A MRI-Compatible Combined Mechanical Loading and MR Elastography Setup to Study Deformation-Induced Skeletal Muscle Damage in Rats

Abstract

Deformation of skeletal muscle in the proximity of bony structures may lead to deep tissue injury category of pressure ulcers. Changes in mechanical properties have been proposed as a risk factor in the development of deep tissue injury and may be useful as a diagnostic tool for early detection. MRE allows for the estimation of mechanical properties of soft tissue through analysis of shear wave data. The shear waves originate from vibrations induced by an external actuator placed on the tissue surface. In this study a combined Magnetic Resonance (MR) compatible indentation and MR Elastography (MRE) setup is presented to study mechanical properties associated with deep tissue injury in rats. The proposed setup allows for MRE investigations combined with damage-inducing large strain indentation of the Tibialis Anterior muscle in the rat hind leg inside a small animal MR scanner. An alginate cast allowed proper fixation of the animal leg with anatomical perfect fit, provided boundary condition information for FEA and provided good susceptibility matching. MR Elastography data could be recorded for the Tibialis Anterior muscle prior to, during, and after indentation. A decaying shear wave with an average amplitude of approximately 2 μm propagated in the whole muscle. MRE elastograms representing local tissue shear storage modulus Gd showed significant increased mean values due to damage-inducing indentation (from 4.2 ± 0.1 kPa before to 5.1 ± 0.6 kPa after, p<0.05). The proposed setup enables controlled deformation under MRI-guidance, monitoring of the wound development by MRI, and quantification of tissue mechanical properties by MRE. We expect that improved knowledge of changes in soft tissue mechanical properties due to deep tissue injury, will provide new insights in the etiology of deep tissue injuries, skeletal muscle damage and other related muscle pathologies.

Full text

RESEARCH ARTICLE
A MRI-Compatible Combined Mechanical
Loading and MR Elastography Setup to Study
Deformation-Induced Skeletal Muscle
Damage in Rats
Jules L. Nelissen
1,2
*, Larry de Graaf
1
, Willeke A. Traa
3
, Tom J. L. Schreurs
1,2
, Kevin
M. Moerman
4
, Aart J. Nederveen
5
, Ralph Sinkus
6
, Cees W. J. Oomens
3
, Klaas Nicolay
1
,
Gustav J. Strijkers
2
1Biomedical NMR, Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The
Netherlands, 2Biomedical Engineering and Physics, Academic Medical Center, Amsterdam, The
Netherlands, 3Soft Tissue Biomechanics and Engineering, Biomedical Engineering, Eindhoven University of
Technology, Eindhoven, The Netherlands, 4Center for Extreme Bionics, Media lab, MIT, Cambridge, MA,
United States of America, 5Department of Radiology, Academic Medical Center, Amsterdam, The
Netherlands, 6Image Sciences & Biomedical Engineering, King’s College London, London, United Kingdom
*j.l.nelissen@gmail.com
Abstract
Deformation of skeletal muscle in the proximity of bony structures may lead to deep tissue
injury category of pressure ulcers. Changes in mechanical properties have been proposed
as a risk factor in the development of deep tissue injury and may be useful as a diagnostic
tool for early detection. MRE allows for the estimation of mechanical properties of soft tissue
through analysis of shear wave data. The shear waves originate from vibrations induced
by an external actuator placed on the tissue surface. In this study a combined Magnetic Res-
onance (MR) compatible indentation and MR Elastography (MRE) setup is presented to
study mechanical properties associated with deep tissue injury in rats. The proposed setup
allows for MRE investigations combined with damage-inducing large strain indentation of
the Tibialis Anterior muscle in the rat hind leg inside a small animal MR scanner. An alginate
cast allowed proper fixation of the animal leg with anatomical perfect fit, provided boundary
condition information for FEA and provided good susceptibility matching. MR Elastography
data could be recorded for the Tibialis Anterior muscle prior to, during, and after indentation.
A decaying shear wave with an average amplitude of approximately 2 μm propagated in
the whole muscle. MRE elastograms representing local tissue shear storage modulus G
d
showed significant increased mean values due to damage-inducing indentation (from 4.2 ±
0.1 kPa before to 5.1 ±0.6 kPa after, p<0.05). The proposed setup enables controlled de-
formation under MRI-guidance, monitoring of the wound development by MRI, and quantifi-
cation of tissue mechanical properties by MRE. We expect that improved knowledge of
changes in soft tissue mechanical properties due to deep tissue injury, will provide new
insights in the etiology of deep tissue injuries, skeletal muscle damage and other related
muscle pathologies.
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 1 / 22
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OPEN ACCESS
Citation: Nelissen JL, de Graaf L, Traa WA,
Schreurs TJL, Moerman KM, Nederveen AJ, et al.
(2017) A MRI-Compatible Combined Mechanical
Loading and MR Elastography Setup to Study
Deformation-Induced Skeletal Muscle Damage in
Rats. PLoS ONE 12(1): e0169864. doi:10.1371/
journal.pone.0169864
Editor: Christophe Egles, Universite de Technologie
de Compiegne, FRANCE
Received: March 7, 2016
Accepted: December 23, 2016
Published: January 11, 2017
Copyright: ©2017 Nelissen et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This research is supported by the Dutch
Technology Foundation STW (www.stw.nl), which
is part of the Netherlands Organisation for
Scientific Research (NWO), and which is partly
funded by the Ministry of Economic Affairs (project
number 12398). The European Cooperation in
Science and Technology (COST) MYO-MRI action

Introduction
Sustained mechanical loading and deformations of skeletal muscle in the proximity of bony
structures may lead to damage in the deep soft tissue layers. This category of injury is recog-
nized as a special type of pressure ulcer by the National and European Pressure Ulcer Advisory
Panel (NPUAP/EPUAP) and is commonly referred to as deep tissue injury [1–5]. The pressure
ulcer may initially stay invisible and remain undetected for days, until the injury becomes visi-
ble as a purple or maroon discolored skin spot, thereafter rapidly developing into a severe and
difficult to heal Stage III or Stage IV pressure ulcer [6,7]. The populations at high risk for
developing deep tissue injury include intensive- and acute-care patients, hospice patients, as
well as people that require long-term care, for example after a spinal cord injury or stroke [8].
Common risk areas are the coccyx, sacrum, buttocks, knee, and heel [9,10]. Deep tissue injury
is related to increased morbidity and mortality [7,11–13]. Cost-of-illness studies for pressure
ulcers report a significant cost burden for society. In particular for deep tissue injuries; with
wound closing times between 127 up to 155 days, typical treatment costs are 10,000–15,000
GBP per ulcer [14–16].
Clinicians and experienced wound nurses have noticed differences in mechanical stiffness
between healthy and injured muscle in early stages of deep tissue injury development by
means of palpation [17,18]. Finite element analysis (FEA) studies have shown that changes in
mechanical properties of the affected tissue may lead to larger deformations and aggravated
wounds [19,20]. Thus, the changes in mechanical properties of soft tissues have been proposed
as one of the risk factors in the development of deep tissue injury, and analysis of tissue stiff-
ness may be useful as a diagnostic tool for early detection [21]. However, the spatial-temporal
changes in muscle mechanical properties from deformation-induced injury have never been
systematically studied and quantified in a controlled setting.
A rat model for deep tissue injury has been studied extensively before, using MRI and FEA
methods [22–32]. This well-established animal model of deep tissue injury involves the com-
pression of the rat Tibialis Anterior (TA) muscle with a custom loading device that can be
placed in an MRI scanner for imaging during the development of the wound [24]. Maps of the
global transversal relaxation time T
2
were used as a readout of muscle edema as a consequence
of damage and inflammation after load release[23,26,31,33]. T
2
is generally accepted as quanti-
tative damage marker in musculoskeletal MRI [34–39]. Animal-specific FEA models, where
the geometry and loading were derived from MRI, supplied estimations of the local tissue
deformations. This combined experimental-numerical approach resulted in new understand-
ings of the mechanical boundary conditions that contribute to the development of deep tissue
injury [32,40].
The animal model raised new questions about the role of the muscle tissue mechanical
properties in the etiology of wound development. Particularly, it is currently not known to
which extent muscle stiffness alters under compression during the development of the injury
and in the early healing stage, nor how stiffness changes contribute to the development of
severe wounds. Since stiffness changes have been reported during tissue injury development,
assessment of changes in tissue stiffness could serve a role in diagnosis of deep tissue injury
[7,19,41–44].
Magnetic Resonance Elastography (MRE) is an MRI technique for measuring mechanical
properties of soft tissue by measuring mechanically induced shear waves [45,46]. The small
strain harmonic shear waves, induced by an external MRI-compatible actuator, are imaged
using a motion-encoded phase contrast MRI sequence. By inversion of the wave image data,
Hookean viscoelastic mechanical properties, such as the shear storage modulus G
d
, can be
quantified [47,48]. In liver diseases MRE proves to be a technique of great diagnostic and
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 2 / 22
supported this research by funding a short term
scientific mission (STSM) (project number
BM1304-34240). The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.

