# Shear Wave Elastography, a New Tool for Diaphragmatic Qualitative Assessment A Translational Study

Yassir Aarab, Aurélien Flatres, Fanny Garnier, Mathieu Capdevila, F.

Raynaud, Alain Lacampagne, David Chapeau, Kada Klouche, Pascal Etienne, Samir Jaber, et al.

► To cite this version:

Yassir Aarab, Aurélien Flatres, Fanny Garnier, Mathieu Capdevila, F. Raynaud, et al. Shear Wave Elastography, a New Tool for Diaphragmatic Qualitative Assessment A Translational Study. American Journal of Respiratory and Critical Care Medicine, 2021, 204 (7), pp.797-806. 10.1164/rccm.202011-4086OC. [https://hal.science/hal-03286291](https://hal.science/hal-03286291)

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# Shear Wave Elastography, a New Tool for Diaphragmatic Qualitative Assessment

## A Translational Study

Yassir Aarab¹,²,³*, Aurelien Flatres¹,³*, Fanny Garnier¹,⁴, Mathieu Capdevila²,³, Fabrice Raynaud³, Alain Lacampagne³, David Chapeau¹, Kada Klouche¹,³, Pascal Etienne⁵, Samir Jaber²,³, Nicolas Molinari⁴,⁶, Lucie Gamon⁶, Stefan Matecki³, and Boris Jung¹,³

¹Medical Intensive Care Department, ²Saint Eloi Anesthesiology and Critical Care Medicine Department, and ³Biostatistics Department, Montpellier University and Montpellier Teaching Hospital, Montpellier, France; ³PhyMedExp, and ⁴Desbrest Institute of Epidemiology and Public Health, IDESP, INSERM, and ⁵Charles Coulomb Laboratory (L2C), Montpellier University, CNRS, Montpellier, France

## Abstract

**Rationale:** Prolonged mechanical ventilation is often associated with either a decrease (known atrophy) or an increase (supposed injury) in diaphragmatic thickness. Shear wave elastography is a noninvasive technique that measures shear modulus, a surrogate of tissue stiffness and mechanical properties.

**Objectives:** To describe changes in shear modulus (SM) during the ICU stay and the relationship with alterations in muscle thickness. To perform a comprehensive ultrasound-based characterization of histological and force production changes occurring in the diaphragm.

**Methods:** Translational study using critically ill patients and mechanically ventilated piglets. Serial ultrasound examination of the diaphragm collecting thickness and SM was performed in both patients and piglets. Transdiaphragmatic pressure and diaphragmatic biopsies were collected in piglets.

**Measurements and Main Results:** We enrolled 102 patients, 88 of whom were invasively mechanically ventilated. At baseline,

SM was 14.3 ± 4.3 kPa and diaphragm end-expiratory thickness was 2.0 ± 0.5 mm. Decrease or increase by more than 10% from baseline was reported in 86% of the patients for thickness and in 92% of the patients for SM. An increase in diaphragmatic thickness during the stay was associated with a decrease in SM (β = -9.34 ± 4.41; *P* = 0.03) after multivariable analysis. In the piglet sample, a decrease in SM over 3 days of mechanical ventilation was associated with loss of force production, slow and fast fiber atrophy, and increased lipid droplets accumulation.

**Conclusions:** Increases in diaphragm thickness during critical illness is associated with decreased tissue stiffness as demonstrated by shear wave ultrasound elastography, consistent with the development of muscle injury and weakness.

Clinical trial registered with www.clinicaltrials.gov (NCT03550222).

**Keywords:** diaphragmatic dysfunction; diaphragmatic ultrasonography; mechanical ventilation; transdiaphragmatic pressure

<footnote>*These authors contributed equally to this work.</footnote>

<footnote>Supported by departmental resources, an INSERM-transfer research grant, the Medical School of the Montpellier University Young Researcher Grant (Y.A.), and RegenFHU PhD Funding (A.F.).</footnote>

<footnote>Author Contributions: A.F. and Y.A. performed all the ultrasound measurements, analyzed the results, and participated in the manuscript editing. F.G. and D.C. contributed to the study design and patient enrollments. M.C. performed all transdiaphragmatic pressure measurements and contributed to the piglet model. F.R. performed all histological analyses. K.K. contributed to the study design. A.L., P.E., and S.M. reviewed some of the measurements and participated in the analysis results. S.J. contributed to the study design, analysis results, and manuscript editing. N.M. and L.G. performed statistical analysis. B.J. designed the study, participated in manuscript editing, and approved its final version.</footnote>

<footnote>Correspondence and requests for reprints should be addressed to Boris Jung, M.D., Ph.D., Medical Intensive Care Unit, Montpellier Lapeyronie Teaching Hospital, Avenue du Doyen Gaston Giraud, 34000, Montpellier, France. E-mail: b-jung@chu-montpellier.fr.</footnote>
**Scientific Knowledge on the**
**Subject:** Diaphragm weakness is
associated with poor outcomes in
critically ill patients. Both decrease
(atrophy) and increase (supposed injury)
in diaphragmatic thickness are
associated with prolonged ventilation.
Shear wave ultrasound elastography
provides additional information about
muscle quality at rest by measuring
shear modulus, a quantifiable
measurement of tissue stiffness.
Diaphragm mechanical properties,
assessed by shear wave ultrasound
elastography, have never been
investigated in the critically
ill population.

