University of Southern California Dissertations and Theses

Electrical signals to assess cardiovascular phenomenon

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Electrical signals to assess cardiovascular phenomenon

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Content ELECTRICAL SIGNALS TO ASSESS
CARDIOVASCULAR PHENOMENON

by

Fei Yu

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
DOCTOR OF PHILOSOPHY
(BIOMEDICAL ENGINEERING)

May 2013

Copyright 2013 Fei Yu
ii
Acknowledgements
Upon completion of the thesis, I would like to express my gratitude to many people and
organizations. Writing this thesis would not have been possible without the support and
help from them.
Foremost, it is difficult to overstate my gratitude to my Ph.D. supervisor, Dr. Tzung Hsiai.
He has been providing me invaluable guidance, advice, encouragement, and lots of good
ideas throughout my Ph.D. training. I am very grateful for his patience, motivation,
enthusiasm, and immense knowledge, all of which had made this thesis possible. I would
also like to thank the rest of my thesis defense and qualifying exam committee members,
Dr. Kirk Shung, Dr. Eun Sok Kim, Dr. Jesse Yen and Dr. Ping Wang for their support
and advice as well.
I am indebted to many colleagues for providing guidance, assistance and help for my
research work, as well as for providing a stimulating, fun environment in which to learn
and grow. I am especially grateful to Dr. Rongsong Li, Dr. Lisong Ai, Dr. Hung Cao,
Nelson Jen, Tyler Beebe, and Juhyun Lee, Elizabeth Parks, and Xiaohu Dai for their great
help in preparing many manuscripts for publication, which had been the foundation of
this thesis.
I am also grateful to all the collaborators from multiple institutions, including Qian Zhang
and Dr. E.S. Kim, Dr. Jinhyoung Park, Dr. Xiang Li, Boon Jin Kang and Dr. K.K. Shung
(USC), Yu Zhao and Dr. Yu-Chong Tai (CalTech), Wangde Dai, Sharon Hale and Dr.
Robert Kloner (Good Samaritan Hospital), Michael Harrison and Dr. Ching-Ling Lien
(CHLA), Dr. Chi (UCSD) and Dr. Hongyu Yu (ASU), and many others who have being
providing assistance to my research.
I wish to thank my entire family for providing a loving environment for me. My parents
and wife were particularly supportive during the hard times.
Lastly but not the least, I would like to thank USC Zumberge Interdisciplinary Research
Award (TKH), NIH (NHLBI) (TKH), AHA Science Development Award (CLL), and
American Heart Association Predoctoral Fellowship (FY) for their financial support.
Alfred E Mann Institute at USC deserves special mention for providing lab and
conference spaces.
iii

Table of Contents
Acknowledgements ii
List of Tables v
List of Figures vi
Abstract viii
Introduction: Overview of Atherosclerosis, Acute Coronary Syndrome and their
Diagnosis and Treatments 1
Atherosclerosis and acute coronary syndrome 1
Diagnosis of mechanically unstable lesions 8
Regeneration Medicine Treatments for myocardium injury after heart attack 12
References 15
Chapter One: Electrochemical Impedance Spectroscopy to Assess Vascular Oxidative
Stress 23
1.1 Chapter One Introduction 24
1.2 Chapter One Materials and Methods 26
1.3 Chapter One Results 30
1.4 Chapter One Discussion 32
1.5 Chapter One References 43
Chapter Two: Characterization of Inflammatory Atherosclerotic Lesions via
Electrochemical Impedance Spectroscopy 48
2.1 Chapter Two Introduction 49
2.2 Chapter Two Materials and Methods 51
2.3 Chapter Two Results 54
2.4 Chapter Two Discussion 57
2.5 Chapter Two References 66
Chapter Three: Elevated Electrochemical Impedance in the Endoluminal Regions with
High Shear Stress: Implication for Assessing Lipid-Rich Atherosclerotic
Lesions 71
3.1 Chapter Three Introduction 72
3.2 Chapter Three Materials and Methods 73
3.3 Chapter Three Results 77
3.4 Chapter Three Discussion 79
3.5 Chapter Three References 96
iv

Chapter Four: Micro-Electrocardiograms to Study Post-Ventricular Amputation of
Zebrafish Heart 102
4.1 Chapter Four Introduction 103
4.2 Chapter Four Materials and Methods 105
4.3 Chapter Four Results 108
4.4 Chapter Four Discussion 110
4.5 Chapter Four References 121
Chapter Five: Electrocardiogram Signals to Assess Zebrafish Heart Regeneration:
Implication of Long QT Intervals 124
5.1 Chapter Five Introduction 125
5.2 Chapter Five Experimental Designs and Methods 127
5.3 Chapter Five Results 130
5.4 Chapter Five Discussion 134
5.5 Chapter Five References 148
Chapter Six: Flexible Microelectrode Arrays to Interface Epicardial Electrical Signals
with Intracardial Calcium Transients in Zebrafish Hearts 151
6.1 Chapter Six Introduction 152
6.2 Chapter Six Materials and Methods 153
6.3 Chapter Six Results 156
6.4 Chapter Six Discussion 158
6.5 Chapter Six References 168
Chapter Seven: Implantable Microelectronic Membranes to Investigate Cardiac Electrical
Phenotypes in Small Animal Models of Heart Regeneration 172
7.1 Chapter Seven Introduction 173
7.2 Chapter Seven Designs and Methods 175
7.3 Chapter Seven Results 178
7.4 Chapter Seven Discussion 180
7.5 Chapter Seven References 191
Conclusion 193
Bibliography 195

v

List of Tables
Table 1 Summary of ISS in Aortas of Rabbits 95
Table 2 Heart Rates in the Presence and Absence of Tricane 113
Table 3 Periodic QTc Assessment. 137

vi

List of Figures
Figure 1. Initiation of atherosclerosis by oxLDL infiltration 3
Figure 2. Development and rupture of atherosclerotic plaques 4
Figure 3. Comparison of mechanically stable plaques and unstable plaques 5
Figure 4. Equivalent circuit and configuration of concentric bipolar electrode 35
Figure 5. Rabbit aorta in response to 8 weeks of high-fed diet 36
Figure 6. Segments of human descending aorta and common carotid artery 37
Figure 7. EIS measurements in relation of the depth and sample orientation 38
Figure 8. Endoluminal EIS measurements in the aortic arch of NZW rabbits 39
Figure 9. Preatherosclerotic aortic arch lesions 40
Figure 10. Endoluminal EIS measurements from the human descending aorta 41
Figure 11. EIS measurements in explants of human carotid arteries 42
Figure 12. Equivalent circuit models to simulate concentric bipolar EIS 60
Figure 13. Endoluminal EIS measurements of fatty streaks 61
Figure 14. Endoluminal EIS measurements of oxLDL-rich fibrous atheroma 62
Figure 15. Endoluminal EIS measurements of oxLDL-absent fibro-atheroma 63
Figure 16. Endoluminal EIS measurements of calcification 64
Figure 17. Sensitivity and specificity of Endoluminal EIS measurements 65
Figure 18. Catheter-based concentric bipolar microelectrodes 82
Figure 19. Comparison of intravascular shear stress profiles with CFD 83
Figure 20. ISS profiles in response of normal diet 84
Figure 21. ISS profiles in response to high-fat diet 85
Figure 22. LDL and blood viscosity at baseline versus after 9 weeks 86
Figure 23. Endoluminal EIS assessment of oxLDL-rich lesions 87
Figure 24. Catheter-based ISS sensor and concentric bipolar microelectrodes 89
Figure 25. Hemodynamic response was measured as voltage output in aorta 90
Figure 26. Time frame of the experimental protocol 91
Figure 27. Intravascular shear stress (ISS) profiles in ND-fed rabbits 92
Figure 28. Intravascular shear stress (ISS) profiles in HD-fed rabbits 93
Figure 29. IVUS and histology images of endoluminal atherosclerotic lesions 94
Figure 30. Zebrafish ECG acquisition and processing 114
Figure 31. Signal processing using Wavelet transform 115
Figure 32. Comparison of ECG signals via two difference filter algorithm 116
Figure 33. Three ECG signals of an amputated zebrafish 117
Figure 34. Representative ECG signals prior to and post amputation 118
Figure 35. ECG signals from 4 different zebrafish at 4 dpa 119
Figure 36. Statistical analyses of ECG features in 6 zebrafish post-amputation 120
vii