etiologic value [49–52], and is also increasingly utilized for use in the kidney, spleen, brain,
breast, heart, lungs, prostate, and skeletal muscle [53–61]. MRE studies of skeletal muscle have
primarily focused on healthy skeletal muscle to better understand the complex anisotropic vis-
coelastic mechanical properties and to assess changes in muscle shear stiffness during active
and passive contraction [62–70]. In a number of studies MRE has been applied to characterize
muscle disease and has proven to have added value in addition to the more conventional MRI
methods; MRE is proposed as objective evaluation method to monitor the efficacy of treat-
ments for pathologic or injured muscle [71–78].
The application of MRE to assess deep tissue injury was proposed in several papers, but it
has, to the best of our knowledge not been performed yet [19,20,41–43,79]. In this paper, we
present an MRI-compatible setup for studying the mechanical properties of rat TA muscle
during the development of deep tissue injury. The setup allows for controlled deformation
under MRI-guidance, monitoring of the wound development by MRI, and quantification of
tissue mechanical properties and potential changes therein by the MRE technique.
Methods
Setup
A schematic illustration of the setup is shown in Fig 1. The setup is fixed in (A) a fiberglass
tube with an outer diameter of 86 mm and a wall thickness of 2 mm. Inside the tube the follow-
ing parts are mounted: (B) a 3D-printed water-circulated heating blanket for maintaining the
rat body temperature, (C) an anesthesia mask with supply and exhaust of anesthesia gas for
sedating the rat, (D) a fixation block to secure the setup in the MRI magnet, (E) the indentor,
and (F) the MRE actuator. (G) A base plate with (H) electromagnetic shaker (LDS V201, Bru¨el
and Kjaer, Royston, UK) is fixed to the wall of the Faraday cage at a safe distance from the
stray field of the 7.0 T MRI magnet. The MRE actuator is brought into motion via (I) a splined
transmission rod (type S518, Sullivan Products, Baltimore, US). The shaker is powered with an
amplifier (QUAD 50E, Huntington, UK) and waveform generator (Agilent 33220A, Santa
Clara, US) placed outside the Faraday cage. The shaker is air cooled with constant airflow. Syn-
chronization of the MRE sequence with the shaker motion is performed via transistor-transis-
tor logic (TTL) triggering. The TTL trigger signal is a 3V voltage level signal which is pulled to
ground in case of a trigger. Signals from TTL triggering and waveform generator were visual-
ized on an oscilloscope (Agilent InfiniVision 2000X oscilloscope, Santa Clara, US) to verify
correct synchronization. The MRE actuator is roughly based on the design of Sinkus et al. that
was used for MRE of colon tumors in mice and for studying an mdx mouse model of muscular
dystrophy [71,80,81]. A photograph of a rat positioned in the setup can be found in the online
supplement (S1 Fig).
Fig 1. Overview of the MR compatible indentation and MRE setup. Following parts are labeled: Fiberglass tube (A), 3D-printed heating
blanket (B), anesthesia mask (C), fixation block (D), indentor (E), MRE actuator (F), shaker base plate (G), electromagnetic shaker (H). Part E
and Fare shown in more detail in Fig 2.
doi:10.1371/journal.pone.0169864.g001
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 3 / 22

The indentation and MRE components (Eand F) are shown in greater magnification and
detail in Fig 2. The rat can be conveniently positioned in the setup using a right-left direction
adjustable u-shaped profile with a cutout for the rat’s groin, which enables positioning of the
right leg of the rat under the indentor (Fig 2 indentor). The indentor can be positioned on the
rat TA muscle using a movable indentor holder and rotatable half arch. The indentor is a cylin-
drical rod (3 mm in diameter, 36.5 mm long) with a rounded head and is composed of two,
screwed-together parts. The indentor assembly contains a hollow compartment, which is filled
with an aqueous solution of 1 g/L CuSO
4
for MRI visualization, inside a rigid solid rod. The
MRE actuator is mounted on a dovetail profile allowing for adjustment in the axial direction.
The height and depth of the indentation can be controlled by removing spacer plates or by
altering the position of the MRE piston (Fig 2 MRE piston). If desired the MRE piston can be
replaced by pistons of a different shape or size. The MRE piston is brought into motion via the
drive rod attached to the electromagnetic shaker (Fig 1H) and cantilever. Detailed overview of
all above described parts of the indentation and MRE components can be found in the online
supplement (S2 Fig).
Indentation of the rat TA muscle is achieved by positioning the indentor above the TA
muscle using the rotatable half-arch, by manually pushing the rod into the muscle, and by fix-
ating it at the desired indentation depth and orientation. The MRE piston is softly coupled to
the tendon at the distal side of the TA muscle. All parts are made of PET-P (polyethylene tere-
phthalate polyester), or from PEEK (polyether ether ketone). Both materials are MRI-compati-
ble, and have high mechanical strength, stiffness and creep resistance.
Fig 2. Detail of indentation and MRE actuator part of setup. Indentor and MRE piston are indicated with a label. Indentor can be positioned on the rats TA
using the movable indentor holder. The MRE piston is coupled to the tendon at the distal side of the TA muscle. Overview figure indicating all parts is shown in
online supplemental S2 Fig.
doi:10.1371/journal.pone.0169864.g002
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 4 / 22

Animal model
To investigate the feasibility of MRE based shear stiffness changes occurring during indenta-
tion induced damage 20 female Sprague-Dawley (SD) rats (11 to 13-week-old, ±250 g, Charles
River, Paris, France) were used. To optimize the settings of the experimental setup and the MR
sequences 13 rats were used (results not shown). MRE measurements pre and post 2 h indenta-
tion was performed in 7 rats. In 1 of these rats MRE measurements were also performed dur-
ing indentation. A duration of 2 h indentation was chosen based on previous studies, in which
different durations of deformation, ischemia and reperfusion were studied [40]. The results of
these experiments showed that deformation, ischemia and reperfusion all contribute to the
damage process but that deformation induced damage is dominant for periods less than 2 h.
Animals were housed under standard laboratory conditions with a 12 h light/dark cycle and
were maintained on a standard diet and with access to water ad libitum. Rats were anesthetized
with isoflurane (4.0 vol% for induction, 1.0–2.0 vol% for maintenance) in 0.6 L/min medical
air. Buprenorphine (0.05 mg/kg s.c.) was administered for analgesia. Eye ointment was applied
to prevent eye dehydration. The rat was placed in supine position in the MR-compatible setup.
Body temperature was maintained at 35–37˚C with the heating blanket and monitored with a
rectal temperature sensor. Respiration was monitored with a balloon pressure sensor placed
on the abdomen and maintained in a physiological range by adjusting the anesthesia. The
right leg of the rat was shaved and positioned in the u-shaped profile filled with alginate mold-
ing substance for firm fixation and susceptibility matching. To assess the TA muscle during
indentation the top of the alginate cast was removed making the TA muscle accessible to the
indentor. After indentation the alginate cast was closed again. Indentation of the TA muscle
for 2 h took place inside the MRI scanner. The MRE actuator was coupled to the tendon at the
distal side of the TA muscle. After the measurements the animals were sacrificed by means of
exsanguination from the inferior vena cava. This procedure was performed under anesthesia
and after administration of analgesia. All animal experiments were approved by the Animal
Care and Use Committee of Maastricht University, Maastricht, The Netherlands (protocol
2013–047, Maastricht University, Maastricht, The Netherlands) and performed in accordance
with the Directive 2010/63/EU for animal experiments of the European Union.
MRI
Measurements were performed with a 7.0 T small animal MRI scanner (Bruker BioSpin MRI
GmbH, Ettlingen, Germany) equipped with a 660 mT/m, 4570 T/m/s gradient coil (BGA-12S
HP, Bruker BioSpin MRI GmbH, Ettlingen, Germany). A 86-mm-inner-diameter quadrature
transmit coil was used in combination with a 20-mm-diameter surface receive coil (Bruker BioS-
pin MRI GmbH, Ettlingen, Germany) placed on top of the TA muscle inside the indentation
part of the setup. Anatomical images were acquired with T
1
-weighted MRI (scan parameters:
sequence = FLASH 3D, field of view (FOV) = 6 x 6 x 6 cm
3
, acquisition matrix (MTX) = 192 x
192 x 192, echo time (TE) = 3.72 ms, repetition time (TR) = 16.84 ms, and acquisition time =
2:35 min). Alginate cast visualization was done with ultra-short echo time MRI (scan parame-
ters: sequence = ultra-short echo time 3D, FOV = 6 x 6 x 6 cm
3
, MTX = 256 x 256 x 256, TE =
21 μs, TR = 2.69 ms, radial undersampling = 3 and acquisition time = 3:04 min) [82]. Elastogra-
phy images were acquired with an in-house developed echo-planar-imaging (EPI) MRE se-
quence (sequence = SE-EPI-MRE 2D with fat suppression, number of slices = 18 slices, slice
thickness = 1 mm, FOV = 3 x 6 cm
2
, MTX = 96 x 192, TE = 26.2 ms, TR = 1000 ms, actuator
and motion encoding gradient (MEG) frequency = 900 Hz, number of EPI segments = 4, num-
ber of MRE offsets = 16, number of encoding directions = 3 (slice, phase, frequency encoding)
plus reference, and acquisition time = 17:04 min). MRE during indentation was performed with
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 5 / 22

