Interventional real-time molecular MRI allows diagnostic targeting of early myocardial injury in a pig model of ischemia and reperfusion

Abstract

Introduction: Inflammation is a hallmark of post-ischemic myocardial injury. Expression of P-selectin by platelets and activated endothelial cells drives recruitment of immune cells to the deprived area and may serve as an early indicator of tissue injury. Due to its high soft tissue contrast, magnetic resonance imaging (MRI) is used for myocardial tissue characterization. Molecular imaging further allows for functional assessment using target-specific contrast agents. In this study, we assessed ischemic cardiac lesions non-invasively within the first hours after ischemia/reperfusion (I/R) in a porcine model using standard and advanced MRI techniques as well as molecular imaging targeting the cell adhesion molecule P-selectin.
Methods: For molecular imaging, a monoclonal P-selectin antibody was functionalized with microparticles of iron oxide (MPIO). Specific binding to the target was confirmed by in vitro flow chamber using activated platelets as well as endothelial cells. In vivo, we used a closed-chest model of I/R of the circumflex artery in juvenile farm pigs by balloon-occlusion for 40 minutes, and real time MRI-guided coronary injection of MPIO-based contrast agents. 3T MRI was performed 2–4 hours after reperfusion, and lesions were characterized using injury (T1 mapping, LGE), edema (T2 mapping) and iron (T2* mapping) sensitive MRI.
Results: Within the first hours after I/R, we detected increased inflammatory activity by means of higher numbers of innate immune cells in the blood. We found T1 mapping to be most sensitive for tissue injury, while no changes were detectable in edema-sensitive T2 mapping this early. Intriguingly, P-selectin MPIO contrast agent selectively enhanced the ischemic area in iron sensitive T2* mapping 4 hours after I/R which was confirmed histologically, while late gadolinium enhancement was always absent.
Conclusion: By using real time MRI-guided coronary intervention, molecular MRI using P-selectin MPIO allows for sensitive detection of early myocardial inflammation after I/R beyond the capabilities of traditional edema sensitive imaging.

Full text

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Interventional real-time molecular MRI allows
diagnostic targeting of early myocardial injury in a
pig model of ischemia and reperfusion
Timo Heidt
Department of Cardiology, Medical Center University of Freiburg,
Simon Reiss
Experimental Physics, Department of Radiology, University Medical Center Freiburg
Julien Thielmann
Department of Cardiology, Medical Center University of Freiburg,
Christian Weber
Department of Cardiology, Medical Center University of Freiburg,
Alexander Maier
Department of Cardiology, Medical Center University of Freiburg,
Thomas Lottner
Experimental Physics, Department of Radiology, University Medical Center Freiburg
Heidi R. Cristina-Schmitz
Center for Models and Transgenic Services - Freiburg (CEMT-FR) - Experimental Surgery
Timon Bühler
Department of Cardiology, Medical Center University of Freiburg,
Diana Chiang
Department of Cardiology, Medical Center University of Freiburg,
Claus Jülicher
Department of Cardiology, Medical Center University of Freiburg,
Carolin Wadle
Department of Cardiology, Medical Center University of Freiburg,
Ingo Hilgendorf
Department of Cardiology, Medical Center University of Freiburg,
Dennis Wolf
Department of Cardiology, Medical Center University of Freiburg,
Gavin Tumlinson
Institute for Experimental Cardiovascular Medicine, Medical Center University of Freiburg
Luis Hortells
Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine
Dirk Westermann

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Department of Cardiology, Medical Center University of Freiburg,
Michael Bock
Experimental Physics, Department of Radiology, University Medical Center Freiburg
Constantin Mühlen
Department of Cardiology, Medical Center University of Freiburg,
Article
Keywords:
Posted Date: April 9th, 2024
DOI: https://doi.org/10.21203/rs.3.rs-4218369/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License
Additional Declarations: No competing interests reported.

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Abstract
Introduction: Inammation is a hallmark of post-ischemic myocardial injury. Expression of P-selectin by
platelets and activated endothelial cells drives recruitment of immune cells to the deprived area and may
serve as an early indicator of tissue injury. Due to its high soft tissue contrast, magnetic resonance
imaging (MRI) is used for myocardial tissue characterization. Molecular imaging further allows for
functional assessment using target-specic contrast agents. In this study, we assessed ischemic cardiac
lesions non-invasively within the rst hours after ischemia/reperfusion (I/R) in a porcine model using
standard and advanced MRI techniques as well as molecular imaging targeting the cell adhesion
molecule P-selectin.
Methods: For molecular imaging, a monoclonal P-selectin antibody was functionalized with
microparticles of iron oxide (MPIO). Specic binding to the target was conrmed by
in vitro
ow chamber
using activated platelets as well as endothelial cells.
In vivo
, we used a closed-chest model of I/R of the
circumex artery in juvenile farm pigs by balloon-occlusion for 40 minutes, and real time MRI-guided
coronary injection of MPIO-based contrast agents. 3T MRI was performed 2–4 hours after reperfusion,
and lesions were characterized using injury (T1 mapping, LGE), edema (T2 mapping) and iron (T2*
mapping) sensitive MRI.
Results: Within the rst hours after I/R, we detected increased inammatory activity by means of higher
numbers of innate immune cells in the blood. We found T1 mapping to be most sensitive for tissue injury,
while no changes were detectable in edema-sensitive T2 mapping this early. Intriguingly, P-selectin MPIO
contrast agent selectively enhanced the ischemic area in iron sensitive T2* mapping 4 hours after I/R
which was conrmed histologically, while late gadolinium enhancement was always absent.
Conclusion: By using real time MRI-guided coronary intervention, molecular MRI using P-selectin MPIO
allows for sensitive detection of early myocardial inammation after I/R beyond the capabilities of
traditional edema sensitive imaging.
Introduction
Myocardial infarction (MI) and heart failure are deadly consequences of atherosclerotic vascular
disease.1 Infarct size denes the risk of functional loss and adverse cardiac remodeling.2, 3 With
increasing duration of atherothrombotic coronary occlusion, cardiomyocytes are permanently lost and a
transmural cardiac lesion is formed. Early reperfusion therefore plays an essential role in preserving
cardiac function, and, thus, constitutes a primary goal in the management of patients with acute MI. Next
to time of ischemia, inammation represents another driving force of wound healing. Dying
cardiomyocytes liberate danger-associated molecular patterns that activate endothelial cells and
platelets to promote the recruitment of innate immune cells for removal of necrotic debris, repair or
degradation of injured cardiac cells.4 P-selectin is among the early cell adhesion molecules expressed by
activated endothelial cells. In resting condition P-selectin is stored in subcellular vesicles. Upon activation

