.
Published in final edited form as: Ultrasound Med Biol. 2022 Aug 25;48(11):2276–2291. doi: 10.1016/j.ultrasmedbio.2022.06.020

Lung Ultrasound Induction of Pulmonary Capillary Hemorrhage in Neonatal Swine

Douglas L Miller 1, Chunyan Dou 1, Zhihong Dong 1
PMCID: PMC9942946  NIHMSID: NIHMS1874469  PMID: 36030131

Abstract

This study investigated induction of pulmonary capillary hemorrhage (PCH) in neonatal pigs (piglets) using three different machines: a GE Venue R1 point-of-care system with C1–5 and L4–12t probes, a GE Vivid 7 Dimension with 7L probe and a Supersonic Imagine machine with SL15–4 probe and shear wave elastography (SWE). Female piglets were anesthetized and each was mounted vertically in a warm bath for scanning at 2–3 intercostal spaces. After aiming at an innocuous output, the power was raised for a test exposure. Hydrophone measurements were used to calculate in situ values of Mechanical Index (MIIS). Inflated lungs were removed and scored for PCH area. For the C1–5 probe at 50% and 100% acoustical output (AO), a PCH threshold of 0.53 MIIS was found by linear regression (r2=0.42). The L4–12t probe did not induce PCH, but the 7L probe induced zones of PCH in the scan planes. The Venue R1 automated B-line tool applied with the C1–5 probe did not detect PCH induced by the C1–5 probe as B line counts. However, when PCH induced by C1–5 and 7L exposures were subsequently scanned with the L4–12t probe using the automated tool, B-lines were counted in association with the PCH. The SWE induced PCH at push-pulse positions for 3 s, 30 s and 300 s of SWE with PCH accumulating at 0.33 mm2/sec and an exponential rise to maximum of 18.4 mm2 (r2=0.61). This study demonstrated induction of PCH by LUS of piglets, and supports the safety recommendation for use of MI < 0.4 in neonatal LUS.

Keywords: Pulmonary ultrasound, B lines, Bioeffects of ultrasound, Ultrasound dosimetry, Diagnostic ultrasound safety

Introduction

Pulmonary capillary hemorrhage (PCH) is a well-known bioeffect of laboratory pulsed ultrasound with single-element transducers activated by function generators and power amplifiers. The induction of PCH by pulsed ultrasound was first reported in 1990 (Child et al. 1990) in mice exposed by a laboratory pulsed ultrasound system. This finding led to significant research attention using pulsed ultrasound exposure of mice and rats (Church et al. 2008). A worst-case threshold was estimated for the Mechanical Index (MI) of exposure at 0.4, well below the diagnostic ultrasound upper limit of MI=1.9. However, at that time, lung ultrasound (LUS) was not thought to be of value clinically, because the nearly total reflection of the ultrasound beam at the tissue-air boundary surface of healthy lungs prevents true images of the pulmonary interior. Only multiple reflection artifacts are displayed beyond the surface. The potential patient risk of PCH posed by actual diagnostic ultrasound exposure remained poorly understood.

Substantial research progress has been made in assessing PCH from diagnostic ultrasound machines in rats, after an initial study of PCH induction by 7.6 MHz B mode ultrasound imaging in rats (Miller, 2012). PCH occurred above a measured in situ MIIS=0.44. Importantly, the ongoing induction of the bioeffect was evident in the diagnostic ultrasound images as growing B-lines (also called comet tail artifacts) at the pleural surface. Research has subsequently revealed an often puzzling range of exposure-response results with ultrasound frequency (Miller et al. 2015), scan duration (Miller et al 2016a), ultrasound modes (Miller et al. 2018a, Miller et al. 2019a, Miller et al. 2019b), and other physical parameters. The physical mechanism for PCH induction remains uncertain (Miller, 2016). Physiological conditions also have profound influences on the PCH, thus complicating the interpretation of results for patients. The bioeffect was enhanced by some anesthetic techniques (Miller et al. 2015c), sedation (Miller et al. 2016b), negative end expiratory pressure (Miller et al. (2018b) or supplemental oxygen (Miller et al. 2020a) in ventilation. In contrast, PCH was diminished by saline infusions (Miller et al. 2018a), hemorrhagic shock (Miller et al. 2021) and positive end expiratory pressure in ventilation (Miller et al. (2018b). The thresholds and magnitudes also depend on animal age (Dalecki et al. 1997; O’Brien et al. 2003).