**What This Study Adds to the Field:**

Our results show that the increase in diaphragmatic thickness in one-third of patients during their ICU stay is associated with a decrease in diaphragmatic shear modulus. Injurious changes in muscle, including muscle fiber atrophy and weakness, are associated with a decrease in shear modulus. These results suggest that shear modulus can detect an alteration in diaphragmatic mechanical properties and are in line with the diaphragmatic myotrauma concept.

Diaphragmatic weakness occurs in 30–60% of critically ill patients (1, 2). It can preexist ICU admission (3) or can appear during the ICU stay (4–6). Prolonged mechanical ventilation (MV) duration (6), increased incidence of weaning failure (3, 5, 6), prolonged ICU stay (3, 7), and long-term mortality (8) are associated with diaphragmatic weakness. MV and sepsis are the main risk factors (6, 9–11), and human biopsies have shown that diaphragmatic weakness is associated with muscle fiber lipid accumulation, inflammatory infiltrate, sarcomere injury, autophagy, and fiber atrophy (4, 12–14).

Ultrasonography is a user-friendly,
noninvasive tool to explore the diaphragm at
the bedside. Ultrasonography has been used
to explore diaphragmatic contractile activity
by measuring its excursion, thickening

fraction, the velocity of diaphragmatic
muscle motion (tissue Doppler imaging),
and diaphragm mass using its thickness as a
surrogate (15–19). In a landmark study,
Goligher and colleagues reported that both
diaphragmatic decrease of thickness and
increase of thickness are associated with poor
outcomes (20, 21). The authors then
developed the “myotrauma” concept in
which abnormal diaphragm function is
associated with either overassistance (disuse
atrophy) or underassistance myotrauma
(muscle overuse leading to inflammation,
edema, and injury) (22).

Shear wave ultrasound elastography (SWE) allows direct and real-time quantification of the mechanical properties of tissues and additional information about muscle quality at rest (23). Briefly, tissue deformation is created by an intrinsic focused push beam generated by the ultrasound probe. This results in the propagation of shear waves parallel to the surface of the probe. The velocity of these shear waves varies with mechanical properties of tissues (i.e., stiffness). Stiffness is then quantified by shear modulus (SM), which is calculated through shear wave propagation (SM = ρ·v², in which ρ is the tissue density, equal to 1,000 kg/m³ in the human body, and v is the shear wave velocity [m²s⁻²]) (24). By using inversion algorithms, this method maps the created wave velocity into elastograms and determines stiffness of the tissue by measuring the SM (25). A higher SM value corresponds to higher tissue stiffness. SWE provides a spatial representation and quantifiable measurement of the mechanical properties of the tissue (23). Although shear wave speed only depends on stiffness for large organs such as breast or liver, for thin structures such as the diaphragm, the shear wave travels approximately 50% faster when the structure's thickness doubles (26). Therefore, if the SWE decreases while thickness increases, it strongly suggests that the muscle's stiffness is decreased. Most of the available SWE studies have been published in the fields of limb muscles and tendons in chronic myopathy and in sports medicine (23, 27). Recently, two studies have evaluated SWE during diaphragmatic contraction and have suggested that SM can be a surrogate for diaphragm contractile activity in healthy volunteers (28, 29). Diaphragmatic SWE exploration is feasible in the critically ill population (30) with a previously reported reference value in this specific population at

admission of 13.7 kPa (±4.4) (31), and
healthy subjects' values at rest ranging from
6.2 kPa (±3.6) to 12 kPa (±2) (29, 30). Fossé
and colleagues investigated changes in SM
during diaphragm contraction in
mechanically ventilated patients as a
potential surrogate to transdiaphragmatic
pressure (Pdi) (31). Changes in diaphragm
SM at rest over time have never been
investigated in the critically ill population.
We hypothesized that change in SM, either
higher or lower, may help to better describe
the histological changes occurring in
diaphragm muscle during critical illness (32).
A drop in stiffness could reflect muscle fiber
atrophy (33) or a combination of muscle
edema, inflammatory or lipid accumulation,
and fiber atrophy (34). An increase in SM
could be due to exacerbated muscle fibrosis
as in late-stage Duchenne's myopathy (35) or
cerebral palsy (36).

The aim of this translational study was first to describe diaphragm thickness and SM changes during the ICU stay and then to explore the relationship between ultrasound measurements, diaphragmatic histology, and force production using diaphragm biopsies in a mechanically ventilated piglet model. Results were partially presented at the American Thoracic Society Annual Meeting (Dallas, Texas, May 17–22, 2019) (37).

Methods

Human Study Population and Setting

This prospective observational study was
conducted in the medical ICU at the
University Hospital of Montpellier, France,
from December 2017 to June 2018 and
included all consecutive patients with at least
one organ failure (defined by the Sepsis-
related Organ Failure Assessment score) (38)
(see online supplement for details). Patients
were included within 24 hours of admission
regardless of the need for MV. Images in
B mode for thickness and SWE mode for SM
were obtained at baseline (Day 0), and
repetitively every other day up to Day 28,
discharge from the ICU, or death. Ultrasound
diaphragm thickness may increase or decrease
according to the combination of changes in
all its components including the muscle fibers
but also connective tissue, the vessels,
inflammatory or lipid accumulation, and
muscle edema. Institutional ethical approval
was obtained (2017-CLER-MTP-09-16). We
followed the STROBE guidelines for
observational studies (39).
# Shear Wave Elastography
## Imaging Procedure

In biomechanics, stiffness is defined by the proportional relationship between stress (the external force or compression) and strain (deformation) applied to it. Transmission of a longitudinal pulse leads to tissue displacement, which is detected by pulse echo ultrasound, and allows for the measurement of shear wave velocity ($V$ in m·s⁻¹) in the tissue. Shear wave velocity is proportional to SM (also named $\mu$ and expressed in kPa) using the formula: $\mu = \rho \cdot V^2$ (in which $\rho$ is the tissue density, equal to 1,000 kg/m³ in the human body). Hard tissues have a higher SM and $V$ than soft tissues (24).

