NOVEL EXPERIMENTAL METHODS FOR THE GENERATION AND MULTI-MODALITY QUANTIFICATION OF MYOCARDIAL INFARCTION

Ph.D. Thesis

by

Ákos Varga-Szemes, M.D.

Head of the Doctoral School: Sámuel Komoly, M.D., Ph.D., D.Sc.

Head of the Doctoral Program: Ákos Koller, M.D., Ph.D., D.Sc.

Supervisor: Tamás Simor, M.D., Ph.D.

Heart Institute, University of Pécs

Pécs

2013

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TABLE OF CONTENTS

1.   INTRODUCTION .................................................................................................... 1  

1.1   Clinical and pathological definition of myocardial infarction ................................ 1  

1.2   Epidemiology of myocardial infarction .................................................................. 1  

1.3   Classification of myocardial infarction ................................................................... 2  

1.4   Brief pathophysiology of myocardial infarction ..................................................... 3  

1.5   Experimental models of myocardial infarction ....................................................... 5  

1.6   Diagnostic imaging in myocardial infarction ......................................................... 6  

2.   OBJECTIVES ......................................................................................................... 10  

2.1   Myocardial infarct model development ................................................................ 10  

2.2   Development of a Gadolinium based infarct quantification tool .......................... 11  

3.   EMBOZENETM MICROSPHERES INDUCED NON-REPERFUSED

MYOCARDIAL INFARCTION IN AN EXPERIMENTAL SWINE MODEL ... 12  

3.1   Introduction ........................................................................................................... 12  

3.2   Materials and Methods .......................................................................................... 13  

3.3   Results ................................................................................................................... 16  

3.4   Discussion ............................................................................................................. 22  

3.5   Conclusions ........................................................................................................... 26  

4.   DETERMINATION OF INFARCT SIZE IN EX VIVO SWINE HEARTS BY

MULTI-DETECTOR COMPUTED TOMOGRAPHY USING GADOLINIUM

AS CONTRAST MEDIUM ................................................................................... 27  

4.1   Introduction ........................................................................................................... 27  

4.2   Materials and methods .......................................................................................... 28  

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4.3   Results ................................................................................................................... 33  

4.4   Discussion ............................................................................................................. 37  

5.   DISCUSSION ......................................................................................................... 42  

5.1   Myocardial infarct model development ................................................................ 42  

5.2   Development of a Gadolinium based infarct quantification tool .......................... 43  

6.   CONCLUSIONS .................................................................................................... 44  

7.   NOVEL FINDINGS ............................................................................................... 45  

8.   REFERENCES ....................................................................................................... 46  

9.   PUBLICATIONS OF THE AUTHOR ................................................................... 59  

9.1   Peer reviewed original research publications related to the thesis ....................... 59  

9.2   Citable peer reviewed abstracts related to the thesis ............................................ 59  

9.3   Peer reviewed abstracts related to the thesis ......................................................... 59  

9.4   Original peer reviewed publications not related to the thesis ............................... 60  

9.5   Citable peer reviewed abstracts not related to the thesis ...................................... 60  

9.6   Peer reviewed abstracts not related to the thesis ................................................... 63  

9.7   Additional presentations/abstracts not related to the thesis .................................. 65  

10.   ACKNOWLEDGEMENTS .................................................................................... 67  

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ABBREVIATIONS

3D Three Dimensional

ACT Activated Clotting Time

ANOVA Analysis Of Variance

ATP Adenosine Triphosphate

au Arbitrary Unit

CA Contrast Agent

CAD Coronary Artery Disease

CM Contrast Medium

CMR Cardiovascular Magnetic Resonance

CNR Contrast to Noise Ratio

CPR Cardiopulmonary resuscitation

CT Computed Tomography

CTA Computed Tomography Angiography

CVD Cardiovascular Disease

DTPA Diethylenetriamine Penta-acetic Acid

EC Extracellular

ECG Electrocardiogram

FoV Field of View

FWHM Full Width at Half Maximum

Gd Gadolinium

Gd-DTPA Gadopentetate Dimeglumine, Magnevist®

GRE Gradient Recalled Echo

H&E Hematoxylin and Eosin

HU Hounsfield Unit

IF Infarct Fraction

IM Image Matrix

IR Inversion Recovery

IV Infarct Volume

v

LAD Left Anterior Descending

LCX Left Circumflex

LGE Late Gadolinium Enhancement

LV Left Ventricle

LVV Left Ventricular Volume

MDCT Multi-Detector Computed Tomography

MI Myocardial Infarction

MO Microvascular Obstruction

MRI Magnetic Resonance Imaging

n-BCA n-Butyl Cyanoacrylate

NSF Nephrogenic Systemic Fibrosis

PVC Premature Ventricular Complex

R1 Longitudinal Relaxation Rate

RCA Right Coronary Artery

RV Right Ventricle

SD Standard Deviation

SI Signal Intensity

SIR Signal Intensity Ratio

SNR Signal to Noise Ratio

SSFP Steady-State Free Precession

ST Slice Thickness

T1 Longitudinal Relaxation Time

T2 Transverse Relaxation Time

TE Echo Time

TI Inversion Time

TR Repetition Time

TTC Triphenyl-Tetrazolium-Chloride

VF Ventricular Fibrillation

WHO World Health Organization

1

1. INTRODUCTION

1.1 Clinical and pathological definition of myocardial infarction

Myocardial infarction (MI) is the condition when there is evidence in a clinical setting

of myocardial necrosis consistent with myocardial ischemia.1 By pathology, MI is

defined as myocardial cell death caused by prolonged ischemia.1

1.2 Epidemiology of myocardial infarction

Cardiovascular disease (CVD) is the number one cause of death in the Western World.

The 2008 overall rate of death attributable to CVD was 244.8 per 100,000. Mortality

data for 2008 show that CVD accounted for 32.8% (811,940) of all 2,471,984 deaths in

2008, or one of every three deaths in the United States. On the basis of 2008 mortality

rate data, more than 2,200 Americans die of CVD each day, an average of one death

every 39 seconds.2

Coronary artery disease (CAD) caused one of every six deaths in the United States in

2008. CAD mortality in 2008 was 405,309. Each year, an estimated 785,000

Americans will have a new coronary attack, and 470,000 will have a recurrent attack. It

is estimated that an additional 195,000 silent, first MI occur each year. Approximately

every 25 seconds an American will have a coronary event, and approximately every

minute someone will die of one.2

Also in Central- and Eastern-European countries, including Hungary, CVD is the

leading cause of total mortality (representing 50-60% of the total mortality).3

According to the World Health Organization (WHO) database, the cardiovascular

mortality rate was 460/100,000 in 2006.3

2

Based on the significant number of heart patients worldwide, experimental and clinical

cardiovascular research is of major importance in the improvement of cardiovascular

diagnostic and therapeutic approaches.

1.3 Classification of myocardial infarction

MI can be classified based on its etiology, transmurality, age, as well as its relation to

reperfusion.

The most recent consensus report classifies MI into five clinical types:1

Type 1 Spontaneous MI related to ischemia caused by a primary coronary

event such as plaque erosion and/or rupture, fissuring, or dissection

Type 2 MI secondary to ischemia caused by either increased oxygen demand

or decreased supply, e.g. coronary artery spasm, coronary embolism,

anemia, arrhythmias, hypertension, or hypotension

Type 3 Sudden unexpected cardiac death, including cardiac arrest, often with

symptoms suggestive of myocardial ischemia, but with death

occurring before blood samples could be obtained, or at a time before

the appearance of cardiac biomarkers in the blood

Type 4a MI associated with percutaneous coronary intervention

Type 4b MI associated with stent thrombosis as documented by angiography

or at autopsy

Type 5 MI associated with coronary artery bypass graft surgery.

Based on its transmurality, MI can be considered either:4

(1) Subendocardial

The ischemia involves only a thin subendocardial layer of the

myocardium. Subendocardial MI usually results from a partial or

transient occlusion of a branch of the coronary artery system.

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(2) Transmural

The ischemia extends through the whole thickness of the myocardium.

Transmural MI is regularly caused by the total, long-lasting occlusion of

a major branch of the coronary artery tree.

MI can also be categorized based on the pathological appearance related to its age as

follows:1

(1) Evolving (<6 hours)  

(2) Acute (6 hours – 7 days)  

(3) Healing (7 – 28 days)  

(4) Healed (29 days and beyond)  

Based on the nature of the occlusion, MI can also be classified as:

(1) Reperfused

Reperfused MI occurs when any revascularization strategy is applied

within a reasonable time following the onset of myocardial ischemia and

the blood supply of the affected area is restored.

(2) Non-reperfused

Non-reperfused MI results when a revascularization strategy is not

available within an appropriate time frame.

1.4 Brief pathophysiology of myocardial infarction

Acute MI is usually caused by the thrombotic occlusion of one or more major branches

of the coronary artery system.5 The reversibility and transmurality of the ischemic

myocardial injury mostly depend on the duration of the coronary artery occlusion; other

factors, however, may also influence the severity of the damage, such as pre-

conditioning or the presence of collateral circulation to the affected myocardial

territory. According to the wavefront phenomenon, necrosis occurs first in the

subendocardium and extends as a wavefront which gradually moves toward the

subepicardium during a period of six hours.4,6 Dog studies have shown that a 15 min

occlusion results in a completely reversible ischemia. Reperfusion after a 40 min

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occlusion, however, always results in focal or confluent subendocardial necrosis.

Reperfusion after three hours produces focal involvement of the mid- or subepicardial

myocardium as well. Beyond six hours, the necrosis becomes nearly transmural.4,7

After the onset of myocardial ischemia, cell death is not immediate but takes a finite

period to develop (as little as 20 min or less).1 The first change within the affected

myocytes can be observed as a shift from the aerobic to the anaerobic metabolic

pathways. As global ischemia develops, mitochondrial electron transport ceases and

anaerobic glycolysis remains the only source of adenosine triphosphate (ATP). The

metabolic shift to the anaerobic glycolytic pathway and the concurrent lysosomal

activation result in ATP depletion and intracellular lactic acid and calcium

accumulation. These changes initiate the development of acidosis, accumulation of free

radicals, and increase in cell osmolarity.8 The ultimate consequence of this complex

series of perturbations is initially a reversible depression of cell function and ultimately

myocardial cell death.9

It takes several hours before myocardial necrosis can be identified by macroscopic or

microscopic post-mortem examination.1 The first microscopic signs of myocardial

ischemia that can be observed after about six hours from the onset of the coronary

artery occlusion, are nuclear shrinkage and nuclear loss. Acute MI is also characterized

by polymorphonuclear leukocyte infiltration, myocyte swelling, and contraction band

necrosis.10 The presence of mononuclear cells and fibroblasts, as well as the absence of

polymorphonuclear leukocytes, characterize healing MI. Healed infarction is

manifested as scar tissue without cellular infiltration. The entire process leading to a

healed MI usually takes at least four weeks.1

Reperfusion of the ischemic myocardium aims to restore the blood supply of the

affected myocardial area. Within less than six hours from the onset of the ischemia

there are still potentially salvageable myocytes at the area at risk that may benefit from

any reperfusion effort. Reperfusion therapy, however, may promote additional injury to

the myocardium, as it can lead to intramyocardial hemorrhage, interstitial edema, and

microvascular obstruction (MO).11-13 Although MO has been described as a reperfusion

injury, it frequently occurs in cases with non-reperfused MI also.14

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1.5 Experimental models of myocardial infarction

Several experimental animal models of MI have been introduced for investigating the

underlying pathophysiology of MI, developing new diagnostic methods, and studying

the effectiveness of new treatment options.15

Based on the size of vertebrate animals used for MI generation, MI models can be

categorized as:

(3) Small animal models

Small animal models of MI (e.g., mouse, rat, rabbit, etc.)16-18 show

better cost effectiveness compared to the large animal models. Small

animals require less complicated surgery, shorter follow up, and less

expensive housing.

