Dual Contrast Molecular Imaging Allows Noninvasive Characterization of Myocardial Ischemia/Reperfusion Injury After Coronary Vessel Occlusion in Mice by mri running title


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DOI: 10.1161/CIRCULATIONAHA.113.008157 



Dual Contrast Molecular Imaging Allows Noninvasive Characterization of 



Myocardial Ischemia/Reperfusion Injury After Coronary Vessel Occlusion in 

Mice by MRI 

Running title: von Elverfeldt et al.; Molecular MRI of myocardial injury 

Dominik von Elverfeldt, PhD

1

*; Alexander Maier, MS



2

*; Daniel Duerschmied, MD

2

*;  


Moritz Braig, MS

1

; Thilo Witsch MD



2

; Xiaowei Wang, PhD

3

;  Maximilian Mauler, MS



 2,4

;  


Irene Neudorfer, MS

2

; Marius Menza, MS



1

; Marco Idzko, MD

5

; Andreas Zirlik, MD



2

;  


Timo Heidt, MD

2,6


; Peter Bronsert, MD

7

; Christoph Bode, MD



2

; Karlheinz Peter, MD, PhD

3#



Constantin von zur Muhlen, MD



2#

 

1



Dept of Radiology–Medical Physics, University Medical Center Freiburg, Germany; 

2

Dept of 



Cardiology I, University Heart Center Freiburg, Germany; 

3

Atherothrombosis and Vascular 



Biology, Baker IDI Heart and Diabetes Institute, Melbourne, Australia; 

4

Faculty of Biology, 



University Freiburg, Germany; 

5

Dept of Pneumology, University Medical Center Freiburg, 



Germany; 

6

Center for Systems Biology, Massachusetts General Hospital, Boston, MA; 



7

Institute 

of Pathology & Comprehensive Cancer Center, University Medical Center Freiburg, Germany 

* these authors contributed equally

these authors contributed equally 



 

Address for Correspondence: 

Constantin von zur Muhlen, MD 

University Heart Center Freiburg 

Hugstetter Strasse 55 

79106 Freiburg, Germany 

Tel: +49-761-270-37835 

Fax: +49-761-270-33884 

E-mail: constantin.vonzurmuehlen@universitaets-herzzentrum.de 

 

Journal Subject Codes: Atherosclerosis:[150] Imaging, Thrombosis:[172] Arterial thrombosis 

Irene Neudorfer, MS

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; Marius Menza, MS



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#

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 by guest on December 22, 2017



http://circ.ahajournals.org/

Downloaded from 

 by guest on December 22, 2017

http://circ.ahajournals.org/

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 by guest on December 22, 2017

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DOI: 10.1161/CIRCULATIONAHA.113.008157 



Abstract



Background—Inflammation and myocardial necrosis play important roles in 

ischemia/reperfusion injury after coronary artery occlusion and recanalization. The detection of 

inflammatory activity and the extent of myocardial necrosis itself are of great clinical and 

prognostic interest. We developed a dual, non-invasive imaging approach using molecular 

magnetic resonance imaging (MRI) in an in vivo mouse model of myocardial ischemia and 

reperfusion. 



Methods and Results—Ischemia/reperfusion injury was induced in 10 week old C57BL/6N mice 

by temporary ligation of the “left anterior descending coronary artery“(LAD). Activated platelets 

were targeted with a contrast agent consisting of microparticles of iron oxide (MPIO) conjugated 

to a single-chain antibody directed against a ligand-induced binding site (LIBS) on activated 

glycoprotein (GP)IIb/IIIa (=LIBS-MPIO). After injection and imaging of LIBS-MPIO, late 

gadolinium enhancement (LGE) was used to depict myocardial necrosis; these imaging 

experiments were also performed in P2Y

12

-/-



 mice. All imaging results were correlated to 

immunohistochemistry findings. Activated platelets were detectable by MRI via a significant 

signal effect caused by LIBS-MPIO in the area of LAD occlusion two hours after reperfusion. In 

parallel, LGE identified the extent of myocardial necrosis. Immunohistochemistry confirmed that 

LIBS-MPIO significantly bound to microthrombi in reperfused myocardium. Only background-

binding was found in P2Y

12

-/-


 mice. 