Figure 37. Histological study of sham versus ventricular resection at 60 dpr 138
Figure 38. Immunohistochemistry for cardiomyocyte gap junction protein 139
Figure 39. Example of signal processing for Fish # ECG recorded at 3 dpa 141
Figure 40. Representative ECG recording from sham operation fish 142
Figure 41. Representative ECG of fish with incomplete regeneration 143
Figure 42. Representative ECG from fish with ventricular regeneration 144
Figure 43. Effect of resection on the variability of RR intervals 145
Figure 44. Dynamic changes in J depression in sham and resected ventricles 146
Figure 45. QTc values between sham and resected ventricles 147
Figure 46. Microfabrication steps for flexible microelectrode arrays 160
Figure 47. Flexible microelectrode arrays for zebrafish ECG recording 161
Figure 48. Wavelet transform and noise-reduction algorithm 162
Figure 49. ECG signals acquired from multi-lead array 163
Figure 50. Calcium transients in the entire zebrafish heart 164
Figure 51. Calcium transients at ventricle apex and isolated cardiomyoctes 165
Figure 52. Comparison between human and zebrafish ECG signals 167
Figure 53. Micro-fabrication of the flexible MEA membrane 183
Figure 54. Flexible MEA membranes for non-planar anatomy 184
Figure 55. Characterization of the MEA impedance 185
Figure 56. Real-time ECG acquisition in zebrafish and neonatal mice 186
Figure 57. Aberrant electric phenotypes in a cryo-injured neonatal mouse 187
Figure 58. Zebrafish ECG signals from implanted MEA over 3 days 188
Figure 59. ST depression in response to cryo-injury 189
Figure 60. Simultaneous four-channel ECG signal acquisition 190

viii

Abstract
Atherosclerosis, particularly mechanically unstable lesions and resulting acute coronary
syndromes remain to be one of the leading causes of death and permanent disabilities in
the world. We hereby aim to address this clinical issue by applying electrical signals to
assess the cardiovascular phenomenon. In particular, we developed novel electrical
approaches to two important issues in early diagnosis and treatment of acute coronary
syndrome: 1) detection of unstable plaque in vivo for early medical intervention, and 2)
assessment of cardiac conduction and mechanical coupling during heart regeneration in
small animal models.
Despite advances in diagnosis and therapy, atherosclerotic cardiovascular disease remains
the leading cause of morbidity and mortality in the Western world. Predicting
metabolically active atherosclerotic lesions has remained an unmet clinical need. We
developed an electrochemical strategy to characterize the inflammatory states of high-risk
atherosclerotic plaques. Using the concentric bipolar microelectrodes, we sought to
demonstrate distinct Electrochemical Impedance Spectroscopic (EIS) measurements for
unstable atherosclerotic plaques that harbored active lipids and inflammatory cells. We
demonstrated increased impedance in response to oxidized low density lipoprotein
(oxLDL)-laden lesions. Using equivalent circuits to simulate vessel impedance at the
electrode-endoluminal tissue interface, we demonstrated specific electric elements to
model working and counter electrode interfaces as well as the tissue impedance. We
hereby assessed the feasibility of integrating EIS with intravascular ultrasound (IVUS)
and shear stress (ISS) to provide a new strategy to assess oxLDL-laden lesions in the fat-
fed New Zealand White (NZW) rabbits. By applying electrochemical impedance in
conjunction with shear stress and high-frequency ultrasound sensors, we provided a new
strategy to identify oxLDL-laden lesions. Our study demonstrated the feasibility of
integrating EIS, ISS, and IVUS for a catheter-based approach to assess mechanically
unstable plaque.
On the other arm, zebrafish (Danio rerio) is an emerging genetic model for regenerative
medicine. In humans, myocardial infarction results in the irreversible loss of
cardiomyocytes. Zebrafish hearts fully regenerate after resection or cryoinjury, without
either scarring or arrhythmias. To study this cardiac regeneration, we developed
microelectrode electrocardiograpm (ECG) platform, and subsequently implantable
flexible multi-electrode membrane arrays that measure the epicardial electrocardiogram
signals of zebrafish in real-time. The microelectrode electrical signals allowed for a high
ix

level of both temporal and spatial resolution. We observed delayed electric repolarization
in either the regenerated hearts or scar tissues. Early regenerated cardiomyocytes lacked
the conduction phenotypes of the sham fish. The electrical signals were in synchrony
with optically measured calcium concentration as well as Doppler Echocardiogram.
These microelectrode devices therefore provide a real-time analytical tool for monitoring
conduction phenotypes of small vertebral animals with a high temporal and spatial
resolution.

1
Introduction: Overview of Atherosclerosis, Acute Coronary
Syndrome and their Diagnosis and Treatments
Atherosclerosis and acute coronary syndrome
a) Development of atherosclerosis
Atherosclerosis is the thickening of artery wall thickens due to accumulation of fatty
materials as a result of chronic inflammatory response [1]. Atherosclerosis is a systemic
disease; however, its manifestations tend to be focal and eccentric [2-11]. The
development of atherosclerosis is dependent upon a complex interplay among numerous
genetic, epigenetic, and environmental risk factors [12-14]. Hemodynamics, specifically,
fluid shear stress or the tangential frictional force acting on the vascular endothelial cells,
is intimately involved in the focal nature of oxidative stress[15] and pro-inflammatory
states [16-19].
Atherosclerosis typically begins in early adolescence, and is usually found in most major
arteries, yet is asymptomatic and not detected by most diagnostic methods for decades
[20]. The development of atherosclerosis starts from low-density lipoprotein molecules
(LDL) present in the blood becoming oxidized by free radicals, particularly reactive
oxygen species (ROS) [15, 21]. When oxidized LDL (oxLDL) are in contact with artery
endothelium, especially at points where re-circulation or oscillatory wall shear stress
(WSS) occurs, the oxLDL will cause damage to endothelial cells, increasing the
permeability of endothelium [22, 23]. oxLDL molecules then can migrate into artery wall
through the endothelium. The initial damage to the blood vessel wall results in an
inflammatory response, recruiting monocytes from the bloodstream to enter the artery
wall and to ingest oxLDL. These white blood cells are not able to process the oxLDL, but
will slowing turn into large foam cells with high lipid content (Fig. 1). Foam cells
eventually die and rupture, further propagating the inflammatory process [10]. Eventually,
fatty substances, cholesterol, waste products, calcium and other substances build up and
form a plaque.
2

The plaque causes the smooth muscle proliferation and migration from tunica media to
intima responding to cytokines secreted by damaged endothelial cells. These fibrous
tissue and form a hard cover over the affected area known as the fibrous cap. Advanced
atherosclerotic lesions can cause stenosis, which is a narrowing of the artery, reducing the
blood flow and increasing blood pressure. But most of the damage occurs when the
fibrous cap ruptures, causing the formation of a thrombus that will rapidly slow or stop
blood flow, leading to death of the tissues fed by the artery in approximately 5 minutes.
This catastrophic event is called an infarction. One of the most common recognized
scenarios is the actue coronary thrombosis, causing myocardial infarction or heart attack
(Fig. 2). The same process in an artery to the brain is commonly called stroke. And if
blood supply to the arms or legs is reduced, it can cause peripheral artery occlusive
disease and eventually lead to gangrene. Alternatively, partial rupture of plaque may only
result in minor thrombosis, which can eventually heal as fibrous organization of the clot
within the lumen ensues, covering the rupture. These complications of advanced
atherosclerosis are chronic, slowly progressive and cumulative. The chronically
expanding atherosclerotic lesions can cause severe stenosis complete closure of the
lumen so that blood supply to downstream tissue is insufficient resulting in ischemia (Fig.
2).

3

Figure 1. Initiation of atherosclerosis by oxLDL infiltration. Atherosclerosis begins
with activation of endothelial cells due to low or oscillatory shear stress, resulting in
increased permeability of endothelium. LDL then penetrates into the intima, which lies
just below the endothelium. Trapped LDL could be oxidized, triggering recruitment of
monocytes into the intima. Several adhesion molecules are involved, including vascular-
cell adhesion molecule (VCAM), integrin, selectin, and others. After entering the intima,
monocytes differentiate into macrophages and ingest oxidized LDL. As atherosclerosis
progresses, T lymphocytes, platelets and smooth muscle cells also join foam cells,
expanding the plaque size. This involves cytokines to activate T lymphocytes and growth
factors to promote proliferation of smooth muscle cells. Platelets can also release
cytokines and growth factors to enhance migration and proliferation of smooth muscle
cells

4

Figure 2. Development and rupture of atherosclerotic plaques. Atherosclerosis
progresses by sequential episodes of plaque rupture and subsequent healing. Alternatively,
rupture or erosion of a vulnerable plaque can cause an acute occlusion owing to the
formation of a thrombus, which manifests clinically as an acute coronary syndrome.
Complete occlusion results in a myocardial infarction with subsequent scar formation.
The equivalent event after a plaque rupture in the carotid artery is an ischemic stroke [24].

5

Figure 3. Comparison of mechanically stable plaques and unstable plaques. (a) A
stable plaque usually has greater stenosis, a thick fibrous cap, relatively small lipid
content and fewer inflammatory cells. (b) In the contrary, an unstable plaque may be
significantly less stenotic, has very thin fibrous cap and much more lipid and macrophage
cells.
a) b)
6

b) Mechanically unstable plaques, thrombosis and myocardium injury
Atherosclerotic lesions or plaques can be classified into two broad categories: Stable and
unstable (also called vulnerable)[10]. The pathobiology of atherosclerosis is very
complicated but generally, stable atherosclerotic plaques are rich in extracellular matrix
and smooth muscle cells; its fatty content, often appear as a lipid pool underneath the
fibrous cap, is comparably small (Fig. 3a). On the other hand, unstable plaques are rich in
inflammatory cells such as macrophages and oxLDL-filled foam cells, has a large lipid
pool and the fibrous cap is usually thin and prone to rupture [25] (Fig. 3b). Ruptures of
the fibrous cap, expose thrombogenic material, such as collagen to the circulation, will
activate cascade events of coagulation and eventually induce thrombus formation in the
lumen[26]. Upon formation, intraluminal thrombi can occlude arteries outright, but more
often they detach, move into the circulation and eventually occlude smaller downstream
branches causing thromboembolism.
Clinically, 75% lumen stenosis used to be considered by cardiologists as the hallmark of
clinically significant disease because it is only at this severity of narrowing of the larger
heart arteries that recurring episodes of angina and detectable abnormalities by stress
testing methods are seen. However, it has been found that there is no direct relationship
between the level of stenosis of a plaque and its vulnerability to rupture. Clinical trials
suggested most severe clinical events do not occur at plaques that produce high-grade
stenosis only 14% of heart attacks occur from artery closure at plaques producing a 75%
or greater stenosis[27]. In fact, the majority of events occur due to plaque rupture at areas
without narrowing sufficient enough to produce any symptoms. From clinical trials, 20%
is the average stenosis at plaques that subsequently rupture with resulting complete artery
closure[28], and the presence of a moderate carotid stenosis (30% to 70%) with a thin
fibrous cap indicates a high risk for plaque rupture[29].
One of the reasons the plaque with high degree stenosis are less vulnerable to rupture is
that most stenoses are formed from repeated minor ruptures, which did not resulting in
total lumen closure, combined with the clot patch over the rupture and the healing
response to stabilize the clot. The stenotic areas tend to become more stable as a result of
high fibrous tissue content, despite increased flow velocities at these narrowings. On the
other hand, most major rupture events occur at plaques, which prior to rupture, produced
very little if any stenosis. Positive remodeling of the artery, the expansion in vessel cross-
sectional area in response to increasing plaque burden is a major contributing factor.
Several studies have indicated that plaques with high fatty content and macrophage count,
7