(4) Large animal models

The generation of large animal models of MI (e.g., dog, sheep, pig,

etc.)15,19,20 is time and cost consuming. Such procedures require more

sophisticated surgical or interventional facilities (similar to the ones

used in human medicine), equipment (e.g., fluoroscope, anesthesia

machine), human resources (dedicated surgeon, interventional

cardiologist), and special housing. Large animal models of MI, however,

show the best pathological and pathophysiological correspondence to

the human disease.

Based on the method of MI induction, animal models can be generated by:

(1) Open chest transthoracic surgical techniques

The main advantage of this approach is the direct access to the heart

with visual control of the occlusion.21 It is also possible to measure

contractile function and coronary blood flow and monitor metabolic

parameters.21 Limitations of this method are the high mortality rate (up

to 55%) and the high complication rate associated with surgery.22,23

Incision of the chest and the pericardium may alter pericardial

regenerative capacity, and may induce pneumothorax, which has major

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hemodynamic effects.24,25 It is also worth mentioning that the

myocardium must be cooled during the procedure. The cooling,

however, is difficult to control and crucially affects the size of the

induced MI.21,26 Moreover, normal cardiac function may be impaired by

incision of the chest wall, and major surgery always affects the local and

global immunological and inflammatory response.21,24,27

(2) Closed chest, minimally invasive percutaneous techniques

Closed chest techniques avoid sternotomy by using a relatively

superficially located artery (radial, femoral or carotid) as a gate of

entrance. Although percutaneous techniques do not provide direct visual

and manual control during the procedure, all segments of the coronary

artery tree, however, can be visualized by fluoroscopy and accessed by

coronary catheters. As a limitation, the average effective radiation dose

delivered during such a procedure is ~8mSv.28 Another shortcoming of

closed chest MI generation techniques is that closed-chest

cardiopulmonary resuscitation (CPR) has been shown to be inferior to

open chest cardiac massage for both hemodynamics produced during

resuscitation and ultimate resuscitation success.29 The rate of

complications and the rate of mortality in closed chest MI models,

however, are still significantly lower than those of open chest MI

models.

1.6 Diagnostic imaging in myocardial infarction

The diagnosis of MI is usually based on the patient’s physical status, electrocardiogram

(ECG), the level of cardiac enzymes (e.g., creatine kinase, troponin I and T) in the

blood, and coronary angiogram. Additional diagnostic imaging methods, however, are

available to confirm the diagnosis, evaluate and quantify the myocardial damage, and

monitor left ventricular (LV) recovery.

Numerous techniques have been developed to distinguish viable myocardium from

irreversibly injured myocardium, and to localize, visualize, and quantify MI.30 Late

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Gadolinium Enhancement Magnetic Resonance Imaging (LGE-MRI), however, has

become the gold standard modality for the assessment of myocardial viability.31

1.6.1 Cardiovascular Magnetic Resonance Imaging for the assessment of myocardial

infarction

Cardiovascular MR (CMR) provides information from different aspects of the infarcted

heart. CMR offers accurate and reproducible tomographic, static, or cine images of

high spatial resolution in any desired plane. Cine gradient recalled echo (GRE) or

steady-state free precession (SSFP) imaging is able to demonstrate segmental regional

wall motion abnormalities, as well as provides global functional assessment.32,33

Dobutamine stress augments wall motion abnormalities, thus improves the diagnostic

value of cine imaging.34 T2-weighted turbo spin echo CMR provides additional

characterization of the myocardium. T2-weighted CMR highlights myocardial edema

related to the ischemic area at risk, and therefore it can differentiate acute from chronic

MI.35 The quantitative assessment of MI is carried out by the gold standard LGE-MRI,

using a T1-weighted inversion recovery (IR) GRE acquisition.36

1.6.2 Gadolinium contrast agents

Gadolinium (Gd)-based contrast agents (CA) result from the complexation of the Gd

cation with a chelating agent minimizing the likelihood of toxic effects from free Gd

cation in the body.37 The first approved Gd CA, gadopentetate dimeglumine (Gd-

DTPA), appeared in 1998, and several other complexes followed.38

Based on their effect, Gd CAs can be classified into two broad groups:38

(1) T1 agents

These CAs alter the longitudinal relaxation time (T1) of tissue protons

(mainly of water) considerably more than their transverse relaxation

time (T2). This dominant T1-shortening effect gives rise to an increase in

signal intensity (SI), and thus these agents are “positive” CAs.

(2) T2 agents

These CAs largely shorten the T2 of tissue protons and cause a reduction

in SI, and thus these agents are known as “negative” CAs.

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Paramagnetic, Gd-based CAs are examples of T1 agents, whereas ferromagnetic (or

superparamagnetic), large iron oxide particles are examples of T2 agents.38

Based on their tissue distribution, Gd-based MRI CAs can be categorized into the

following groups:39

(1) Extracellular (EC) agents

EC agents (e.g. gadopentetate, gadoterate, gadodiamide, gadoteridol,

gadobutrol) are very hydrophilic and they quickly and freely distribute

from the blood stream into the EC space.38 Chemically, these

compounds exhibit three features: they all contain Gd, they all contain a

ligand that is bound to Gd, and they all contain a single water molecule

inner coordination site to Gd. The ligand encapsulates the Gd, resulting

in a high thermodynamic stability. This enables the CA to be excreted

intact. These compounds are eliminated almost exclusively via the

kidneys.38

(2) Blood pool agents

Blood pool agents are tailored for vascular imaging. They remain in the

vascular compartment and do not leak out into the EC space.38 They are

significantly larger in size (>20kDa) than the EC agents and have higher

T1 relaxivities.39 The most successful types of blood pool agents are

albumin-binding Gd complexes (e.g. gadofosveset) and polymeric Gd

complexes (e.g. gadomer-17). Blood pool agents are also eliminated

primarily through the renal system.39

For the evaluation of myocardial perfusion and LGE assessment of myocardial

viability, EC T1 agents became the most frequently used CAs in CMR.

1.6.3 Quantification of myocardial infarct using late gadolinium enhancement

magnetic resonance imaging

The quantification of MI is based on the gold standard imaging technique, late

gadolinium enhancement.36 The LGE phenomenon can be explained by the difference

between the CA kinetics in the healthy vs. the infarcted myocardium. In the blood and

the normal myocardium, EC agents show rapid elimination kinetics due to renal

9

washout.38 The elimination of the CA from the infarcted myocardium, however, is

restricted. Therefore, a specific time window (10-20 minutes) exists after CA

administration, when CA has already been eliminated from the normal myocardium,

but the infarcted myocardium is still highlighted.36

The T1-weighted, segmented IR pulse sequence, used for the LGE protocol, acquires

images in mid-diastole when cardiac motion is minimal. It employs a non-selective

180° pulse and a delay before imaging, called inversion time (TI). With the appropriate

choice of TI and imaging 10-20 minutes after Gd CA administration, the SI of the

normal myocardium is nulled (i.e. black), but the infarcted tissue becomes very

bright.36

1.6.4 Off label use of gadolinium contrast

Since adverse effects related to iodine affect a significant number of patients,

alternative X-ray contrast materials have become frequently investigated targets.40-44 Of

the contrast media (CM) investigated to date, mostly Gd-chelates have been shown to

be viable alternatives for selected X-ray applications.45 Gd-chelates as CM have been

used in several clinical settings such as quantitative coronary angiography,

percutaneous coronary intervention and 3D Computed Tomography (CT)

angiography.46-48 It is clear that Gd-chelates at the dose approved for MRI (0.1-0.2

mmol/kg in case of Gd-DTPA) provide an image quality that is inferior to iodine CM,

thus are not useful for the evaluation of the cardiac vasculature.45 The literature

suggests that a dose of 0.3-0.4 mmol/kg is necessary for angiography and intravenous

or intraarterial interventional procedures.45 Different structures such as certain tumors

or MIs, however, accumulate Gd-chelates and can be sufficiently highlighted by a

regular dose of Gd.

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2. OBJECTIVES

The overall goal of our study was to develop a reliable and MRI compatible method for

the generation of non-reperfused MI, and to investigate the feasibility of Gd-based

Multi-Detector CT (Gd-MDCT) for the detection and quantification of MI.

2.1 Myocardial infarct model development

2.1.1 Significance

Early reperfusion therapy is the most important aim in the treatment of acute MI.

Universal achievement of this purpose, however, is almost impossible due to several

reasons, e.g. contraindications, non-availability of revascularization sites, belatedly

recognized ischemic events, etc.49 Based on the assumed number of patients

worldwide, generating a model for such situations (i.e. non-treated infarcts) is of major

importance in the development and preclinical testing of new diagnostic and

therapeutic strategies for patients missing the appropriate timeframe suitable for

revascularization following acute MI.50,51 As most of the available techniques have

some limitations, in this study we introduce an easily applicable, cost effective, MRI

compatible, non-reperfused MI model.

2.1.2 Objectives

Our aim has been (1) the development of a percutaneous technique for the generation

of non-reperfused MI, (2) the investigation of the MRI compatibility of the novel

model, and (3) the confirmation of the non-reperfused nature of the model by

histopathology.

2.1.3 Strategy

Pigs were used for the development of the model. Non-reperfused MI was generated by

900µm microspheres injected into the coronary artery via a microcatheter advanced

11

through the femoral artery. The existence of MI was confirmed by in vivo LGE-MRI,

as well as by ex vivo triphenyl-tetrazolium-chloride (TTC) staining at infarct ages of 2,

4, 14, and 56 days. Hematoxylin-Eosin (H&E) and Masson’s Trichrome staining were

used to study the histological characteristics of the MI.

2.2 Development of a Gadolinium based infarct quantification tool

2.2.1 Significance

Based on the high frequency of adverse effects related to iodine CM administration, the

use of an alternative CM for X-ray and CT studies may be required in certain cases.40

During the past decade, Gd-chelates have been used for various non-MRI radiology

examinations as an alternative CM.45-48,52,53 Coronary CT Angiography (CTA), a well-

established diagnostic tool for the non-invasive evaluation of coronary artery anatomy

and pathomorphology, has been validated using Gd-enhanced MDCT.54-56 Since the

LGE-MRI phenomenon is based on the accumulation of Gd in the MI, it can be

hypothesized that the LGE phenomenon can also be observed by CT due to the fact that

CT detects Gd. Thus Gd-enhanced CTA supplemented with the LGE protocol could

evaluate coronary status and myocardial viability noninvasively in a single imaging

session.

2.2.2 Objectives

We aimed (1) to develop an ex vivo heart model suitable for the performance of

repeated and/or multi-modality imaging, when a stable position of the sample is

required, (2) to investigate the ability of Gd-MDCT for the detection of MI, (3) to study

the feasibility of Gd-MDCT for the quantification of MI, (4) to investigate the accuracy

of the different MRI quantification methods for the evaluation of MI, and (5) to

compare the accuracy of each imaging method to the reference TTC technique.

2.2.3 Strategy

Reperfused MI was induced in male swine. One week later, the animals received

Gd-DTPA and were sacrificed. On the excised hearts, Gd-MDCT, LGE-MRI, and TTC

staining were then conducted. MIs were quantified and results were compared among

the methods.