a spin-echo based MRE sequence (sequence = SE-MRE 2D, number of slices = 10, slice thick-
ness = 1 mm, FOV = 2.5 x 2.5 cm
2
, MTX = 128 x 128, TE = 21.1 ms, TR = 1000 ms, actuator and
MEG frequency = 900 Hz, number of MRE offsets = 8, number of encoding directions = 1 (slice
direction), and acquisition time = 17:05 min). Skeletal muscle edema was assessed with T
2
map-
ping in both axial and coronal orientations (axial: sequence = MSME 2D with fat suppression,
number of slices = 20, slice thickness = 1 mm, FOV = 2.5 x 2.5 cm
2
, MTX = 256 x 256, TE = 6.9–
180.7 ms, number of echoes = 26, TR = 4000 ms, and acquisition time = 12:48 min; coronal:
sequence = MSME 2D with fat suppression, number of slices = 18, FOV = 6 x 3 cm
2
, MTX =
512 x 256, TE = 10.2–203.5 ms, number of echoes = 20, TR = 3994 ms, and acquisition time =
12:54 min). All measurements were performed up to 90 min after the end of 2 h indentation.
Data analysis
The MRE acquisitions provide phase data for the harmonic vibrations which are proportional
to the harmonic displacements. By using an inversion algorithm the displacement data can be
converted to elastograms, i.e. images representing the local tissue linear elastic shear storage
modulus G
d
(real part of the complex shear modulus G). The inversion process assumes lin-
ear (visco-) elasticity, isotropy, and local homogeneity for all tissues, and aims to solve the fol-
lowing partial differential equation:
ro2qðxÞ ¼ Gr2qðxÞ1
Where Gis the complex shear modulus, ρis the density, qis the curl of the complex displace-
ment vector (q(x) = r×u(x)) derived from the MRE phase data, and ωis the known angular
frequency. In case of a continuous monochromatic wave the displacement is defined as:
uðxÞ ¼ Aeikx2
with A the amplitude, and kthe complex wave number. Combination of Eqs 1and 2produces:
Goð Þ ¼ ro2
k23
Combining Eq 3 with the fact that k=β+iα, the complex shear modulus Gcan be split in
terms of shear storage modulus G
d
and shear loss modulus G
i
. Full details of the inversion
algorithm implemented in the ROOT data analysis framework (ROOT 5.34/17, CERN,
Meyrin, Switzerland) were previously described by Sinkus et al [83–85].
Quantitative T
2
maps were obtained by pixel-wise fitting of the MR signal to S¼S0eTE=T2
(Mathematica 10, Wolfram Research, Champaign, USA). Pixels with goodness of fit R
2
<0.7
and SNR <4 were excluded from further analysis.
Region-of-interest (ROI) based analysis of the elastograms and T
2
maps of the 6 rats mea-
sured before and after 2 h of indention was performed (Matlab R2016a, The Mathworks, Inc.,
Natick, Massachusetts, USA). An ROI was defined by manually outlining the TA muscle in the
center slice. Mean values of G
d
, displacement amplitude A
tot
and T
2
were determined in this
ROI before and after indentation. The percentages of significantly elevated G
d
and T
2
pixels
were calculated by thresholding of pixels with values increased by 2 standard deviations as
compared to the mean values pre-indentation. Mean, standard deviation (sd), and coefficient
of variation (CV) were calculated to quantify the group average and variation at baseline, dur-
ing and after indentation. Mean values of G
d
and T
2
were also determined in a circular ROI of
4 x the diameter of the indentor, positioned at the center of indentation. Paired sampled t-tests
were conducted to test for significant differences between before and after indentation (Origin
2015, OriginLab Corporation, Northampton, USA).
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 6 / 22

Results
The experimental methods presented here combine soft tissue indentation with MRE to assess
the skeletal muscle shear storage modulus G
d
before, during and after deep tissue injury devel-
opment. Both the indentation device as well as the MRE actuator are designed to allow flexible
positioning on top of the TA muscle in the hind leg of a rat and can be used before, during,
and after indentation inside a small animal MRI scanner. The combination of an RF surface
receiver coil and volume transmission coil resulted in high-quality MRI and MRE images of
the whole TA muscle before, during, and after 2 h indentation. Fixation with alginate assures a
good leg fixation with a perfect anatomical fit. In addition, it improves susceptibility matching
enabling fast imaging with EPI-based sequences.
MRI
The manually set indentation depth can accurately be determined since the indentor is visible
in the MR images (Fig 3B). Indentation depth was varied between rats resulting in different
degrees of deep tissue injury in the TA muscles. Fig 3 shows representative axial anatomical
T
1
-weighted images of a rat leg (A) pre, (B) during, and (C) post indentation together with the
corresponding quantitative (D-F) T
2
maps of a slice through the center of the region of defor-
mation. The indentor pressing in the TA muscle is clearly visible in the T
1
-weighted images
(arrow). The tibia bone, fibula bone, and skin contour provide anatomical landmarks from
which indentation depth can be estimated. Compression of the TA muscle between the inden-
tor and tibia bone is seen in (B). Elevated T
2
values compared to pre indentation are seen in
Fig 3. Anatomical images and T
2
maps. Axial anatomical images (A-C) and T
2
maps (D-F) of the hind limb before, during, and after indentation,
respectively.
doi:10.1371/journal.pone.0169864.g003
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 7 / 22

the T
2
map post indentation (Fig 3F). These increased T
2
values are indicative for the forma-
tion of edema and skeletal muscle damage as validated before [25,26,40].
Several axial slices of the 3D ultra-short echo time images of the alginate cast around the leg
before, during, and after indentation are shown in Fig 4. Due to its short T
2
relaxation time, the
alginate is only visible on the ultra-short echo time scans and not on the regular T
1
-weighted
and T
2
-weighted scans. The presence of alginate therefore does not interfere with the anatomi-
cal scans and the MRE measurements, and improves image quality by providing good suscepti-
bility matching between the leg and its surroundings. During indentation (Fig 4 during) the top
of the alginate cast was removed. Newly applied alginate is brighter on the post-indention ultra-
short echo time scan (Fig 4 post). The cast closely follows the anatomy of the leg and little dis-
placement of the leg between the three conditions is seen. The contours and the rigid fixation
by the cast provide essential boundary conditions for FEA calculations of deformation related
strains, as previously demonstrated [86].
Magnetic resonance elastography
Fig 5A displays a representative displacement wave in non-damaged TA muscle along the blue
line in the displacement field of Fig 5B. A sinusoidal displacement wave with decaying ampli-
tude was observed along the entire path of the travelling wave. Mean (A
tot
) and maximum
(A
max
) TA displacement amplitudes for all 6 animals measured before and at all time points
after indentation are listed in Table 1. The displacement amplitude was in the range of 0.5–
10 μm (mean 2 μm).
In Fig 6 eight offsets of coronal SE-MRE magnitude images (mag) and wave images (θ
1
—
θ
8
) are shown before, during, and after indentation. Distinct wave propagation was observed
during the equally distributed time offsets θ
1
—θ
8
of the 900 Hz wave with a time resolution of
0.1389 ms. In Fig 6B the dark spot in the TA corresponds to the location of the indentor. The
wave pattern in (Fig 6B1–6B8) during indentation was distinctly different compared to pre
indentation (Fig 6A1–6A8). The changes in the wave patterns relate to changes in tissue elas-
ticity and geometry due to indentation, and also by reflection of shear waves at the indentor
surface. Post indentation (Fig 6C1–6C8) the waves show a different pattern compared to pre
indentation (Fig 6A1–6A8).
Fig 7 shows one offset of a 16 offsets SE-EPI-MRE data set acquired (A) before and (B) after
2 h indentation, respectively. A movie of all offsets is added in the online supplemental of this
article (S2 Fig). A distinct change in the wave pattern was observed due to altered tissue stiff-
ness of the wound caused by indentation.
In Fig 8, representative MRE elastograms (Fig 8A) before, as well as (Fig 8B) after 2 h of
indentation are shown together with the corresponding T
2
-maps (Fig 8C and 8D). Increased
shear storage modulus G
d
was observed in the TA in the elastogram that was measured follow-
ing 2 h of indentation. T
2
-maps after indentation revealed a large region of elevated T
2
values
in the TA muscle, indicative for edema and muscle injury. The region of increased shear stor-
age modulus G
d
was less diffuse and smaller compared to the region of elevated T
2
. The region
of high shear storage modulus G
d
highlights a central focus with severe muscle damage,
whereas T
2
elevation occurs in a larger region with edema surrounding the injury. The MRE
elastograms of the other analyzed animals show the same consistent results.
For all 6 animals measured before and after end of indentation, the shear storage modulus
G
d
maps of the TA muscle were reconstructed at four time points: before, 30 min after, 60 min
after, and 90 min after end of indentation. Two animals had missing data at the 90 min after
time point. The T
2
maps were calculated at two time points: before and 45 min after end of
indentation. T
2
and G
d
cannot be measured at the same time, particularly because of the long,
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 8 / 22

Fig 4. Alginate cast visualization. Axial 3D ultra-short echo time images of the alginate cast surrounding the
rat leg, pre, during and post indentation. FLASH 3D rendering shows anatomical landmarks and location of
selected 3D ultra-short echo time images.
doi:10.1371/journal.pone.0169864.g004
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 9 / 22