both markers of plaque vulnerability, tend to cause more positive remodeling but minimal
luminal stenosis[30, 31].
Among all the complications resulted from vulnerable plaque rupture, myocardial
infarction and stroke are two of the most severe scenarios. Myocardial infarction, also
known as heart attack, is the death of heart muscle from deprivation of blood and oxygen,
as a result of coronary artery blockage. If blood flow is not restored to the heart muscle
within 20 to 40 minutes, irreversible death of the heart muscle will begin to occur [32].
Muscle continues to die for six to eight and the dead heart muscle is eventually replaced
by scar tissue[33]. The scar tissue on the heart may further predispose the patient to
cardiac arrhythmia such as ventricular fibrillations, during which the cardiac output drops
to zero, and, unless remedied promptly, death usually ensues within minutes with
survival rate of less than 2%.
8

Diagnosis of mechanically unstable lesions
a) Significance
Approximately one million Americans suffer a heart attack each year. Four hundred
thousand of them die as a result of their heart attack. It is the leading cause of death in
developed countries, and third to AIDS and lower respiratory infections in developing
countries [34, 35]. Therefore, detection of atherosclerotic lesions prone to rupture is of
utmost importance in the management of patients for acute coronary syndromes or stroke
[36].
At current stage, while a single ruptured plaque can be identified during autopsy as the
cause of a coronary event, there is currently no way to identify a culprit lesion before it
ruptures. Additionally, because these lesions do not produce significant stenoses, they are
typically not considered "critical" and/or interventionable by interventional cardiologists,
even though research indicates that they are the more important lesions for producing
heart attacks. Thus, the quantitative assessment of vascular oxidative stress in
atherosclerotic lesions and the potential to distinguish mechanically unstable plaque from
non-inflammatory or fibrotic lesions holds promise to identify patients for selective
intervention. These studies are relevant to public health because better understanding of
the mechanically unstable plaque has the potential to reduce acute coronary syndromes
and stroke events.

9

b) Approaches
At present, despite imaging modalities [37] and pharmacological stress tests [38],
characterizing the focal metabolic states of a stenotic lesion prone to rupture during
angiograms remains a clinical challenge. Because positive remodeling of the artery walls
in response to enlarging plaques, these plaques do not usually produce much stenosis of
the artery lumen. Therefore, they are not detected by cardiac stress tests or angiography,
the tests most commonly performed clinically with the goal of predicting susceptibility to
future heart attack.
Over the last couple of decades, methods other than angiography and stress-testing have
been increasingly developed as ways to better detect atherosclerotic disease before it
becomes symptomatic. These have included both anatomic detection methods and
physiologic measurement methods. For both, clinicians and third party payers have been
slow to accept the usefulness of these newer approaches [39].
Examples of anatomic methods include: (1) coronary calcium scoring by CT, (2) carotid
IMT (intimal media thickness) measurement by ultrasound, and (3) intravascular
ultrasound (IVUS). The anatomic methods directly measure some aspect of the actual
atherosclerotic disease process itself, thus offer potential for earlier detection, including
before symptoms start, disease staging and tracking of disease progression. However,
they are also generally more expensive and several are invasive, such as IVUS[39].
Examples of physiologic methods include: (1) lipoprotein subclass analysis, (2) HbA1c,
(3) hs-CRP, and (4) homocysteine. The physiologic methods are often less expensive and
safer and changing them for the better may slow disease progression, in some cases with
marked improvement, but most of them do not quantify the current state of the disease or
directly track progression[39].
The tests most commonly performed clinically with the goal of testing susceptibility to
future heart attack include several medical research efforts, starting in the early to mid-
1990s, using intravascular ultrasound (IVUS), thermography, near-infrared spectroscopy,
careful clinical follow-up and other methods, to predict these lesions and the individuals
most prone to future heart attacks. Imaging modalities including magnetic resonance
imaging (MRI) and electron beam computed tomography (EBCT) have also been
10

proposed to predict unstable plaque [40, 41]. These efforts remain largely research with
no useful clinical methods to date[42].
Although oxLDL and macrophage count have been identified as markers for plaque
vulnerability [43], up to date, there are no diagnostic tools to detect intravascular
oxidative stress and inflammatory states when patients undergo angiograms. Serum
biomarkers have been proposed for predicting pro-inflammatory states. Specifically,
circulating Lp-PLA2 is bound predominantly to low-density lipoprotein (LDL) (~80%)
which transmigrated to the subendothelial space of atherosclerosis-prone regions [36].
However, assessing stable versus unstable atherosclerotic lesions during catheterization
remains an unmet clinical need.

11

c) Application of Electrochemical Impedance Spectroscopy
Electrical impedance (Z) is a measure of the opposition to electrical flow through a
substance, a complex quantity combining resistance and reactance as a function of
frequency when an alternative current (AC) was applied. The value of impedance is
conventionally represented as a complex number (Z = R + iXc), consisting of the real
number R for the resistance and the complex number iXc for the reactance [44].
Investigations on the electrochemical properties of the biological tissue can be dated to as
early as 1871 [45].
Biological tissue such as blood vessels harbor resistance and store charges, exhibiting
complex electric impedances as a function of frequency. Atherosclerotic lesions harbor
pro-inflammatory substrates; namely, oxLDL and macrophage-derived foam cells
infiltrates, which engender distinct electrochemical properties [46, 47]. Hence, we were
able to determine the lesions’ frequency-dependent electrical and dielectrical behavior by
recording the electric impedance of a tissue over a frequency range, otherwise quantified
as electrochemical impedance spectroscopy (EIS)[48]. Previously, changes in bulk
resistance were prevalent in the atherosclerotic lesions as measured by the linear 4-point
probe [46, 47, 49]. Electric impedance (Z) develops as a function of frequency in
response to the applied alternative current (AC) to the biological tissue. Recently, a
catheter-based linear 4-point microelectrode was reported to assess EIS in New Zealand
White (NZW) rabbits.
In our study, we introduced concentric bipolar microelectrodes to further address the non-
uniform and complex tissue current distribution, uneven endoluminal topography and
non-uniform current distribution, in both rabbit aortas and human descending aortas with
high spatial resolution.
12

Regeneration Medicine Treatments for myocardium injury after heart attack
a) Significance
Understanding heart regeneration in a vertebrate model system is highly relevant to
public health. Myocardial infarction results in irreversible loss of cardiomyocytes in the
heart[33]. Injured human hearts heal by scarring, which leads to remodeling and heart
failure[50]. Nearly 5 million people in the US are afflicted with heart failure with an
additional 550,000 new cases diagnosed each year. Despite current treatment regimens,
heart failure remains the leading cause of morbidity and mortality in the US and
developed world due to failure to adequately replace lost ventricular myocardium from
ischemia-induced infarct. Though heart regeneration was recently discovered in neonatal
mouse, this capability soon disappears in one-week old animals[51]. The adult
mammalian ventricular cardiomyocytes have a limited capacity to divide, but this
proliferation is insufficient to overcome the significant loss of myocardium from
ventricular injury[52-54].
The concept of regenerative medicine holds the promise of regenerating damaged tissues
and organs in the body by stimulating previously irreparable organs to heal themselves. It
is defined as the process of creating living, functional tissues to repair or replace tissue or
organ function lost due to damage, or congenital defects. Importantly, regenerative
medicine has the potential to solve the problem of the shortage of organs available for
donation compared to the number of patients that require life-saving organ
transplantation, as well as solve the problem of organ transplant rejection, since the
organ's cells will match that of the patient [55, 56].
While heart regeneration in adult mammals has not been achieved yet, a number of
studies have been done in the field of tissue engineering, which is a process of growing
functional cells in vitro and transplant them back into the damaged organ with the aid of a
biodegradable scaffold. However a major bottleneck for the realization of cell
transplantation therapies for human heart failure has been the lack of integration and
coupling of regenerate cells (cardiac or skeletal muscle) to recipients’ hearts resulting in a
pro-arrhythmic substrate in patients’ heart[57, 58]. Therefore studying a viable heart
regeneration animal model would not only provide mechanistic information for the
regeneration process, but also provide insights into the functional integration of the newly
formed cells and existing tissue.
13

b) Zebrafish as a model for heart regeneration
Zebrafish (Danio rerio) possess the remarkable capacity to regenerate a significant
amount of myocardium in injured hearts, and thus represent an emerging vertebrate
model for regenerative medicine and cardiovascular research[59], in part, due to the
relatively ease of maintenance and breeding and its significant promise for high
throughput drug-screening [60]. Because cardiac development, structure, and function are
relatively conserved between lower vertebrates and mammals[61-64], further mechanistic
investigations into this regeneration and integration of myocardium into damaged hearts
may yield future insights into cellular therapies for human heart failure and myocardial
infarctions.
Despite a two-chambered heart and a lack of pulmonary vasculature [65], the zebrafish
heart electrocardiogram (ECG) is fundamentally similar to that of humans in terms of P
waves, QRS complexes, and T waves [61, 64]. The critical conduction pathways of the
zebrafish in cardiovascular development also parallel that of higher vertebrates [66]. The
zebrafish heart is encased by a pericardial sac in the thoracic cavity below the pectoral
bone, and the atrium is medially dorsal and posterior to the ventricle. The bulbous
arteriosus (BA) is analogous to the human aortic arch with thick contractile smooth
muscle. Deoxygenated blood returns to the sinus venosus (SA), a structure analogous to
the vena cava in humans. Thus, zebrafish is a viable model for developmental biology,
cardiac arrhythmia, and drug discovery[62].