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3. EMBOZENETM MICROSPHERES INDUCED NON-REPERFUSED

MYOCARDIAL INFARCTION IN AN EXPERIMENTAL SWINE MODEL

3.1 Introduction

Coronary heart disease is the number one cause of death in the Western World.57

Several animal species have been used to model MI, it, however, has been proven that

the anatomy of the pig heart and its coronary circulation are the closest to those of

humans.58

MI models have been created by either open chest transthoracic surgical methods or

closed chest percutaneous catheter techniques.59,60 In an open chest procedure the

temporary ligation of a coronary artery can be used to model reperfused MI, while the

permanent ligation of a coronary artery provides non-reperfused MI.59 The closed chest

models of the large animals are usually generated by catheter manipulation of the

coronary arteries through the femoral (rarely the carotid) artery.21,61 Trans-femoral

introduction and inflation of an angioplasty balloon catheter in the coronary artery is

used to create temporary occlusion and the reperfusion of the myocardial area supplied

by the occluded vessel occurs following the deflation of the balloon. Creating closed-

chest, non-reperfused MI model requires special techniques and/or materials that allow

advancing and leaving an item or substance in the coronary artery providing direct or

indirect mechanical obstruction and usually consequent thrombus formation. Several

techniques have been developed to model non-reperfused MI, e.g., inflatable-

detachable balloon occlusion62; flexible plug63, open-cell sponge64, tungsten spiral21,

agarose gel bead65, and microsphere (42µm)66 embolization; ethanol67, thrombin68, and

n-butyl cyanoacrylate (n-BCA) injection69. As many of those techniques have some

shortcoming, in this work we introduce a new, easily applicable, cost effective, MRI

compatible non-reperfused MI model in pigs using percutaneously administered

Embozene microspheres.

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3.2 Materials and Methods

3.2.1 Animal model

Study protocol was approved by the Institutional Animal Care and Use Committee and

complied with the Guidelines for the Care and Use of Laboratory Animals (National

Institutes of Health).

3.2.2 Embozene preparation

Color advanced (purple) 900µm (900µm±75µm) diameter EmbozeneTM microspheres

(CeloNova BioSciences, Inc., Newnan, GA) were purchased in 1ml prefilled syringes.

Embozene microspheres are based on the company’s proprietary material

poly[bis(trifluroexthoxy)phosphazene], or Polyzene®-F, described as “lubricious and

flexible,” as well as “versatile, durable, and highly biocompatible.” Following vendor

recommendations, Embozene was mixed with 7.0ml iohexol (Omnipaque 350, GE

Healthcare, Princeton, NJ) to make the solution visible under fluoroscopy.

3.2.3 Myocardial infarct model

Anesthesia was initiated in male swine (n=31, 25±8.2kg) with an intramuscularly

administered mixture of telazol (4.4mg/kg) and xylazine (4.4mg/kg). Following

intubation, animals were ventilated mechanically (Model 2000, Hallowell EMC,

Pittsfield, MA) and anesthesia was maintained by continuous administration of

Isoflurane (2.0–2.5%V/V). Normal body temperature was maintained using a heating

pad. Heart rate and blood oxygen saturation were monitored, and ECG was recorded.

The right femoral artery was surgically prepared and cannulated using a 6F arterial

sheath (Pinnacle, Terumo Medical Co, Elkton, MD). Heparin (100 IU/kg) was

administered intravenously and the activated clotting time (ACT) was monitored and

adjusted as needed with additional heparin to maintain ACT above 300s. A 6F coronary

guide catheter (RunWay Kimny Mini, Boston Scientific, Natick, MA) was introduced

to cannulate the ostium of the left main, an initial coronary angiography (Philips BV

Pulsera, Best, The Netherlands) was performed (Fig. 1A), and then a 2.9F straight tip

microcatheter (Merit Maestro, Merit Medical, South Jordan, UT) was introduced over a

coronary guide wire into the Left Anterior Descending (LAD) or the Left Circumflex

(LCX) coronary arteries. After determining the proper position for the occlusion using

14

the radiopaque tip of the microcatheter (Fig. 1B), the coronary guide wire was pulled

out and the Embozene mixture was flushed into the coronary artery under fluoroscopic

control (Fig. 1C).

Fig. 1 The main steps of the catheterization procedure are shown (fluoroscopy, antero-posterior view). A: The left main is cannulated by a 6F coronary guide catheter (empty arrow). The left coronary artery system is visualized by angiography. The target vessel (LAD) is shown by white arrows. Note that the coronary guide wire was advanced to the LAD. B: The delivery catheter was advanced over the coronary guide wire, then the wire was pulled out. At the center of the white circle the radiopaque tip of the delivery microcatheter is shown, indicating the site of microsphere administration. C: As microspheres are diluted in iodine contrast material, the administration can be followed under fluoroscopy. White arrows show the injected solution in the coronary artery. Note that contrast material is temporarily entrapped among the microspheres due to a reduced anterograde flow. D: Repeated angiography confirms the total occlusion of the targeted coronary artery. The contrast flow stops at the point of the planned occlusion (white arrow). The distal part of the LAD is unseen in panel D compared to panel A

15

When the lumen of the coronary artery distal to the tip of the microcatheter was

completely filled with Embozene, the injection was stopped and the occlusion was

confirmed by repeated angiography (Fig. 1D). Obvious signs of myocardial ischemia

(hyper-acute T-wave, ST segment elevation and Q-wave in the corresponding leads)

were accepted as signs of MI. Then the microcatheter was removed and the femoral

artery was decannulated, surgically ligated, and the wound was closed. The animal was

monitored for an additional 90 minutes.

3.2.4 Magnetic Resonance Imaging

MRI studies were carried out using a 1.5T GE Signa-Horizon CV/i scanner (GE

Healthcare, Milwaukee, WI) equipped with a cardiac phase-array coil. Pigs were

anesthetized and ventilated mechanically as described above. Imaging was performed

during breath-hold at end-inspiration using the following parameters: field of view

(FoV) = 300mm, image-matrix (IM) = 256x256, slice thickness (ST) = 10mm.

Following angulation, cine and T2-weighted MRI were performed. Cine images were

obtained using a fast GRE, cine sequence. T2-weighted, double IR fast spin-echo

images were generated using the following parameters: flip angle = 90°, TE = 60ms,

echo train length = 24, and TR = two cardiac cycles. No fat saturation pulse was used.

After T2-weighted imaging, a bolus of 0.2mmol/kg Gd-DTPA (Magnevist®, Bayer

HealthCare Pharmaceuticals Inc, Wayne NJ) was administered as CA. Segmented,

180º-prepared, IR, fast GRE short-axis oriented images were generated at 5, 15 and 30

minutes after the administration of bolus CA to assess viability. Imaging parameters

were: flip angle = 25°, TE = 3.2ms, views per segment = 16 and TR = two cardiac

cycles. At the end of their planned in vivo MRI session (2, 4, 14, and 56 days following

infarct generation), animals were sacrificed using a 100mg/kg sodium pentobarbital

followed by 100ml of 2M potassium chloride solution. Euthanasia was ascertained by

ECG and auscultation above the thorax. Hearts were excised following euthanasia,

rinsed with saline, and prepared for further studies.

3.2.5 Image analysis

Localization of MI was assessed using the standardized 17-segment model.70 Visual

assessment of the quality of the MRI images was performed to study possible

occurrence of image artifacts caused by the administered microspheres.

16

3.2.6 Triphenyl-Tetrazolium-Chloride staining

TTC staining was used as a post-mortem gold standard to confirm the existence of MI.

Hearts were bread-sliced using a commercial meat slicer, and subsequently the slices

were incubated for 20 min with a buffered (pH 7.4) 1.5% TTC solution at a

temperature of 37°C. Finally, both surfaces of each slice were digitally scanned with a

Lexmark X1270 (Lexmark International Inc, Lexington KY) image scanner.

Localization of MI was determined by the same method as used in the MRI images.

3.2.7 Microscopic histology

Tissue samples were prepared from the infarct, peri-infarct, and healthy myocardial

regions of the LV to confirm the existence of MI by microscopic histopathology. The

samples were fixed in 10% formalin, embedded in paraffin, sectioned at 5µm thickness,

and stained with H&E and Masson’s Trichrome. The slides were evaluated using an

Olympus BX51 video microscope (Olympus America Inc., Center Valley, PA).

3.3 Results

Microsphere administration was successfully applied in all 31 pigs. The total duration

of the procedure including the surgery (25±6 min), catheter intervention (26±13 min),

and post-occlusion observation was 144±15 min (more procedural details are shown in

Table 1).

The first animal that underwent microsphere embolization of the LCX died in the cage

one day after the generation of MI. The autopsy and TTC staining of the heart showed

that the LCX was overfilled with microspheres and microspheres were regurgitated into

several branches of the LAD resulting in multiple infarcted tissue areas at various loci

of the LV (Fig. 2A).

17

Table 1 Procedural details of the 31 pigs underwent microsphere embolization

Swine #

Target vessel

Infarct location by MRI

Infarct location by TTC Survival

Cause of

death

Procedure time (min) Adverse events

S C O T

1ex LCX N/A multiple 1 day UN 20 60 100 180

2ex LAD N/A AS, MAS, MIS 27 min VF 30 40 N/A N/A VF, CPR†

3ex LAD N/A AA, AS, MAS, MAI 30 min VF 20 30 N/A N/A VF, CPR†

4 LAD Ax, AS, AA, MAS Ax, AS, AA, MAS 4 days EU 30 40 90 160 PVC

5 LAD AS Ax, AS 56 days EU 40 20 90 150 2º AV-block, CPR

6 LAD AA Ax, AA 56 days EU 30 40 95 165

7ex LAD N/A AA, AS, AI, MAS 10 min VF 40 30 N/A N/A VF, CPR†

8 LCX AI, MI AI, MI 56 days EU 30 20 100 150

9 LAD AS, MAS AS, MAS 56 days EU 30 20 90 140

10 LAD AS, AA, MAS, MIS Ax, AS, AA, MAS, MIS 4 days EU 30 30 100 160

11 LAD AS AS 56 days EU 30 30 90 150

12 LCX AI, MI, BI AI, MI, BI 4 days EU 20 20 100 140

13 LAD AA, AS, MAS AA, AS, MAS 4 days EU 30 30 90 160 PVC, VT

14 LCX MI, MIL, BIL MI, MIL, BIL 4 days EU 30 10 110 150 PVC

15 LCX MI, BI MI, BI 56 days EU 30 30 90 150

16 LAD Ax, AS, MA, MAS Ax, AS, MA, MAS 14 days EU 20 10 90 120 PVC, VT

17 LCX MIL, BIL MIL, BIL 4 days EU 20 50 90 160

18 LCX MAL, MIL, BAL, BIL MAL, MIL, BAL, BIL 2 days EU 30 30 90 150

19ex LAD N/A N/A 1 day UN 30 20 90 140

20 LAD AA, AS, MA, MAS Ax, AA, AS, MA, MAS 2 days EU 40 10 90 140 PVC, VT

21 LCX MIL, BIL MIL, BIL 14 days EU 20 10 90 120

22 LCX MI, MIL, BI, BIL MI, MIL, BI, BIL 2 days EU 20 20 90 130 PVC, VT, VF, CPR

23 LCX MIL, BIL MIL, BIL 14 days EU 20 10 90 120

24 LCX MAL, MIL, BAL, MIL MAL, MIL, BAL, MIL 14 days EU 20 20 90 130

25 LCX MIL, BAL, BIL MIL, BAL, BIL 2 days EU 20 10 100 130

26 LCX MIL, BIL MIL, BIL 14 days EU 20 40 90 150 PVC

27ex LCX N/A N/A 30 min VF 20 50 N/A N/A VF, CPR†

28ex LCX N/A N/A 5 min VF 20 10 N/A N/A VF, CPR†

29 LAD AS AS 2 days EU 20 30 90 140

30 LAD Ax, AA, AS, MAS Ax, AA, AS, MAS 14 days EU 20 30 90 140 PVC, VT

31 LCX MI, MIL, BI, BIL MI, MIL, BI, BIL 2 days EU 20 10 90 120

LAD, Left Anterior Descending; LCX, Left Circumflex; S, Surgery; C, Catheterization; O, Observation; T, Total; ex, excluded from the study; UN, Unknown; VF, Ventricular Fibrillation; EU, Euthanasia, AV-block, atrioventricular block; CPR, cardio-pulmonary resuscitation; CPR†, unsuccessful CPR Cardiac segmentation: BA, BAS, BIS, BI, BIL, BAL (basal anterior, anterospetal, inferoseptal, inferior, inferolateral, anterolateral); MA, MAS, MIS, MI, MIL, MAL (mid anterior, anterospetal, inferoseptal, inferior, inferolateral, anterolateral); AA, AS, AI, AL (apical anterior, septal, inferior, lateral); and Ax (Apex)