17 min, G
d
acquisition. As a result fewer time points were chosen for T
2
than G
d
as the latter is
of more interest in this study. G
d
and T
2
values in the circular ROI around the indentor are
shown in Fig 9. The percentage of significant elevated G
d
and T
2
pixels measured in the whole
TA ROI are displayed in Fig 10. Measurements of mean (±sd) and percentage elevated pixels
of G
d
and T
2
are also summarized for each animal in Tables 2and 3. G
d
Standard deviation
(0.1 kPa) and coefficient of variation (3%) were low at baseline in non-damaged TA muscle.
Fig 5. Displacement field of z-direction with line profile. (A) Shows the displacement in μm along the line profile illustrated as blue line in the (B)
displacement field image of the z direction in the TA muscle.
doi:10.1371/journal.pone.0169864.g005
Table 1. Mean and maximum displacement amplitude A
tot
before, 30, 60 and 90 min after end of indentation per animal and mean ±sd of the
group.
animal before 30 min after 60 min after 90 min after
A
tot
A
max
A
tot
A
max
A
tot
A
max
A
tot
A
max
[μm] [μm] [μm] [μm] [μm] [μm] [μm] [μm]
11.5 7.2 1.6 4.2 1.9 5.1
23.7 9.9 2.8 8.0 2.3 5.2
31.1 5.1 1.3 3.9 1.5 3.9 1.6 3.2
42.2 6.9 1.8 6.9 1.7 7.9 1.8 8.4
52.5 8.9 2.8 7.3 2.7 9.7 3.1 7.8
61.3 5.8 1.8 5.3 1.8 4.2 1.6 3.9
mean 2.0 7.3 2.0 5.9 2.0 6.0 2.0 5.8
sd 1.0 1.8 0.6 1.7 0.5 2.3 0.7 2.7
doi:10.1371/journal.pone.0169864.t001
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 10 / 22

Averaged across all animals, the mean T
2
of the TA muscle at 45 min after end of indenta-
tion was significantly higher than before indentation (p <0.01). A trend of increased mean
shear storage modulus G
d
after indentation was apparent. Mean shear modulus G
d
at 30 min
after indentation was significantly higher than before indentation (p <0.05).
The percentage significantly elevated T
2
and G
d
pixels after end of indentation was signifi-
cantly higher compared to before indentation (T
2
; p <0.01, G
d
; p <0.05 at 30 min after).
The percentage elevated T
2
pixels after indentation was significantly higher compared to the
percentage elevated G
d
pixels after indentation at all three time points (p <0.01, p <0.01,
p<0.05). In both T
2
and G
d
maps before indentation a comparable percentage of significantly
elevated pixels was found (3.6 ±0.6% for G
d
and 4.0 ±0.7% for T
2
).
Discussion
In this paper we have presented a combined Magnetic Resonance (MR) compatible indenta-
tion setup and MR Elastography (MRE) setup to study the spatial-temporal changes in
mechanical properties that result from deformation-induced injury. The setup was designed
in such way that tissue indentation and the MRE methods can either be used separately or
simultaneously. The setup was successfully tested in a deep tissue injury rat model by damage-
inducing indentation and MR Elastography measurements of the rat TA muscle. Results show
that it is possible to quantitatively measure muscle mechanical properties over time. The elas-
tograms after 2 h of indentation revealed a local increase in shear storage modulus G
d
after
indentation. The spatial extent of change in stiffness was also evaluated by quantifying the
percentage pixels with significantly enhanced G
d
values with respect to baseline. Quantifica-
tion of G
d
values in a circular ROI around the indentation showed a significant elevation of G
d
values 30 min after indentation. At 60 and 90 min after indentation a trend of elevated G
d
was
observed. The determined mean shear moduli values in non-damaged condition are in range
with reported values of MRE measurements and ex vivo biomechanical tests [23,71,87–89].
Fig 6. MRE pre, during post indentation. SE-MRE magnitude (mag) and wave (θ
1
—θ
8
) images before, during and after indentation. Wave images θ
1
,θ
2
,
θ
3
,θ
4
,θ
5
,θ
6
,θ
7
, and θ
8
correspond to 0
0
, 45
0
, 90
0
, 135
0
, 180
0
, 225
0
, 270
0
, 315
0
wave phase, respectively.
doi:10.1371/journal.pone.0169864.g006
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 11 / 22

These results show the high potential of the setup to measure non-invasively the quantitative
mechanical properties related to deep tissue injury in vivo.
There are still options to further improve the indentation setup. Firstly, the setup does not
allow for an indentation controlled from outside the scanner; i.e. currently the setup has to be
taken out of the scanner bore to apply, adjust or remove the indentor. This manual operation,
especially if multiple indentation depths are of interest, is time consuming and repositioning
of the setup following indentor adjustment may cause misalignment of image data sets. Image
registration can be employed to correct for such misalignment. However, a controlled indenta-
tion device would be preferred. Also, the indentor has no force sensor incorporated in the cur-
rent implementation. Although indentation control and force sensing can be achieved inside
an MRI scanner (e.g. see [90] for a clinical scanner set-up) these were not implemented in the
current study due to the space challenges in a small animal MR scanner. Knowledge of the
indentation force boundary conditions, combined with MRI data of the undeformed and
deformed configuration would enable inverse FEA based investigation of hyperelastic tissue
mechanical properties [91].
Fig 7. MRE pre and post indentation. Snapshot of a 16 offset 900 Hz SE-EPI-MRE (A) before and (B) after indentation.
doi:10.1371/journal.pone.0169864.g007
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 12 / 22

Fig 8. Elastograms and T
2
maps before and after indentation. (A, B) Elastograms representing shear storage
modulus G
d
and (C, D) T
2
-maps (A, C) before, and (B, D) after indentation.
doi:10.1371/journal.pone.0169864.g008
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 13 / 22

The MRE part was designed to be flexible in feet-head and anterior-posterior direction, but
is limited in left-right direction due to the fixed position of the electromagnetic shaker driving
the MRE part. In the positioning of the leg of the animal this limitation was taken into account,
which resulted in successful coupling of the MRE actuator piston in all animals. Although only
MRE measurements of 900 Hz were presented here, higher and lower frequencies (100 Hz–
Fig 9. Mean (±sd) T
2
and G
d
measured in the circular ROI of 4x indentor’s diameter positioned around the center of indentation of 6 animals
before and after end of indentation. (A) Mean (±sd) T
2
before and 45 min after end of indentation. T
2
45 min after end of indentation was significantly
higher (***p<0.01) than before indentation. (B) Mean (±sd) G
d
before, 30, 60 and 90 min after end of indentation. G
d
is increased at all time points after
indentation. G
d
at 30 min after end of indentation was significantly higher than before indentation (**p<0.05).
doi:10.1371/journal.pone.0169864.g009
Fig 10. Percentage elevated T
2
and G
d
pixels of ROI in the TA muscle of 6 animals before and after end of indentation. Pixels were selected as
significantly elevated, if the value was higher as mean value before indentation + 2 x sd. (A) Percentage elevated T
2
pixels before and 45 min after end
of indentation. Percentage elevated T
2
pixels 45 min after end of indentation was significant higher (***p<0.01) than before indentation. (B) Percentage
elevated G
d
pixels before, 30, 60 and 90 min after end of indentation. Percentage elevated G
d
pixels 30 min after end of indentation was significant higher
than before indentation (**p<0.05).
doi:10.1371/journal.pone.0169864.g010
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 14 / 22