Revolutionary developments in clinical LUS have completely reversed the perception of the clinical value of LUS. Thoracic ultrasound had been used for many years to evaluate pulmonary conditions, such as pulmonary embolism or pleural effusion for thoracentesis. However, clinical value has been found also for the LUS image artifacts (Lichtenstein et al. 1997). LUS is now commonly used for clinical pulmonary examinations (Ahmad and Eisen, 2015; Dietrich et al. 2017) and is valuable in the diagnosis of pneumonia, pulmonary edema, pulmonary embolism, atelectasis, diffuse parenchymal disease, respiratory distress syndrome, and lung cancer (Sartori and Tombesi, 2010; Soldati et al 2019)). LUS is often used with curved or linear array probes operated in B mode. Various ultrasound elastographic methods are also used to assess lung stiffness in fibrosis, pulmonary edema and lung lesions (Zhou et al. 2020; Liu et al 2021). The ultrasound images of the lung surface often are examined for the presence and configuration of B-lines, which extend inward from the bright reflection of the pleura (A lines are multiple reflections displayed parallel to the lung surface). The B-lines are indicative of edema or interstitial lung disease (Copetti et al. 2008; Ahmad and Eisen, 2015). The appearance of the B-lines is somewhat subjective and depends on the machine settings (Matthias et al. 2020). However, these distinctive image signs are amenable to quantitative scoring, for example, by simple manual counting of lines. The scoring has even been automated using image analysis methods (Brattain et al. 2013; Brusasco et al. 2019; Short et al. 2019; Baloescu et al. 2021).

The assessments of B-lines and other image features are valuable particularly in neonatal examinations because of the simplicity of point-of-care LUS, the avoidance of ionizing radiation and the presumed safety of LUS (Lichtenstein and Mauriat 2012). Neonatal LUS is valuable for diagnosis of respiratory distress syndrome (Chen et al 2017), assessing surfactant treatment (Oktem et al 2019; Razak and Faden 2020), and pneumothorax (Liu et al 2017). The clinical value of LUS in newborns has been enhanced through use of quantitative scoring methods (De Martino et al. 2018; Corsini et al. 2020; Mongodi et al. 2021) and scores are associated with later clinical outcomes (Raimondi et al. 2021; Gunes et al. 2022).

The potential for induction of PCH in neonates may be greater than for adults with thicker chest walls (Patterson and Miller, 2020), owing to the lower ultrasonic attenuation. However, the intercostal spaces of human neonates typically are thicker than those of the adult rats commonly used for LUS PCH research, with the exception of some pre-term newborns). The possible induction of PCH by laboratory pulsed ultrasound in neonatal swine was investigated early on as a more pertinent model of human LUS. Baggs et al. (1996) performed PCH testing on 1 d old piglets using 2.3 MHz focused ultrasound with 10 μs pulse duration and 100 Hz pulse repetition frequency (PRF). Exposures were performed with the piglet in a warm water bath at several positions for 16 min exposure durations. Some motion of the animal was noted, possibly reducing the dwell time at any fixed point. A threshold was located at a peak rarefactional pressure amplitude (PRPA) of 0.9 MPa in situ (water measurements adjusted for chest wall attenuation to approximate the value at the lung surface), corresponding to an in situ MIIS of 0.6. This group also studied 10 d old piglets under similar conditions and obtained an in situ threshold of 0.6 MPa (MIIS=0.46) (Dalecki et al, 1997). O’Brien et al. (2003) studied 5 d old piglets, weighing 2.2 kg, using 3.1 MHz focused ultrasound with 1.2 μs pulse duration and 1 kHz PRF for 10 s exposure duration. An in situ occurrence threshold of 2.7 MPa was determined for neonatal pigs, corresponding to MIIS=1.5. The threshold results of this study seem quite different from those of (Dalecki et al. 1997), but the threshold difference might be attributable primarily to the substantial differences in pulse duration and exposure duration. Another important observation was that older pigs (58 d) were found to have a low in situ occurrence threshold of 1.2 MPa, corresponding to MIIS = 0.7.

This study was designed to investigate LUS induction of PCH in piglets using clinical diagnostic ultrasound systems for exposures. Three different machines were employed, a GE Venue R1 point-of-care system with automated B line scoring, a GE Vivid 7 dimension with simple linear array probe and a Supersonic Imagine Aixplorer machine with elastography capability. The study primarily sought the occurrence of PCH, or not, with these clinical machines. The influence of physical parameters of exposure, including acoustic output and duration, was also evaluated.

Methods

Neonatal animal model

All in vivo animal procedures were conducted with the approval and guidance of the Institutional Animal Care and Use Committee, University of Michigan, Ann Arbor, MI. Female piglets (neonatal pigs) were acquired from Michael Fanning Farms (Howe, IN) or Oak Hill Genetics (Ewing, IL) and maintained in the animal housing rooms of the Unit of Laboratory Animal Medicine, University of Michigan. Seven piglets that were used for preliminary testing or had problems in testing, were excluded from the study. The 34 piglets included in this study were 8.8 ± 1.8 d old and weighed an average of 2.7 ± 0.5 kg at the time of testing. Anesthesia was induced in the housing area by injection of Telazol (6 mg/kg) plus Dexdomitor (Dexmedetomidine, 0.1 mg/kg) or Xylazine (4.4 mg/kg) IM and maintained with supplemental Ketamine (20 mg/kg) plus Dexdomitor (0.1 mg/kg) or Xylazine (4.4 mg/kg). In a rat study, Dexmedetomidine (used clinically) and Xylazine (used in veterinary practice) appeared to be essentially interchangeable in the LUS PCH research (Miller et al. 2016).