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it is rapidly translocated to the cell membrane to promote leukocyte rolling prior to rm adhesion and
transmigration. In platelets, P-selectin participates in platelet-leukocyte complex formation that enhance
endothelial attachment and migration.5 After transmigration, monocytes transform to pro-inammatory
macrophages to breakdown debris and injured cells. Based on the local microenvironment immune cells
may thereby repair or replace cardiomyocytes. While some degree of inammation is essential for wound
healing, exaggerated presence of immune cells in the lesion may also increase cardiomyocyte removal
with detrimental consequences for cardiac function.6 Assessment of inammatory activity may therefore
enable cardiovascular risk assessment after MI.7
Due to the excellent soft tissue contrast, magnetic resonance imaging (MRI) has become the gold
standard for non-invasive myocardial tissue characterization. T2-weighted imaging, advanced
relaxometric mapping techniques (T1-, T2-, T2* mapping) and contrast-enhanced MRI (late gadolinium
enhancement (LGE)) allow for detection of tissue edema and structural abnormalities as myocardial
brosis or scarring.8, 9 After MI, LGE depicts the extent and transmurality of the scar.10 Furthermore, T2-
weighted uid sensitive imaging or advanced mapping helps to delineate the previously ischemic tissue
by means of highlighting excess extracellular uids. While these MRI techniques are well established in
clinical routine, they only represent indirect indicators of cardiac inammation.
Molecular imaging constitutes an intriguing approach to improve diagnostic capabilities of non-invasive
imaging tools. Functionalizing gadolinium or iron oxide with target specic receptor-ligands enables
localization of specic contrast deposition thus tissue characterization.11, 12 Previously, molecular MRI
has successfully been used in several small animal models targeting diverse receptors of the
inammatory cascade of atherothrombotic disease including cell adhesion molecules.13–15 With
increasing appreciation of the role of inammation in human cardiovascular disease 16, methods would
be highly desirable that combine the standard assessment of edema and brosis with molecular imaging
of inammatory targets in a hybrid imaging approach. So far, transition of this concept to a larger model
or the patient has been restricted to nuclear imaging due to challenges with sensitivity.17
In this study we demonstrate that molecular MRI with P-selectin allows to detect early signs of cardiac
inammation after ischemia and reperfusion injury in a pig model.
Methods
Animals
All experiments were conducted in accordance with FELASA and GV-SOLAS standards for animal
welfare. Experiments were approved by the local ethics committee of Freiburg University and the regional
council of Freiburg, Baden-Wuerttemberg, Germany (licence number 35-9185.81/G-21/008).
In total, experiments were performed on 7 juvenile (3 months of age) domestic landrace pigs (body
weight 50–70 kg). For premedication, the pigs received an intramuscular injection of midazolam (0.5