1500 Hz) were also evaluated. Frequencies between 1000 and 1500 Hz did not always result in
sufficient displacement (<0.5 μm displacement) to produce waves through the whole TA
muscle. At 900 Hz a decaying wave with displacement in the range of 0.5–10 μm (mean 2 μm)
was induced. This displacement amplitude is comparable to other MRE studies [87,92]. We
observed that the MRE actuation system behaves frequency dependent; less power was needed
with lower frequencies to generate sufficient displacement at the MRE piston. In addition,
tests with a prototype of the current MRE actuator showed eigenfrequencies with different
designs of cantilevers resulting in higher MRE piston amplitudes [80]. Altogether, measure-
ments at each frequency in the range 100 Hz– 1500 Hz are possible but further optimization to
obtain the correct amplitude at the MRE piston is required.
Table 2. Measured mean (±sd) shear storage modulus G
d
, and percentage elevated pixels of shear storage modulus G
d
elev. in ROI of whole TA
muscle, and mean shear storage modulus G
d
in circular ROI of 4x indentor’s diameter.
animal before 30 min after 60 min after 90 min after
Whole TA Whole TA Ind. Whole TA Ind. Whole TA Ind.
G
d
G
d
G
d
elev. G
d
Gd G
d
elev. G
d
G
d
G
d
elev. G
d
[kPa] [kPa] [%] [kPa] [kPa] [%] [kPa] [kPa] [%] [kPa]
14.4 (1.4) 4.5 (1.8) 8.2 4.4 4.5 (1.6) 7.3 4.3
24.2 (1.6) 4.6 (1.6) 7.4 4.7 4.4 (1.7) 6.0 4.5
34.1 (1.8) 4.3 (1.9) 7.8 4.9 3.9 (1.6) 2.3 4.3 4.0 (1.5) 2.5 4.4
44.2 (1.7) 4.8 (2.8) 12.2 5.3 4.4 (2.1) 6.8 4.8 4.4 (2.1) 8.2 4.5
54.1 (1.8) 5.8 (2.9) 25.0 6.2 5.9 (3.0) 26.0 6.5 5.7 (3.0) 21.8 6.4
64.1 (2.1) 5.1 (2.1) 5.8 5.4 5.0 (2.3) 11.5 5.3 4.8 (2.3) 8.6 5.2
mean 4.2 4.8 11.1** 5.1** 4.7 10.0 4.9 4.7 10.3 5.1
sd 0.1 0.6 7.2 0.6 0.7 8.4 0.9 0.7 8.2 0.9
CV 3 12 65 13 15 84 12 15 80 18
G
d
elev., percentage of pixels with elevated G
d
(>mean G
d
before + 2 x sd); Ind.,circular ROI of 4x indentor’s diameter
** p<0.05 with paired sampled t-test.
doi:10.1371/journal.pone.0169864.t002
Table 3. Measured mean (±sd) and percentage elevated pixels of T
2
map in ROI of whole TA muscle, and mean T
2
in circular ROI of 4x indentor’s
diameter.
animal before 45 min after
Whole TA Whole TA Ind.
T
2
T
2
T
2
elev. T
2
[ms] [ms] [%] [ms]
143 (6) 47 (11) 15.2 49
238 (4) 65 (20) 79.4 69
338 (5) 57 (23) 65.7 57
439 (5) 54 (22) 47.4 52
540 (5) 69 (36) 78.8 60
639 (4) 47 (14) 32.0 49
mean 39 57*** 53.1*** 56***
sd 2 9 26.2 8
CV 4 16 49 14
T
2
elev., percentage of pixels with elevated T
2
(>mean T
2
before + 2 x sd); Ind.,circular ROI of 4x indentor’s diameter
*** p<0.01 with paired sampled t-test.
doi:10.1371/journal.pone.0169864.t003
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 15 / 22

The alginate cast allowed proper fixation of the animal leg with anatomical perfect fit, pro-
vided boundary condition information for FEA and provided good susceptibility matching.
The use of alginate has advantages over plaster [93–95]. Alginate does not clot, is easy to pre-
pare, can be molded anatomically around the rat leg and is very easy to remove without any
remnants. In addition, the surrounding alginate provided appropriate susceptibility matching
beneficial for MRI, i.e. it reduced artefacts and improved image quality. For this reason, the
MRE measurements during indentation, where the top of the alginate cast was removed, was
performed with SE-MRE instead of SE-EPI-MRE. SE-MRI is less sensitive to field inhomoge-
neities but more time consuming. A typical SE-MRE scan with three encoding directions plus
reference will take approximately 1h. To capture dynamic changes in tissue mechanical prop-
erties SE-EPI-MRI is therefore preferred.
After application of the indentor to the TA muscle inside the MRI scanner, increased signal
intensity in the TA muscle was observed on T
2
maps as a result of edema, resulting from the
induced damage and associated inflammatory response [22–26]. ROI analysis showed a signif-
icant increase in T
2
after end of indentation. A significant increase in the percentage of signifi-
cantly elevated T
2
pixels was observed after indentation. The area of pixels with elevated T
2
at
45 min after indentation was significantly larger than those with elevated G
d
at 30, 60 and 90
min after indentation. Using T
2
alone as specific damage area indicator might result in overes-
timation of the size of the damaged area, as in the initial injury phase fluids might diffuse
between the fibers proximally and distally from the wound. The size of the affected region in
T
2
in comparison to G
d
may provide insights in the causes and consequences of deformation
induced muscle damage.
In the current study deep tissue injury and skeletal muscle tissue were of interest. Shear
moduli data for the more superficial adipose tissue were not derivable in the current study
because fat suppression techniques were employed for the MR acquisitions, enabling minimi-
zation of chemical shift artefacts. If such data is however desired non-EPI MRE sequences can
be employed without the use of fat suppression.
The employed MRE inversion algorithm assumes Hooke’s law of linear (visco) elasticity (as
is valid for the small strain vibrational motions) and assumes isotropic tissue behavior. How-
ever, as is the case for muscle tissue, the epi-, peri- and endomysium structure causes the tissue
to be anisotropic. Furthermore, damage and deformation may independently cause changes in
elastic symmetry and therefore induce anisotropy. Since the inversion algorithm assumes isot-
ropy the reconstructed shear moduli information may potentially be affected by any underlying
anisotropy. Exactly how anisotropy is reflected in analyses based on isotropic inversion methods
is currently unknown, and may also relate to the directions wave happen to locally follow. How-
ever, when combined with MRI-compatible force sensing, diffusion tensor-based fiber architec-
ture assessment, and inverse FEA, the presented methods do offer a means to assess such effects
in the future [86,90,96–98]. This is because the initial anisotropy and non-linear elastic behavior
would be accurately reflected in FEA based on such measurements, and elastic tensor data for
any state of deformation can be directly compared to MRE findings. In principle such analysis
can be coupled with FEA incorporating constitutive formulations of damage and swelling.
In conclusion, we have presented an MRI-compatible setup for studying the mechanical
properties of rat TA muscle. The setup allows for controlled deformation under MRI-guid-
ance, monitoring of the wound development by MRI, and quantification of tissue mechanical
properties by MRE. We expect that improved knowledge of changes in soft tissue mechanical
properties due to deep tissue injury, will provide new insights in the etiology of deep tissue
injuries, skeletal muscle damage and other related muscle pathologies. Future studies will
focus on the pathophysiological processes behind the changes in mechanical properties of
damaged skeletal muscle and evaluation of the changing mechanical properties over time.
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 16 / 22

Ethical approval
All animal experiments were approved by the Animal Care and Use Committee of Maastricht
University, Maastricht, The Netherlands (protocol 2013–047, Maastricht University, Maas-
tricht, The Netherlands) and performed in accordance with the Directive 2010/63/EU for ani-
mal experiments of the European Union.
Supporting Information
S1 Fig. Photograph of a rat positioned in the setup. Rat, Indentation and MRE component
are indicated with arrows. In A, the indentor, put through the surface RF coil, positioned on
top of TA muscle in rats hindleg, and the MRE piston attached at distal side of TA muscle are
shown. Indentation component is removed in B, revealing the MRE component and surface
coil. Pre-amplifier block of the surface coil, the respiratory sensor and the rectal temperature
probe are also visible. Anesthesia mask and rat’s head are underneath the pre-amplifier block.
(TIF)
S2 Fig. Detail of indentation and MRE actuator part of setup. Following parts are labeled: u-
shaped profile (a), cutout for the rat’s groin (b), indentor (c), movable indentor holder (d),
rotatable half arch (e), dovetail profile (f), spacer plates (g), MRE piston (h), drive rod (i), can-
tilever (j).
(TIF)
S3 Fig. MRE pre and post indentation movie. Movie of a 16 offsets 900 Hz SE-EPI-MRE (A)
before and (B) after indentation.
(AVI)
Acknowledgments
The authors thank Leonie Niesen, Jo Habets, and David Veraart for biotechnical assistance
and dr. ir. Sandra Loerakker for her help with starting up the animal model and discussions.
VDL ETG Quick Service and EPC (TU/e) workshops for building the setup.
Author Contributions
Conceptualization: JLN LdG WAT TJLS KMM AJN RS CWJO KN GJS.
Formal analysis: JLN LdG RS GJS.
Funding acquisition: AJN CWJO KN GJS.
Investigation: JLN LdG WAT TJLS.
Methodology: JLN LdG WAT TJLS KMM AJN RS CWJO KN GJS.
Project administration: KN GJS.
Resources: JLN LdG WAT TJLS KMM AJN RS CWJO KN GJS.
Software: JLN LdG WAT TJLS KMM RS GJS.
Supervision: AJN CWJO KN GJS.
Validation: JLN LdG WAT TJLS KMM AJN RS CWJO KN GJS.
Visualization: JLN LdG.
Writing – original draft: JLN LdG WAT TJLS KMM AJN RS CWJO KN GJS.
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 17 / 22