In preparation for ultrasound, the anesthetized piglets were shaved and depilated over the right thorax. Each piglet was mounted on a metal frame using Velcro strips and soft belts. The piglets were mounted vertically in a 38–39 °C physiological saline bath with immersion to the shoulder level. Breathing was spontaneous, and no supplemental oxygen or ventilation was used. The saline bath setup allowed precise aiming of the diagnostic ultrasound probes, and maintenance of the body temperature of the anesthetized piglets. Heart rate and SpO2 were measured with a pulse oximeter probe (SurgiVet V3395 TPR, Smiths Medical Inc. St Paul, MN USA) on the tongue. At testing, the average heart rate was 137 ± 39 bpm, SpO2 was 89 ± 5 % and respiration was 85 ± 12 bpm.

Five min after scanning, the piglet was sacrifice by exsanguination at the abdominal vessels, the trachea was tied closed, and the inflated lungs removed for examination. The fresh lungs were photographed for measurement of PCH on the LUS scanned lobes using a stereomicroscope with digital camera (Spot Flex, Diagnostic Instruments Inc., Sterling Heights, MI USA). The PCH effect appeared as bright red spots or zones and was limited to the scan plane, with other (essentially sham-exposed) lung outside the ultrasound beam area unaffected. The PCH areas were outlined manually in the photomicrographs for quantification to obtain the calibrated PCH area using image analysis software (Spot v. 5.1, Diagnostic Instruments, Inc., Sterling Heights, MI USA).

Ultrasound

Three different diagnostic ultrasound machines were employed. A GE Venue R1 point-of-care system (GE Medical Systems, Ultrasound & Primary Care Diagnostics LLC, Wauwatosa, WI USA) with automated B line scoring, was used with the C1–5 and L4–12t probes. This system has been used clinically with the C1–5 probe (Short et al. 2019). The B line counting tool required a depth of 12 cm for the C1–5, and 8 cm for the L4–12t. A B-line clearly extending toward the bottom of the image was needed for the automated tool to accept a perturbation in the lung surface image as a B-line count. A GE Vivid 7 Dimension (GE Ultrasound AS, Horten, Norway) with 7L linear array probe, that had been used previously in a rat PCH study (Miller et al. 2015), was used as an alternative to the Venue L4–12t probe, which produced puzzling results. Finally, a SuperSonic Imagine Aixplorer machine (SuperSonic Imagine, 13857 Aix-en-Provence (France)) with shear wave elastography (SWE) capability was used with the SL15–4 probe. It should be noted that the acronym SWE is used for two types of elastography system (Zhou et al. 2020); the shear wave elastography ultrasound push pulse system for generating shear waves and measuring them inside the tissue (Liu et al 2021) and the surface wave elastography system generating a lung surface wave with a transthoracic shaker and measuring the surface waves (Zhou et al. 2019). In this study SWE refers to the Aixplorer push-pulse system, that also was previously used for a study of PCH generation in rats (Miller et. al. 2019). Ultrasound parameters for these systems are listed in Table I.

Table 1.

Ultrasound parameters used for the three ultrasound systems for separate intercostal spaces. Probe designations of a, b or c indicate different conditions in different ICS.

US Depth Focus Lung Freq. Output Duration Pulse PRP FPS
System Probe cm cm cm MHz AO, dB s ns μs Hz
Venue C1–5 a 12 4.9 3.5 3.1 100 % 300 797 192 121
C1–5 b 12 4.9 3.5 3.1 50 % 300 797 192 121
C1–5 c 6 3.6 1.5 3.1 100 % 300 697 114 204
Venue L4–12t a 8 2.4 1.2 7.2 100 % 300 227 611 29
L4–12t b 3 2.4 1.2 7.2 100 % 300 228 219 76
L4–12t c 3 2.4 1.2 7.2 100 % 30 228 219 76
Vivid 7 7L a 3 1.6 1.5 4.8 0 dB 300 413 67 74.7
7L b 3 1.6 1.5 4.8 0 dB 300 413 160 32.1
SSI SL15–4 a 3 0.4–2.2 1.2 5.0 0 dB 300 153000 164 1
SL15–4 b 3 0.4–2.2 1.2 5.0 0 dB 30 153000 164 1
SL15–4 c 3 0.4–2.2 1.2 5.0 0 dB 3 153000 164 1
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PRP, pulse repetition period; FPS, frames per second.

To maximize information and minimize use of piglets, an exposure was performed in each of two or three adjacent intercostal spaces (ICS) which were ~1 cm apart, starting at the most superior ICS with an acceptable image. The intervening rib isolated the adjacent exposures and all spaces usually presented a similar ultrasound view. However, the third ICS had an intrusive partial view of the diaphragm and liver in some piglets, and these third ICS tests were excluded from the study. A final study group of 8 piglets (see below) were limited to 2 ICS. Each exposure testing day, one probe setup was used for 2 piglets, and changed to another probe setup for the next day. The specific exposures for each ICS are listed in Table I.