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mg/kg body weight (bw)) and ketamine (20 mg/kg bw). After preoxygenation, anaesthesia was induced
by propofol injection (2–4 mg/kg bw) via a peripheral vein catheter and maintained with a mixture of
isourane (1.5–2%) and oxygen/air (FiO2 > 0.3) as well as intravenous (iv) administration of vecuronium
(0.2–0.4 mg/kg bw per hour). Fluid loss was compensated at a dose of 5–10 ml/kg bw ringer’s
solutionper hour. Analgesia was maintained by intravenous application of fentanyl at a dose of 0.002–
0.004 mg/kg bw per hour. Mechanical ventilation (IPPV) was adjusted to keep parameters within
physiological range. Oxygen level, electrocardiogram and concentration of carbon dioxide were
monitored.
Closed chest model of myocardial ischemia/reperfusion
injury
For angiography, a 10 French arterial access sheath was introduced into the right femoral artery using
ultrasound needle-guidance. To reduce the risk of sudden fatal arrhythmias, potassium and magnesium
was supplemented and amiodarone (10 mg/kg bw) was intravenously administered prior to the
procedure. Coronary angiography was performed using a C-arm x-ray (Philips Medical) and standard
coronary catheters. The left coronary artery was depicted with bolus injection of diluted iodinated
contrast agent (Accupaque™ 300mg, GE Healthcare). A coronary wire was thus mounted with a balloon
catheter and inserted into the circumex coronary artery. The balloon was then inated in the mid-
segment of the coronary artery to trigger ischemia, and contrast agent was injected to ensure tight
sealing of the vessel. After 40 min, the balloon was deated and removed from the coronary vessel.
CMR
Cardiac MR imaging and MR-guided coronary catheterization was performed at a clinical 3 T system
(PrismaFit, Siemens) with the animals in head-rst supine position and the heart at magnet iso-center. A
32-channel spine coil and an anterior 18-channel thorax coil array were used for signal reception. An ECG
with four leads was attached and to enable cardiac gating of the imaging sequences. All acquisitions
except the cine and real-time sequences were gated to end-diastole. Real-time images for catheter
guidance were displayed on an in-room monitor (BOLD Screen 24, Cambridge Research Systems Ltd)
positioned close to the patient table and communication between the cardiologist and the system
operator was established via the conventional headphones and in-room microphone of the MRI system. A
custom-made active coronary guiding catheter equipped with a single-loop receive coil at the tip was
used for catheterization of the left coronary artery. The catheter was connected to the MRI system via a
custom-made tuning/matching circuit with variable signal attenuation.
Functional Imaging
Cardiac MRI started with the acquisition of a set of localizer images in the three orthogonal standard
views and denition of the main axes of the hearts. Then, a multi-slice 2D cine bSSFP sequence was
acquired in short-axis view for functional and volumetric imaging (TE/TR = 1.5/3.0 ms, ip angle (FA) =
42°, BW = 970 Hz/px, FoV = 340x273 mm², matrix: 224x126, slice thickness (SL) = 8 mm, number of
slices: 6–8, retrospective cardiac gating with 20 reconstructed phases, multiple breath-holds).

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T1, T2 and T2* mapping
Parametric mapping of the relaxation times T1, T2, and T2* was performed in 3 or 4 mid-ventricular short
axis slices depending on the size of the heart. T1 maps were acquired with an inversion recovery bSSFP
sequence (TE/TR = 1.2/2.7 ms, FA = 35°, BW = 1085 Hz/px, FoV = 360x307 mm², matrix: 192x132, SL = 5
mm, 8 different TI values from 100 to 4500 ms depending on the heart rate, single breath-hold). T2 maps
were acquired with a T2-prepared FLASH sequence with varying T2 preparation times (TE/TR = 1.3/3.1
ms, TET2prep: [0, 30, 40] ms, FA = 12°, BW = 1185 Hz/px, FoV = 360x247 mm², matrix: 192x132, SL = 6
mm, single breath-hold). The inline motion correction and calculation of T1/T2 values provided by the
vendor were used for both T1 and T2 mapping. T2* mapping was performed with a multi-echo FLASH
sequence (TE/TR = [2.4, 6.0, 9.5, 13.0]/16.2 ms, FA = 12°, BW = 590 Hz/px, FoV = 260² mm², matrix: 256²,
SL = 5 mm, single breath-hold) and T2* maps were calculated oine in Matlab by a pixel-wise linear t of
the logarithm of the signal intensities. The T2* mapping sequences was acquired again after injection of
MPIOs under real-time guidance.
Real-time imaging
After functional and parametric imaging a non-contrast 3D compressed-sensing accelerated prototype
whole heart FLASH sequence (TE/TR = 2.3/5.2 ms, FA = 15°, BW = 250 Hz/px, FoV = 320x310x139 mm³,
matrix: 256 x 248 x 104, navigator gating to end-expiration) was acquired for coronary angiography and
planning of the imaging planes for the MR-guided catheterization. Therefore, three planar views were
extracted from the 3D dataset to cover the aortic arch and the left coronary ostium in a short-axis and
long-axis view. Catheterization of the LCA was performed under imaging with a real-time FLASH
sequence (TE/TR = 1.3/3.4 ms, FA = 10°, BW = 790 Hz/px, FoV = 289² mm², matrix: 192x144, SL = 8 mm).
Successful intubation was veried by imaging the perfusion of a small amount of 1% Gd-solution
injected via the catheter in a single short-axis slice with an inversion recovery FLASH sequence (TE/TR =
1.0/2.0 ms, TI = 95 ms, FA = 10°, BW = 1185 Hz/px, FoV = 360x270 mm², matrix: 192x106, SL = 8 mm,
single breath-hold). The MPIO contrast agent was injected after successful intubation and the injection
was imaged with a FLASH sequence in short-axis view (TE/TR = 4.0/5.0 ms, FA = 12°, BW = 1185 Hz/px,
FoV = 280² mm², matrix: 128x102, SL = 6 mm, single breath-hold).
LGE imaging
Late gadolinium enhancement (LGE) image data were acquired after the coronary catheterization and 10
min after intravenous injection of 2.5 mmol/kg Gd. A TI scout was rst acquired in short axis view to
determine the optimal inversion time. The LGE sequence was then acquired with phase sensitive
inversion recovery FLASH sequence in short-axis view (TE/TR = 1.4/3.7 ms, FA = 20°, BW = 465 Hz/px,
FoV = 360² mm², matrix: 144x141, SL = 10 mm, single breath-hold).
Ex vivo T2* mapping
Ex vivo
imaging was performed after the hearts were removed and stored in the xative for at least 7
days to allow for the xative to fully diffuse through the heart. The hearts were imaged at the same 3T