Writing – review & editing: JLN LdG WAT TJLS KMM AJN RS CWJO KN GJS.
References
1. Bouten C V., Oomens CWJ, Baaijens FP, Bader DL. The etiology of pressure ulcers: skin deep or mus-
cle bound? Arch Phys Med Rehabil. 2003/04/12. 2003; 84: 616–9. doi: 10.1053/apmr.2003.50038
PMID: 12690603
2. Stekelenburg A, Gawlitta D, Bader DL, Oomens CW. Deep tissue injury: how deep is our understand-
ing? Arch Phys Med Rehabil. 2008/07/01. 2008; 89: 1410–1413. doi: 10.1016/j.apmr.2008.01.012
PMID: 18586145
3. Black J. National Pressure Ulcer Advisory Panel’s updated pressure ulcer staging system. Adv Skin
Wound Care. 2007; 20: 269–274. doi: 10.1097/01.ASW.0000269314.23015.e9 PMID: 17473563
4. Beeckman D, Schoonhoven L, Fletcher J, Furtado K, Gunningberg L, Heyman H, et al. EPUAP classifi-
cation system for pressure ulcers: European reliability study. J Adv Nurs. 2007; 60: 682–91. doi: 10.
1111/j.1365-2648.2007.04474.x PMID: 18039255
5. Ankrom M. Pressure-Related Deep Tissue Injury under Intact Skin and the current pressure ulcer stag-
ing systems. Adv Skin Wound Care. 2005; 18: 35–42. PMID: 15714036
6. Ankrom MA, Bennett RG, Sprigle S, Langemo D, Black JM, Berlowitz DR, et al. Pressure-related deep
tissue injury under intact skin and the current pressure ulcer staging systems. Adv Skin Wound Care.
2005; 18: 35–42. PMID: 15714036
7. Gefen A, Farid KJ, Shaywitz I. A review of deep tissue injury development, detection, and prevention:
shear savvy. Ostomy Wound Manage. 2013; 59: 26–35.
8. Fleck CA. Suspected deep tissue injury. Adv Skin Wound Care. 2007; 20: 413–5. doi: 10.1097/01.
ASW.0000280206.96378.5b PMID: 17620743
9. Honaker J, Brockopp D, Moe K. Suspected Deep Tissue Injury Profile. Adv Skin Wound Care. 2014; 27:
133–140. doi: 10.1097/01.ASW.0000443267.25288.87 PMID: 24531520
10. Salcido R. Heel Pressure Ulcers: Purple Heel and Deep Tissue Injury. Adv Skin Wound Care. 2012; 24:
374–380.
11. Fleck CA. Deep Tissue Injury: What, Why and When? Wound Care Canada. 2007; 5: 10–13.
12. Richbourg L, Smith J, Dunzweiler S. Suspected deep tissue injury evaluated by North Carolina WOC
nurses: a descriptive study. J Wound Ostomy Continence Nurs. 2011; 38: 655–60. doi: 10.1097/WON.
0b013e31823429e7 PMID: 22011819
13. Brown G. Long-term outcomes of full-thickness pressure ulcers: healing and mortality. Ostomy Wound
Manage. 2003; 49: 42–50. Available: http://europepmc.org/abstract/MED/14652420
14. Bennett G, Dealey C, Posnett J. The cost of pressure ulcers in the UK. Age Ageing. 2004; 33: 230–5.
doi: 10.1093/ageing/afh086 PMID: 15082426
15. Dealey C, Posnett J, Walker A. The cost of pressure ulcers in the United Kingdom. J Wound Care.
2012; 21: 261–2, 264, 266. doi: 10.12968/jowc.2012.21.6.261 PMID: 22886290
16. Severens. The cost of illness of pressure ulcers in the Netherlands. Adv Ski wound care. 2002; 15: 72–
77.
17. Black J, Baharestani M, Black S, Cavazos J, Conner-Kerr T, Edsberg L, et al. An overview of tissue types
in pressure ulcers: a consensus panel recommendation. Ostomy Wound Manage. 2010; 56: 28–44.
18. Black JM, Brindle CT, Honaker JS. Differential diagnosis of suspected deep tissue injury. Int Wound J.
2016; 13: 531–539. doi: 10.1111/iwj.12471 PMID: 26123043
19. Gefen A, Gefen N, Linder-Ganz E, Margulies SS. In Vivo Muscle Stiffening Under Bone Compression
Promotes Deep Pressure Sores. J Biomech Eng. 2005; 127: 512. PMID: 16060358
20. Linder-Ganz E, Gefen A. Stress analyses coupled with damage laws to determine biomechanical risk
factors for deep tissue injury during sitting. J Biomech Eng. American Society of Mechanical Engineers;
2009; 131: 11003.
21. NPUAP/EPUAP/PPPIA. Prevention and treatment of pressure ulcers: clinical practice guideline. Haes-
ler E, editor. Osbrone Park, Western Australia: Cambridge Media; 2014.
22. Bosboom EMH, Bouten CVC, Oomens CWJ, Van Straaten HWM, Baaijens FPT, Kuipers H. Quantifica-
tion and localisation of damage in rat muscles after controlled loading; a new approach to study the aeti-
ology of pressure sores. Med Eng Phys. 2001; 23: 195–200. PMID: 11410384
23. Bosboom EMH, Bouten CVC, Oomens CWJ, Baaijens FPT, Nicolay K. Quantifying pressure sore-
related muscle damage using high-resolution MRI. J Appl Physiol. 2003; 95: 2235–40. doi: 10.1152/
japplphysiol.01023.2001 PMID: 12819217
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 18 / 22

24. Stekelenburg A, Oomens CWJ, Strijkers GJ, de Graaf L, Bader DL, Nicolay K. A new MR-compatible
loading device to study in vivo muscle damage development in rats due to compressive loading. Med
Eng Phys. 2005/08/25. 2006; 28: 331–338. doi: 10.1016/j.medengphy.2005.07.005 PMID: 16118060
25. Stekelenburg A, Oomens CWJ, Strijkers GJ, Nicolay K, Bader DL. Compression-induced deep tissue
injury examined with magnetic resonance imaging and histology. J Appl Physiol. 2006/02/18. 2006;
100: 1946–1954. doi: 10.1152/japplphysiol.00889.2005 PMID: 16484364
26. Loerakker S, Stekelenburg A, Strijkers GJ, Rijpkema JJM, Baaijens FPT, Bader DL, et al. Temporal
effects of mechanical loading on deformation-induced damage in skeletal muscle tissue. Ann Biomed
Eng. 2010/03/17. 2010; 38: 2577–2587. doi: 10.1007/s10439-010-0002-x PMID: 20232152
27. Stekelenburg A, Strijkers GJG, Parusel H, Bader DL, Nicolay K, Oomens CW. Role of ischemia and
deformation in the onset of compression-induced deep tissue injury: MRI-based studies in a rat model.
J Appl Physiol. 2007/01/27. 2007; 102: 2002–2011. doi: 10.1152/japplphysiol.01115.2006 PMID:
17255369
28. Ceelen KK, Stekelenburg A, Loerakker S, Strijkers GJ, Bader DL, Nicolay K, et al. Compression-
induced damage and internal tissue strains are related. J Biomech. 2008/11/18. 2008; 41: 3399–3404.
doi: 10.1016/j.jbiomech.2008.09.016 PMID: 19010470
29. Stekelenburg A. Compression induced deep tissue injury examined with MRI and histology. J Appl Phy-
siol. 2006; 100.
30. van Nierop BJ, Stekelenburg A, Loerakker S, Oomens CW, Bader DL, Strijkers GJ, et al. Diffusion of
water in skeletal muscle tissue is not influenced by compression in a rat model of deep tissue injury. J
Biomech. 2010; 43: 570–5. doi: 10.1016/j.jbiomech.2009.07.043 PMID: 19897200
31. Loerakker S, Manders E, Strijkers GJ, Nicolay K, Baaijens FPT, Bader DL, et al. The effects of deforma-
tion, ischemia, and reperfusion on the development of muscle damage during prolonged loading. J Appl
Physiol. 2011; 111: 1168–77. doi: 10.1152/japplphysiol.00389.2011 PMID: 21757578
32. Coleman S, Nixon J, Keen J, Wilson L, McGinnis E, Dealey C, et al. A new pressure ulcer conceptual
framework. J Adv Nurs. 2014;
33. Loerakker S, Oomens CWJ, Manders E, Schakel T, Bader DL, Baaijens FPT, et al. Ischemia-reperfu-
sion injury in rat skeletal muscle assessed with T2-weighted and dynamic contrast-enhanced MRI.
Magn Reson Med. 2011/03/02. 2011; 66: 528–537. doi: 10.1002/mrm.22801 PMID: 21360588
34. Carlier PG, Marty B, Scheidegger O, Loureiro de Sousa P, Baudin P-Y, Snezhko E, et al. Skeletal Mus-
cle Quantitative Nuclear Magnetic Resonance Imaging and Spectroscopy as an Outcome Measure for
Clinical Trials. J Neuromuscul Dis. 2016; 3: 1–28. doi: 10.3233/JND-160145 PMID: 27854210
35. Prompers JJ, Jeneson JAL, Drost MR, Oomens CCW, Strijkers GJ, Nicolay K. Dynamic MRS and MRI
of skeletal muscle function and biomechanics. NMR Biomed. 2006; 19: 927–953. doi: 10.1002/nbm.
1095 PMID: 17075956
36. Bryant ND, Li K, Does MD, Barnes S, Gochberg DF, Yankeelov TE, et al. Multi-parametric MRI charac-
terization of inflammation in murine skeletal muscle. NMR Biomed. 2014; 27: 716–25. doi: 10.1002/
nbm.3113 PMID: 24777935
37. Hollingsworth KG, de Sousa PL, Straub V, Carlier PG. Towards harmonization of protocols for MRI out-
come measures in skeletal muscle studies: Consensus recommendations from two TREAT-NMD NMR
workshops, 2 May 2010, Stockholm, Sweden, 1–2 October 2009, Paris, France. Neuromuscul Disord.
Elsevier B.V.; 2012; 22: S54–S67. doi: 10.1016/j.nmd.2012.06.005 PMID: 22980769
38. Heemskerk AM, Drost MR, van Bochove GS, van Oosterhout MFM, Nicolay K, Strijkers GJ. DTI-based
assessment of ischemia-reperfusion in mouse skeletal muscle. Magn Reson Med. 2006; 56: 272–81.
doi: 10.1002/mrm.20953 PMID: 16826605
39. Arpan I, Forbes SC, Lott DJ, Senesac CR, Daniels MJ, Triplett WT, et al. T2 mapping provides multiple
approaches for the characterization of muscle involvement in neuromuscular diseases: A cross-sec-
tional study of lower leg muscles in 5-15-year-old boys with Duchenne muscular dystrophy. NMR
Biomed. 2013; 26: 320–328. doi: 10.1002/nbm.2851 PMID: 23044995
40. Oomens CWJ, Bader DL, Loerakker S, Baaijens FP. Pressure Induced Deep Tissue Injury Explained.
Ann Biomed Eng. 2014; 43: 297–305. doi: 10.1007/s10439-014-1202-6 PMID: 25480479
41. Loerakker S, Solis LR, Bader DL, Baaijens FP., Mushahwar VK, Oomens CWJ. How does muscle stiff-
ness affect the internal deformations within the soft tissue layers of the buttocks under constant load-
ing? 2012; doi: 10.1080/10255842.2011.627682 PMID: 22300480
42. Gehin. Which techniques to improve the early detection and prevention of pressure ulcers? Proc IEEE.
2006; 28: 6057–6060.
43. Deprez J-F, Brusseau E, Fromageau J, Cloutier G, Basset O. On the potential of ultrasound elastogra-
phy for pressure ulcer early detection. Med Phys. 2011; 38: 1943–1950. doi: 10.1118/1.3560421 PMID:
21626927
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 19 / 22