18

Fig. 2 Postmortem TTC-stained myocardial slices are shown. A: During our first experiment with Embozene, reflux of the microspheres was detected with consequent multiplex infarcted spots. Black arrows show the infarct of the target area supplied by the LCX. In addition, infarcted spots are found in the anterior papillary muscle (black arrowhead), and in the septum (white arrow), of the LAD myocardial territory. B: A right ventricular (RV) free wall infarct was detected in two animals (black arrowhead) in addition to the targeted infarct of the LAD territory

19

An additional animal died in the cage one day after the induction of MI due to

unknown reasons. During the observation period, three pigs with LAD and two pigs

with LCX occlusion had ventricular fibrillation (VF) and all of them died after

unsuccessful resuscitation efforts. Extensive MI was described by autopsy as the cause

of death of those five animals. All seven animals detailed above were excluded from

the study. Twenty-four animals reached their planned study endpoint resulting in a

mortality rate of 22.6%. As an irregular finding, unexpected MI in the free wall of the

right ventricle (RV) occurred in two surviving animals (Fig. 2B). Except for the first

animal described above, reflux of the microspheres to any non-target vessel was not

observed on fluoroscopy and no MI in unexpected territories in the LV was detected by

MRI or by TTC.

In eleven pigs the LAD, and in thirteen other pigs the LCX, were occluded. During the

observation period arrhythmia was observed in nine of the surviving animals. In seven

of these nine pigs, five with LAD occlusion and two with LCX occlusion, isolated

premature ventricular complexes (PVC) and/or short ventricular tachycardias were

detected and successfully treated with intravenously administered lidocaine. In the

eighth, a pig with LAD occlusion, a 2nd degree atrioventricular block occurred followed

by sudden asystole. This animal was successfully treated with epinephrine, atropine,

and chest compressions. In the ninth pig, one with LCX occlusion, VF developed after

repeated episodes of ventricular tachycardia. This animal was successfully treated with

a single electric shock applied at 200 Joule.

Existence of MI was confirmed in the different phases with in vivo MRI, macroscopic

pathology and histology. The MI localizations determined by MRI and TTC

corresponded with the territories supplied by the target vessels. Representative cine,

T2- and T1-weighted MRI and TTC images are shown in Figure 3. No artifacts with

significant influence on image assessment were observed. Microscopic evaluation of

the microsphere-induced MI tissue is shown in Figure 4.

20

Fig. 3 Corresponding cine (A), T2-weighted (B) and post-contrast T1-weighted images obtained at 5 (C), 15 (D) and 30 (E) minutes following CA administration, and TTC image (F) from a representative heart are shown. Contrast enhanced MRI, and TTC, localize the MI in the LCX myocardial territory. The large unenhanced region in the center of the infarct (C-D) is considered MO. The area of the MO appears darker then myocardial areas with normal perfusion (C) and remains mostly unenhanced for 15 minutes (D). The area of the MO becomes highlighted by 30 minutes after contrast administration (E), probably due to the relatively slow interstitial diffusion of the CA. The TTC image (F) confirms the existence and localization of MI. No artifacts influencing the image quality and MI evaluation were observed

21

Fig. 4 Shown are H&E and Masson’s Trichrome-stained histological slides. A: whole-slice histological map of the LV (1.25x). The B and C panels show magnifications of the area of the microsphere occlusion (20x and 100x, respectively). The microspheres shrink during sample preparation (~500µm in the slide) causing the separation of the microspheres from the endothelial wall. Note that the shape (C) of the microspheres corresponds to the outline of the occluded vessel. The D and E panels show the longitudinal section of the same coronary artery. The artery is filled with microspheres and the gaps located between the microspheres are filled with thrombi and blood cell remnants. (H&E and Trichrome staining, respectively, 40x magnification). Panel F shows a single microsphere surrounded by thrombus formation (Masson’s Trichrome, 100x)

22

3.4 Discussion

In this study, we reported the use of percutaneously administered Embozene

microspheres to generate non-reperfused MI in a swine model. MI was successfully

induced in all 31 animals. Seven pigs, however, died during the post-occlusion period.

The mortality rate in this study (22.6%) was better than in some other closed-chest

models and was similar to other authors’ experiences with coil embolization

models.21,69

The procedure can be easily controlled under fluoroscopy since the microspheres are

diluted in iodine contrast material. The administration, however, requires some

experience. The procedure requires slow injection while the syringe is kept in a

horizontal position, providing constant rate of distribution of the microspheres. The

injection becomes increasingly harder when the coronary artery is being filled with

microspheres, indicating that the anterograde flow in the artery is being significantly

reduced. At this point, the artery is temporarily outlined by the entrapped iodine

contrast under the fluoroscopy and the occlusion can be confirmed by angiography

(Fig. 1).

While open chest MI models have some benefits (direct visual control, avoiding the use

of radiation, etc.), these models also have many limitations. The increased length of the

procedure, the higher mortality rate, the high occurrence of complications due to the

thoracotomy, issues with MRI compatibility (wires and air in the chest) etc., have

motivated the development of closed chest techniques. Some of these techniques,

mentioned in the Introduction, have significant limitations (Table 2). The inflatable,

detachable balloons62 have been removed from the U.S. market since safety issues were

reported (e.g., early detachment, early deflation, rupture of the balloon, and problems

with the balloon valve mechanism).71 Introduction of large items, such as flexible plugs

or sponges, requires cannulation of the carotid artery.63,64 Administration of liquids

(ethanol, n-BCA, etc.) may cause accidental embolization into non-target vessels due to

the reflux of the agents.67,69 Among the techniques available to date, tungsten spiral

embolization seems to be a promising method.21 Tungsten spirals are MRI safe, but the

MRI compatibility of tungsten-containing materials regarding artifacts, however, is not

clear. A prosthetic heart valve composed of tungsten showed an artifact slightly larger

than the size of the device when imaged with a T1-weighted spin-echo pulse sequence

23

and also showed an artifact larger than twice the size of the device when imaged with a

GRE sequence.72 The above described prosthetic valve, however, contains Delrin,

Dacron, and silicone. Thus the reason for the artifacts may not be necessarily associated

with tungsten. In addition, Peukert et al. reported that MRI images of animals

undergone tungsten spiral embolization did not show any artifacts.21 Using the

microsphere embolization technique, the MRI image quality was not influenced either,

and the assessment of T1- and T2-weighted images did not show any artifacts

interfering with MI visualization.

Table 2 Techniques for generating non-reperfused MI

Technique Benefits Limitations

Open chest

Ligation Visual control, proper place of occlusion

Procedure length, complications of thoracotomy, MRI incompatibility

Closed chest

Inflatable-detachable balloon Low mortality rate (10%), the balloon can be relocated during the procedure

Safety issues (self-detachment, rupture)

Flexible plug Cost-effective, occlusion exclusively at the epicardial level of the coronary artery

Requires large lumen for introduction (e.g. carotid)

Ethanol Low mortality rate (8%) Reflux, arrhythmogenic lesions

Agarose gel bead (75-150µm) Cost effective, easy to perform

Mortality rate (>35%), occlusion at the level of arterioles, patchy infarcts

Thrombin / Fibrinogen Suitable to study thrombolytic therapies

Spontaneous reperfusion within three days

Microsphere (42µm) Suitable to study coronary micro-embolization

Occlusion at the level of arterioles, patchy infarcts

Open-cell sponge Occlusion at the epicardial level

Requires large lumen for introduction (e.g. carotid), retrograde thrombosis

n-BCA Proven long-lasting occlusion

Reflux, high mortality, foreign body reaction

Spiral Proper place of occlusion MRI compatibility may need further investigation

24

The methods for infarct generation described above can be classified into two well

defined approaches: (1) advancing a solid material into the coronary artery21,62-64 to

create immediate occlusion or induce thrombus formation, or (2) flushing a liquid into

the coronary artery65,67-69 generating tissue changes, thrombus formation, and

consequent occlusion. Our method introduced here could be considered a mixture of

both, similarly to previously described techniques.73,74 Embozene microspheres can be

easily injected as a liquid through a delivery catheter of the proper size, using a 6F

sheath inserted in the femoral artery. The size of the microspheres (~900µm in

diameter) provides a plug-like occlusion. As a result, the column of microspheres stays

in place at the site of administration for up to at least 54 days.

This method has advantages and also possible shortcomings compared to the other

available techniques. The main advantages of Embozene microsphere embolization are

the easy applicability, the cost effectiveness, and the MRI compatibility. The

administration, however, requires some experience, and the level of occlusion differs

from that of the other techniques. (Figure 5)

Fig. 5 Schematic illustration of a branch of the bifurcating coronary artery system is shown. The average diameter of the 1st, 2nd, 3rd and 4th bifurcations of the pig heart is 3176±654 (I), 1430±379 (II), 730±129 (III) and 438±64µm (IV), respectively. The blue line indicates the endovascular catheter (A, C) or wire (B). A: The administration of liquids (n-BCA, ethanol) to the coronary artery (yellow) illustrates that liquids can reach the capillary system, thus they interact with the whole coronary artery system below the point of injection. B: Solid materials (coils, plugs) advanced to the coronary artery produce an occlusion at a well defined target point. C: Microspheres are trapped in the coronary artery system at the level of the 3rd bifurcation and above. Microspheres create a pearl necklace-like column in the target artery. Such embolization particles can reach the smallest epicardial coronary arteries but never reach the capillary circulation

25

Liquids, such as n-BCA or ethanol, reach the microvascular level of the coronary artery

circulation, while plugs or coils (spirals) cause an occlusion at the level of placement.

Embozene microspheres work similarly to the latter as they get trapped in the target

artery at the level of ~900µm diameter and they fill the artery as a pearl necklace up to

right below the place of administration. It is clear that the place of occlusion is not as

well defined as with, e.g., coil embolization. The occlusion, however, does not reach

the microvascular level, but rather affects the epicardial level of the coronary

circulation.

We report a case with overfilled LCX. It is explained by our lack of experience during

the initial period of this study. The most sensitive step during microsphere

administration is the above detailed speed of injection. Overfilling is caused when the

injected amount of microspheres causes retrograde flow and thus patchy infarcts in

unexpected loci in the LV (Figure 2A). We also report two survival cases when an

unexpected MI of the RV free wall occurred (Figure 2B). The reason for the RV free

wall infarcts could be potentially explained by either of two major causes: the reflux of

microspheres all the way back to the entrance to the right coronary artery (RCA), or an

anatomic variant of coronary circulation. The chance for reflux from the LAD to the

RCA without affecting the LCX, however, is really small. The other possible

explanation for RV free wall infarct could be a dominant LAD supplying partially the

RV also. It is evident in swine that the RCA is almost always dominant (78%), or a

balanced blood supply exists (17%).58 Left dominant supply occurs only in 5% of

swine hearts. Intracoronary dye injections showed that 27.6% of the RV mass is

supplied by the LAD.58 Based on the literature and the coronary angiographies of those

two animals we concluded that our above described observation of RV free wall

infarcts can be explained by left dominancy.