The probes were held with a gimbal mount in the heated water bath filled with vacuum degassed saline. Exposimetric parameters are given in Table 2. The on-screen MIOS, an indicator of pulse-peak exposure, and TIOS, related to temporal average intensity and heating during exposure, are listed for each test condition. The ultrasound pulse parameters were measured in the exposure water bath using a calibrated hydrophone with a 0.2 mm sensitive spot (model HGL-0200, Onda Corp., Sunnyvale, CA). The image depth position of the maximum pulse amplitude was located from the saline bath measurements and used for the position of the lung surface during exposure. The measurements were used to calculate the pulse duration, peak rarefactional pressure amplitude (PRPA), MI (PRPA divided by the square root of the fundamental pulse frequency f) and to estimate the spatial peak pulse average intensity (ISPPA). These ultrasound exposimetric parameters are listed in Table 2. The saline measurements were derated by an attenuation factor to estimate the in situ exposure values at the lung surface. The chest wall thickness (CWT) was measured for each piglet during exposure and the average obtained for each probe is listed in Table 2. The CWT was similar to human neonatal CWT. The chest wall attenuation was found from the frequency dependent attenuation coefficient A determined by Miller RJ et al. (2002) to be A=1.94f0.090, which ranged from 1.6 dB/cm to 1.7 dB/cm. The derating value in -dB was found for each probe condition as the product of CWT times the value of A, are listed in Table 2. The attenuation was substantial; for example, the 7L probe at 4.8 MHz, 0.8 cm CWT and −6.4 dB attenuation gave a PRPA reduction factor of 0.48 and an ISPPA reduction factor of 0.23.

Table 2.

Ultrasound exposimetric parameters measured for the three ultrasound systems and probes for separate intercostal spaces (a,b,c).

US On Screen Freq. CWT Atten PRPASV ISPPASV PRPSIS ISPTAIS In situ
System Probe MIOS TIOS MHz cm dB MPa W/cm2 MPa W/cm2 MIIS
Venue C1–5 a 1.4 0.4 3.1 0.92 5.4 3.3 352 1.8 113 1.0
C1–5 b 1.0 0.2 3.1 0.92 5.4 2.3 176 1.3 56 0.73
C1–5 c 1.1 0.4 3.1 0.92 5.4 2.6 221 1.5 71 0.84
Venue L4–12t a 1.4 0.1 7.2 0.78 8.9 4.1 622 1.5 81 0.54
L4–12t b 1.4 0.4 7.2 0.78 8.9 4.8 859 1.7 112 0.86
L4–12t c 1.4 0.4 7.2 0.78 8.9 4.8 859 1.7 112 0.86
Vivid 7 7L a 1.2 1.7 4.8 0.80 6.4 3.9 700 1.9 161 0.86
7L b 1.2 0.7 4.8 0.80 6.4 3.9 700 1.9 161 0.86
SSI SL15–4 a 1.6 0.9 5.0 0.83 6.9 4.8 996 2.2 209 1.0
SL15–4 b 1.6 0.9 5.0 0.83 6.9 4.8 996 2.2 209 1.0
SL15–4 c 1.6 0.9 5.0 0.83 6.9 4.8 996 2.2 209 1.0
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OS, on screen value, SV, saline value; IS, in situ value.

Experimental Plan and Statistics

The exposure method progressed by obtaining a good LUS view at an innocuous output level, then raising the level quickly and manually timing of the desired exposure duration before lowering the output level again. The Venue system aiming was performed at 4 % acoustic output (AO). For the Vivid 7 Dimension, aiming was performed at −12 dB. The Aixplorer machine was aimed in B mode at −15 dB, then switched to SWE mode at 0 dB. Images were recorded before, during and after the exposure period. Visually identified B-lines were observed on the final images and automated B line scores were noted when counted by the B-line tool. The actual counts were variable depending on the image clip used and the image changes from lung sliding; therefore, the counts were not taken as a unique characterization datum for the test.

The study was conducted in three parts. The initial experimental plan included the use of 4 piglets for each of 4 probe setups. Several piglets became severely ill soon after arrival and the testing was stopped. Only 9 piglets were completed for this initial part of the study, including three each for the C1–5, L4–12t and SL15–4 probes. The research was restarted with a reduced plan, as listed in Table 1 (including the initial tests), and 6, 5 and 6 piglets were completed for the C1–5, L4–12t and SL15–4 probes, respectively. This plan included tests of two outputs, and two depths for the C1–5 probe. Two depths and two durations were tested for the L4–12t probe. Three durations were used for the SWE SL15–4 probe.

The results were puzzling for the L4–12t probe, in that there was no PCH induction. In addition, the automated B-line counting system was too insensitive to register the lung surface image changes from PCH induction by the C1–5 probe, that were seen by visual inspection of the images. An additional final part of the study was conducted using 8 piglets to clarify these puzzling results. The 7L linear array probe, known to be effective for PCH in rats (Miller et al. 2015), was used to test 6 piglets for comparison to the tests with the L4–12t linear array probe. The L4–12t probe and B-line tool then was used to examine the ICS scanned with the 7L probe. In addition, two piglets were exposed using the C1–5 probe (3.1 MHz) and then scanned using the L4–12t probe with its higher resolution (7.2 MHz). That is, the L4–12t probe with automated B-line tool was used to assess the PCH signs on the lung surface that were induced by the 7L and C1–5 probe exposures. In total, the C1–5, L4–12t, 7L and SL15–4 probes were used for 11, 8, 6 and 9 piglets, respectively.