An Aixplorer ultrasonic scanner (SuperSonic Imagine) was used with a 4- to 15-MHz linear transducer (SL15-4; SuperSonic Imagine) in both B and SWE modes with musculoskeletal preset. All images and measurements were performed by two operators (Y.A. and A.F.) according to a standardized and reproducible protocol (30) (see Methods, Figure E1, and Video E1 in the online supplement).

Patients were assessed in supine position, half sitting at 30°. Conscious subjects were instructed to remain relaxed and to breathe as calmly as possible throughout the procedure. We performed all measurements in triplicate on the right hemidiaphragm. Because contractile activity increases SM, we performed all the measurements at the end of expiration when the diaphragm is at rest. Conscious subjects were instructed to remain relaxed, to breathe as calmly as possible throughout the procedure, and to remain apneic at FRC for SWE acquisition as long as possible. For mechanically ventilated patients, an end-expiratory pause was applied during SWE acquisition. When a stable image with good quality criteria (homogeneous SWE color map) was not possible to collect, owing to high respiratory rate for example, the measured SM was not used for data analysis. Proofreading of the images was done offline and blinded by the two operators (A.F. and Y.A.). To enable replication of image location on repeated ultrasound assessments, a mark was carefully drawn on the subject's chest during the first measurement procedure and a standardized protocol was applied.

During image acquisition, transducers were placed with minimal compression on top of a generous amount of at least 5 mm of

coupling gel to avoid distortion of the underlying tissue (40). The transducer was placed via an intercostal approach at the zone of apposition, in the sagittal plane perpendicular to the diaphragm muscle, and between the right anterior and midaxillary lines. The diaphragm was identified as a three-layered structure comprising two hyperechoic lines (pleural and peritoneal membranes) and a middle hypoechoic layer (diaphragm muscle fibers). Subsequently, the probe was oriented approximately 90° and finely adjusted to obtain a longitudinal view with the probe (and acoustic beam) parallel to the direction of the diaphragm muscle fascicles, and to obtain maximal echo intensity from diaphragm membranes. When activating the SWE mode, a color-coded box representing the region of interest was superimposed on the image. The widest area possible was drawn in the most homogeneous zone of the region of interest using built-in software of the ultrasound system (Q-Box trace) to avoid measurement bias, and SM was collected (Figure E1 and Video E1).

All acquired images and data were stored and secondarily analyzed as recommended by Nijholt and colleagues

using Osirix DICOM Viewer software (Pixmeo) (41). The whole procedure, with image acquisition in B-mode frames and SM elastograms in triplicate, required 4–5 minutes at the patient's bedside

# Mechanically Ventilated Piglet Studies

After approval by the University of Montpellier Institutional Ethics Committee for Animal Research and Care and according to the recommendations of the Helsinki Declaration, we conducted a piglet study to describe the association between diaphragm thickness and SM. Twelve piglets (25–35 kg) were sedated and mechanically ventilated in volume-controlled mode for 3 days as previously described (42). The detailed protocol is provided in the electronic supplement. Briefly, we used Pdi as the reference method for the assessment of diaphragm function (1). Pdi was calculated from esophageal and gastric pressure measurements. Pressure-frequency curves (Day 0–3) were measured daily by stimulating diaphragm contractions at end-expiration and using transvenous pacing catheters to achieve phrenic nerve stimulation with frequencies ranging from 20 to 100 Hz in a serial manner (43).

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Study flow chart of critically ill patients. This figure shows the flow chart of patients admitted to our unit during the study. Consecutive patients with at least one organ failure and an expected duration of ICU stay $\ge$3 days were enrolled. Of 412 adults, 138 were enrolled, 36 were excluded, and 102 completed the study evaluation. Ninety-four patients were available for analysis with at least three diaphragm evaluations. SM = shear modulus.</figcaption></figure>
Diaphragm ultrasound-acquired images for thickness and SM measurement were recorded daily at the end of expiration when the diaphragm was at rest for all 12 piglets. Finally, diaphragm biopsies were obtained before euthanasia at Day 3 for 10 piglets (see Methods in the online supplement). Briefly, segments of dissected diaphragm strips were either immediately fixed for electronic microscopy examination and the quantification of intramuscular lipid droplets

accumulation, or stained with hematoxylin/ eosin for inflammatory cells infiltration and fiber types determination. Computer images captured from randomly selected microscopic fields were analyzed.

## Endpoints

The two coprimary endpoints were to describe the evolution of diaphragm SM during the ICU stay and the association between diaphragm SM (as a surrogate for

diaphragm stiffness) and thickness. Diaphragm thickness is a surrogate for the whole diaphragm muscle mass including the muscle fibers but also connective tissue, the vessels, inflammatory or lipid accumulation, and muscle edema.