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MRI system as the
in vivo
experiments. For imaging, the hearts were placed in a plastic container
containing the xative positioned at iso-center inside a 64 channel head/neck coil. Three-dimensional R2*
maps were acquired via a multi-echo spoiled gradient echo sequence with 0.58 mm isotropic resolution
covering the whole heart (TE/TR = [3.4, 9.8, 17.1]/23 ms, FA = 12°, BW = 260 Hz/px, FoV = 129×129×84
mm³, matrix: 224×224×144, averages = 2). R2* maps were calculated oine in the same way as the
in
vivo
R2* maps.
Real-time MRI catheter guidance
Using an in-room video monitor (BOLD Screen 24, Cambridge Research Systems Ltd, Rochester, UK) next
to the patient table of the MRI system, the real-time images were presented to the interventionalist during
MRI. Conventional headphones and the in-room microphone of the MRI system were used for
communication between the interventionalist and the system operator. The animal was positioned on the
MR table in supine position with the heart located in the magnet’s isocenter. For a full coverage of the
vascular system, a posterior 32-channel spine coil and an anterior 18-channel thorax coil array were used.
The wireless ECG system supplied by the vendor was attached with hydrogel electrodes.
After the acquisition of an initial set of localizer images, a 3D whole-heart ECG-triggered gradient echo
(FLASH) data set was acquired with the following imaging parameters: fat saturation, TE/TR = 1.6/3.5
ms, FA = 16°, FoV = 282x282x102 mm³, matrix: 176x176x64, T2-preparation with TET2prep = 40 ms,
GRAPPA acceleration factor
R
= 2. The ECG-triggered FLASH sequence was repeated later during the
experiment to conrm the position of the interventional instruments.
First, a guidewire (standard Terumo 0.018") was inserted via the arterial sheath to guide a modied 6F
guiding catheter (Terumo; Optitorque Radial Tig II 4.0) to the aortic root. After intubation of the LCA,
proper position of the guiding catheter was conrmed by injection of 5 ml diluted gadolinium contrast
agent (1:20, Gd-DTPA, Magnevist, Bayer, Germany) via the guiding catheter. A 2D real-time radial bSSFP
sequence with the following imaging parameters was used to monitor the advancement of the
instruments: TE/TR = 1.4/2.8 ms, #spokes = 105, FA = 40°, FoV = 275x275x7 mm³, matrix: 160x160, fat
saturation. Real-time image slice orientations and positions were dened using the localizer and 3D
FLASH images acquired prior to the catheter advancement. Molecular contrast agents were injected via
the guiding catheter in a volume of 20 ml and ushed with saline.
Molecular contrast agent
Construction of the molecular contrast agent was performed using pelleted MyOne™ Tosylactivated
MPIOs with a size of 1 µm. MPIO pellets were washed in 0.1M natrium borate buffer and resuspended in
ammonium sulfate buffer containing 200 microgram of antibody to reach a nal concentration of 1M 20
hours at 37°C. Constant rotation insured separation of pelleted beats. Afterwards, residual active tosyl-
remnants were removed using a blocking buffer and contrast agent was resuspended in a storage buffer
with constant rotation.
Post-processing

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Dedicated MRI post-processing software (CVI 42, Circle Imaging) was used to quantify T2* times from 3
short axis slices of the basal, mid and apical left ventricle. Regions of interest (ROI) were dened in the
area of LAD, LCX or RCA on each slice. For analysis, the mean T2* time in each area, i.e. LAD, LCX or RCA,
was used.
FACS
For ow cytometry full blood was stained with antibodies targeting cell specic epitopes after red blood
cell lysis. Neutrophils were identied according to SSC and FSC, classical-type monocytes were identied
as SWC3+, CD163neg, CD14+, non-classical-type monocytes were identied as SWC3+, CD163+, CD14int.
Analysis was performed using FACS Diva software.
In vitro ow chamber
Cell culture dishes (35mm; CytoOne) were coated with 1ml brinogen (100µg/ml) and stored overnight at
4°C. The following day human blood was collected in a citrate tube and separated in various blood
components via centrifugation at 150G for 5 minutes. The supernatant (platelet rich plasma; PRP) was
then transfered into a Falcon tube and 1ml of PRP was applied to each of the previously prepared
brinogen coated dishes to allow platelet adhesion to the surface of the dish. Platelet activation was
induced using a 1:10 dilution of 20 µg of adenosine diphosphate (ADP). The activation process was
carried out for a precisely controlled duration of 30 min at room temperature.
The parallel plate ow chamber kit (GlycoTech, Rockville, Maryland, USA) consists of a transparent
chamber with two parallel plates, one plate is coated with a monolayer of platelets, and the other plate is
a continous uid ow channel. A syringe pump connected to the inlet port is used to deliver a precise and
constant ow of contrast agent through the chamber and thus over the platelets. The outlet port is used
to allow the left over contrast agent to exit the chamber into a waste container. The chamber was
observed using a microscope (Zeiss Vert. A1) at 20x magnication connected to a digital imaging system
(AxioCam ICc1, Carl Zeiss AG, Feldbach, CH).
IgG-MPIO served as a non-specic control group, whereasanti-CD62P-MPIO was used as contrast
agentwith specic binding properties for P-selectin. The binding properties of both contrast agents were
evaluated on the platelet monolayer over a specic duration of 60 seconds. The video recording
commenced upon the appearance of the initial MPIO within the designated eld of view, measuring 450
µm x 350 µm. Only MPIOs that demonstrated adhesion for a minimum of 10 seconds were deemed to
have established a bond with the platelets.
Incubation Assay
Porcine endothelial cells were cultured according to the manufacturer's protocol (Sigma Aldrich Chemie
GmbH, P300-05). In short after thawing the cryovials in a 37°C water bath, the cells were resuspended a
10% dulbecco’s modied eagle medium (DMEM). The cells were centrifuged, washed three times with
DMEM, and nally resuspended in Porcine Endothelial Growth Medium. The cell suspension was