14

c) Regeneration of electrical conduction in zebrafish heart
Despite recent electrophysiology studies in zebrafish revealing that regenerating
myocardium may electrically couple with uninjured myocardium[67], it remains unclear
as to whether structurally regenerated zebrafish hearts exhibit functionally normal
physiologic phenotypes with complete integration of regenerated myocardium with host
myocardium. Zebrafish cardiac propagation in the regenerated cardiac tissue is a
complex process governed

by the excitable properties of the tissue and its macroscopic

and microscopic architecture. Encouraging results from our laboratory [68] and others
[69] showed the feasibility of monitoring zebrafish heart regeneration by the use of
microelectrodes. In this context, we assessed the histology-conduction relationship in
response to ventricular resection[68]. Our in vivo regeneration model provides a non-
invasive approach to assess cardiac conduction with relevance to future assessment of
genetically[62], epigenetically[60], or pharmacologically[64] induced cardiac
phenotypes.

15

References
[1] A. Maton, R. L. J. Hopkins, C. W. McLaughlin, S. Johnson, M. Q. Warner, D.
LaHart, et al., Human Biology and Health, 3 ed. Englewood Cliffs, NJ, USA:
Prentice Hall, 1997.
[2] C. K. Zarins, D. P. Giddens, B. K. Bharadvaj, V. S. Sottiurai, R. F. Mabon, and S.
Glagov, "Carotid bifurcation atherosclerosis. Quantitative correlation of plaque
localization with flow velocity profiles and wall shear stress," Circulation
research, vol. 53, pp. 502-514, 1983.
[3] P. F. Davies, C. F. Dewey Jr, S. R. Bussolari, E. J. Gordon, and M. A. Gimbrone
Jr, "Influence of hemodynamic forces on vascular endothelial function: in vitro
studies of shear stress and pinocytosis in bovine aortic cells," Journal of Clinical
Investigation, vol. 73, pp. 1121-1129, 1984.
[4] N. DePaola, M. A. Gimbrone Jr, P. F. Davies, and C. F. Dewey Jr, "Vascular
endothelium responds to fluid shear stress gradients," Arteriosclerosis and
Thrombosis, vol. 12, pp. 1254-1257, 1992.
[5] C. F. Dewey Jr, S. R. Bussolari, M. A. Gimbrone Jr, and P. F. Davies, "The
dynamic response of vascular endothelial cells to fluid shear stress," Journal of
biomechanical engineering, vol. 103, pp. 177-185, 1981.
[6] J. A. Frangos, T. Y. Huang, and C. B. Clark, "Steady shear and step changes in
shear stimulate endothelium via independent mechanisms--superposition of
transient and sustained nitric oxide production," Biochemical and Biophysical
Research Communications, vol. 224, pp. 660-665, 1996.
[7] J. J. Chiu, D. L. Wang, S. Chien, J. Skalak, and S. Usami, "Effects of disturbed
flow on endothelial cells," Journal of biomechanical engineering, vol. 120, pp. 2-
8, 1998.
[8] J. Hwang, A. Saha, Y. C. Boo, G. P. Sorescu, J. S. McNally, S. M. Holland, et al.,
"Oscillatory Shear Stress Stimulates Endothelial Production of O 2- from p 47
phox-dependent NAD (P) H Oxidases, Leading to Monocyte Adhesion," Journal
of Biological Chemistry, vol. 278, pp. 47291-47298, 2003.
16

[9] J. Hwang, H. Michael, A. Salazar, B. Lassegue, K. Griendling, M. Navab, et al.,
"Pulsatile versus oscillatory shear stress regulates NADPH oxidase subunit
expression: implication for native LDL oxidation," Circulation research, vol. 93,
pp. 1225-1232, 2003.
[10] R. Ross, "Atherosclerosis--an inflammatory disease," N Engl J Med, vol. 340, pp.
115-26, Jan 14 1999.
[11] D. D. Heistad, "Unstable coronary-artery plaques," The New England journal of
medicine, vol. 349, pp. 2285-2287, 2003.
[12] L. K. Curtiss, "Reversing atherosclerosis," N Engl J Med, vol. 360, pp. 1144-6,
Mar 12 2009.
[13] B. C. Berk, R. W. Alexander, T. A. Brock, M. A. Gimbrone Jr, and R. C. Webb,
"Vasoconstriction: a new activity for platelet-derived growth factor," Science, vol.
232, pp. 87-90, 1986.
[14] M. M. Brooks, S. C. Chung, T. Helmy, W. B. Hillegass, J. Escobedo, K. A.
Melsop, et al., "Health Status After Treatment for Coronary Artery Disease and
Type 2 Diabetes Mellitus in the Bypass Angioplasty Revascularization
Investigation 2 Diabetes Trial," Circulation, vol. 122, pp. 1690-1699, 2010.
[15] N. R. Madamanchi, A. Vendrov, and M. S. Runge, "Oxidative stress and vascular
disease," Arterioscler Thromb Vasc Biol, vol. 25, pp. 29-38, Jan 2005.
[16] Topper JN, Cai J, Falb D, and G. M. Jr., "Identification of vascular endothelial
genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2,
manganese superoxide dismutase, and endothelial cell nitric oxide synthase are
selectively up-regulated by steady laminar shear stress," Proc Natl Acad Sci U S
A., vol. 93, pp. 10417-22, 1996 Sep 17 1996.
[17] R. M. Nerem, R. W. Alexander, D. C. Chappell, R. M. Medford, S. E. Varner, and
W. R. Taylor, "The study of the influence of flow on vascular endothelial
biology," Am J Med Sci, vol. 316, pp. 169-75, 1998.
17

[18] O. Traub and B. C. Berk, "Laminar shear stress: mechanisms by which
endothelial cells transduce an atheroprotective force," Arterioscler Thromb Vasc
Biol, vol. 18, pp. 677-85, 1998.
[19] L. K. Curtiss, "Reversing atherosclerosis?," N Engl J Med, vol. 360, pp. 1144-6,
Mar 12 2009.
[20] R. Ross, "The pathogenesis of atherosclerosis: a perspective for the 1990s,"
Nature, vol. 362, pp. 801-809, 1993.
[21] V. Fuster, "Atherosclerosis, thrombosis, and vascular biology," in Cecil Medicine,
L. Goldman and D. Ausiello, Eds., 23rd ed Philadelphia, PA, USA: Saunders
Elsevier, 2007.
[22] L. Ai, M. Rouhanizadeh, J. C. Wu, W. Takabe, H. Yu, M. Alavi, et al., "Shear
stress influences spatial variations in vascular Mn-SOD expression: implication
for LDL nitration," American Journal of Physiology- Cell Physiology, vol. 294,
pp. C1576-C1585, 2008.
[23] C. Cheng, D. Tempel, R. van Haperen, H. C. de Boer, D. Segers, M. Huisman, et
al., "Shear stress-induced changes in atherosclerotic plaque composition are
modulated by chemokines," Journal of Clinical Investigation, vol. 117, pp. 616-
26, Mar 2007.
[24] H. Watkins and M. Farrall, "Genetic susceptibility to coronary artery disease:
from promise to progress," Nature Reviews Genetics, vol. 7, pp. 163-173, 2006.
[25] A. V. Finn, M. Nakano, J. Narula, F. D. Kolodgie, and R. Virmani, "Concept of
vulnerable/unstable plaque," Arteriosclerosis, Thrombosis, and Vascular Biology,
vol. 30, pp. 1282-1292, 2010.
[26] A. Didangelos, D. Simper, C. Monaco, and M. Mayr, "Proteomics of acute
coronary syndromes," Current atherosclerosis reports, vol. 11, pp. 188-195, 2009.
[27] A. Maseri and V. Fuster, "Is there a vulnerable plaque?," Circulation, vol. 107, pp.
2068-2071, 2003.
18