The exploratory endpoints were to describe the association between diaphragm SM and the patients' clinical outcomes, and between diaphragm SM, fiber size, muscle inflammatory cells infiltration, muscular

<table><caption>**Table 1.** Demographics, Clinical Characteristics, and Outcomes of the Study Population</caption><thead><tr><th rowspan="2">Characteristics</th><th colspan="4">Change in Diaphragm Shear Modulus during the First Week</th><th rowspan="2">P Value</th></tr><tr><th>Study Population (n = 92)</th><th>>10% Increase (n = 47)</th><th><10% Change (n = 7)</th><th>>10% Decrease (n = 38)</th></tr></thead><tbody><tr><td>Age, yr</td><td>60 ± 17</td><td>62 ± 17</td><td>59 ± 16</td><td>55 ± 17</td><td>0.46</td></tr><tr><td>Sex, M</td><td>63 (70)</td><td>34 (72)</td><td>3 (43)</td><td>26 (72)</td><td>0.29</td></tr><tr><td>Body mass index, kg/m²</td><td>26 ± 16</td><td>24 ± 5</td><td>24 ± 7</td><td>24 ± 6</td><td>0.64</td></tr><tr><td>SAPS II</td><td>58 ± 19</td><td>62 ± 20</td><td>50 ± 10</td><td>55 ± 18</td><td>0.11</td></tr><tr><td>SOFA score at admission</td><td>8 (5–11)</td><td>9 (5–11)</td><td>6 (5–6)</td><td>8 (6–11)</td><td>0.22</td></tr><tr><td>Comorbidities</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>COPD</td><td>16 (17)</td><td>9 (19)</td><td>1 (14)</td><td>6 (16)</td><td>0.90</td></tr><tr><td>Ischemic heart disease</td><td>11 (12)</td><td>5 (11)</td><td>0 (0)</td><td>2 (5)</td><td>0.46</td></tr><tr><td>Chronic heart failure</td><td>17 (19)</td><td>5 (11)</td><td>0 (0)</td><td>12 (32)</td><td>0.02</td></tr><tr><td>Diabetes mellitus</td><td>20 (22)</td><td>7 (15)</td><td>1 (14)</td><td>12 (32)</td><td>0.16</td></tr><tr><td>Chronic kidney disease</td><td>13 (14)</td><td>9 (19)</td><td>1 (14)</td><td>3 (8)</td><td>0.33</td></tr><tr><td>Cancer</td><td>31 (34)</td><td>13 (28)</td><td>2 (29)</td><td>16 (42)</td><td>0.36</td></tr><tr><td>Cirrhosis</td><td>5 (5)</td><td>1 (2)</td><td>2 (29)</td><td>2 (5)</td><td>0.02</td></tr><tr><td>Inflammatory disease</td><td>7 (8)</td><td>5 (11)</td><td>0 (0)</td><td>2 (5)</td><td>0.47</td></tr><tr><td>Chronic corticosteroid treatment</td><td>21 (23)</td><td>13 (28)</td><td>1 (14)</td><td>7 (18)</td><td>0.20</td></tr><tr><td>Malnutrition</td><td>22 (24)</td><td>9 (19)</td><td>2 (29)</td><td>11 (29)</td><td>0.35</td></tr><tr><td>Sepsis-3 criteria within first week</td><td>72 (78)</td><td>35 (74)</td><td>6 (86)</td><td>31 (82)</td><td>0.87</td></tr><tr><td>Primary reason for admission</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Respiratory distress</td><td>52 (56)</td><td>27 (57)</td><td>4 (57)</td><td>21 (55)</td><td>0.98</td></tr><tr><td>Cardiac arrest</td><td>9 (10)</td><td>4 (8)</td><td>1 (14)</td><td>4 (11)</td><td>0.87</td></tr><tr><td>Acute heart failure</td><td>3 (3)</td><td>2 (4)</td><td>0</td><td>1 (3)</td><td>0.41</td></tr><tr><td>Coma – Encephalopathy</td><td>10 (11)</td><td>5 (11)</td><td>2 (29)</td><td>3 (8)</td><td>0.27</td></tr><tr><td>Sepsis</td><td>12 (13)</td><td>5 (11)</td><td>0</td><td>7 (18)</td><td>0.32</td></tr><tr><td>Other</td><td>6 (7)</td><td>4 (9)</td><td>0</td><td>2 (5)</td><td>0.64</td></tr><tr><td>Clinical management during ICU stay</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Use of NMBA</td><td>55 (60)</td><td>28 (60)</td><td>6 (86)</td><td>21 (55)</td><td>0.32</td></tr><tr><td>Duration of NMBA infusion, d*</td><td>3 (2–5.5)</td><td>3 (2–4.5)</td><td>8 (6–18)</td><td>3 (2–6)</td><td>0.30</td></tr><tr><td>Use of corticosteroids</td><td>51 (55)</td><td>32 (68)</td><td>3 (43)</td><td>16 (42)</td><td>0.35</td></tr><tr><td>Duration of corticosteroid infusion, d*</td><td>8 (4–12.3)</td><td>8 (3.8–11)</td><td>8 (4–12.5)</td><td>5 (4.3–8)</td><td>0.62</td></tr><tr><td>Use of aminoglycosides</td><td>29 (32)</td><td>14 (30)</td><td>0 (0)</td><td>15 (39)</td><td>0.11</td></tr><tr><td>Use of sedatives</td><td>79 (86)</td><td>40 (85)</td><td>6 (86)</td><td>33 (87)</td><td>0.50</td></tr><tr><td>Duration of deep sedation, d*</td><td>5 (3–9)</td><td>5.5 (2.3–8)</td><td>8 (4–8.5)</td><td>4 (3–9)</td><td>0.41</td></tr><tr><td>Duration of invasive MV, d*</td><td>7 (4–15)</td><td>7.2 (2.8–13.5)</td><td>8 (7.4–18)</td><td>7 (4–15.2)</td><td>0.41</td></tr><tr><td>Duration of CMV, d*</td><td>2.8 (1.1–5.7)</td><td>2.7 (0.9–5.4)</td><td>4.4 (3.3–6)</td><td>2.8 (1.6–5.6)</td><td>0.41</td></tr><tr><td>No need for ventilation</td><td>13 (14)</td><td>7 (15)</td><td>1 (14)</td><td>5 (13)</td><td>0.38</td></tr><tr><td>Need for vasopressors</td><td>78 (85)</td><td>41 (87)</td><td>4 (57)</td><td>33 (87)</td><td>0.11</td></tr><tr><td>Duration of vasopressor infusion, d*</td><td>4 (3–7)</td><td>4 (3–6)</td><td>4 (2.8–7.8)</td><td>4 (3–7)</td><td>0.41</td></tr><tr><td>Need for dialysis</td><td>24 (26)</td><td>14 (30)</td><td>1 (14)</td><td>9 (24)</td><td>0.62</td></tr><tr><td>Duration of dialysis, d*</td><td>6 (3–8.5)</td><td>7 (4–12.5)</td><td>1 (1–1)</td><td>4 (3–7)</td><td>0.28</td></tr><tr><td>Cumulative fluid balance at Day 3, L</td><td>4.7 ± 4.4</td><td>4.4 ± 4.7</td><td>3.7 ± 2.6</td><td>5.2 ± 4.3</td><td>0.83</td></tr><tr><td>Cumulative fluid balance at Day 7, L</td><td>5.5 ± 5.4</td><td>5.1 ± 5.7</td><td>4.4 ± 4.9</td><td>6.1 ± 5.3</td><td>0.81</td></tr></tbody></table>