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transferred to a T-75 cell culture ask, and daily medium changes were performed until the cells reached
60% conuency. The volume of the culture medium was then doubled, and after further incubation, the
cells were split onto 12-well-dishes (Thermo Fisher Scientic, 168844). For proper comparison of the
specically targeting P-Selectin-MPIO contrast agent we compared the binding properties to MPIO with
unspecic targeting. For each contrast agent six dishes were seeded with a total of 4000 cells each. Each
contrast agent was prepared identically as described above. 10µl of each contrast agent was diluted with
990 ml of PBS and incubated for 30 seconds. After,, each dish was washed thoroughly with PBS. For
subsequent analysis photographic documentation was performed to capture the different binding
properties of each contrast agent. Per dish 10 photos of evenly distributed cells were taken randomly
yielding a total of 60 photos per group. Each MPIO located exactly next to or on top of a cell was
considered as bound to the endothelial cell layer.
Immunouorescence staining
For each heart sample, three specimens of each supply areas of the RCA, LCX, and LAD (nine in total)
were collected from porcine myocardium and cryo-embedded for further histologic processing. Using a
microtome, cryosections of 6 µm were extracted and stained with hematoxylin (25%). 10 µm sections
were cut for immunouorescence imaging (for quantication, at least 9 sections per heart were used).
Slices were permeabilized with 0.1%Triton X-100 (Invitrogen, Waltham, MA, USA) at RT for 15 minutes.
Antigen retrieval was performed by boiling in 1× citrate buffer-based antigen retrieval solution (H-3300,
Vector laboratories, USA). Unspecic antibody binding was blocked with 6% donkey serum (Sigma-
Aldrich, Waltham, MA, USA), and 2.5% bovine serum albumin (BSA, Sigma-Aldrich, Waltham, MA, USA) at
RT for one-hour. Samples were incubated with a primary antibody anti-P-selectin (NB100-65392, Novus
Biologicals, C), USA) at 4 oC overnight. After washing, samples were incubated with Alexa Fluor 555
conjugated donkey anti-mouse secondary antibody at 1:400 for one-hour at RT. DAPI was applied at
1:500 concentration in PBS for 10 min. Samples were mounted with Fluoromount-G (ThermoFisher,
Waltham, MA, USA). Samples were imaged with an automated slide scanner (AxioScan.Z1 Zeiss, Jena,
Germany).
For image analysis, thresholds were manually selected in Zen Blue microscopy software (Zeiss, Jena,
Germany). To acquire a baseline for uorescence, a value eliminating approximately 95% of nonzero p-
selectin pixels remote sample images was chosen and applied. Autouorescence from the green channel
was used to determine the total area of the tissue. A custom Python script using the packages czile
(Christoph Gohlke, University of California, Irvine) and NumPy was used to quantify uorescent area.
Positive pixels for autouorescence were compared to positive pixels for p-selectin (red channel) in a
given sample. Coverage was determined by dividing the sum of pixels both positive for p-Selectin and
autouorescence by the sum of all total membrane pixels positive for autouorescence.
Hematoxylin staining (HE)
For analysis of MPIO, HE staining was performed using a 25% hematoxylin solution for 50 seconds.
Further washing steps with saline were held to a minimum to avoid loss of MPIO binding. For analysis a