The tissue access of the contrast agent to the non-reperfused MI can be explained by

the study of Wang et al.14 Non-reperfused infarcts start to show hyperenhancement as

early as eight hours after occlusion due to the gradual establishment of the collateral

circulation. Hyperenhancement gradually appears from the border to the center of the

MI. The CA reaches the MO area by a slow interstitial diffusion mechanism with a

characteristic time-delay (~30min after administration).14

26

3.5 Conclusions

We have demonstrated the feasibility of the generation of MI using Embozene

microspheres and the MRI compatibility of this method. Our method can be used as an

alternative technique to induce non-reperfused MI. Modeling such a clinical situation

(i.e. non-treated MI) has major importance in the development and preclinical testing of

new therapeutic strategies (e.g., myocardial edema reduction, anti-inflammatory

therapy, and other possible drug therapies) for patients missing the time window

suitable for revascularization following MI.

27

4. DETERMINATION OF INFARCT SIZE IN EX VIVO SWINE HEARTS BY

MULTI-DETECTOR COMPUTED TOMOGRAPHY USING GADOLINIUM AS

CONTRAST MEDIUM

4.1 Introduction

Iodine-based CM can be used safely for X-ray and CT studies in most patients. Adverse

effects caused by iodine exist in 0.15% of cases, as described in a retrospective review

based on almost 300,000 patients.40 In such patients, the use of either a different

imaging technique or an alternative CM may be required. As alternatives to iodine CM,

Carbon-dioxide, Xenon, and Gd-chelates have been studied and successfully applied in

select X-ray and CT examinations.41,75 In the field of cardiovascular radiology, Gd-

chelates have become the most investigated potential CM.

Gd is the element with atomic number 64 (atomic weight 157), while iodine's atomic

number is 53 (atomic weight 127). X-ray attenuation increases with atomic number and

decreases with the energy of the X-ray photons in a nonlinear fashion. For CT studies,

the maximum X-ray photon energy is 140keV. The attenuation by Gd in this setting is

twice that of iodine, but since there are three iodine atoms in each iodine-based CM

molecule (e.g. Omnipaque), the iodine CM attenuates 1.5 times more radiation than

does a typical Gd-chelate CM.76

The first use of Gd-chelates as radiographic CM in CT studies was reported in 1989 by

Janon and Bloem et al.52,53 Since then, several cases using Gd-chelates in various

radiographic procedures have been published. Furuichi et al. reported the use of

Gd-DTPA, in the course of percutaneous coronary intervention.46 Gupta et al. studied

the thoracic aorta, and cervical and abdominal vessels, by 3D CTA (280mAs, 120-

140kV) using 0.3mmol/kg Gd-DTPA and found sufficient contrast to clearly define

these vessels.47 Kälsch et al. studied 19 patients with contraindication to iodinated CM

and found reduced but acceptable image quality for diagnostic purposes using

Gd-based CM in coronary angiography.48 A review article by Strunk et al. on the use of

28

Gd-DTPA for non-MRI applications concludes that Gd-based CM could be safely used

for radiography. It is, however, presently approved for MRI use only.45

Using CT with Gd as CM for the detection or quantification of MI has not yet been

reported. The aim of this ex vivo study was to investigate the ability of MDCT using

Gd-DTPA as CM (Gd-MDCT) to evaluate MI, and compare the results with data

obtained by the in-vivo gold standard LGE-MRI and the ex-vivo gold standard TTC

staining method.

4.2 Materials and methods

4.2.1 Experimental model

Study protocol was approved by the Institutional Animal Care and Use Committee

(IACUC, University of Alabama at Birmingham) and complied with the Guidelines for

the Care and Use of Laboratory Animals (National Institutes of Health). MI was

generated in six male swine (n=6, 25-30kg) under Isoflurane anesthesia (2.0–3% V/V)

by a 90-minute long percutaneous balloon occlusion of the LAD coronary artery.

Reperfusion of the coronary artery was confirmed by repeated angiography. One week

later, the animals received 0.2mmol/kg Gd-DTPA (Magnevist®, Bayer HealthCare

Pharmaceuticals Inc, Wayne NJ) by bolus injection and were sacrificed 20 minutes

later using 100mg/kg sodium pentobarbital and 2mmol/kg KCl. Euthanasia was

monitored by ECG and auscultation above the thorax. Hearts were immediately excised

following the successful euthanasia. Excised hearts were rinsed in physiologic saline

and hung up on a stand to set the annulus fibrosus in a horizontal position. (Fig. 6) To

provide a horizontal reference plane for angulation and reconstruction, 1ml of a 10%

V/V Gd-DTPA solution was mixed with a 20ml agar solution, layered into the bottom

of a 1000ml beaker, and separated by a 50µm thick plastic foil to prevent free upward

diffusion of Gd-DTPA. Hearts were then placed in the beaker and embedded in 4.2%

agar gel (Nutrient Agar, Remel Inc., Lenexa KS) providing a stable position for the

heart structures during the imaging sessions. Same day Gd-MDCT, LGE-MRI, and

TTC-staining were then conducted.

29

Fig. 6 Embedding procedure of the heart. Lateral (a, c) and superior (b, d) views of the beaker.

The heart is hanged on a stand using four silk surgical sutures (a, b). Red arrows show the

Gd-DTPA containing agar layer, which provides the horizontal reference plane during the

angulation (MRI). After setting the heart to the proper position, the beaker is filled with 40°C

agar solution (c, d)

30

4.2.2 Multi-Detector Computed Tomography

MDCT studies were carried out immediately following the embedding procedure of the

heart using a Philips Brilliance 64 scanner (Philips Healthcare, Best, The Netherlands).

Images were collected covering the entire beaker using the following parameters: tube

voltage = 80/120/140kV, tube current = 600mA, IM = 512x512; FoV = 160mm; ST =

1mm; increment = 0.5mm. Short axis oriented reconstruction process (ST = 1mm) was

aided by the bottom of the radiopaque beaker providing the same image orientation as

obtained by MRI.

4.2.3 Magnetic Resonance Imaging

MRI acquisitions were conducted following the MDCT sessions using a 1.5T GE

Signa-Horizon CV/i scanner (GE Healthcare, Milwaukee, WI) equipped with a head

coil. Since the glass beaker itself is invisible by MRI, the Gd-DTPA-containing bottom

layer was used to aid the angulation process by serving as a reference plane. Using an

IR, 180°-prepared, fast GRE pulse sequence, multi-slice short axis oriented LGE

images (TE=3.1ms; TR=2000ms; flip angle=25º; IM=256x256; FoV=160mm;

ST=5mm) were acquired covering the entire heart. The TI was selected from the range

of 200-400ms, where the signal was nearly null in the viable myocardium.

4.2.4 Triphenyl-Tetrazolium-Chloride staining

Following the imaging sessions, hearts were frozen and bread-sliced (5mm), based on

the orientations of the tomographic slices, using a commercial meat slicer.

Subsequently, the slices were incubated for 20min with a buffered (pH=7.4) 1.5% TTC

solution at 37°C, as described by Fishbein et al.77 Following staining, slices were

immersed in 10% formalin for 20 minutes to increase the contrast between healthy and

infarcted myocardium. Finally, both surfaces of each slice were scanned with a

Lexmark X1270 digital scanner (Lexmark International Inc., Lexington KY) connected

to a personal computer.

4.2.5 Histopathology

After scanning the TTC stained slices, tissue samples were prepared from the infarct,

peri-infarct, and healthy myocardial regions of the LV to confirm the existence of MI

31

by microscopic histopathology. The samples were fixed in 10% formalin, embedded in

paraffin, sectioned at 5µm thickness, and stained with H&E.

4.2.6 Image analysis

MDCT and MRI dicom images were imported as image sequences with the use of

ImageJ v1.42 software (Wayne Rasband, NIH). Images were cropped and vertical

stacks were created. The mean MRI signal intensity (SI) or CT attenuation of the

remote (i.e. viable) myocardium (SIremote), the mean SI (or attenuation) of the most

intense portion of the hyperenhanced area corresponding to the infarcted myocardium

(SIinfarct), and the mean SI of the background noise (area on the image but outside the

beaker) (SInoise) were measured with multiple regions of interest.

To compare the image quality provided by MDCT vs. MRI, the signal to noise ratio

(SNR), contrast to noise ratio (CNR), and signal intensity ratio (SIR) were calculated

using the following definitions:78,79

noise

mean

SDSISNR =

(1)

CNR = SNRinfarct – SNRremote (2)

remote

infarct

SISISIR =

(3)

where SImean is SIinfarct or SIremote (or CT attenuation), and SDnoise is the standard

deviation (SD) of the signal intensity of background air. Due to the Rayleigh

distribution of the background noise in magnitude images, equation 1 was corrected

with the factor of π42−

(≈1.53) in case of MRI images.78

For further analysis, the endocardial and epicardial contours of the LV were traced

manually to delineate the total myocardial area. Myocardial pixels were counted, and

based on the pixel dimensions the myocardial volume of each slice was determined,

from which the total LV myocardial volume was also calculated (LVV). To avoid

observer bias, instead of manual contouring of the infarct region, the thresholding

technique was used to delineate MI as follows.

32

Since there is no agreement in the literature which thresholding technique provides the

most accurate evaluation of MI, we used several methods for infarct quantification in

the MRI images. Mean SI of the remote plus 2, 3, 4, 5 and 6 times the SD, and the full-

width at half-maximum (FWHM) method were used to define the threshold limit.36,80-84

In each case, pixels with SI above the specific threshold limit were considered infarct

pixels.

For MDCT images, infarct pixels were observed as Hounsfiled Unit (HU) elevations

due to the increased presence of Gd-DTPA in such pixels. Mean attenuation of the

remote areas plus two times the SD was used to threshold the images. Each 1mm thick

slice was evaluated.

In the thresholded images infarct pixels were counted and the infarct volume of each

slice was determined, from which the total infarct volume of each heart (IV) was

calculated and expressed as a percentage (Infarct Fraction, IF) of the LVV.

Both the apical and basal surfaces of each TTC stained slice were analyzed. The LV

myocardium and the infarct area were manually contoured and quantified. LVV, IV and

IF were determined in each heart.

4.2.7 Statistical analysis

Statistical analysis was carried out using SigmaStat v2.03 (SPSS Inc, Chicago IL). Data

were expressed as mean±SD. Normality and equal variance tests were used to

determine whether a parametric or nonparametric statistical method should be applied.

One-way analysis of variance (ANOVA) was used to compare results among the

imaging methods and the TTC-staining, and Holm-Sidak multiple comparison analysis

was applied for pairwise comparison. Bland-Altman plots were used to analyze the

accuracy of each technique. When a significant difference was observed among

parameters (IVs, IFs) obtained by the different techniques, the value of over- or

underestimation was calculated by comparing the results to the reference TTC.

Comparison of CNR, SNR, and SIR among the different MDCT settings and MRI was

carried out using one-way ANOVA. Post hoc statistical power was calculated using the

power analysis for ANOVA with a type-I error rate (α) of 0.05. Rejecting the null

hypothesis at this level of α with a P-value <0.05 was interpreted to indicate a

significant difference.

33

4.3 Results

Raw and post-processed corresponding images from a representative heart are shown in

Fig. 7 and 8. All three techniques provided sufficient image quality to visualize MI.

Microscopic evaluation of the tissue samples confirmed the existence of MI. The

average attenuation, SI, the calculated SNR, CNR, and SIR values are shown in Table

3.

Fig. 7 Corresponding ex-vivo, short-axis oriented, raw and post-processed MDCT images are

shown. Raw MDCT images obtained by 80 (a), 120 (b) and 140kV (c) (same window/level) are

shown in the first row. The area of the MI delineated by the thresholding method is highlighted

in the bottom row (d-f).

34

Fig. 8 Raw (a), thresholded LGE-MRI (b), and corresponding TTC images are shown. The area

of the MI delineated by various thresholds of the MRI image (b) is shown superimposed on the

image. The color codes are the following: black (remote), white (2SD), red (3SD), yellow

(4SD), blue (5SD), green (6SD), and orange (FWHM).