Definition of abbreviations: CMV = controlled mechanical ventilation; COPD = chronic obstructive pulmonary disease; MV = mechanical ventilation; NMBA = neuromuscular blockade; SAPS II = Simplified Acute Physiology Score; SOFA = Sequential Organ Failure Assessment. Data are presented as n (%), mean ± SD, or median (interquartile range). Two out of 94 patients could not have shear modulus measurements and were therefore removed from this table.

*Results are presented for patients who received the mentioned therapy.
lipid droplets accumulation, and force production (measured by Pdi) in mechanically ventilated piglets.

## Statistical Analysis

The study population was divided into three groups (increase, decrease, or stable) based on the overall change in diaphragm thickness (thickness groups) or SM (SWE groups), from the baseline measurement to the last measurement obtained during the first week in the ICU using a 10% cutoff value (20). This cutoff was chosen because it was clinically relevant according to previous studies about SWE in limb muscles (23, 35) and was in agreement with the measurement of variance identified in our previous study (30).

Descriptive statistics are reported as numbers (%), means ± SD, or medians (interquartile range) and compared as appropriate. A mixed linear regression was used for the analysis. Repeated measures correlation coefficient (R) was used for determining overall relationships between variables. Covariates were tested in the model (if P < 0.15 in the univariate analysis), selected in a backward selection procedure, and then presented as a regression coefficient with 95% confidence intervals. A nonlinear effect for continuous variables was tested using a polynomial model. Akaike information and Bayesian information criteria were used to assess the nonlinearity. For all final comparisons, a P value ≤0.05 was considered statistically significant. Statistical analysis was conducted using SAS version 9.3 (SAS Institute).

## Results

### Demographics and Ultrasound Measurements at Baseline

Between December 2017 and June 2018, 102 critically ill patients were enrolled and at least three ultrasound evaluations of thickness and SM were respectively available for 94 and 92 of them (Figure 1). Demographic data and clinical characteristics are reported in Table 1. At Day 0, 67 patients (71%) were mechanically ventilated. The duration of MV was 7 (4–15) days, 2.8 (1.1–5.7) days being spent in controlled mode. We collected 637 and 631 ultrasound measurements for thickness and SM, respectively, after excluding 42 and 38 images for poor quality criteria. On ICU admission, diaphragm end-expiratory thickness was 2.0 ± 0.5 mm and

SM was 14.3 ± 4.3 kPa. In the 12 mechanically ventilated piglets, baseline end-expiratory thickness and SM were 2.3 (2.1–2.5) mm and 16.4 ± 4.3 kPa, respectively.

### Change in Diaphragm Thickness and Diaphragm SM Over Time

In critically ill patients overall, diaphragm thickness did not significantly change during the first week of the ICU stay (2.0 ± 0.5 mm on admission vs. 1.8 ± 0.4 mm at Day 7; P = 0.24). Diaphragm thickness change showed heterogeneous patterns in the critically ill patients. It remained unchanged in 13 patients (14%), decreased by more than 10% in 46 patients (49%), and increased by more than 10% in 35 patients (37%) (Figure E2). In critically ill patients, diaphragm SM remained unchanged in 7 patients (8%), decreased by more than 10% in 38 patients (41%), and increased by more than 10% in 47 (51%) patients (Figures 2 and E3). In multivariable analysis, diaphragm SM dropped over time in older patients (P < 0.05), in patients treated for sepsis (P = 0.03) or with steroids (P = 0.01), and in patients showing increased

diaphragmatic thickness during the ICU stay (P = 0.03) (Table 2). Time spent under controlled MV was associated with a decrease in SM in comparison with time spent under pressure support ventilation or without ventilatory assistance (P < 0.05) (Table 2).