Statistical analysis and graphing was performed using SigmaPlot for Windows V. 14.0 (Systat Software Inc., San Jose CA, USA). The PCH measurements for each specific exposure of intercostal spaces where averaged as listed in Table 3. The significance of PCH occurrence was found for each exposure condition relative to zero occurrence outside the scanned area (considered as shams). The Mann-Whitney Rank Sum test was used to compare means of measured PCH areas between exposure groups with statistical significance assumed at p<0.05. A threshold was estimated from the C1–5 exposure results at 50% and 100% AO by linear regression of individual piglet results. The exposure duration trend was assessed for the SWE exposures for 3 s, 30 s and 300 s at 1 fps was evaluated with non-linear curve fitting by the two-parameter equation for the exponential rise to maximum.

Table 3.

PCH measured for the three ultrasound systems and probes for separate intercostal spaces (a,b,c). The p value vs 0 is the occurrence significance and the p vs a is the comparison between PCH in conditions b and c vs a.

US Freq. In situ Occur Area vs. 0 vs. a
System Probe MHz MIIS Variable n n mm2 p p
Venue C1–5 a 3.1 1.0 12 cm,100 % 13 13 47 ± 22 <0.001
C1–5 b 3.1 0.73 12 cm, 50 % 7 6 11 ± 11 0.004 0.005
C1–5 c 3.1 0.84 6 cm, 100 % 6 5 16 ± 15 0.03 0.02
Venue L4–12t a 7.2 0.54 8 cm,300 s 8 0 0 - -
L4–12t b 7.2 0.86 3 cm, 300 s 9 0 0 - -
L4–12t c 7.2 0.86 3 cm, 30 s 6 0 0 - -
Vivid 7 7L a 4.8 0.86 74.7 fps 6 5 31 ± 29 0.015
7L b 4.8 0.86 32.1 fps 6 4 17 ± 25 0.065 0.4
SSI SL15–4 a 5.0 1.0 300 s 11 11 18 ± 8 <0.001
SL15–4 b 5.0 1.0 30s 8 6 8 ± 7 0.01 0.01
SL15–4 c 5.0 1.0 3s 8 3 0.6 ± 0.6 0.23 0.001
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SV, saline value; IS, in situ value.

Results

Venue R1 Results

The initial study began with a test of the consistency of the three ICS method of exposure. Three ICS were scanned with the C1–5 probe for 300 s each starting at a superior ICS with a good view of the lung surface and then moving downward ~1 cm using similar aiming technique at each position. The initial and final images for the middle ICS using the B-line tool are shown in Fig. 1. The lung surface image line is thicker, and the multiple reflection of the chest wall is obscured in the post-exposure image due to PCH induction. The B-line counting tool shows a score of 2, indicating 5 or more B lines or coalescent B-lines, and a count of ≥5, for both pre-and post-exposure images. The initial image has one large B-line area on the anterior side, while another strong B line artifact appears on the post-exposure image.

Figure 1.

Figure 1.

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LUS images for the C1–5 probe using the B-line tool. The top image was taken at aiming, before the high output exposure at the second ICS. The B line tool has identified a line on the left (anterior) side which is not related to LUS induced PCH. After 100 AO exposure for 300 s, the bottom image shows an additional B line, and other indications of lung surface PCH (unfocused image at the lung surface, small artifacts below the surface image.

A photomicrograph of the fresh lung sample is shown in Fig. 2 with a distinct PCH band along each scan line. The upper two lines are on the middle lobe, while the third is on the inferior lobe. The PCH bands are quite similar, although the lowest PCH region is somewhat reduced in length at the anterior boundary of the lung. The PCH areas cross numerous lobules with irregular boundaries (unlike rat lungs, which do not have lobules), and appear to be widened toward the anterior regions by lung sliding. In comparison to the LUS images, changes in the lung surface image (Fig. 1) were associated with the PCH on the lung (Fig. 2). However, the specific features associated with the initial B-line count mark, the strong post-exposure B-line with a count mark and the automated counts is not completely clear on the lung surface. The automated tool did not detect any B-lines with the C1–5 probe that were definitively associated with the image features of PCH on the lungs.

Figure 2.

Figure 2.

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A photomicrograph of the lung that was imaged in Fig. 1 showing three distinct PCH bands for the three ICS exposures. The middle band does not show any PCH feature which corresponds with the B line shown in Fig. 1. The bands appear to be widened, possibly due to lung sliding. Scale bar: 1 cm.

The C1–5 scanning at the 12 cm depth was performed both at 100 % and 50% AO and 6 cm depth at 100% AO. Data on the PCH occurrence and measured areas are listed in Table 3. All three results with the C1–5 were statistically significantly greater than zero. The two results for 12 cm depth were significantly different (p=0.005), indicating a strong exposure response trend. Using the individually measured chest wall thicknesses to determine attenuation, PCH area results at 12 cm depth for the 13 tests at 100% AO and 7 tests at 50 % AO were evaluated individually for each piglet to calculate the MIIS, and the exposure-responses are plotted in Fig. 3. There are large variations in PCH between individual piglets, suggesting that some unidentified differences between the individual piglets strongly influence PCH results. A linear regression was performed on the PCH area data and is shown in Fig. 3 to indicate the threshold trend. This linear trend toward a threshold is imprecise as indicated by the coefficient of determination of r2 = 0.42. The regression line based on the 20 results indicates a threshold at 0.53 MIIS (0.9 MPa at 3.1 MHz), with a standard error of 0.30.