[28] S. Glagov, E. Weisenberg, C. K. Zarins, R. Stankunavicius, and G. J. Kolettis,
"Compensatory enlargement of human atherosclerotic coronary arteries," New
England Journal of Medicine, vol. 316, pp. 1371-1375, 1987.
[29] Z. Y. Li, S. P. S. Howarth, T. Tang, and J. H. Gillard, "How critical is fibrous cap
thickness to carotid plaque stability?: A flow-plaque interaction model," Stroke,
vol. 37, pp. 1195-1199, 2006.
[30] A. M. Varnava, P. G. Mills, and M. J. Davies, "Relationship between coronary
artery remodeling and plaque vulnerability," Circulation, vol. 105, pp. 939-943,
2002.
[31] P. Schoenhagen, K. M. Ziada, D. G. Vince, S. E. Nissen, and E. M. Tuzcu,
"Arterial remodeling and coronary artery disease: the concept of," Journal of the
American College of Cardiology, vol. 38, pp. 297-306, 2001.
[32] K. Thygesen, J. S. Alpert, and H. D. White, "Universal definition of myocardial
infarction," Circulation, vol. 116, pp. 2634-2653, 2007.
[33] J. L. Reeve, A. M. Duffy, T. O'Brien, and A. Samali, "Don't lose heart--
therapeutic value of apoptosis prevention in the treatment of cardiovascular
disease," J Cell Mol Med, vol. 9, pp. 609-22, Jul-Sep 2005.
[34] C. Murray and A. Lopez, "WHO World Health Report " World Health
Organization, Geneva, Switzerland2002.
[35] E. Boersma, N. Mercado, D. Poldermans, M. Gardien, J. Vos, and M. L. Simoons,
"Acute myocardial infarction," The Lancet, vol. 361, pp. 847-858, 2003.
[36] R. Ross, "Atherosclerosis is an inflammatory disease," American Heart Journal,
vol. 138, pp. S419-20, 1999.
[37] J. Sanz and Z. A. Fayad, "Imaging of atherosclerotic cardiovascular disease,"
Nature, vol. 451, pp. 953-957, 2008.
19

[38] J. L. Anderson, C. D. Adams, E. M. Antman, C. R. Bridges, R. M. Califf, D. E.
Casey Jr, et al., "ACC/AHA 2007 Guidelines for the Management of Patients
With Unstable Angina/Non-ST-Elevation Myocardial Infarction--Executive
Summary," Journal of the American College of Cardiology, vol. 50, pp. 652-726,
2007.
[39] Wikipedia. (Mar 24). "Atherosclerosis":
http://en.wikipedia.org/wiki/Atherosclerosis. Available:
http://en.wikipedia.org/wiki/Atherosclerosis
[40] R. P. Choudhury, V. Fuster, J. J. Badimon, E. A. Fisher, and Z. A. Fayad, "MRI
and characterization of atherosclerotic plaque: emerging applications and
molecular imaging," Arterioscler Thromb Vasc Biol, vol. 22, pp. 1065-1074, 2002.
[41] A. Schmermund and R. Erbel, "Unstable coronary plaque and its relation to
coronary calcium," Circulation, vol. 104, pp. 1682-1687, 2001.
[42] "American Heart Association, "Heart Disease and Stroke Statistics - 2006
Update"," American Heart Association2006.
[43] S. Zeibig, Z. Li, S. Wagner, H. P. Holthoff, M. Ungerer, A. Bultmann, et al.,
"Effect of the oxLDL Binding Protein Fc-CD68 on Plaque Extension and
Vulnerability in Atherosclerosis," Circulation research, vol. 108, pp. 695-703,
2011.
[44] K. R. Aroom, M. T. Harting, C. S. Cox, R. S. Radharkrishnan, C. Smith, and B. S.
Gill, "Bioimpedance Analysis: A Guide to Simple Design and Implementation,"
Journal of Surgical Research, vol. 153, pp. 23-30, 2009.
[45] L. A. Geddes, "Historical evolution of circuit models for the electrode-electrolyte
interface," Ann Biomed Eng, vol. 25, pp. 1-14, 1997.
[46] I. Streitner, M. Goldhofer, S. Cho, H. Thielecke, R. Kinscherf, F. Streitner, et al.,
"Electric impedance spectroscopy of human atherosclerotic lesions,"
Atherosclerosis, vol. 206, pp. 464-468, 2009.
20

[47] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an intravascular
impedance catheter for detection of fatty lesions in arteries," IEEE transactions
on medical imaging, vol. 16, pp. 439-446, 1997.
[48] K. R. Foster and H. P. Schwan, "Dielectric properties of tissues," Handbook of
biological effects of electromagnetic fields, pp. 25-102, 1996.
[49] T. Suselbeck, H. Thielecke, J. Kochlin, S. Cho, I. Weinschenk, J. Metz, et al.,
"Intravascular electric impedance spectroscopy of atherosclerotic lesions using a
new impedance catheter system," Basic research in cardiology, vol. 100, pp. 446-
452, 2005.
[50] C. Hahn and M. A. Schwartz, "The role of cellular adaptation to mechanical
forces in atherosclerosis," Arterioscler Thromb Vasc Biol, vol. 28, pp. 2101-7,
Dec 2008.
[51] E. R. Porrello, A. I. Mahmoud, E. Simpson, J. A. Hill, J. A. Richardson, E. N.
Olson, et al., "Transient Regenerative Potential of the Neonatal Mouse Heart,"
Science, vol. 331, pp. 1078-1080, 2011.
[52] P. C. Hsieh, V. F. Segers, M. E. Davis, C. MacGillivray, J. Gannon, J. D.
Molkentin, et al., "Evidence from a genetic fate-mapping study that stem cells
refresh adult mammalian cardiomyocytes after injury," Nature Medicine, vol. 13,
pp. 970-4, Aug 2007.
[53] O. Bergmann, R. D. Bhardwaj, S. Bernard, S. Zdunek, F. Barnabe-Heider, S.
Walsh, et al., "Evidence for cardiomyocyte renewal in humans," Science, vol. 324,
pp. 98-102, Apr 3 2009.
[54] K. Bersell, S. Arab, B. Haring, and B. Kuhn, "Neuregulin1/ErbB4 signaling
induces cardiomyocyte proliferation and repair of heart injury," Cell, vol. 138, pp.
257-70, Jul 23 2009.
[55] C. Mason and P. Dunnill, "A brief definition of regenerative medicine,"
Regenerative Medicine, vol. 3, pp. 1-6, 2008.
21

[56] "Regenerative medicine glossary," Regenerative Medicine, vol. 4 (4 Suppl), pp.
S1 - 88, 2009.
[57] C. E. Murry, H. Reinecke, and L. M. Pabon, "Regeneration gaps: observations on
stem cells and cardiac repair," Journal of the American College of Cardiology,
vol. 47, pp. 1777-1785, 2006.
[58] W. Roell, T. Lewalter, P. Sasse, Y. N. Tallini, B. R. Choi, M. Breitbach, et al.,
"Engraftment of connexin 43-expressing cells prevents post-infarct arrhythmia,"
Nature, vol. 450, pp. 819-824, 2007.
[59] K. D. Poss, L. G. Wilson, and M. T. Keating, "Heart regeneration in zebrafish,"
Science, vol. 298, pp. 2188-90, Dec 13 2002.
[60] N. C. Chi, R. M. Shaw, B. Jungblut, J. Huisken, T. Ferrer, R. Arnaout, et al.,
"Genetic and physiologic dissection of the vertebrate cardiac conduction system,"
Public Library of Science - Biology, vol. 6, pp. 1006 - 1019, 2008.
[61] D. Sedmera, M. Reckova, A. deAlmeida, M. Sedmerova, M. Biermann, J.
Volejnik, et al., "Functional and morphological evidence for a ventricular
conduction system in zebrafish and Xenopus hearts," Am J Physiol Heart Circ
Physiol, vol. 284, pp. H1152-60, Apr 2003.
[62] R. Arnaout, T. Ferrer, J. Huisken, K. Spitzer, D. Y. R. Stainier, M. Tristani-
Firouzi, et al., "Zebrafish model for human long QT syndrome," Proceedings of
the National Academy of Sciences of the United State of America, vol. 104, pp.
11316 - 11321, 2007.
[63] R. Kopp, T. Schwerte, and B. Pelster, "Cardiac performance in the zebrafish
breakdance mutant," Journal of Experimental Biology, vol. 208, pp. 2123-2134,
2005.
[64] D. J. Milan, I. L. Jones, P. T. Ellinor, and C. A. MacRae, "In vivo recording of
adult zebrafish electrocardiogram and assessment of drug-induced QT
prolongation," Am J Physiol Heart Circ Physiol, vol. 291, pp. H269 - H273, 2006.
22

[65] C. Nusslein-Volhard (Editor) and R. Dahm (Editor), Zebrafish. New York:
Oxford University Press, 2002.
[66] D. Y. Stainier, "Zebrafish genetics and vertebrate heart formation," Nat Rev Genet,
vol. 2, pp. 39-48, Jan 2001.
[67] K. Kikuchi, J. E. Holdway, A. A. Werdich, R. M. Anderson, Y. Fang, G. F.
Egnaczyk, et al., "Primary contribution to zebrafish heart regeneration by gata4+
cardiomyocytes," Nature, vol. 464, pp. 601-605, 2010.
[68] P. Sun, Y. Zhang, F. Yu, E. Parks, A. Lyman, Q. Wu, et al., "Micro-
electrocardiograms to study post-ventricular amputation of zebrafish heart," Ann
Biomed Eng, vol. 37, pp. 890-901, May 2009.
[69] D. J. Milan and C. A. MacRae, "Animal models for arrhythmias," Cardiovasc Res,
vol. 67, pp. 426-37, Aug 15 2005.