Table 3 Average attenuation (HU) and SI (arbitrary units, au) values, and calculated SNR, CNR and

SIR in MDCT and MRI images, respectively (mean±SD, n=6)

MDCT MRI

P-value

MDCT vs.

MRI 80kV 120kV 140kV

Background -1009±2.5 -1010±2.1 -1009±2.6 144±10.2

Remote 43±1.9 42±1.9 42±2.0 168±12.5

Remote SD 16±0.9 8±0.5 6±0.5 16±4.2

Infarct 91±6.2 81±5.1 80±5.0 734±118.1

Infarct peak 133±16.1 109±12.3 100±5.4 921±42.4

SNR remote 4.0±0.3 4.5±0.6 4.6±0.5 10.7+4.5 <0.05

SNR infarct 8.5±1.0 8.8±1.1 8.8±1.2 46.9+11.2 <0.05

CNR 4.5±0.7 4.3±0.8 4.2±0.7 36.2+6.1 <0.05

SIR 2.1±0.2 1.9±0.1 1.9±0.1 4.4+0.7 <0.05

35

Comparing the MDCT images obtained by the three different kV settings, the mean of

the background air and the remote myocardium showed statistically the same average

attenuation independently of tube voltage. The SD of the remote myocardium (i.e. the

noise of the remote areas) showed a strong linear relationship (R2=0.9999) with

(kV)-1.3.85 The background noise showed a similar correlation (R2=0.9786). Mean MI

attenuation values at 80kV were significantly higher (P<0.01) than at 120 or 140kV,

while no statistical difference was observed between the latter two. SNRremote was

significantly lower at 80kV (p<0.05) than at the other two tube voltages, while no

difference was observed between 120 and 140kV. There was no statistical difference

among the SNRinfarct, CNR and SIR values measured at the different kV settings. All

these ratios were significantly lower than ratios measured by MRI (P<0.001, power of

0.902).

Average LVV, IV and IF values measured by MDCT, MRI and TTC are shown in

Table 4. Average LVVs obtained by the different techniques were in good agreement

(P=N.S.) with each other indicating that all these techniques provide comparable

volumetric data.

Table 4 Average LVV, IV, and IF (%) values (mean±SD, n=6) measured by each technique. Results

were compared to the reference TTC

Modality Tube voltage (kV) LVV

(ml)

IV

(ml)

IF

(%)

MDCT 80 72.06±13.23* 1.65±1.24 2.34±2.11

120 3.80±1.78 5.36±2.01

140 5.06±1.64 7.15±2.28

Threshold

MRI 2SD 74.72±13.15* 8.91±2.99 12.03±3.81

3SD 8.07±1.15 10.80±1.89

4SD 7.22±1.31 9.66±1.49

5SD 5.87±1.26 7.86±2.03

6SD 5.47±1.23 7.36±1.57

FWHM 4.36±1.16 5.86±1.55

TTC 75.42±14.85 5.36±1.57 7.34±2.30

*The LV volume was calculated only once with each method, since the same endo- and epicardial

contours were used for each kV (MDCT) or threshold (MRI)

36

The multiple comparison method showed that MDCT80kV, LGE-MRI with 2 and 3SD

thresholds are significantly different from the reference TTC (p<0.05), while no

statistical difference was observed among IVs and IFs obtained by MDCT120kV,

MDCT140kV, MRI4SD, MRI5SD, MRI6SD, MRIFWHM and TTC. (Fig. 9)

Fig. 9 IFs (n=6) obtained by the different methods are shown by a bar diagram. IFs were

compared to the reference TTC. Significant difference is indicated by an asterisk.

Bland-Altman plots of IF obtained by MDCT and MRI vs. TTC are shown in Fig. 10.

The plots indicate good agreement for the IF values obtained by MDCT120kV,

MDCT140kV, MRI5SD, MRI6SD and MRIFWHM vs. the gold standard TTC method. Note,

that the mean of differences (i.e. bias) is less than 1% in the case of MDCT140kV and

MRI6SD, and less than 2% with MDCT120kV, MRI5SD and MRIFWHM. The Bland-Altman

analysis of MDCT80kV showed a bias of -5.07% (~3.7ml) indicating a significant

37

underestimation of the IF by this method. Plots of the MRI data with 2, 3 and 4SD

thresholding showed overestimation of the IF with a bias of 4.68% (~3.41ml), 3.50%

(~2.61ml) and 2.31% (~1.72ml), respectively.

Fig. 10 Bland-Altman plots of IF obtained by MDCT with various tube voltages (first raw) and

MRI with various thresholds (second and third rows) vs. TTC are shown. Dashed lines indicate

the upper and lower 1.96SD interval.

4.4 Discussion

We have demonstrated in an ex-vivo experimental model that quantification of MI

using the Gd-MDCT technique is feasible, providing accurate evaluation of MI size as

38

judged by the in-vivo gold standard LGE-MRI, and the ex-vivo gold standard TTC

method.

We have also demonstrated that the agar embedded ex-vivo heart model is suitable for

the performance of repeated, or multi-modality, imaging, when a stable position of the

sample is required. A similar technique was used by Dahele et al to study small lung

specimens.86

The SNR, CNR, and SIR results of our ex-vivo MRI studies were in good agreement

with other investigators' in-vivo findings.87,88 Measurement of these parameters in

infarcted myocardium by Gd-MDCT has not been published previously. Our results

(Table 3) showed that all three parameters (SNR, CNR, and SIR) obtained from

Gd-MDCT images were significantly lower than those obtained from LGE-MRI

images. CNR was ~8 times higher in MRI than in Gd-MDCT. SIR in MDCT images

was also inferior to that in LGE-MRI. Thus, the dynamic range of attenuation (the

range between the remote and infarcted myocardium) is restricted in Gd-MDCT

compared to MRI. Nevertheless, Gd-MDCT still provided sufficient contrast to

distinguish between normal and infarcted myocardium.

The MI was visually detectable by Gd-MDCT at all settings. The measured MI size,

however, depended on the tube voltage used. The mean attenuation of the MI was

highest at 80kV, but the increase of the noise in the remote myocardium, was a much

more dominant change caused by the reduction in voltage. The noise in the remote

myocardium influenced the accuracy of the MI quantification because the thresholding

level depended on the myocardial noise. The noise in the remote myocardium was

linearly proportional to (kV)-1.3, thus the image noise decreased with higher kV

settings.85

In this study, we found that MI size measured by Gd-MDCT at 140kV showed good

correlation with the reference TTC. Applying 80 or 120 kV tube voltages, however,

underestimated MI size. The underestimation is surprising at first, since the peak and

mean attenuation of the MI are highest at the level of 80kV, and iodine studies also

indicate that the accuracy of MI size determination is better with a low tube voltage.79

This finding, however, can be explained by the fact, that the noise of the remote

myocardium is increased at lower tube voltages (detailed above). There are areas in the

39

MI where the accumulation of the Gd is restricted or decreased: areas of MO in the

center of the infarct or peri-infarct zones with patchy infarct and normal perfusion. In

those areas, the amount of Gd is either below the detectable limit, or the attenuation

associated with the Gd is within the remote noise limit. When the noise of the remote is

increased, more areas with moderate Gd accumulation are considered as noise and only

the infarct core areas with high Gd content are highlighted.

In our study, the LGE-MRI method, using the higher 5 or 6SD thresholds, provided

accurate determination of MI, as also described previously.80-82,84 LGE-MRI6SD

provided the closest result to the reference TTC value. LGE-MRI using the other

threshold limits overestimated (2, 3, 4 SD) or underestimated (FWHM) the infarct size.

The reason for infarct overestimation using DE may be due to the partial volume effects

mostly presenting in the patchy peripheral zone of the infarct, while underestimation is

the likely result of excess filtering out of useful infarct data.

Our finding that Gd-MDCT at high tube voltage with 2SD thresholding yielded more

accurate results than the widely used LGE-MRI with the same thresholding, may be

due to several reasons. The spatial resolution of MDCT is higher than that of MRI. The

voxel volume [(FoV2/IM)*ST] in our study was 0.1mm3 (MDCT) vs. 1.95mm3 (MRI),

resulting in a negligible partial volume effect in MDCT, but not in MRI. The areas

affected by partial volume effect usually consist of the patchy peri-infarct zones where

the Gd distribution is highly inconsistent.89 Another reason is the difference between

the mechanisms of the contrast effect of Gd-DTPA in these two imaging modalities. In

MRI, Gd-chelates act as a contrast agent, since they exert an effect on the protons of

the tissue water molecules, and this effect is observed as SI enhancement by MRI. SI

enhancement induced by a Gd CA depends nonlinearly on the concentration of the CA

in the myocardial voxel observed.90,91 In CT, Gd works as a contrast medium. The high

atomic weight of Gd (157) makes it radiopaque, thus its simple physical presence, and

not an indirect effect on another molecule, is measured. Infarct determination by Gd-

MDCT is straightforward since Gd concentration in any voxel is related to the

attenuation in a linear fashion, as described in phantom studies.92 The literature

suggests that the diagnostic value of Gd-based CT techniques is inferior to the iodine

based methods.45,47,48,56 Although it is evident that Gd-MDCT provides less contrast

than Gd-MRI or iodine CT, our results, nevertheless, proved the presence of sufficient

40

contrast for distinguishing between normal and infarcted myocardium using

0.2mmol/kg Gd-DTPA.

In conclusion, the Gd-MDCT technique has been found suitable for the evaluation of

MI, at least in an ex-vivo experimental setting. Gd-MDCT has the ability to detect MI

even at low kV settings with accuracy being limited by high image noise due to

reduced photon flux. LGE-MRI showed its best accuracy at the threshold level of 6SD.

4.4.1 Practical implications

The ability of Gd-MDCT to detect MI has been proven here in an ex vivo animal

model. The usefulness of this method in in vivo circumstances, however, depends on

several additional factors (e.g. imaging a rapidly moving heart) whose evaluation

requires in vivo experiments.

A further issue with Gd-MDCT is the lack of approval of Gd-chelates for non-MRI

purposes which is strongly influenced by the question of the safety of the

administration of Gd containing materials. Nephrogenic Systemic Fibrosis (NSF) is a

potentially severe systemic disease characterized by fibrosis of the skin and connective

tissues. Based on a review, 89% of patients with NSF underwent Gd injection prior to

the onset of the disease, thus Gd CM can be considered as a trigger of NSF.93 It is

hypothesized that the prolonged presence of the agent in tissue increases the risk of

de-chelation, initiating fibroblastic reaction. Since the rate of de-chelation highly

depends on the stability of the complex, the less stable, non-ionic linear complexes (e.g.

gadodiamide) are the most highly associated with the development of NSF.93 Although

ionic linear complexes (e.g. Gd-DTPA) are more stable, they are also responsible for

some NSF cases. The odds ratio of the risk of NSF with gadodiamide is 13.2 times

greater than with Gd-DTPA.93 Cyclic Gd-chelates (e.g. gadoteridol) have extremely

high stability, and indeed they are not associated with NSF. Also, NSF only occurs in

patients with severe renal impairment (82% of NSF patients were on dialysis, 27% had

a renal transplant), thus the administration of linear complexes is contraindicated in

such patients (GFR<30).94 Based on the safety of the cyclic Gd-chelates suggesting by

the presently available literature, those chelates may have a chance to get approval for

non-MRI applications.