Figure 3.

Figure 3.

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PCH area results at 12 cm depth for the 13 tests at 100% AO and 7 tests at 50% AO plotted against the individually calculated in situ MIIS for each piglet. The linear regression indicates a threshold of 0.53 MIIS (r2=0.42).

Exposure-response tests were conducted also with the L4–12t probe. The ultrasound images before and after 300 s exposure for one piglet test at 100% AO are shown in Fig. 4, and the lung photograph is presented in Fig. 5. The LUS exposure from the L4–12t probe did not produce any PCH effect, as listed in Table 3. There were no B-line counts, except one set of bright multiple A-line artifacts led to a B-line count from the automated tool as shown in Fig. 4.

Figure 4.

Figure 4.

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Ultrasound images for the L4–12t probe before (top) and after (bottom) 300s of scanning at 100 % AO. No perturbation of the lung surface image was identified. The bottom image shows a result for the B-line tool, which incorrectly counted the central series of A-line reflections as a B line.

Figure 5.

Figure 5.

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A photomicrograph of the lung shown in the ultrasound images (Fig. 4) for the L4–12t probe. There was no indication of PCH for this lung, nor was there any definitively identified PCH for the other 7 piglets tested with 8 cm depth and 100 % AO. Scale bar: 1 cm.

Additional B-Line Tool Results

As noted above, the negative PCH result for the 7.2 MHz L4–12t probe was unsatisfying in regard to establishing an exposure-response trend. An additional series of 6 piglet tests was conducted with the 7L probe from the Vivid 7 Dimension machine. This probe, like the L4–12t, is a linear array but with a lower frequency of 4.8 MHz, see Table 1. From Table 2, these two probes have similar MIOS values, but the 7L probe has longer pulses and shorter PRP (Table 1), which gives a higher temporal average exposure, as indicated by the TIOS. Before and after ultrasound images at 74.7 fps are shown in Fig. 6, and the lung image with PCH is shown in Fig. 7. The 7L exposures gave PCH areas that were similar to those of the Venue C1–5 probe, see Table 3. The post-exposure 7L image produced several B-line artifacts associated with the PCH. When the L4–12t probe was used to scan the same ICS used for the 7L exposure, the B line tool counted two lines as shown in Fig. 6, that were clearly associated with the image features and PCH effect.

Figure 6.

Figure 6.

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Ultrasound images with the Vivid 7 Dimension 7L linear array probe before (top) and after (middle) 300 s of scanning at 0 dB output setting. The post-exposure image shows numerous B-line artifacts extending inward from the lung surface image, in contrast to the results for the L4–12t linear array probe (Fig. 4). The L4–12t probe was used to scan the same ICS shown in the 7L image. The bottom L4–12t image shows B-line artifacts similar to the 7L image, and the B-line tool correctly counts two of the prominent artifacts.

Figure 7.

Figure 7.

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A photomicrograph of the lung showing the PCH generated at two ICS exposures (only two ICS were tested for this piglet) for exposure with the 7L probe. The images shown in Fig. 6 were associated with the second PCH band. Scale bar: 1 cm.

In the additional final plan, two piglets were exposed with the C1–5 to produce PCH. The ultrasound images shown in Fig. 8 have some thickening of the bright lung surface image, but do not have clear indications of PCH B-lines. The one count from the B line tool was associated with the artifacts commonly seen at the anterior edge of the lung, which were not associated with the PCH. However, these exposures at two ICS positions for each piglet gave quite distinct PCH on the lungs as shown in Fig. 9, that was quite similar to the PCH shown in Fig. 2 (as expected from the replicate exposures). The 7.2 MHz L4–12t probe was use for scans with the automated B-line tool to gauge its ability to count PCH-associated B-lines, which were not counted with 3.1 MHz C1–5 probe. The higher resolution probe did display clear B-line features for both piglets and some longer lines were counted by the B line tool, see Fig. 8. A notable feature of the auto B-line display in these tests was that the image quality indicator (the colored horizontal line at the bottom of the images) showed yellow (“average”) for the counts not associated with PCH but green (“ideal”) for the PCH-associated counts.

Figure 8.

Figure 8.

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Images from C1–5 probe before (top) and after (middle) 300 s scanning at 100 % AO. The post exposure image shows only subtle indications of PCH related perturbation of the lung surface image, and the one B-line detected was the non-PCH artifact on the anterior (left) side, which was often seen with the C1–5 probe. However, re-imaging with the L4–12t probe (bottom) revealed B-line artifacts and one correct PCH B-line count.

Figure 9.

Figure 9.

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A photomicrograph of the lung showing the PCH generated at the two ICS exposures for this piglet by exposure with the C1–5 probe. The boundaries of the lobules seem to influence the uniformity of the PCH bands. The images shown in Fig. 8 were associated with the second PCH band. Scale bar: 1 cm.