23

Chapter One: Electrochemical Impedance Spectroscopy to
Assess Vascular Oxidative Stress
1

1
Disclaimer: the content of this chapter has been published as “Electrochemical
Impedance Spectroscopy to Assess Vascular Oxidative Stress”, Annals of Biomedical
Engineering, 39(1): 287-296 (2011).
Acknowledgement: Co-authors of this manuscript include: Fei Yu, Rongsong Li, Lisong
Ai, Collin Edington, Hongyu Yu, Mark Barr, Eun Sok Kim, Tzung K. Hsiai. The author
of this thesis credits other co-authors for their significant input in the process of preparing
the manuscript for publication.
24
1.1 Chapter One Introduction
Oxidized low density lipoprotein (oxLDL) and macrophage infiltrates contribute to pro-
inflammatory states relevant to the initiation of atherosclerotic lesions [70]. These lesions
can be classified as stable or unstable plaque. The latter is often non-obstructive by
conventional X-ray angiography or intravascular ultrasound (IVUS), and is prone to
rupture, leading to acute coronary syndromes or stroke [71-73]. Unstable plaque or
otherwise known as thin-cap fibroatheroma, represents a transitional plaque that is
characterized by a thin fibrous cap (<65 µ m), a large necrotic core, an abundance of
macrophages, and limited luminal narrowing [70]. However, detection and diagnosing
the non-obstructive, albeit pro-inflammatory, lesions during catheterization remains a
clinical challenge.
Up to date, there are no diagnostic tools to detect intravascular oxidative stress and pro-
inflammatory states when patients undergo angiograms. Serum biomarkers have been
proposed for predicting pro-inflammatory states. Examples include C-reactive protein,
matrix metalloproteinases, CD40 ligand, Lp-PLA2 (lipoprotein-associated phospholipase
A2) and myeloperoxidase. Specifically, circulating Lp-PLA2 is bound predominantly to
low-density lipoprotein (LDL) (~80%) which transmigrated to the subendothelial space
of atherosclerosis-prone regions [74]. Imaging modalities including magnetic resonance
imaging (MRI) and electron beam computed tomography (EBCT) have also been
proposed to predict unstable plaque [75, 76]. However, assessing stable versus unstable
atherosclerotic lesions during catheterization remains an unmet clinical need.
Atherosclerotic lesions display distinct electrochemical properties [77, 78]. Pre-
atherosclerotic lesions harbor pro-inflammatory substrates; namely, oxLDL and
macrophage-derived foam cells infiltrates, which engender distinct endoluminal
electrochemical impedance or otherwise quantified as electrochemical impedance
spectroscopy (EIS) [78, 79]. Previously, changes in bulk resistance were prevalent in the
atherosclerotic lesions as measured by the linear 4-point probe [77-79]. Electric
impedance (Z) develops as a function of frequency in response to the applied alternative
current (AC) to the biological tissue. Recently, a catheter-based linear 4-point
microelectrode was reported to assess EIS in New Zealand White rabbits [77, 78, 80]. In
our study, we introduced concentric bipolar microelectrodes to further address the non-
uniform and complex tissue current distribution, uneven endoluminal topography and
non-uniform current distribution, in both rabbit aortas and human descending aortas. Our
findings indicate the potential application of concentric bipolar microelectrodes to
25
measure electrochemical impedance in regions of pro-inflammatory states with high
spatial resolution.
26
1.2 Chapter One Materials and methods
1.2.1 Rabbit aortas and explants of human descending aortas
Two male New Zealand White (NZW) rabbits (10-week-old) were acquired from a local
breeder (Irish Farms, Norco, CA) and maintained by the USC vivaria in accordance with
the National Institutes of Health guidelines. After a 7-day quarantine period, they were
fed with a high fat, high cholesterol diet (Newco® 1.5% cholesterol & 6% peanut oil)
for 8 weeks. The rabbits were then euthanized with an overdose of intramuscular
injection of ketamine (Fort Dodge Laboratories, Inc) combined with 1 mg/kg
Acepromazine (Aveco Co.). The hearts and aorta were resected for ex vivo study and
immunohistochemistry. All experimental procedures were performed in compliance with
the Institutional Animal Care and Use Committee in the Heart Institute of the Good
Samaritan Hospital, Los Angeles, which is accredited by the American Association for
Accreditation for Laboratory Animal Care.
Also, six explants of human descending aortas and common carotid arteries were
collected from cardiac transplant patients or National Disease Research Interchange
(NDRI) for study in compliance with the University Institutional Review Board.
1.2.2 Operating principle and equivalent circuit model
Electrical impedance (Z) is a measure of the opposition to electrical flow through a
substance, a complex quantity combining resistance and reactance as a function of
frequency when an alternative current (AC) was applied. The value of impedance is
conventionally represented as a complex number (Z = R + iXc), consisting of the real
number R for the resistance and the complex number iXc for the reactance [81]. Blood
vessels harbor resistance and store charges, exhibiting complex electric impedances as a
function of frequency. Hence, we were able to determine the lesions’ frequency-
dependent electrical and dielectrical behavior by recording the electric impedance of a
tissue over a frequency range[82].
Biological tissue impedance measured by metallic electrodes entailed electrode-
electrolyte interface. Investigations on the electrochemical properties at the interface can
be dated to as early as 1871 [83], and various equivalent circuit models have been
27
proposed. To date, a simple yet efficient model for the electrochemical impedance in
tissue is represented as a parallel interface capacitance impedance, C P, shunted by a
charge transfer resistance RP, in series with the tissue resistance RS (Fig. 4a) [81]. A
more sophisticated model replaces CP with a constant phase angle impedance ZCPA (Fig.
4b), a measurement for the non-Faraday impedance arising from the interface
capacitance and non-homogeneities [84], which can be expressed by the empirical
relation:
(1)
with n being a constant (0 < n < 1) for the non-homogeneities of the surface and ω being
angular frequency equal to 2πf. If n = 1, the Q is equal to CP, and ZCPA represents a
purely capacitive impedance element corresponding to the interface capacitance [84, 85].
In the case of concentric bipolar electrode experiments (Fig. 4c), the RS is mainly
dependent on the tissue composition and the geometric dimension of the tissue between
the inner working electrode and the outer counter electrode, whereas the RP and ZCPA are
predominantly dependent on the tissue dielectric and conductive properties as well as the
geometric dimension between the two electrodes.
1.2.3 Ex vivo electrochemical impedance spectroscopy (EIS) measurements
The rabbit aorta was flushed with physiological saline solution and resected
longitudinally to expose the inner lumen. Tissue specimens at approximately 2cm in
length were isolated from the aortic arch, thoracic aorta and abdominal aorta,
respectively. Gross histology of atherosclerotic lesions was identified for the individual
specimens (Fig. 5). For the human artery specimens, en face arteries were sectioned
longitudinally to reveal endoluminal surface. Two specimens were observed to have
mild to prevalent fatty streaks (Fig. 6). The tissues were maintained in the phosphate
buffered saline (PBS).
Endoluminal EIS measurements were performed at multiple sites associated with the
plaque lesions and compared to the healthy arterial lumens and to PBS. More than 3
replicates were performed at each site. The concentric bipolar microelectrodes (FHC co.
ME, USA) consisted of working and reference electrodes; the former was the inner pole
made of the platinum at 75µ m in diameter and the latter was the stainless steel outer
28
shell made of a at 300µ m in diameter. An Ag/AgCl electrode immersed in the PBS
solution was used as a reference electrode. EIS measurements were performed by using
a Gamry Series G 300 potentiostat (Gamry Instruments, PA) installed in a Dell desktop
computer. An input of 10mV peak-to-peak AC voltage and a frequency decay ranging
from 300 kHz to 100 Hz were delivered to the sites. The magnitudes and phases of the
impedance were acquired at 20 data points per frequency decade. To test whether the
depth of PBS solution and orientation of contact with the specimens would interfere with
the EIS recordings, we mounted the concentric bipolar microelectrode on a micro-
manipulator (World Precision Instruments Inc., FL, USA). EIS measurements were
performed at the identical point of interest for the electrodes submersed in 4 various
depths in PBS solution while in contact with the specimen, or the specimens were
rotated around the electrodes by 90
o
.
1.2.4 Immunohistochemistry
Vascular rings or stripes were cut from the rabbit aorta or human artery immediately
after the specimens were collected and measured, and immersed in 4%
paraformaldehyde for 24 hours. They were then frozen in optical cutting temperature
compound (Sakura Finetek, Torrance, Calif) for histopathological analysis. Serial 5-µ m
cryosections were cut. Immunostaining was performed with standard

techniques in
frozen vascular tissue using biotinylated secondary

antibodies and streptavidin-
conjugated horse radish peroxidase (HRP). Chromogen Diaminobenzidine (DAB) was
used as the substrate of HRP and the sections were counterstained

with hematoxylin for
the visualization of intima, media, smooth muscle cells and adventitia. Plaque
macrophages, or foam cells, were identified. Tissue sections were viewed with a
microscope (Leica DM LB2, Leica Microsystems, Germany) and images were captured
with a CCD digital camera (Spot RT-KE, Diagnostic Instruments, MI, USA). Evaluation
of the plaque histology was performed according to the modified AHA classification of
atherosclerotic lesions [78, 86-88]. For further evaluation of the local oxidation stress,
foam cells were identified by Sudan black stain [89-91], macrophages with anti-CD68
antibody, and oxLDL with mAb4E6 [91]. H&E and von Kossa staining were used to
demonstrate calcification. A color intensity threshold mask for immunostaining was
defined to detect foam cells and oxLDL.