41

The experimental benefit of Gd-MDCT is the ability to measure the exact Gd

concentration of a specific voxel. It has been proven that MI is not a solid, confluent

non-viable mass; rather it is a mixture of viable and non-viable islets with varying

density of the viable components.89,95,96 CT phantom studies showed that Gd

concentration of any voxel is related to the attenuation in a linear fashion, i.e. the Gd

concentration of the voxel observed can be determined by the attenuation measured in

the same voxel.92 MRI phantom experiments have proven that R1 (the inverse of T1) of

an infarct voxel is related to the Gd concentration of the same voxel in a linear

fashion.91 Along with proper co-registration, the Gd-MDCT method has the potential to

enable the study of the in vivo correspondence between the Gd concentration (i.e. CT

density) and the relaxation rate (i.e. MRI R1 intensity) in the infarcted heart.

Presently, the clinical benefits of Gd-MDCT are mostly theoretical. Although,

Gd-MDCT has the ability to detect MI, numerous alternative techniques (e.g.

LGE-MRI, 201Tl, 99mTc-sestamibi or 99mTc-tetrafosmin SPECT, 18F-deoxy-glucose PET

etc.) have been developed to noninvasively distinguish viable myocardium from

irreversibly injured myocardium, and to localize, visualize, and quantify MI.

4.4.2 Limitations

Our study has some limitations. The presented data were obtained from ex-vivo, agar

embedded hearts. This model does not fully mimic the in-vivo environment of the

heart, thus our results may not be completely applicable for in-vivo situations. Also, MI

was studied 7 days following reperfusion, thus the obtained data are not necessarily

applicable for all phases of the evolution of MI. There are superior, multiple identical

acquisition-based methods available to calculate SNR from MRI data, those methods,

however, were not applicable to our studies as we carried out single image acquisitions.

A further limitation is the lack of approval at the present time of Gd-chelates for

non-MRI purposes.

42

5. DISCUSSION

5.1 Myocardial infarct model development

In spite of the effort to provide revascularization therapy within a reasonable time for

patients with acute MI, a significant number of infarcts remain untreated mostly due to

belatedly recognized or non-recognized ischemic events. Thus generating an

experimental model of the clinical scenario of non-treated infarcts is of major

importance for the development of novel diagnostic approaches and preclinical testing

of new therapeutic strategies for patients missing the appropriate timeframe suitable for

revascularization following acute MI. Since CMR has become the appropriate

diagnostic tool in several cardiovascular indications, such as myocardial perfusion,

function, and viability assessment, therefore an animal model should be MRI safe (i.e. a

high magnetic field would have no effect on the material placed into the coronary

artery) and MRI compatible (i.e. the material would not cause any image artifact).97 As

the currently available techniques for the generation of non-reperfused MI raised many

concerns regarding safety, reliability and MRI compatibility (Table 2), the development

of an easy and cost effective model suitable for MRI studies was highly desired.

In this study, we have introduced a novel animal model of non-reperfused MI. The

percutaneously administered Embozene microsphere technique we used in the swine

model can be easily controlled under fluoroscopy, although the procedure requires the

presence of an experienced interventional cardiologist. The non-reperfused nature of

the model has been confirmed at the end of the infarct generation procedure by repeated

coronary angiography, as well as at 2, 4, 14, or 56 days following infarct generation by

histopathology.

The main advantages of our microsphere embolization method are the easy

applicability and the cost effectiveness. The only method reported in the literature

having comparable reliability is coil embolization.21 The MRI compatibility of coils

and spirals advanced into the coronary artery, however, is still a question. Since our

43

procedure avoids the placement of any metallic material in the coronary artery, it can

be considered MRI safe and compatible, as proven in a relatively large number of our

MRI experiments.

5.2 Development of a Gadolinium based infarct quantification tool

In this study we reported the development of an agar embedded ex-vivo heart model of

MI that is suitable for the performance of repeated, or multi-modality, imaging, as well

as for the co-registration of imaging methods with each other and with gross pathology

(e.g., TTC). Employing this model we demonstrated the feasibility of quantification of

MI by the MDCT technique using Gd as CM. Based on our experiments, MI size

measured by Gd-MDCT at 140kV showed the best correlation with the reference TTC.

Presently, the applicability of Gd-MDCT in the clinical diagnosis of MI is theoretical

only. Initially, further studies will be required to evaluate the feasibility of this method

to detect MI in in vivo circumstances. Furthermore, the safety of Gd contrast materials

has to be clarified, especially in patients with severe renal impairment who may

develop NSF due to the de-chelation of the less stable linear chelates.93,94 Since cyclic

Gd chelates have extremely high stability, those compounds may have a better chance

to get approval for such off label use.

For experimental purposes, Gd-MDCT is capable to determine the concentration of Gd

accumulated in a specific volume element. The Gd-MDCT method combined with

LGE-MRI may be feasible to correlate the Gd concentration (as reflected in CT

density) and the relaxation rate (as MRI R1) in any tissue accumulating Gd (e.g. MI,

tumors), providing an independent tool for the validation of Gd contrast enhanced MRI

results.

44

6. CONCLUSIONS

We have demonstrated the feasibility of the generation of MI using Embozene

microspheres and the MRI compatibility of this method. Our method can be used as an

alternative technique to induce non-reperfused MI.

We have also demonstrated the feasibility of the Gd-MDCT technique for the

evaluation of MI, at least in an ex-vivo experimental setting. Gd-MDCT is able to

quantify MI with the same accuracy as the ex vivo gold standard does.

45

7. NOVEL FINDINGS

1. We have worked out a novel, cost effective, percutaneous technique for the

generation of non-reperfused MI.

2. We have proven in a large number of MRI experiments that our novel infarct

generation method is fully suitable for MRI studies.

3. We have also proven by postmortem histopathology that Embozene microspheres

create total occlusion in the target vessel and initiate consequent thrombosis. The

occlusion is reliably maintained up to 56 days after the induction of MI.

4. We have developed an ex vivo method, the agar embedded heart model, that is

suitable for the performance of repeated and/or multi-modality imaging, when a

stable position of the sample is required.

5. We have demonstrated that MDCT enhanced by Gd-DTPA, an MRI contrast agent,

is able to detect MI.

6. We have also demonstrated that Gd-MDCT, performed with appropriate settings,

provides the accurate assessment of MI.

7. We have found that Gd-MDCT provides the same accurate evaluation of MI as the

in vivo gold standard LGE-MRI (with appropriate post-processing) and the ex vivo

gold standard TTC staining do.

46

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9. PUBLICATIONS OF THE AUTHOR

Impact factor of original papers: 20.928

9.1 Peer reviewed original research publications related to the thesis

1. Varga-Szemes A, Kiss P, Brott BC, Wang D, Simor T, Elgavish GA: EmbozeneTM

Microspheres Induced Non-reperfused Myocardial Infarction in an

Experimental Swine Model. Catheter Cariovasc Interv 2012 (In Press) IF: 2.398

2. Varga-Szemes A, Ruzsics B, Kirschner R, Singh SP, Kiss P, Brott BC, Simor T,

Elgavish A, Elgavish GA: Determination of Infarct Size in Ex Vivo Swine

Hearts by Multi-detector Computed Tomography Using Gadolinium as

Contrast Medium. Invest Radiol 2012;47(5):277-283 IF: 4.665

9.2 Citable peer reviewed abstracts related to the thesis

1. Varga-Szemes A, Ruzsics B, Kirschner R, Singh SP, Simor T, Elgavish A, Elgavish

GA: Determination of Infarct Size In Ex Vivo Swine Hearts Using Gadolinium-

Enhanced Multi-Detector Computed Tomography. J Cardiovasc Comput

Tomogr 2010;45:S53. 5th Annual Scientific Meeting of the Society of

Cardiovascular Computed Tomography, 2010, Las Vegas, NV, USA

9.3 Peer reviewed abstracts related to the thesis

1. Varga-Szemes A, Ruzsics B, Kirschner R, Singh SP, Simor T, Elgavish A, Elgavish

GA: Gadolinium-Enhanced Multi-Detector Computed Tomography for the

Evaluation of Myocardial Infarct. 38th Annual Meeting of the North American

Society for Cardiovascular Imaging, 2010, Seattle, WA, USA

60

2. Varga-Szemes A, Kiss P, Brott BC, Simor T, Elgavish GA: Embozene

microspheres for the generation of non-reperfused myocardial infarction. 39th

Annual Meeting of the North American Society for Cardiovascular Imaging, 2011,

Baltimore, MD, USA

9.4 Original peer reviewed publications not related to the thesis

1. Faludi R, Toth L, Komocsi A, Varga-Szemes A, Simor T, Papp L: Chronic

Postinfarction Pseudo-pseudoaneurysm Diagnosed by Cardiac MRI. J Magn

Reson Imaging 2007;26:1656-1658 IF: 2.209

2. Kirschner R, Toth L, Varga-Szemes A, Simor T, Suranyi P, Ruzsics B, Kiss P, Toth

A, Baker R, Brott BC, Litovsky S, Elgavish A, Elgavish GA: Differentiation of

Acute and Four-week Old Myocardial Infarct with Gd(ABE-DTTA)-enhanced

CMR. J Cardiovasc Magn Reson 2010;12:22 IF: 4.328

3. Simor T, Suranyi P, Ruzsics B, Toth A, Toth L, Kiss P, Brott BC, Varga-Szemes A,

Elgavish A, Elgavish GA: Percent Infarct Mapping for Delayed Contrast

Enhancement MR Imaging to Quantify Myocardial Viability by Gd(DTPA). J

Magn Reson Imaging 2010;32:859-868 IF: 2.747

4. Kirschner R, Varga-Szemes A, Simor T, Suranyi P, Kiss P, Ruzsics B, Brott BC,

Elgavish A, Elgavish GA: Acute Infarct Selective MRI Contrast Agent. Int J

Cardiovasc Imaging 2012;28(2):285-93 IF: 2.539  

5. Kirschner R, Varga-Szemes A, Brott BC, Litovsky S, Elgavish A, Elgavish GA,

Simor T: Quantification of myocardial viability distribution with Gd(DTPA)

bolus-enhanced, signal intensity-based percent infarct mapping. Magn Reson

Imaging 2011;29(5):650-8 IF: 2.042

9.5 Citable peer reviewed abstracts not related to the thesis

1. Toth L, Faludi R, Fodi E, Knausz M, Varga-Szemes A, Papp L, Simor T: Evidence

Based, MRI Strengthened Risk Stratification Strategy for Hypertrophic

Cardiomyopathy Patients – A Follow Up Study. Eur J Echocardiogr

61

2006;7:S205. 10th Annual Meeting of the European Association of

Echocardiography, 2006, Prague, Czech Republic

2. Toth L, Varga-Szemes A, Faludi R, Toth A, Papp L, Simor T: MRI Study in

Isolated Left Ventricular Noncompaction. J Magn Reson Imaging 2007;9:406-

407. 10th Annual Meeting of the Society for Cardiovascular Magnetic Resonance,

2007, Rome, Italy

3. Varga-Szemes A, Toth L, Faludi R, Toth A, Repa I, Papp L, Simor T: MRI Study

in Isolated Left Ventricular Noncompaction. Cardiologia Hungarica

2007;37:A72. Scientific Congress of the Hungarian Society of Cardiology, 2007,

Balatonfured, Hungary

4. Toth L, Horvath I, Varga-Szemes A, Kantor M, Repa I, Papp L, Simor T: MRI

Measurement of Infarct Size in Patients with Chronic Coronary Occlusion.

Cardiologia Hungarica 2007;37:A52. Scientific Congress of the Hungarian Society

of Cardiology, 2007, Balatonfured, Hungary

5. Toth L, Faludi R, Toth A, Varga-Szemes A, Repa I, Papp L, Simor T: Correlation

of the Extent of Left Ventricular Noncompaction and Left Ventricular

Function. Eur J Heart Failure 2007;6:S161. Heart Failure Congress, 2007,

Hamburg, Germany

6. Varga-Szemes A, Toth L, Faludi R, Papp L, Simor T: Assessment of ECG

Abnormalities in Patients with Isolated Left Ventricular Noncompaction.