Shear Wave Elastography Results

The SWE exposures with the SL15–4 probe of the SSI machine delivered push pulse sequences to each of 4 positions. The first test with this machine was performed at three ICS positions with 300 s durations and PCH bands on the right lobe are shown in Fig. 10. This photograph, made with a cellular telephone camera, gives the perspective of the PCH on the entire right lung (about 12 cm long by 7 cm wide) with three sets of PCH spots. One ICS was skipped owing to aiming problems for the boundary between the middle and inferior lobes.

Figure 10.

Figure 10.

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A photomicrograph showing the PCH generated by the first SWE exposures with the SL15–4 probe of the SSI machine. The entire right lung is shown for perspective. The same 300 s exposure was performed at three ICS positions, each of which generated four PCH spots associated with the 4supersonic push-pulse positions. Scale bar: 1 cm.

The PCH results are listed in Table 3 for the 3s, 30s and 300s duration exposures that were performed at 3 different ICS for each piglet. In Fig. 11, the SWE image, post exposure image and lung photomicrograph are shown for the 3 different ICS exposures of one piglet. The SWE image boxes during exposure show some elasticity results below the lung surface image, which are apparently multiple reflection artifacts similar to the multiple chest wall reflections. The PCH appears as growing B-lines during exposure and in the final post SWE images. The lung image for 3 s exposure (3 SWE push shots) has small spots, one of which produced a clear B line in the post SWE image (another small B line was evident before exposure on the anterior side). The 30 s and 300 s images and PCH results were similar in magnitude. This exposure response trend is consistent with an accumulation of injury from each SWE push pulse series tending toward a maximum as the exposed surface area is filled with PCH spots. This trend is shown in Fig. 12 with the data points (Table 3) well fitted by a 2 parameter equation for exponential rise to maximum. This nonlinear regression gave an initial rate of increase of 0.33 mm2/sec and a maximum of 18.4 mm2 (r2=0.61).

Figure 11.

Figure 11.

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The SL15–4 probe was used to perform SWE exposures of 3 s (left), 30 s (middle) and 300 s (right) durations shown for one piglet. The SWE image of each exposure is shown on the top, the post exposure B mode image is shown in the middle, and the resulting PCH band is shown in the bottom of the sets of images. The SWE images apparently show multiple reflection artifacts within the elastography box that are not true elasticity determinations: for these SWE image, red indicates ~ 180 kPa elasticity according to the associated scale (not included in the images). The 3 s (three SWE push pulse series delivered at 1 fps) produced clear PCH spots with PCH-related B-line in the middle (left) image. The PCH from the 30 s and 300 s of SWE exposure were comparable. Scale bars: 1 cm.

Figure 12.

Figure 12.

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A plot of the mean PCH results for the three SWE exposure durations with an exposure response trend that is consistent with incremental PCH from each SWE determination accumulating toward a maximum as the exposed surface area is filled with PCH spots. This trend was fitted to the data by a 2 parameter equation for exponential rise to maximum with initial rate of rise of 0.33 mm2/sec and a maximum of 18.4 mm2 (r2=0.61).

Discussion

This study was performed to assess the induction of PCH from LUS in neonatal piglets. The piglet is an established animal model for neonatal acute respiratory distress syndrome (Spengler et al. 2019) and other clinical problems in neonates. There are several clinical indications for LUS assessment in neonates (Liu et al. 2019). The LUS is often conducted as point-of-care examinations by observation of B-line artefacts, and several authoritative recommendations and guidelines have been published (Dietrich et al. 2016; Kurepa et al. 2018; Singh et al. 2020; Dietrich et al. 2021).

PCH is an established bioeffect of pulsed ultrasound. We have studied PCH induction in rats by diagnostic ultrasound machines and find that its occurrence and magnitude depend on the pulse parameters, exposimetry and various physiological conditions. This study investigated the PCH exposure response in the piglet model of neonatal ultrasound for a Venue R1 point-of-care ultrasound machine. Using the 3.1 MHz C1–5 probe at 50% AO and 100% AO, a threshold was determined at MIIS =0.53 (Fig. 3). In contrast, no PCH was produced by the 7.2 MHz L4–12t linear array probe (Table 3). However, PCH was produced by the 4.8 MHz 7L linear array probe from a Vivid 7 Dimension machine, with magnitudes commensurate with the C1–5 probe results. Tests also were conducted with a Supersonic Imagine Aixplorer with SL15–4 probe in SWE mode. This exposure produced PCH in 4 spots, corresponding to the positions of the supersonic push pulses (Fig. 10). For exposure durations of 3 s, 30 s and 300 s (Fig. 11), the PCH accumulated with each SWE image-measurement (1 fps) at ~0.33 mm2/s, increasing to a maximum area of 18.4 mm2 (Fig. 12).