Page 10/21
bright-eld microscopy at 100x magnication was used. 20 elds of view were recorded in a standardized
fashion and analyzed to count individual MPIO. In total, 3 slides per region of interest (RCA, LCX, and
LAD) were analyzed covering the epicardium, mid-section and subendocardium.
Statistics
GraphPad Prism software (GraphPad Software, Inc.) was used for statistical analyses. Results are
depicted as mean ± standard error of mean. For two-group comparison a Mann-Whitney U test for
nonparametric data or students-t test for parametric data was used. For a comparison of more than two
groups an ANOVA, followed by a Bonferroni test for multiple comparison, was applied. P values of p <
0.05 indicate statistical signicance.
Results
40 minutes of I/R induces early injury only visible by myocardial T1 mapping.
To investigate early changes after myocardial injury we used a closed-chest model of I/R placing an
obstructing coronary balloon into the circumex (LCX) coronary artery (Fig.1A) Ischemia was conrmed
by documentation of signicant ST-segment elevation on the corresponding monitor ECG (Fig.1B). In
addition, echocardiography conrmed ischemia by wall motion abnormalities (Supplement Movie 1/2).
Reperfusion was obtained after 40 minutes of ischemia and animals were directly transferred to the MRI.
Due to the time of ischemia and transportation rst images were acquired 2 hours after onset of
ischemia. Performing advanced MR imaging, T1 mapping indicated elevated T1 times within the lesion
compared to remote areas (p < 0.001, Fig.1C). However, edema sensitive T2 mapping remained
unchanged (p = 0.11, Fig.1D). Likewise, late gadolinium enhancement was absent four hours after onset
of ischemia in all animals (Fig.1E).
Inammation responds early after myocardial ischemia.
To characterize the early inammatory response after I/R, we analyzed repetitive blood samples at
baseline and after ischemia. Flow cytometry analysis of innate immune cells indicated an immediate and
steady increase of pro-inammatory neutrophilic granulocytes and inammatory “classical-type” CD14+
monocyte within the rst 3 hours after ischemia before reaching a plateau at 4 hours. Other “non-
classical type” monocyte subsets (CD14neg, CD163+) remained unchanged (Fig.2A). In accordance with
increased myocardial T1 mapping times and a proinammatory state in circulation, we detected elevated
expression of the leukocyte binding integrin P-selectin within the myocardial lesion area when compared
to remote myocardial tissue (p = 0.02, Fig.2B).
P-selectin expression by platelets and endothelial cells can be selectively targeted with a functionalized
molecular imaging contrast agent.
After MPIO-labelling of P-selectin antibody (P-selectin MPIO) or unspecic IgG antibody (Control MPIO),
we tested binding eciency of the contrast agents using an
in vitro
ow chamber model for platelets or

Page 11/21
an incubation assay for endothelial cells. In comparison to labelled IgG control, P-selectin contrast agent
selectively bound to endothelial cells
in vitro
and indicated a higher binding eciency to platelets in a
ow chamber model (p < 0.0001 for each, Fig.3).
In vivo molecular imaging of P-selectin in myocardial I/R
Molecular imaging in large animal models is especially challenging due to the large blood pool of the
animal. We therefore used interventional MRI to selectively inject MPIO contrast agent into the left
coronary artery. Figure4A depicts the interventional MRI setup. After intubation of the LCA, selective
injection of diluted gadolinium allowed to separate the area of the perfused left and none-perfused right
coronary artery (Fig.4B). After injection of P-selectin MPIO, we detected a visible signal decrease in T2*
maps indicating shorter T2* times in the lesion area. Comparing this signal to the non-perfused RCA area,
P-selectin-MPIO lead to a stronger reduction of T2* times than Control-MPIO within the lesion (p = 0.03,
Fig.4C). This difference, however, was absent when comparing the equally perfused but non-injured LAD
area (p = 0.34, Fig.4D).
Ex vivo MRI and histology conrms selective contrast deposition
After termination of the experiment we excised the porcine heart and repeated T2* mapping of the xed
heart
ex vivo
. Signal inversion (R2*) helped to identify contrast agent deposition in myocardial tissue. In
support of our
in vivo
ndings, we detected a signicant increase in the signal intensity within the lesion
area in comparison to the perfused but non-injured LAD area and the non-perfused RCA area (p = 0.03,
Fig.5A). Similar ndings were observed when counting MPIO particles in histology sections. In animals
injected with P-selectin MPIO we detect highest amounts of MPIO in the lesion area in comparison to the
perfused but non-injured LAD area and the non-perfused RCA area, while MPIO target binding was equally
low in all areas after injection of Control-MPIO (Fig.5B).
Discussion
In this study, we demonstrate that molecular targeting of early markers of inammation can complement
existing MRI tools for tissue characterization after ischemic injury providing target-specic information.
Importantly, in our study the application of molecular contrast agents was performed using a real time
MRI-guided intervention by selectively injecting the contrast agent into the coronary artery.
Ischemic cardiac injury usually forms a continuum with increasing severity over time.18 As
cardiomyocytes fully depend on oxygen to cover their excessive energy demand, even short durations of
MI may induce cell death and apoptosis.19 With increasing time of impaired myocardial blood ow, injury
may expand and reach a critical mass of cardiomyocyte loss for permanent impairment of cardiac
function and formation of a non-contractile scar. However, this detrimental development can be
interrupted at any time by reperfusion to salvage injured cardiac cells.20 Next to the necrotic core zone,
this consequently leaves an area of unknown fate with cells that are unequivocally lost intermingling with
cells that could potentially be saved and repaired, termed ‘area at risk’.21 Innate immune cells recruited to