Conclusions—Dual molecular imaging of myocardial ischemia/reperfusion injury allows 

characterization of platelet-driven inflammation by LIBS-MPIO as well as myocardial necrosis 

by LGE. This non-invasive imaging strategy is of clinical interest for both diagnostic and 

prognostic purposes, and highlights the potential of molecular MRI for characterizing 

ischemia/reperfusion injury. 

 

Key words: reperfusion, thrombosis, platelets, magnetic resonance imaging 

 

 

 



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 by guest on December 22, 2017

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Downloaded from 


DOI: 10.1161/CIRCULATIONAHA.113.008157 



Introduction 

During cardiac ischemia and after reperfusion, platelets play an important role not only as a 

mediator of acute macrovessel closure, but also by causing microvascular obstruction (MVO) as 

well as inflammation in the ischemic/reperfused myocardium, which both have a deleterious 

effect on myocardial damage 

1, 23

. Platelet inhibition is a key factor in acute myocardial 



infarction management, and increasingly potent antiplatelet drugs targeting the platelet ADP-

receptor P2Y

12

 have improved patient outcome 



4, 5

. Also the amount of platelets in the 

ischemic/reperfused myocardium has an important prognostic value 

6

. Studies have shown that a 



high degree of MVO that involves platelet microthrombi results in a poor outcome 

7, 8


.  In 

addition, the extent of myocardial necrosis is widely accepted to be an important prognostic 

factor 

9, 10


 and cardiac magnetic resonance imaging (MRI) can be used to characterize wall 

motion and myocardial necrosis (late gadolinium enhancement = LGE) after acute myocardial 

infarction (AMI) 

9, 11


Therefore, it would be highly desirable to noninvasively characterize the extent of platelet 

involvement in ischemia/reperfusion injury, as well as the extent of myocardial necrosis. LGE 

specifically detects necrotic myocardial tissue, causing a signal increase in cardiac MRI 

12, 13



However, detection of platelets needs a targeted molecular imaging approach 



14

. We have 

previously developed a unique contrast agent, which specifically detects activated platelets by 

targeting a ligand-induced binding site (LIBS) of the activated glycoprotein (GP)IIb/IIIa-receptor, 

using a single-chain antibody directed against these sites 

15

. This antibody was conjugated to 



microparticles of iron oxide (MPIO), resulting in the unique activation-specific antiplatelet 

contrast agent (LIBS-MPIO). These LIBS-MPIOs cause a signal decrease in T

2

* weighted MRI 



at areas of platelet accumulation with an excellent sensitivity, since the 1 μm-sized MPIOs cause 

addition, the extent of myocardial necrosis is widely accepted to be an important

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 by guest on December 22, 2017

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DOI: 10.1161/CIRCULATIONAHA.113.008157 

a magnetic field disturbance exceeding their own diameter by far. Using this approach, we were 



able to detect even small amounts of activated platelets in coronary and carotid artery thrombosis, 

plaque rupture, and cerebral inflammation in mice 

16-18



In this study, we induced ischemia/reperfusion injury in C57BL/6N mice by temporary 



ligation of the left anterior descending coronary artery (LAD) and thereafter performed 

molecular magnetic resonance imaging of activated platelets by LIBS-MPIO to characterize 

platelet accumulation contributing to MVO and ischemia/reperfusion-associated inflammation. 

Furthermore, in a dual magnetic resonance imaging approach, myocardial necrosis was directly 

assessed by LGE in the same animals. The central role of platelets in myocardial 

ischemia/reperfusion injury as well as the clinical relevance of the respective therapeutic receptor 

inhibition was confirmed in dual contrast MRI and histology in P2Y

12

 knockout mice undergoing 



temporary LAD ligation.   



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