44. Loerakker S. The relative contributions of muscle deformation and ischaemia to pressure ulcer develop-
ment. PhD thesis, Eindhoven Univ Technol. 2011;
45. Muthupillai R, Ehman RL. Magnetic resonance elastography. Nat Med. 1996/05/01. 1996; 2: 601–3.
PMID: 8616724
46. Ophir J. Elastography a quantitative method for imaging the elasticity of biological tissues. Ultrason
Imaging. 1991; 13: 111–134. PMID: 1858217
47. Doyley MM. Model-based elastography: a survey of approaches to the inverse elasticity problem. Phys
Med Biol. 2012; 57: R35–R73. doi: 10.1088/0031-9155/57/3/R35 PMID: 22222839
48. Sinkus R, Tanter M, Catheline S, Lorenzen J, Kuhl C, Sondermann E, et al. Imaging anisotropic and vis-
cous properties of breast tissue by magnetic resonance-elastography. Magn Reson Med. 2005; 53:
372–387. doi: 10.1002/mrm.20355 PMID: 15678538
49. Rouvière O, Yin M, Dresner MA, Rossman PJ, Burgart LJ, Fidler JL, et al. MR elastography of the liver:
preliminary results. Radiology. 2006; 240: 440–448. doi: 10.1148/radiol.2402050606 PMID: 16864671
50. Huwart L, Peeters F, Sinkus R, Annet L, Salameh N, ter Beek LC, et al. Liver fibrosis: non-invasive
assessment with MR elastography. NMR Biomed. 2006/03/08. 2006; 19: 173–179. doi: 10.1002/nbm.
1030 PMID: 16521091
51. Bohte AE, Niet A De, Jansen L, Bipat S. Non-invasive evaluation of liver fibrosis: a comparison of ultra-
sound-based transient elastography and MR elastography in patients with viral hepatitis B and C. 2014;
638–648. doi: 10.1007/s00330-013-3046-0 PMID: 24158528
52. Runge JH, Bohte AE, Verheij J, Terpstra V, Nederveen AJ, Nieuwkerk KMJ Van, et al. Comparison of
interobserver agreement of magnetic resonance elastography with histopathological staging of liver
fibrosis. 2014; 283–290. doi: 10.1007/s00261-013-0063-z PMID: 24366108
53. Sack I, Beierbach B, Hamhaber U, Klatt D, Braun J. Non-invasive measurement of brain viscoelasticity
using magnetic resonance elastography. NMR Biomed. 2008; 21: 265–271. doi: 10.1002/nbm.1189
PMID: 17614101
54. Garteiser P, Doblas S, Daire JL, Wagner M, Leitao H, Vilgrain V, et al. MR elastography of liver tumours:
value of viscoelastic properties for tumour characterisation. Eur Radiol. 2012/05/11. 2012; 22: 2169–
2177. doi: 10.1007/s00330-012-2474-6 PMID: 22572989
55. Sinkus R, Siegmann K, Xydeas T, Tanter M, Claussen C, Fink M. MR elastography of breast lesions:
Understanding the solid/liquid duality can improve the specificity of contrast-enhanced MR mammogra-
phy. Magn Reson Med. 2007; 58: 1135–1144. doi: 10.1002/mrm.21404 PMID: 17969009
56. Elgeti T, Beling M, Hamm B, Braun J, Sack I. Cardiac magnetic resonance elastography: toward the
diagnosis of abnormal myocardial relaxation. Invest Radiol. 2010/09/11. 2010; 45: 782–787. doi: 10.
1097/RLI.0b013e3181ec4b63 PMID: 20829709
57. Mariappan YK, Kolipaka A, Manduca A, Hubmayr RD, Ehman RL, Araoz P, et al. Magnetic resonance
elastography of the lung parenchyma in an in situ porcine model with a noninvasive mechanical driver:
correlation of shear stiffness with trans-respiratory system pressures. Magn Reson Med. 2011/05/19.
2012; 67: 210–217. doi: 10.1002/mrm.22976 PMID: 21590723
58. Kemper J, Sinkus R, Lorenzen J, Nolte-Ernsting C, Stork A, Adam G. MR elastography of the prostate:
initial in-vivo application. Rofo. 2004/09/04. 2004; 176: 1094–1099. doi: 10.1055/s-2004-813279 PMID:
15346284
59. Rouviere O, Souchon R, Pagnoux G, Menager JM, Chapelon JY. Magnetic resonance elastography of
the kidneys: feasibility and reproducibility in young healthy adults. J Magn Reson Imaging. 2011/07/20.
2011; 34: 880–886. doi: 10.1002/jmri.22670 PMID: 21769970
60. Mannelli L, Godfrey E, Joubert I, Patterson AJ, Graves MJ, Gallagher FA, et al. MR elastography:
Spleen stiffness measurements in healthy volunteers—preliminary experience. AJR Am J Roentgenol.
2010/07/24. 2010; 195: 387–392. doi: 10.2214/AJR.09.3390 PMID: 20651194
61. Bilston LE, Tan K. Measurement of Passive Skeletal Muscle Mechanical Properties In Vivo: Recent
Progress, Clinical Applications, and Remaining Challenges. Ann Biomed Eng. 2015; 43: 261–273. doi:
10.1007/s10439-014-1186-2 PMID: 25404536
62. Uffmann K, Maderwald S, Ajaj W, Galban CG, Mateiescu S, Quick HH, et al. In vivo elasticity measure-
ments of extremity skeletal muscle with MR elastography. NMR Biomed. 2004/07/02. 2004; 17: 181–
190. doi: 10.1002/nbm.887 PMID: 15229931
63. Dresner MA, Rose GH, Rossman PJ, Muthupillai R, Manduca A, Ehman RL. Magnetic resonance elas-
tography of skeletal muscle. J Magn Reson Imaging. 2001; 13: 269–276. PMID: 11169834
64. Klatt D, Papazoglou S, Braun J, Sack I. Viscoelasticity-based MR elastography of skeletal muscle.
Phys Med Biol. 2010; 55: 6445–6459. doi: 10.1088/0031-9155/55/21/007 PMID: 20952814
65. Chen Q. Characterization of Skeletal Muscle Elasticity Using Magnetic Resonance Elastography.
Touch Briefings. 2008; 60–63.
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 20 / 22