Page 12/21
the lesion have been recognized to be essential in repair or replacement after injury and excessive
inammation has been linked to increased cardiomyocyte loss. 22, 23 Likewise, several preclinical studies
have demonstrated a benecial role of immunomodulation to improve wound healing and prevent from
adverse cardiac remodeling.24, 25 Highly sensitive and quantitative assessment as well as
characterization of inammation could thus set grounds for individualized risk prediction and novel
therapeutic strategies after ischemic injury.
MRI evolved as the gold-standard for cardiac tissue characterization due to its capabilities of tissue
specic measurement of T1, T2 and T2* relaxation times as well as assessment of gadolinium contrast
distribution.26–28 These techniques have proven to be of value in assisting the non-invasive detection of
inammatory cardiac disease like myocarditis. In myocarditis, T1 mapping demonstrated highest
sensitivity for cardiac injury while edema-sensitive T2 mapping can be used to differentiate acute from
healing myocarditis.29 After MI, T1/T2 mapping or late gadolinium enhancement (LGE) are used for
quantication of the ischemic scar and the area at risk.30 In our model we allowed for reperfusion after
40 minutes of MI. Interestingly, after this time we neither found signs of LGE nor edema specic increase
in T2 times in the lesion up to 4 hours after reperfusion which is an indicator of the absence of
myocardial scarring or relevant edema. Only T1 mapping detected reproducible alterations in T1 times
within the lesion supporting existing evidence of the high sensitivity of T1 mapping in cardiac injury. Yet,
reasons for T1 alterations are manifold including brosis and inltrative diseases as well as myocardial
edema. Therefore, more specic information is needed.
Following the increase of innate immune cells in the blood early after ischemia, we investigated if
activation of inammatory pathways could be associated to changes in T1. Intriguingly, we found higher
expression of the integrin P-selectin in areas with elevated T1 time. P-selectin is mainly expressed by
endothelial cells and platelets and is involved in the recruitment process of immune cells to the site of
injury.31, 32 In endothelial cells P-selectin induces rolling of leukocytes at the vascular surface prior to rm
adhesion and transmigration. In platelets, P-selectin contributes to complex-formation with leukocytes to
boost immune cell recruitment.33 Non-invasive detection of P-selectin in cardiac lesions could thus serve
as an early and highly specic marker of cardiac inammation.
Using a covalent binding strategy, we functionalized MPIO with antibodies targeting P-selectin or
unspecic IgG as a Control compound. The concept of targeted contrast agents for molecular imaging
emerged from nuclear imaging techniques and was later transferred to MRI in several preclinical settings.
Iron oxide compounds are suitable contrast agents due to the strong eld distortions that surpass the
actual particle size and thus allow for visualization of even smallest moieties. T2* mapping or its inverted
R2* map are sensitive to display iron-induced eld distortions. In general, MR relaxometry has been
applied for various cardiac applications such as the detection of changes in the microvascular structure
in hypo-perfused areas by T2* measurements34,35, or the quantication of the degree of amyloidosis in
the myocardium in T1 and T2 parameter maps.36 Cardiac relaxometry is challenging as the relaxation
times are calculated from a series of images that need to be acquired in the same cardiac and respiratory

Page 13/21
phase which can lead to long measurement times and can make the measurement sensitive to other
types of patient motion. Here, T1 and T2 maps were measured with breath held bSSFP protocols, where
the acquisition time was short enough to acquire all relevant information in a single breath hold, and
additional motion compensation during post-processing allowed to correct for the cardiac motion. T2*
maps were measured using a breath held and ECG-triggered multi-echo FLASH sequence which is even
less sensitive to residual motion. However, as the parameter T2* is sensitive to both microscopic eld
distortions caused by the iron-containing contrast agents and the meso- and macroscopic eld variations
as a consequence of magnetic susceptibility changes between tissues, this parameter is varying along
the myocardium. To overcome this limitation, in this work T2* was measured before and after infusion of
the molecular contrast agents; thus, the macroscopic eld variations were the same before and after
administration, so that the T2* differences could solely be attributed to the contrast agent.
As already discussed, we facilitated interventional MRI to selectively inject the contrast agents into the
left coronary artery in order to reach sucient concentration at the site of interest. Next to cost reductions,
this approach added an additional internal control as it allowed to compare the lesion not only to remote
healthy tissue (here: the area of the RCA), but also to the area of the LAD which was equally exposed to
contrast agent, but not injured.
Limitations
A few limitations of the current study must be acknowledged. Despite all measures taken to optimize
contrast deposition and imaging, the
in vivo
MRI signal still had a very low SNR with variations in
absolute T2* times between single experiments. Therefore, comparison of absolute T2* times was
impeded. Next to low binding eciency of the compound under arterial shear stress, dislocation of the
guiding catheter from the coronary ostium during injection of the contrast agent owing to anatomic
variations of the pigs could have contributed to this nding. Forming an individual T2* relative ratio for
the lesion and control area allowed us to compare results from individual experiments.
Microvascular structural changes in hypoperfused tissue are known to inuence T2* times. Unspecic
T2* signal changes in the lesion despite the absence of relevant moieties of MPIO as conrmed by
histology most likely originated from such microscopic eld distortions. Repetition of R2* = 1/T2*
mapping
ex vivo
in a non-beating heart clearly depicted specic deposition of MPIO in the lesion as
compared to the LAD and RCA area.
Last, injection of molecular contrast agents into the coronary artery does not fulll the criteria of non-
invasive imaging that would be favorable in this setting. In future approaches increasing the target signal
either by enhancing target specic binding or replacing the signal giving contrast agent will be necessary.
Fluorine-19 is selectively enhanced with a separate coil which reduces unspecic background noise.
However, target-specic labelling of uorine yet has to be explored.
Conclusion