In piglets, diaphragm SM decreased between Day 0 and Day 3 (16.4 ± 4.3 kPa vs. 14.1 ± 3.1 kPa [P = 0.07]), when diaphragm thickness did not (2.3 ± 0.4 mm vs. 2.3 ± 0.4 mm, P = 0.18) (Figure 3A). When using the same 10% cutoff change in SM as in critically ill patients, SM remained unchanged in six piglets and decreased by more than 10% in six piglets. No correlation was found between absolute diaphragm thickness and SM values (Figure 3B).

### SM, Muscle Fiber Size, Inflammatory Cells Infiltration, Lipid Accumulation, and Pdi

To better understand the drop in SM over time, we evaluated the relation between SM and diaphragm ultrastructure in mechanically ventilated healthy piglets for 3 days. A decrease in SM during the 3-day experiment was associated with a smaller fast- and slow-twitch fiber cross-sectional

<table><caption>**Table 2.** Factors Associated with Changes in Diaphragm Shear Modulus and Thickness during ICU Stay: Adjusted Multivariable Analysis</caption><thead><tr><th rowspan="2">Risk Factors</th><th rowspan="2">(/ Reference)</th><th colspan="3">Diaphragm Shear Modulus as a Quantitative Value</th></tr><tr><th>β</th><th>SD</th><th>P Value</th></tr></thead><tbody><tr><td>Sepsis</td><td>Yes / No</td><td>-1.79</td><td>0.82</td><td>0.03</td></tr><tr><td>Ventilation mode</td><td></td><td></td><td></td><td>&lt;0.01</td></tr><tr><td></td><td>Assisted / Controlled</td><td>1.45</td><td>0.40</td><td></td></tr><tr><td></td><td>No MV / Controlled</td><td>2.85</td><td>0.47</td><td></td></tr><tr><td>Age</td><td></td><td>-0.05</td><td>0.02</td><td>&lt;0.05</td></tr><tr><td>Days under deep sedation</td><td></td><td>0.14</td><td>0.07</td><td>0.05</td></tr><tr><td>Days on corticosteroids</td><td></td><td>-0.11</td><td>0.04</td><td>0.01</td></tr><tr><td>Diaphragm thickness</td><td></td><td>-8.92</td><td>4.46</td><td>0.03</td></tr></tbody></table><table><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th colspan="3">Diaphragm Thickness as a Quantitative Value</th></tr><tr><th>β</th><th>SD</th><th>P Value</th></tr></thead><tbody><tr><td>Sex</td><td>M / F</td><td>0.03</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Diaphragm shear modulus</td><td></td><td>&lt;-0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr><tr><td>Days of invasive MV</td><td></td><td>&lt;-0.01</td><td>&lt;0.01</td><td>&lt;0.01</td></tr></tbody></table>

Definition of abbreviation: MV = mechanical ventilation.

Adjusted on day of measurement, and global number of days under MV. Deep sedation was defined as a Richmond agitation sedation scale of -3 to -5. Variables included in the model are those with P < 0.15. See Table E1 for thickness and Table E2 for shear modulus. Shear modulus model variables: sepsis, difficult weaning, ventilation mode, age, ventilation-free days, days under vasopressors, days under deep sedation, days on corticosteroids, and diaphragm thickness. Thickness model variables: sex, ventilation mode, diaphragm shear modulus, days under deep sedation, days of invasive MV, and septic shock.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Variation in diaphragm shear modulus over time obtained in critically ill patients. Percentage of change from baseline (enrollment day) is represented on the y-axis and days of follow-up on the x-axis. Subjects are categorized into three groups according to the magnitude and direction of change in diaphragm shear modulus during the first week of the ICU stay using a 10% cutoff value. Mean and SD are plotted for each group on each study day. Numbers shown next to each data point indicate the number of measurements on each study day. Shared area indicates confidence intervals. Trend line and confidence intervals (shaded area) were fitted by Loess smoothing.</figcaption></figure>

area demonstrating diaphragm fiber atrophy (Figures 3C and 3D) and with higher lipid vacuoles accumulation (Figures 3F and 3G). No difference was found with respect to inflammatory cells infiltration (Figures 3H and 3I).

Furthermore, the decrease in SM was associated with diaphragm loss of force measured by Pdi (Figures 3E and E5).

## Patient Outcome

Ventilator-free days, ICU length of stay, weaning difficulty, and mortality at Day 28 were not significantly associated with change in diaphragm SM or thickness (Tables 3 and E5).

## Discussion

The study demonstrates a heterogeneous pattern of changes in SM during the first

week of ICU stay, in which SM does not change in 8%, increases in 51%, and decreases in 41% of patients. Changes in SM were negatively related to changes in diaphragm thickness in critically ill patients after multivariate analysis. Decrease in SM was associated with diaphragm fiber atrophy, diaphragm lipid accumulation, and loss of force production in a healthy mechanically ventilated piglet model. The apparent increase in diaphragm thickness during the ICU stay may therefore be linked with an alteration of diaphragmatic ultrastructural and mechanical properties, as suggested in the diaphragm myotrauma concept.

SWE provides a reliable quantifiable measurement of muscle mechanical properties and muscle stiffness (40, 44) and therefore has been suggested as a tool to evaluate muscle quality and changes of *in vivo* mechanical properties over time (23, 40).