66. Qin EC, Sinkus R, Geng G, Cheng S, Green M, Rae CD, et al. Combining MR elastography and diffu-
sion tensor imaging for the assessment of anisotropic mechanical properties: A phantom study. J Magn
Reson Imaging. 2013; 37: 217–226. doi: 10.1002/jmri.23797 PMID: 22987805
67. Papazoglou S, Rump J, Braun J, Sack I. Shear wave group velocity inversion in MR elastography of
human skeletal muscle. Magn Reson Med. 2006/08/09. 2006; 56: 489–497. doi: 10.1002/mrm.20993
PMID: 16894586
68. Dresner MA, Rose G, Rossman PJ, Muthupillai R. Magnetic resonance elastography of skeletal mus-
cle. J Magn Reson Imaging. 2001; 13: 269–276. PMID: 11169834
69. Green M a, Sinkus R, Gandevia SC, Herbert RD, Bilston LE. Measuring changes in muscle stiffness
after eccentric exercise using elastography. NMR Biomed. 2012; 25: 852–8. doi: 10.1002/nbm.1801
PMID: 22246866
70. Barnhill E, Kennedy P, Hammer S, van Beek EJR, Brown C, Roberts N. Statistical mapping of the effect
of knee extension on thigh muscle viscoelastic properties using magnetic resonance elastography. Phy-
siol Meas. 2013; 34: 1675–98. doi: 10.1088/0967-3334/34/12/1675 PMID: 24254405
71. Qin EC, Juge
´L, Lambert S a, Paradis V, Sinkus R, Bilston LE. In Vivo Anisotropic Mechanical Proper-
ties of Dystrophic Skeletal Muscles Measured by Anisotropic MR Elastographic Imaging: The mdx
Mouse Model of Muscular Dystrophy. Radiology. 2014; 132661.
72. Basford JR, Jenkyn TR, An KN, Ehman RL, Heers G, Kaufman KR. Evaluation of healthy and diseased
muscle with magnetic resonance elastography. Arch Phys Med Rehabil. 2002; 83: 1530–1536. PMID:
12422320
73. Bensamoun SF, Ringleb SI, Chen Q, Ehman RL, An K-NN, Brennan M. Thigh muscle stiffness
assessed with magnetic resonance elastography in hyperthyroid patients before and after medical treat-
ment. J Magn Reson Imaging. 2007/08/31. 2007; 26: 708–713. doi: 10.1002/jmri.21073 PMID:
17729336
74. Ringleb SI, Bensamoun SF, Chen QS, Manduca A, An K- NN, Ehman RL. Applications of magnetic res-
onance elastography to healthy and pathologic skeletal muscle. J Magn Reson Imaging. 2007/01/30.
2007; 25: 301–309. doi: 10.1002/jmri.20817 PMID: 17260391
75. Brauck K, Galban CJ, Maderwald S, Herrmann BL, Ladd ME. Changes in calf muscle elasticity in hypo-
gonadal males before and after testosterone substitution as monitored by magnetic resonance elasto-
graphy. Eur J Endocrinol. 2007/05/31. 2007; 156: 673–678. doi: 10.1530/EJE-06-0694 PMID:
17535867
76. McCullough MB, Domire ZJ, Reed AM, Amin S, Ytterberg SR, Chen Q, et al. Evaluation of muscles
affected by myositis using magnetic resonance elastography. Muscle Nerve. 2011/02/15. 2011; 43:
585–590. doi: 10.1002/mus.21923 PMID: 21319167
77. Chen Q, Basford J, An KN. Ability of magnetic resonance elastography to assess taut bands. Clin Bio-
mech (Bristol, Avon). 2008/01/22. 2008; 23: 623–629.
78. Bensamoun SF, Charleux F, Debernard L, Themar-Noel C, Voit T. Elastic properties of skeletal muscle
and subcutaneous tissues in Duchenne muscular dystrophy by magnetic resonance elastography
(MRE): A feasibility study. Irbm. Elsevier Masson SAS; 2015; 36: 4–9.
79. Linder-Ganz E, Gefen A. Stiffening of Muscle Tissue Under Bone Compression is a Key Factor in For-
mation of Pressure Sores. Proc IEEE. 2003; 2: 1839–1842.
80. Schreurs TJL. A small animal Magnetic Resonance Elastography (MRE) setup. Journal Article, MSc.
Thesis, Eindhoven University of Technology. 2012.
81. Juge L, Doan BT, Seguin J, Albuquerque M, Larrat B, Mignet N, et al. Colon Tumor Growth and Antivas-
cular Treatment in Mice: Complementary Assessment with MR Elastography and Diffusion-weighted
MR Imaging. Radiology. 2012/06/14. 2012; 264: 436–444. doi: 10.1148/radiol.12111548 PMID:
22692038
82. van Nierop BJ, Bax NAM, Nelissen JL, Arslan F, Motaal AG, de Graaf L, et al. Assessment of Myocar-
dial Fibrosis in Mice Using a T2*-Weighted 3D Radial Magnetic Resonance Imaging Sequence. PLoS
One. 2015; 10: e0129899. doi: 10.1371/journal.pone.0129899 PMID: 26115443
83. Sinkus R, Lorenzen J, Schrader D, Lorenzen M, Dargatz M, Holz D. High-resolution tensor MR elasto-
graphy for breast tumour detection. Phys Med Biol. 2000/06/28. 2000; 45: 1649–64. PMID: 10870716
84. Sinkus R, Tanter M, Xydeas T, Catheline S, Bercoff J, Fink M. Viscoelastic shear properties of in vivo
breast lesions measured by MR elastography. Magn Reson Imaging. 2005; 23: 159–165. doi: 10.1016/
j.mri.2004.11.060 PMID: 15833607
85. Green MA, Bilston LE, Sinkus R. In vivo brain viscoelastic properties measured by magnetic resonance
elastography. NMR Biomed. 2008/05/07. 2008; 21: 755–764. doi: 10.1002/nbm.1254 PMID: 18457350
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 21 / 22

86. Loerakker S, Bader DL, Baaijens FPT, Oomens CWJ. Which factors influence the ability of a computa-
tional model to predict the in vivo deformation behaviour of skeletal muscle? Comput Methods Biomech
Biomed Engin. 2013; 16: 338–45. doi: 10.1080/10255842.2011.621423 PMID: 22300425
87. Ipek-Ugay S, Drießle T, Ledwig M, Guo J, Hirsch S, Sack I, et al. Tabletop magnetic resonance elasto-
graphy for the measurement of viscoelastic parameters of small tissue samples. J Magn Reson. Else-
vier Inc.; 2015; 251: 13–18. doi: 10.1016/j.jmr.2014.11.009 PMID: 25554945
88. Qin EC, Sinkus R, Geng G, Cheng S, Green M, Rae CD, et al. Combining MR elastography and diffu-
sion tensor imaging for the assessment of anisotropic mechanical properties: A phantom study. J Magn
Reson Imaging. 2012/09/19. 2013; 37: 217–226. doi: 10.1002/jmri.23797 PMID: 22987805
89. Bosboom EM, Hesselink MK, Oomens CW, Bouten C V, Drost MR, Baaijens FP. Passive transverse
mechanical properties of skeletal muscle under in vivo compression. J Biomech. 2001; 34: 1365–8.
PMID: 11522318
90. Moerman KM, Sprengers AMJ, Nederveen AJ, Simms CK. A novel MRI compatible soft tissue indentor
and fibre Bragg grating force sensor. Med Eng Phys. 2013; 35: 486–99. doi: 10.1016/j.medengphy.
2012.06.014 PMID: 22819569
91. Moerman KM, Holt C a, Evans SL, Simms CK. Digital image correlation and finite element modelling as
a method to determine mechanical properties of human soft tissue in vivo. J Biomech. 2009; 42: 1150–
3. doi: 10.1016/j.jbiomech.2009.02.016 PMID: 19362312
92. Riek K, Klatt D, Nuzha H, Mueller S, Neumann U, Sack I, et al. Wide-range dynamic magnetic reso-
nance elastography. J Biomech. 2011; 44: 1380–6. doi: 10.1016/j.jbiomech.2010.12.031 PMID:
21295305
93. Strobel K, Bergmann R, Meister S, Van Den Hoff J, Pietzsch J. Improved multimodality imaging using
alginate molding in xenograft tumor models. J Magn Reson Imaging. 2010; 31: 747–752. doi: 10.1002/
jmri.22090 PMID: 20187223
94. Rommel D, Abarca-Quinones J, Christian N, Peeters F, Lonneux M, Labar D, et al. Alginate moulding:
an empirical method for magnetic resonance imaging/positron emission tomography co-registration in a
tumor rat model. Nucl Med Biol. 2008; 35: 571–577. doi: 10.1016/j.nucmedbio.2008.04.002 PMID:
18589301
95. Landuyt W, Sunaert S, Farina D, Meijerink M, Be
´atse E, Van Hecke P, et al. In vivo animal functional
MRI: improved image quality with a body-adapted mold. J Magn Reson Imaging. 2002; 16: 224–7. doi:
10.1002/jmri.10144 PMID: 12203772
96. Heemskerk AM, Strijkers GJ, Vilanova A, Drost MR, Nicolay K. Determination of mouse skeletal muscle
architecture using three-dimensional diffusion tensor imaging. Magn Reson Med. 2005; 53: 1333–40.
doi: 10.1002/mrm.20476 PMID: 15906281
97. Froeling M, Nederveen AJ, Nicolay K, Strijkers GJ. DTI of human skeletal muscle: the effects of diffu-
sion encoding parameters, signal-to-noise ratio and T2 on tensor indices and fiber tracts. NMR Biomed.
2013; 26: 1339–52. doi: 10.1002/nbm.2959 PMID: 23670990
98. Oudeman J, Nederveen AJ, Strijkers GJ, Maas M, Luijten PR, Froeling M. Techniques and applications
of skeletal muscle diffusion tensor imaging: A review. J Magn Reson Imaging. 2016; 43: 773–788. doi:
10.1002/jmri.25016 PMID: 26221741
A Combined Mechanical Loading and MR Elastography Setup to Study Skeletal Muscle Damage in Rats
PLOS ONE | DOI:10.1371/journal.pone.0169864 January 11, 2017 22 / 22