Page 14/21
Our study represents a proof-of-concept study providing evidence that antibody-directed molecular
imaging strategies using MRI are generally translatable from rodents to large animal models. By using
real time MRI-guided coronary interventions, target-specic imaging of early markers of inammation
was possible, providing a deeper understanding of post-ischemic cardiac lesions.
Declarations
Acknowledgments
Ethics
All applicable institutional and/or national guidelines for the care and use of animals were followed.We
acknowledge the SCI-MED imaging facility at the Institute for Experimental Cardiovascular Medicine in
Freiburg for access to the slide scanner and support with image acquisition.
Consent for publication
Not applicable
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author
on reasonable request.
Conict of Interest
The authors declare that they have no conict of interest.
Funding sources
This work was supported by institutional funds.
Ingo Hilgendorf, Dennis Wolf, Dirk Westermann, Alexander Maier, Constantin von zur Mühlen and Timo
Heidt are members of SFB1425, funded by the Deutsche Forschungsgemeinschaft (DFG, German
Research Foundation) – Project #422681845. Alexander Maier was funded by the Berta-Ottenstein-
Programme for Advanced Clinician Scientists, Faculty of Medicine, University of Freiburg.
Authors’ contribution
All coauthors provided intellectual content, reviewed, and approved the manuscript prior to submission.
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Figures
Figure 1
Ischemia and reperfusion injury. A Coronary angiography of the left coronary artery. Left Overview of the
left anterior descending (LAD) and left circumex artery (LCX) Middle A standard coronary wire (red
arrow) and obstructive coronary balloon (black arrow) are introduced into the LCX. Then the balloon is
inated to block blood the supply to the distal myocardium. RightRed box indicates the blocked LCX
artery during contrast deposition. After 40 min of ischemia the balloon is removed for reperfusion. B ECG
showing ST-segment elevations during ischemia C T1 mapping after reperfusion indicates a signicantly
elevated T1 time in the “Lesion” area of the LCX as compared to “Remote” (LAD/RCA area); p < 0.001 D
T2 mapping after reperfusion shows no difference between “Lesion” and “Remote”; p = 0.11 E 40 minutes
after ischemia no LGE can be detected in the “Lesion” area. * P<0.05, Mann-Whitney U test.

Page 18/21
Figure 2
Post-ischemic inammation. A Flow cytometry characterization of innate immune cells in the blood after
I/R. Above FACS plots indication the gating scheme for neutrophils and monocyte subsets. Below
Timeline analysis of innate immune cells from baseline to 4 hours after onset of ischemia for neutrophils,
classical-typed CD14+CD163neg monocytes and nonclassical-typed CD14negC163+ monocytes. * p < 0.05;
indicates the comparison of timepoints 3 hours to baseline; p = 0.04 for neutrophils and for CD14+
monocytes. B Immunouorescence staining of cardiac tissue from “Injury” and “Remote” targeting P-
selectin expression (red) and nuclei (dapi-blue). After injury, P-selectin expression is signicantly
increased in the “Injury” area compared to remote; p = 0.02. * p < 0.05; Mann-Whitney U test.

Page 19/21
Figure 3
Targeting P-selectin
in vitro
. Binding of functionalized MPIO targeting P-selectin (P-selectin (Psel) –
MPIO) to platelets and endothelial cells was evaluated using an incubation essay (endothelial cells,
above) or a ow chamber model (platelets, below)
in vitro
. For comparison we used functionalized MPIO
targeting unspecic IgG (Control-MPIO). Both essays conrmed enhanced binding of P-selectin MPIO to
platelets and endothelial cells in comparison to Control MPIO. For MPIO binding to endothelial cells p <
0.0001, for platelet binding eciency p < 0.0001. * p < 0.05; Mann-Whitney U test.

Page 20/21
Figure 4
Targeting P-selectin in the ischemic lesion
in vivo
. A Interventional MRI setup for selective contrast
injection into the left coronary artery. B Perfusion map of the porcine heart. Cine image (corner) shows
midventricular short axis slice of the heart. Gadolinium injection into the left coronary artery (LCA) depicts
the supply area of the left circumex (LCX) and left anterior decending (LAD) arteries as well as the non-
perfused area of the right coronary artery (RCA). C T2* map of the left ventricular short axis after injection
of the MPIO contrast agent (above: P-selectin MPIO; below: Control MPIO). Injection of P-selectin MPIO
induced a signicant decrease in the LCX (lesion) to RCA ratio in comparison to Control MPIO, p = 0.03 D
while no difference was observed when comparing the equally perfused but non-injured LAD area to the
RCA area, p = 0.34.* p < 0.05; Mann-Whitney U test.

Page 21/21
Figure 5
Ex vivo
analysis of heart specimens. A/B Ex vivo, high resolution inverse T2* (R2*) map of the left
ventricle after I/R of the LCX and P-selectin MPIO perfusion via the left coronary artery. P-selection MPIO
selectively enriched in the area of the LCX as compared to the LAD. * p=0.03; standard t-test. C We
randomly assessed tissue sections taken from specimens resected from the LCX, LAD or RCA area. P-
selectin MPIO were found most frequent in the area of tissue injury compared to perfused, non-injured
LAD area and non-perfused, non-injured RCA area. Only minimal MPIO moieties were found in the tissue
after injection of Control-MPIO with unspecic binding. * p<0.05 one-way ANOVA.
Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.
SuplementMovie2postIR.mov
SupplementMovie1preIR.mov