SWE is routinely used to evaluate tendon injuries in sports medicine and muscle mechanical properties in muscular diseases (45, 46). According to muscle stiffness change occurring during diseases, SM would be either increased or decreased. For instance, increased SM has been observed in late-stage Duchenne's myopathy, which is associated with muscle fibrosis (35), or in the affected and stiff limb consecutive to cerebral palsy (36). Conversely, conditions associated with inflammation, edema, and muscle swelling (e.g., ultramarathons, myositis) have been associated with loss of stiffness and decreased SM (34, 46). Decreased SM has also been associated with denervated muscle atrophy (33). Although recent studies reported that SM of the diaphragm was correlated with diaphragm force production during inspiratory efforts (28, 29, 31), no study has evaluated SWE of the diaphragm in the
<figure><img src="image_4.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>F</figcaption></figure>

<10% SM loss at day 3

>10% SM loss at day 3

<figure><img src="image_10.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>H</figcaption></figure>

<10% SM loss at day 3

>10% SM loss at day 3

<figure><img src="image_12.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 3.** (A–I) Relationship between diaphragm shear modulus, (A and B), diaphragm thickness, (C and D) diaphragm muscle fiber size, (E) diaphragm force production, (F and G) diaphragm lipid accumulation, and (H and I) diaphragm inflammatory infiltration obtained in mechanically ventilated healthy piglets. (A) Quantitative analyses of the diaphragm thickness (left panel) and the diaphragm shear modulus (SM) (right panel) at Day 0 (circles) and after 3 days of mechanical ventilation (MV) (squares). (B) Relationship between thickness and SM for all</figcaption></figure>
<table><caption>**Table 3.** Univariate Analysis of Outcomes According to Change in Diaphragm Shear Modulus over Time</caption><thead><tr><th rowspan="2">Outcomes</th><th colspan="5">Change in Diaphragm Shear Modulus during the First Week</th></tr><tr><th>Study Population (n = 92)</th><th>&gt;10% Increase (n = 47)</th><th>&lt;10% Change (n = 7)</th><th>&gt;10% Decrease (n = 38)</th><th>P Value</th></tr></thead><tbody><tr><td>Ventilator-free days to Day 28*</td><td>18.4 (3.8-24.1)</td><td>19.2 (4.1-25.1)</td><td>5.3 (0-20)</td><td>15 (3.8-24)</td><td>0.21</td></tr><tr><td>Ventilator-free days to Day 28† (n = 79)</td><td>15.5 (3.6-22.2)</td><td>16.9 (3.7-22.6)</td><td>5.2 (0.1-19.8)</td><td>13.7 (3.5-22.7)</td><td>0.48</td></tr><tr><td>Prolonged or difficult weaning† (n = 70)</td><td>35 (50)</td><td>16 (44)</td><td>4 (80)</td><td>15 (52)</td><td>0.30</td></tr><tr><td>Tracheostomy*</td><td>4 (4.4)</td><td>1 (2)</td><td>1 (14)</td><td>2 (5)</td><td>0.31</td></tr><tr><td>ICU mortality*</td><td>21 (23)</td><td>9 (19)</td><td>2 (29)</td><td>9 (24)</td><td>0.74</td></tr><tr><td>Day-28 mortality*</td><td>19 (20)</td><td>9 (19)</td><td>2 (29)</td><td>8 (22)</td><td>0.83</td></tr><tr><td>ICU length of stay, d*</td><td>12.4 (7.1-19)</td><td>12.2 (7.5-18.6)</td><td>12 (8.5-39)</td><td>13.1 (6.3-19.6)</td><td>0.82</td></tr></tbody></table>

Data are presented as n (%) or median (interquartile range).

*Results are presented for the total study population (92 patients).

†Results are presented for patients who received the mentioned therapy.

critically ill population as a surrogate for muscle quality or to follow up its architectural changes over time.

In the present study, diaphragm SM as well as thickness changes over time did present different patterns (Figures 2 and E2). After adjustment for day of measurement and number of days on MV, thickness and SM changes over time appeared inversely associated (Table 2). Assuming the theoretical increase in shear wave speed when thickness increases (26), a positive correlation was expected if mechanical properties and stiffness of the analyzed tissue remained similar. Thus, the present negative correlation between diaphragm thickness and SM changes over time is even more valuable. These results indeed demonstrate that the diaphragm's stiffness is reduced because the shear wave speeds decrease with increasing thickness. After multivariable analysis, aging, sepsis, steroids, duration of MV, and the absence of spontaneous breathing cycles were associated with a decrease in diaphragm SM (Table 2). Conversely, one-fourth of the patients did show an increased SM and decreased thickness, which may

represent a combination of decreased muscle mass, myofibrillar destruction, fiber atrophy, and muscle fibrosis (Table E9 and Figure E7).

Aging is associated with sarcopenia, myosteatosis, and myofibrosis. In a clinical study involving different age groups of volunteers, aging was associated with a decline in skeletal SM correlated with muscle weakness, which is in line with the present study (47). The decrease of SM in patients with sepsis in our study may be the consequence of the combination of atrophy, inflammation, edema, and lipid accumulation that reduce diaphragm stiffness (48). The effects of short-term corticosteroids on the diaphragm in critically ill patients is complex because corticosteroids both downregulate protein synthesis and promote atrophy but may also limit disease-related myositis thus with an unpredictable effect on muscle stiffness (11). A recent study showed that SWE detected decreased muscle stiffness associated with weaker muscles over time in patients on long-term glucocorticoid (49). Interestingly, we observed that changes in diaphragm thickness and in SM occurred in 80% of the 14 nonventilated patients, confirming that both MV and critical illness

target the respiratory muscles before (3) or during the ICU stay (50).

To better understand the link between diaphragm histology and SM, we performed a 3-day MV piglet study and biopsied the diaphragm at the end of the experiment. We report herein for the first time that the decrease in SM was associated with both slow and fast fiber atrophy (Figures 3C and 3D), higher intramuscular lipid vacuoles content (Figures 3F and 3G), and diaphragm loss of force production (Figures 3E and E4).