29
1.2.5 Statistical Analysis
Histological plaque characteristics were qualitatively ranked in four groups
(no/minor/moderate/severe oxidative stress). Statistical analyses were performed using
two-tailed T-test for two groups of data, or one-way ANOVA for multi-group
comparison with the statistical software package SPSS 15.0 (SPSS Inc. Chicago, Ill). P
values of less than 0.05 were considered statistically significant. Power analysis
determined sample size (N) for rabbits.
30
1.3 Chapter One Results
1.3.1 Endoluminal EIS measurements at various microelectrode submersion and
tissue orientation
En face segments of rabbit aorta revealed prevalent pre-atherosclerotic lesions. After 8
weeks of high fat diet, pre-atherosclerotic lesions were prominent in the aortic arch in
which disturbed flow occurred (Fig. 5a), whereas the segment distal to the aortic arch
was spared of visible lesions (Fig. 5b). Next, EIS measurements were performed in
relation to the depth of microelectrode submersion and orientation of the specimens.
Both EIS and phase measurements were identical as the electrode was positioned from 0
to 4 mm below PBS solution surface (Fig. 7a). At 300 kHz, the lowest impedance was
measured at ~ 4000 , corresponding to a phase value of about -15˚, indicating near-
ohmic resistance. Furthermore, impedance was inversely proportional to the frequencies,
indicating the effect of double layer capacitance at the electrode/electrolyte interface.
Moreover, EIS measurements were compared at two different sites of the endoluminal
surface (Fig. 7b). Both EIS and phase measurements remained unchanged despite
rotation of the specimens at 90
o
. Hence, the impedance readings acquired by the
concentric bipolar microelectrodes were unaffected by the depth of electrode submersion
and orientation of specimens.
1.3.2. Endoluminal EIS measurements between healthy and atherosclerotic lesions
in rabbits
EIS measurements were compared between the healthy and atherosclerotic tissues from
the two rabbits. Five representative healthy sites and atherosclerotic sites were assessed
(from Fig. 2a) and significant differences in EIS measurements were observed between
10 kHz to 100 kHz in the aortic arch (Fig. 8a). Bar graphs further corroborated
statistically significant differences in EIS values (P < 0.01, n = 10) (Fig. 8b). The
impedances of the lesions were nearly 2-fold higher than those of the healthy sites.
Hence, concentric bipolar microelectrodes offered a reliable entry point to assess
endoluminal EIS for plaque diagnosis.

31
1.3.3. Linking electrochemical impedance with immunohistochemistry
We demonstrated EIS measurements in the non-obstructive, albeit pro-inflammatory,
pre-atherosclerotic lesions in both aortic arch and descending aortas isolated from the
fat-fed rabbits. The pre-atherosclerotic lesions, occupying less than 20% of the luminal
diameter, were notable for both intimal thickening and intimal xanthoma (Fig. 9), which
corresponded to the EIS measurements as illustrated in figures 4. Intimal thickening
consisted of mainly smooth muscle cells in a proteoglycan-rich matrix, whereas intimal
xnathoma or otherwise known as a fatty streak primarily contained macrophage-derived
foam cells (Fig. 9c), T lymphocytes (not stained), and varying degrees of smooth muscle
cells (SMC) [87]. Initmal thickening further revealed positive staining for oxidized low
density lipoprotein (oxLDL) (Figs. 9d). Calcification was absent as evidenced by the
negative von Kossa staining. These lesions possessing minor to medium oxidative stress
were considered clinically silent and were previously reported to have no significantly
different impedance spectrum compared healthy aorta [77]. However, concentric bipolar
microelectrodes enabled the detection of endoluminal impedance spectrum changes in
the presence of oxLDL and macrophage/foam cell infiltrates.
1.3.4 Linking electrochemical impedance with fatty streaks in human descending
aortas
Fatty streak or otherwise known as early atheromas harbor oxLDL and
microphages/foam cell infiltrates [87]. En face segments of human descending aorta and
carotid arteries revealed numerous fatty streaks for which endoluminal EIS
measurements were performed (Fig. 6). Significant differences in EIS measurements
were observed between 10 kHz to 100 kHz in the descending aortas (Fig. 10a and 11a).
Bar graphs further corroborated statistically significant differences in EIS values (Fig.
10b and 11b) (p < 0.05, n = 3, and p < 0.01, n=6, respectively). The impedances of the
fatty streaks were nearly 1.5-fold higher than those of the healthy sites.
Immunohitochemistry analysis revealed presence of oxLDL (Fig. 11c and 11d) in the
lesion region. Hence, endoluminal EIS measurements provided distinct electrochemical
impedance in regions properties of pre-atheromas from explants of human arteries.

32
1.4 Chapter One Discussion
To better characterize the biochemical properties of non-obstructive, albeit pro-
inflammatory, lesions, we demonstrated the application of endoluminal electrochemical
impedance spectroscopy (EIS). EIS measures the dielectric properties of a specimen as a
function of frequency [92]. It is based on the interaction of an external field with the
electric dipole moment of the samples, often expressed by permittivity [93]. The novelty
of our current study was to address biological tissues that harbor both energy storing and
dissipation properties. The configuration of concentric bipolar microelectrodes allowed
for consistent and reliable electrical impedance measurement despite the non-
homogeneous composition and non-uniform current distribution in the lesions.
We showed that EIS measurements were significantly elevated in the pre-atherosclerotic
lesions or otherwise known as fatty streaks or deposition in which oxLDL and
macrophage/foam cell infiltrates were prevalent. Thus, linking endoluminal EIS
measurements with pre-atherosclerotic lesions holds promise to develop intravascular-
based sensors and to predict pro-inflammatory lesions when the patients are undergoing
catheterization.
The use of a linear array of four microelectrodes based on microfibrication and
semiconductor technology was recently reported [78]. A balloon catheter system with an
integrated polyiminde-based microelectrode structure was introduced into the aorta and
the impedance was measured at frequency range of 1 kHz - 10 KHz. Then the
corresponding histomorphometric data of the aortic segments were compared [77]. The
investigators provided the first catheter-based four point electrodes arranged axially with
a diameter of 100 m and a spacing of 333 µ m apart for impedance measurement in the
New Zealand White rabbits on high fat diet.
Unlike the linear array of electrodes, concentric configuration further allowed for EIS
measurement independent of the depth of the PBS submersion and orientation of the
tissues. Electrical currents flow preferably via the least resistive pathways; that is, the
shortest conducting paths between the central electrode and the outer shell of the
concentric bipolar microelectrodes. Because of the micro-scale of the concentric
electrodes, the impedance measurement is mainly sensitive to the electrochemical
properties of the tissue at close proximity, and changes in volume of saline solution
would not alter the impedance reading. Our ex vivo investigation can be extrapolated to
33
perform in vivo investigation in which the impedance measurements will be independent
of lumen diameters, blood volumes and flow rates as long as the contact is made between
microelectrodes and endoluminal surface.
Up to date, little is known about the numerous variables affecting assessment of
atherosclerotic lesions. Investigations have been centered around atherosclerotic lesion’s
lipid pool content, calcification, fibrous cap, and intima/media thickness [72]. The
emerging vascular oxidative hypothesis of unstable plaque is that atherosclerotic lesion is
deemed a dynamic process [94, 95]. Reactive oxygen species via NADPH oxidase
enzyme system and the release of matrix metalloproteinase (MMP’s) by the
inflammatory cells contributes to the vulnerability of a rupture-prone plaque [96]. These
macrophage-derived foam cells are trapped by interaction with oxidized low-density
lipoprotein (LDL) and can be mobilized by dynamic exposure in response to key
antioxidants such as apocynin, N-acetylecysteine (NAC) as well as resveratrol, a
polyphenolic compound in grapes and red wine [97].
In this study, we focused on the specific content of oxidative stress; namely, oxLDL and
foam cell infiltrates, and assessed both aortas of New Zealand White rabbits and explants
of human arteries. The ability to obtain reliable EIS measurements with high special
resolution provides a basis to further deploy the catheter-based concentric bipolar
microelectrodes for endoluminal EIS assessment in vivo. Ideally, EIS sensors can be
incorporated onto a steerable catheter accompanied with intravascular ultrasound (IVUS)
to scan the circumferential profile of the atherosclerotic [98]. In fact, EIS measurement
can be performed at multiple sites for a single lesion to generate a contour map
containing both topographical and electrochemical information. In addition, EIS
measurements can be potentially incorporated with intracardiac echocardiogram [99],
optical coherence tomography (OCT) [100, 101], and/or micro-thermal sensors [102] to
further enhance the sensitivity and specificity for the assessment of pro-inflammatory
states or unstable plaque.
Distinct from the linear four point electrode arrays, our methodology employed the
concentric bipolar electrodes (FHC
®
), allowing for reproducible assessment for vascular
regions harboring vascular oxidative stress in terms of oxLDL and foam cells [103]. The
unique feature of the concentric electrodes included the constant and symmetric
displacement between the working and counter electrodes. Similar to the previously
reported EIS [77, 78], specimens that harbor oxidative stress generated distinctly higher
EIS values compared to the healthy tissues over a range of frequency from 10 KHz to
34
100 kHz. Moreover, the EIS measurements were independent of the surrounding
medium and orientation of the specimens.
Severe stenosis reduces arterial blood flow and causes high tensile and compressive stress
in the stenotic plaque [104]. Oxidative stress and pro-inflammatory states modulate
mechanical vulnerability of plaque [105]. Oxidative stress is involved in the oxidation /
modification of low-density lipoprotein (oxLDL) [106-108], which occurs at high levels
in the atherogenic prone regions of aorta and plays a critical role in pro-inflammatory
states [109]. Mounting evidence supports that oxLDL and macrophage/foam cell rich
shoulder areas are more prone to disruption, leading to thrombus formation and embolic
events [110]. Hence, the application of EIS to assess pre-atherosclerotic lesions will
likely provide a means to measure electrochemical properties in regions of pro-
inflammatory states that harbor both oxLDL and foam cells. Further characterization of
EIS in relation to histology is warranted to stratify the extent of oxidative stress and pro-
inflammatory states to identify patients in whom selective intervention may be indicated.
In summary, we demonstrated a link between electrochemical properties and
vascular oxidative stress in the pre-atherosclerotic lesions in the aortas of New Zealand
White (NZW) rabbits and explants of human descending aortas. These distinct
electrochemical properties of the lesions correlated with the substrates of vascular
oxidative stress; namely, oxLDL and macrophage/foam cell infiltrates. It has been
demonstrated that the fat present in the plaques have high specific electrical resistivity
compared to other possible plaque compounds [111]. Atheromas at various stages
contain differential levels of lipid-rich components, rendering a differential level of
oxidative stress that can be potentially identified by the magnitude of tissue impedance.
35