The assessment of LUS B-lines has become an important diagnostic aid. Qualitatively, these seem to present a clear polar question: are there B lines or not. These signs could be even more useful if quantified and reproducible tools or apps were available to the clinician. The B line tool included in the Venue R1 system was used in this study in relation to B-lines resulting from causation of PCH. For the C1–5 probe, which caused PCH on the piglet lungs, the B line tool did not provide useful B line detection (Fig. 1). The L4–12t probe did not cause PCH in our tests. The L4–12t B-line tool was used to search for B lines related PCH caused by the C1–5 probe and the 7L probe of the Vivid 7 Dimension machine. This was successful for both the 7L probe (Fig. 6) and the C1–5 probe (Fig. 8). The automated search for the B lines was somewhat user dependent in choosing the view to score, but some longer, obvious B lines seen visually in images were counted. Small B lines or the broadening of the lung surface image with reduction of multiple chest wall reflection was not detected. This automated tool may be useful for providing a quantitation of the B line sign that could document findings and enhance patient care. This method should be validated clinically for each LUS indication.

In this study, a threshold was found at PRPA = 0.9 MPa, or MIIS =0.53 for 3.1 MHz imaging for 300 s. As noted in the introduction, early research on PCH using laboratory pulsed ultrasound also found thresholds for piglets. Baggs et al. (1996) found a 0.9 MPa threshold at 2.3 MHz, or MIIS = 0.6, for 16 min exposures of 1 d piglets, and Dalecki et al. (1997) found a 0.6 MPa threshold, or MIIS = 0.46 under similar conditions for 10 d piglets. The comparable results for this study 25 y later with a clinical device is remarkable. The worst case value of the threshold MI ~ 0.4 has been validated for different animal models, LUS modes and probes and physiological conditions (one caveat is the MIIS = 0.24 found for pulsed doppler exposure of lung (Miller et al 2018a), but this non-imaging mode is not recommended for LUS (Richards et al. 2017). For 10 s exposure duration, O’Brien et al. (2003) exposed 5 d piglets to 3.1 MHz pulses and found a 2.7 MPA threshold, or MIIS = 1.5. Here, the finding of no PCH for the L4–12t probe at MIOS = 1.4 is encouraging for the safety of similar probes, though different from the 7L probe that induced PCH for MIOS = 1.2 at 0 dB. Thus, the specific use of MI as the exposimetric parameter for LUS PCH seems to be problematical, although the on screen indices MIOS and TIOS are critical parameters for clinical safety guidance. Possibly, the TIOS could be useful as an additional exposimetric gauge of risk because, as noted above (Table 2), the Venue L4–12t probe with zero PCH had TIOS values of 0.1 and 0.4, while the Vivid 7 Dimension 7L probe with substantial PCH had TIOS values of 0.7 and 1.7. However, uncertainty is increased further because PCH results are strongly dependent of physiological conditions, noted in the introduction, and vary between individuals (large standard deviations) even for carefully repeated tests (Fig. 3). This uncertainty complicates the definition of a universal dose parameter for LUS that might proactively indicate PCH magnitude.

This work did not address possible health impacts of LUS PCH. Potential health impacts could arise from the leakage of blood into the alveolar space with increases in surfactant surface tension and alveolar flooding. Filling of alveolar space with fluid can lead to exacerbation of ventilator-induced injury (Wu et al. 2014). Another potential consideration is the use of high FiO2 of 85–95% in neonates (Sweet et al. 2019). In previous work we have shown exacerbation of the PCH effect by elevated FiO2 (Miller et al. 2020). However, use of positive end expiratory pressure in ventilation (or use of CPAP) may reduce or eliminate the risk of PCH from LUS (Miller et al. 2018). Overall, LUS induction of PCH represents an adverse bioeffect, and the risk should be minimized.

Conclusion

This study shows that consideration of the LUS PCH safety issue is warranted for neonatal LUS examinations. Some reviews of perinatal ultrasound safety mention this issue but without giving specific recommendations for the on-screen indices, for example Jagla et al. (2018) and Sande et al. (2021). Point-of-care LUS is a new aspect of safety assurance considerations (Miller et al. 2020b). Two medical ultrasound societies have addressed the issue with recommendations for use of MI < 0.4 by the American Institute of Ultrasound in Medicine (Church et al. 2008; AIUM, 2021) and MI < 0.3 by the British Medical Ultrasound Society (BMUS, 2021). The numerical value of the worst-case PCH threshold has been validated in different animal models and comparable thresholds have been found for 25 y. The recommended upper limits are valuable worst-case exposure metrics to guide the sonographer. For exams with the lung surface imaging through thin chest walls or at higher MI, the image of the lung surface actually can be improved by reducing power to avoid saturation and loss of image detail at the surface (Soldati et al. 2020). The operator can adjust the MI on many systems and should set lung applications to start at or below the worst-case limits. In many point-of-care examinations, the person performing the exam may not be knowledgeable about the MI nor how to change it: in that situation, the assurance of safety requires that a MI ≤ 0.4 output setting has been used in the lung application employed for the exam. For many patients and conditions, such as for obese patients, there likely is little or no risk of the PCH bioeffect, and therefore output power above the guideline limit should be utilized if needed for acquisition of diagnostically acceptable images.

Acknowledgments:

This study was supported by the US National Heart Lung and Blood Institute via grant number HL116434. The information contained herein does not necessarily reflect the position or policy of the US government, and no official endorsement should be inferred. We thank Drs. Krishnan Raghavendran and Suresh Madathilparambil, Department of Surgery, Michigan Medicine, for their help with this research project.

Footnotes

Conflict of Interest Statement:

The authors have no conflicts of interest in regard to this research report.

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