The lack of difference in inflammatory cells infiltration between SM groups in piglets may be related with the healthy state of this model, with less systemic inflammation like sepsis or circulatory shock (51). These findings are in line with the compelling diaphragm myotrauma concept in which underassistance may lead to muscle injury, edema, and inflammation (22). The model of healthy mechanically ventilated piglet is a model of diaphragm overassistance that may explain the increased diaphragm lipid content. Picard and colleagues showed that resting diaphragm during controlled MV may indeed be associated with a state of relative energetic oversupply versus demand that would trigger mitochondrial dysfunction

**Figure 3.** (Continued). values during 72 hours of MV. (C) Representative images of transverse frozen sections stained with antibodies directed against slow isoforms of myosin heavy chain (red), according to SM decrease at Day 3: specimens with a decrease in the SM of <10% (left panels) and a decrease in the SM of >10% (right panels). Scale bars, 50 µm. (D) Quantitative analyses of diaphragm fiber cross-sectional area (median and quartiles) according to SM decrease at Day 3. (E) Relationship between transdiaphragmatic pressure after supramaximal phrenic nerve stimulation at a frequency of 100 Hz and diaphragm SM for all values during 72 hours of MV. (F) Representative electron micrographs showing diaphragm lipid droplets accumulation according to SM decrease at Day 3: SM decrease <10% (left panel) and SM decrease >10% (right panel). Scale bars, 2 µm. (G) Quantitative analyses of diaphragm lipid droplets accumulation (mean ± SD) according to SM decrease at Day 3. (H) Representative images of hematoxylin and eosin-stained sections for inflammatory cells infiltrate according to SM decrease at Day 3: SM decrease <10% (left panel) and SM decrease >10% (right panel); extracellular nuclei of an inflammatory cell are colored in purple. (I) Quantitative analyses of diaphragm inflammatory cells infiltrate (mean ± SD) according to SM decrease at Day 3. All extracellular nuclei outside the muscle fibers were assumed as inflammatory cells, and their presence was expressed as a percentage of the extracellular nuclei out of total nuclei in the area.
and oxidative stress (13). Diaphragm
inflammation and edema may be the main
hypothesis for decreased SM in critically ill
patients with overuse injury (34, 52). We
summarized the different hypothesis for
diaphragm SM and thickness changes in
Table E9.

The perspective of the present results
and the recent literature (29, 31) is that
diaphragm SM evaluation in critically ill
mechanically ventilated patients may be used
as a tool in future interventional studies that
would aim to promote diaphragm protective
strategies. It may also help in phenotyping
patients according to their diaphragm profile
using both diaphragm mass surrogate
(thickness) and diaphragm mechanical
properties (SM). Finally, SM has been
described as a surrogate of inspiratory muscles
effort in spontaneously breathing patients.

Our study presents limits that should be discussed. First, we did not perform serial biopsies of the diaphragm in patients, a dangerous procedure outside the operating room. We therefore could only describe the association between ultrasound measurements and biopsy findings in the piglet model. Diaphragm fiber atrophy was associated with a decreased SM in piglets. Second, to ensure the highest reproducibility, we performed the ultrasound measurements at the end of expiration (and carefully checked the absence of diaphragm

contraction) (20). However, because this
study was designed as a “real life” study, we
did not use Pdi monitoring to make sure
none of the measurements were performed
above the FRC. Third, we performed more
than 600 ultrasound examinations from ICU
admission to discharge, increasing the
statistical power of the analysis, but we
analyzed a combination of different
ventilatory conditions (Figure E5). To date,
longitudinal studies have been
heterogeneous, with follow-up being cut off
right after extubation (53) at Day 5 (50) or at
Day 14 (20). Our study design is a limit but
also better reflects the journey of the ICU
stay. Finally, although the present
combination of both critically ill patients
mechanically ventilated for 7 days and
healthy piglet model ventilated for 3 days
illustrate the myotrauma concept, it may be
difficult to extrapolate some data from one
model to the other.

In summary, we show for the first time
that both diaphragm thickness and stiffness
change vary heterogeneously during the
ICU stay. The increase in global diaphragm
thickness during the ICU stay in one-third
of the patients is associated with a drop in
diaphragm stiffness. In a mechanically
ventilated piglet model, we report that the
decrease in diaphragm stiffness from Day 1
to Day 3 is associated with fast and slow
fiber atrophy, diaphragm lipid

accumulation, and loss of diaphragm force
production. Our results could be another
building block to corroborate the
myotrauma concept in which increase in
diaphragm thickness marks a different yet
not independent type of diaphragm injury
than the better-known decreased thickness.
Further studies focusing on better
understanding cellular and molecular
changes associated with changes in
diaphragm thickness and stiffness over time
are warranted to investigate whether
increased thickness associated with
decreased stiffness is a noninvasive
indicator of diaphragmatic inflammation,
lipid accumulation, injury, and edema,
associated with the fiber atrophy previously
described in piglets.

**Author disclosures** are available with the
text of this article at www.atsjournals.org.

**Acknowledgment:** The authors thank Claudine Gniadek (R.N.) and the members of the medical and nursing team for their participation in the present study. They also thank the animal laboratory facility at the medical school (Plateau Technique de Recherche Experimentale, Nimes, France) for their technical support and Mrs. Rama Levin for her help in English editing for the manuscript. They thank Christophe Cassinoto (M.D., Radiology Unit, Montpellier Teaching Hospital) for his help with the short video presented in the online supplement.

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