Cardiologia Hungarica 2007;37:C3. 6th Congress of the Hungarian Society of

Cardiology Working Group on Arrhythmia, 2007, Szeged, Hungary

7. Toth L, Varga-Szemes A, Faludi R, Sepp R, Nagy V, Repa I, Varga A, Forster T,

Papp L, Simor T: Which are the Determinant Factors Altering Left Ventricular

Function and Clinical Outcome of Patients with Isolated Noncompact

Cardiomyopathy? Eur J Echocardiogr 2007;8:S165. 11th Annual Meeting of the

European Association of Echocardiography, 2007, Lisbon, Portugal

8. Varga-Szemes A, Toth L, Faludi R, Papp L, Simor T: Characterization of Left

Ventricular Regional Function in Isolated Left Ventricular Noncompaction.

62

Cardiologia Hungarica 2008;38:B7 Scientific Congress of the Hungarian Society

of Cardiology, 2008, Balatonfured, Hungary

9. Manfai B, Faludi R, Rausch P, Tahin T, Fodi E, Toth L, Varga-Szemes A, Papp L,

Simor T: Reverse-remodeling of the Left Atrium After Catheter Ablation of

Atrial Fibrillation. Cardiologia Hungarica 2008;38:B43 Scientific Congress of

the Hungarian Society of Cardiology, 2008, Balatonfured, Hungary

10. Varga-Szemes A, Toth L, Faludi R, Papp L, Simor T: Assessment of Regional

Left Ventricular Function in Isolated Left Ventricular Noncompaction. Eur J

Heart Failure 2008;7:S33. Heart Failure Congress, 2008, Milan, Italy

11. Varga-Szemes A, Toth L, Faludi R, Papp L, Simor T: Assessment of ECG

Abnormalities in Patients with Left Ventricular Noncompaction. Eur J

Echocardiogr 2008;9:S96-97. 12th Annual Meeting of the European Association of

Echocardiography, 2008, Lion, France

12. Faludi R, Toth L, Varga-Szemes A, Fodi E, Simor T: Correlations Between

Systolic and Diastolic Function in a Group of Subjects with Variable Degrees

of Left Ventricular Diastolic and Systolic Dysfunction. Eur J Heart Failure

2009;8:S1456. Heart Failure Congress, 2009, Nice, France

13. Kirschner R, Varga-Szemes A, Toth L, Simor T, Suranyi P, Ruzsics B, Kiss P, Toth

A, Baker R, Brott BC, Litovsky S, Elgavish A, Elgavish GA: Reinfarction-

Specific Magnetic Resonance Imaging Contrast Agent. J Am Coll Cardiol

2010;55:A84. 59th Annual Scientific Session of the American College of

Cardiology, 2010, Atlanta, GA, USA

14. Singh SP, Varga-Szemes A, Goetze S, Nath H: Feasibility of combined adenosine

augmented SPECT and MDCT for evaluation of myocardial perfusion and

coronary artery morphology. J Cardiovasc Comput Tomogr 2010;45:S71. 5th

Annual Scientific Meeting of the Society of Cardiovascular Computed

Tomography, 2010, Las Vegas, NV, USA

15. Kirschner R, Varga-Szemes A, Toth L, Simor T, Suranyi P, Kiss P, Ruzsics B, Toth

A, Brott BC, Elgavish A, Elgavish GA: Acute Infarct Selective MRI Contrast

63

Agent. J Am Coll Cardiol 2010;56:B88. Transcatheter Cardiovascular Therapeutics

Conference, 2010, Washington, DC, USA

16. Pump A, Rausch P, Varga-Szemes A, Tahin T, Papp L, Simor T: Efficacy of

Pulmonary Vein Isolation in Patients with Persistent Atrial Fibrillation.

Cardiologia Hungarica 2010;37:C3. 7th Congress of the Hungarian Society of

Cardiology Working Group on Arrhythmia, 2010, Budapest, Hungary

17. Pump A, Simor P, Rausch P, Varga-Szemes A, Tahin T, Papp L: Efficacy of

pulmonary vein isolation for persistent atrial fibrillation regarding some

specific parameters. J Cardiovasc Electrophysiol 2011;22:S105. 12th International

Workshop on Cardiac Arrhythmias, 2011, Venice, Italy

18. Kirschner R, Varga-Szemes A, Simor T, Elgavish GA. Differentiation of

myocardial infarct age using Gd(ABE-DTTA), an MRI contrast agent.

Cardiologia Hungarica 2011;41:F57, Scientific Congress of the Hungarian Society

of Cardiology, 2011, Balatonfured, Hungary

19. Kirschner R, Varga-Szemes A, Simor T, Brott BC, Litovsky S, Elgavish A,

Elgavish GA: Quantification of infarct size and mixing of necrotic and viable

myocardial tissue with signal intensity-based percent infarct mapping. Eur

Heart J 2011;32:S46. Annual meeting of the European Society of Cardiology,

2011, Paris, France

9.6 Peer reviewed abstracts not related to the thesis

1. Toth L, Faludi R, Fodi E, Knausz M, Varga-Szemes A, Repa I, Papp L, Simor T:

Cardiac MRI for the Assessment of Risk of Sudden Cardiac Death in Patients

with Hypertrophic Cardiomyopathy. Abstract Book p136. 5th Annual Meeting of

the European Society of Cardiology Working Group on Cardiovascular Magnetic

Resonance Imaging, 2006, Vienna, Austria

2. Varga-Szemes A, Toth L, Faludi R, Papp L, Simor T: Novel Parameter for the

Evaluation of Left Ventricular Noncompaction. Abstract CD #54. 6th Annual

Meeting of the European Society of Cardiology Working Group on Cardiovascular

Magnetic Resonance Imaging, 2008, Lisbon, Portugal

64

3. Kantor M, Toth L, Varga-Szemes A, Horvath I, Papp L, Simor T: The

Relationship Between of the Extent of Myocardial Infarction and Global

Cardiac Function in Chronic Total Occlusion in the Presence and Absence of

Coronary Collateral Circulation. Abstract CD #35. 6th Annual Meeting of the

European Society of Cardiology Working Group on Cardiovascular Magnetic

Resonance Imaging, 2008, Lisbon, Portugal

4. Varga-Szemes A, Kirschner R, Toth L, Brott BC, Simor T, Elgavish A, Elgavish

GA. In Vivo R1 Based Percent Infarct Mapping Using Continuous Gd(DTPA)

Infusion Aided Magnetic Resonance Imaging. 38th Annual Meeting of the North

American Society for Cardiovascular Imaging, 2010, Seattle, WA, USA

5. Singh SP, Varga-Szemes A, Goetze S, Nath H: Quantitative Evaluation of

Myocardial Perfusion Abnormalities Using Adenosine Augmented MDCT and

SPECT. 38th Annual Meeting of the North American Society for Cardiovascular

Imaging, 2010, Seattle, WA, USA

6. Kirschner R, Varga-Szemes A, Toth L, Brott BC, Simor T, Elgavish A, Elgavish

GA: Accurate Determining of Myocardial Viability Distribution with Percent

Infarct Mapping. 38th Annual Meeting of the North American Society for

Cardiovascular Imaging, 2010, Seattle, WA, USA

7. Kirschner R, Varga-Szemes A, Toth L, Brott BC, Simor T, Elgavish A, Elgavish

GA: Acute Infarct Selective Magnetic Resonance Imaging Contrast Agent. 38th

Annual Meeting of the North American Society for Cardiovascular Imaging, 2010,

Seattle, WA, USA

8. Singh SP, Varga-Szemes A, Goetze S, Nath H: Feasibility of Combined

Adenosine Augmented SPECT and MDCT for Evaluation of Myocardial

Perfusion and Coronary Artery Morphology. Abstract# LL-CAS-TH2B. 96th

Scientific Assembly and Annual Meeting of the Radiological Society of North

America, 2010, Chicago, IL, USA

9. Varga-Szemes A, Kirschner R, Brott BC, Simor T, Elgavish A, Elgavish GA:

Percent Infarct Mapping for the evaluation of chronic myocardial infarction.

65

39th Annual Meeting of the North American Society for Cardiovascular Imaging,

2011, Baltimore, MD, USA

10. Varga-Szemes A, Kirschner R, Brott BC, Simor T, Elgavish A, Elgavish GA:

Myocardial infarct density distribution by MRI. 40th Annual Meeting of the

North American Society for Cardiovascular Imaging, 2012, Pasadena, CA, USA

11. Varga-Szemes A, Kirschner R, Brott BC, Simor T, Elgavish GA: Contrast uptake

kinetics of microvascular obstruction in a reperfused and a non-reperfused

acute infarct model. 40th Annual Meeting of the North American Society for

Cardiovascular Imaging, 2012, Pasadena, CA, USA

9.7 Additional presentations/abstracts not related to the thesis

1. Tormasi I, Varga-Szemes A: Stress Perfusion MRI. Conference of Research

Student Fellowship, 2005, University of Pecs, Medical School, Hungary (2nd prize)

2. Varga-Szemes A: MRI Study in Isolated Left Ventricular Noncompaction.

Conference of Research Student Fellowship, 2006, University of Pecs, Medical

School, Hungary (1st prize)

3. Varga-Szemes A: Assessment of Left Ventricular Function in Patients with Left

Ventricular Noncompaction Using Cardiovascular MRI. Conference of

Research Student Fellowship, 2007, University of Pecs, Medical School, Hungary

(2nd prize)

4. Varga-Szemes A: Assessment of Left Ventricular Function in Patients with Left

Ventricular Noncompaction Using Cardiovascular MRI. 28th Nationwide

Conference of Research Student Fellowship, 2007, Semmelweis University,

Medical School, Budapest, Hungary (Semmelweis special award)

5. Varga-Szemes A, Toth L, Simor T: Diagnosis of Left Ventricular

Noncompaction Using MRI. 21st Ultrasound Days, 2007, Sopron, Hungary

6. Rausch P, Tahin T, Varga-Szemes A, Simor T: Difficult Decision in Case of an

Acute Electrophysiology Intervention. 15th Annual Meeting of Young

Cardiologists, 2008, Lillafured, Hungary

66

7. Varga-Szemes A: Diagnosis of Noncompaction Cardiomyopathy. Dice Section:

Controversies in Cardiac Imaging. Scientific Congress of the Hungarian Society of

Cardiology, 2008, Balatonfured, Hungary

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10. ACKNOWLEDGEMENTS

First and foremost I would like to express my gratitude to my mentor, Dr. Tamás

Simor, for his encouragement and support throughout my Ph.D. studies.

I would also like to acknowledge the help, constant guidance and support of Dr.

Gabriel A. Elgavish, the head of the “Elgavish Lab” at the University of Alabama at

Birmingham. He created a perfect atmosphere for learning and working in science and

gave me invaluable advises to both academic and personal life.

I am grateful for the hard work of my immediate colleagues, Drs. Tamás Bodnár,

Róbert Kirschner, Pál Kiss, Balázs Ruzsics, Pál Surányi and Levente Tóth, and I thank

my former professor, Dr. Lajos Papp, as well as my current chief, Dr. Sándor Szabados

for their continuing support.

I am thankful for the help of our collaborators, Drs. Brigitta C. Brott, Ada Elgavish,

Satinder P. Singh, Dezhi Wang and the staff at the University of Alabama at

Birmingham Animal Resources Program (Drs. Robert A. Baker, Eric D. Dohm, Cheryl

R. Killingsworth, as well as Deidra H. Isbell).

I would also like to acknowledge the support from the National Institutes of Health,

National Heart, Lung and Blood Institute for the following research grants that made

this work possible: R41 HL-080886, R41 HL-084844.

Last but far not the least, I would like to thank my parents Éva and Gábor Varga-

Szemes, as well as my wife and daughter, Évi and Dora, for their faithful support

throughout the years.