Figure 4. Equivalent circuit and configuration of concentric bipolar electrode. (a)
Equivalent circuit model of electrode-tissue interface, with interface reactance
represented by a single capacitor Cp. Rp: charge transfer resistance, Rs:
Tissue/electrolyte resistance. (b) Equivalent circuit model of electrode-tissue interface,
with interface reactance represented by a constant phase element (Zcpa). (c) Illustration
of experimental setup. Ag/AgCl reference electrode, Platinum core of the concentric
bipolar electrode and Stainless steel outer shell of the bipolar electrode were connected
to the potentiostat as the reference, working, and counter electrode, respectively. (d)
Illustration of the geometry and dimension of the bipolar electrode tip.
36

Figure 5. Rabbit aorta in response to 8 weeks of high-fed diet. (a) En face segment of
aortic arch. Gross pathology revealed atherosclerotic lesions on the endoluminal surface.
(b) En face segment from the descending aorta. Small lesions with a diameter less than
1mm were present proximal to the aortic arch. The remaining endoluminal surface was
grossly spared of visible lesion.
37

Figure 6. Ex vivo segments of human descending thoracic aorta and common
carotid artery. (a) En face thoracic aorta, revealing several orifices to the intercostal
branches (black arrows) and fatty streaks (red circles) corresponding to the EIS readouts
in Figure 7a. (b) Numerous fatty streaks (red circles) and apparently normal endothelium
(black circles) corresponded to the EIS readouts in Figure 8a. Cross-sectional
histological analyses were performed along blue dashed lines (Figure 8c and 8d).
38

Figure 7. EIS measurements in relation of the depth of microelectrode submersion
and orientation of the specimens. (a) EIS and phase data of rabbit artery tissue
submerged in PBS solution. The lowest impedance was about 4000 Ohm at 300 kHz
(left panel). The corresponding phase value was close to -1.5
o
indicating that the
impedance was mostly in the ohmic resistance range (right panel). (b) EIS and phase
data were compared between two sites. The impedance and phase readings were
independent of the orientation of the specimens.
39

Figure 8. Endoluminal EIS measurements in the aortic arch of NZW rabbits (a)
EIS measurements were performed on 5 normal endoluminal sites and 5 plaque sites of
the aortic arch tissue from two high-fat diet fed rabbits. Average impedance values at
frequency range 100 Hz to 300 kHz for healthy and plaque tissue from Rabbit 1 and
Rabbit 2 were presented as Normal 1, Normal 2 (healthy tissue) and Plaque 1 and Plaque
2 (Plaque tissue), respectively (n = 5 for each group). Vertical bar denotes standard
deviation. Baseline impedance measured from PBS solution alone was illustrated as a
reference. (b) Difference in measured impedance from normal and plaque tissue were
the most significant between operating frequency 10 kHz to 100 kHz (*P < 0.01, n =
10).
40

Figure 9. Preatherosclerotic aortic arch lesions. (a) Aortic arch isolated from the
rabbit on normal diet with absence of foam cells. (b) Corresponding
immunohistochemiscal staining showed negative mAb4E6 stain for oxidized low density
lipoprotein (oxLDl). (c) Intimal thickening and intimal xanthoma were prevalent in the
aortic arch isolated from the rabbit with exposure to high fat diet. Intimal thickening
consists of mainly smooth muscle cells in a proteoglycan-rich matrix, whereas intimal
xnathoma or otherwise known as a fatty streak primarily contains macrophage-derived
foam cells, T lymphocytes, and varying degrees of smooth muscle cells (SMC). (d)
Subendothelial layers were stained positive with mAb4E6 for oxLDL. Von Kossa
staining was negative for calcification.
41

Figure 10. Endoluminal EIS measurements from the human descending aorta. (a)
EIS data results were performed on the thoratic aorta. Significantly higher impedance
values were recorded from the fatty streaks over the frequency range from 10 to 100 kHz
compared to the healthy endoluminal surface. (b) T-test verified the statistically
significant difference in impedance measured at 95kHz, 30kHz and 9.5kHz between the
healthy and plaque tissues (* P < 0.05, n = 3).
42

Figure 11. EIS measurements in explants of human carotid arteries. (a) EIS
measurements were performed on 6 control endoluminal sites and 6 lesion sites of
human carotid arteries (n = 6). Average impedance values at frequency range 1000 Hz
to 300 kHz for control and lesion sites were presented as control versus lesion,
respectively (n = 6). Vertical bar denotes standard deviation. Baseline impedance
measured from PBS solution alone was illustrated as a reference. (b) Difference in
measured impedance from control and lesion sites were the most significant between
operating frequency 10 kHz to 100 kHz (*P < 0.01, n = 6). (c) Immunohistochemistry
was performed at the control site. (d) At the lesion site, subendothelial layers were
stained positive with mAb4E6 for oxidized low density lipoprotein (oxLDL).
43
References
[70] Y. Vengrenyuk, S. Carlier, S. Xanthos, L. Cardoso, P. Ganatos, R. Virmani, et al.,
"A hypothesis for vulnerable plaque rupture due to stress-induced debonding
around cellular microcalcifications in thin fibrous caps," Proc Natl Acad Sci U S
A, vol. 103, pp. 14678-83, Oct 3 2006.
[71] J. A. Ambrose, M. A. Tannenbaum, D. Alexopoulos, C. E. Hjemdahl-Monsen, J.
Leavy, M. Weiss, et al., "Angiographic progression of coronary artery disease and
the development of myocardial infarction," Lead Article, vol. 12, pp. 184-202,
1988.
[72] V. Fuster, L. Badimon, J. J. Badimon, and J. H. Chesebro, "The pathogenesis of
coronary artery disease and the acute coronary syndromes," The New England
Journal of Medicine, vol. 326, pp. 242-250, 1992.
[73] W. C. Little, M. Constantinescu, R. J. Applegate, M. A. Kutcher, M. T. Burrows,
F. R. Kahl, et al., "Can coronary angiography predict the site of a subsequent
myocardial infarction in patients with mild-to-moderate coronary artery disease?,"
Circulation, vol. 78, pp. 1157-1166, 1988.
[74] R. Ross, "Atherosclerosis is an inflammatory disease," Am Heart J, vol. 138, pp.
S419-420, 1999.
[75] R. P. Choudhury, V. Fuster, J. J. Badimon, E. A. Fisher, and Z. A. Fayad, "MRI
and characterization of atherosclerotic plaque: emerging applications and
molecular imaging," Arterioscler Thromb Vasc Biol, vol. 22, pp. 1065-1074, 2002.
[76] A. Schmermund and R. Erbel, "Unstable coronary plaque and its relation to
coronary calcium," Circulation, vol. 104, pp. 1682-1687, 2001.
[77] T. Suselbeck, H. Thielecke, J. Kochlin, S. Cho, I. Weinschenk, J. Metz, et al.,
"Intravascular electric impedance spectroscopy of atherosclerotic lesions using a
new impedance catheter system," Basic research in cardiology, vol. 100, pp. 446-
452, 2005.
44
[78] I. Streitner, M. Goldhofer, S. Cho, H. Thielecke, R. Kinscherf, F. Streitner, et al.,
"Electric impedance spectroscopy of human atherosclerotic lesions,"
Atherosclerosis, vol. 206, pp. 464-468, 2009.
[79] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an intravascular
impedance catheter for detection of fatty lesions in arteries," IEEE transactions
on medical imaging, vol. 16, pp. 439-446, 1997.
[80] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an intravascular
impedance catheter for detection of fatty lesions in arteries," IEEE Transac Med
Imaging, vol. 16, pp. 439-446, 1997.
[81] K. R. Aroom, M. T. Harting, C. S. Cox, R. S. Radharkrishnan, C. Smith, and B. S.
Gill, "Bioimpedance Analysis: A Guide to Simple Design and Implementation,"
Journal of Surgical Research, vol. 153, pp. 23-30, 2009.
[82] K. R. Foster and H. P. Schwan, "Dielectric properties of tissues," Handbook of
biological effects of electromagnetic fields, pp. 25-02, 1996.
[83] L. A. Geddes, "Historical evolution of circuit models for the electrode-electrolyte
interface," Ann Biomed Eng, vol. 25, pp. 1-14, 1997.
[84] W. Franks, I. Schenker, P. Schmutz, and A. Hierlemann, "Impedance
characterization and modeling of electrodes for biomedical applications," IEEE
Transac Biomed Eng, vol. 52, pp. 1295-1302, 2005.
[85] E. T. McAdams, A. Lackermeier, J. A. McLaughlin, D. Macken, and J. Jossinet,
"The linear and non-linear electrical properties of the electrode-electrolyte
interface," Biosensors and Bioelectronics, vol. 10, pp. 67-74, 1995.
[86] H. C. Stary, "Natural history and histological classification of atherosclerotic
lesions: an update," Arterioscler Thromb Vasc Biol, vol. 20, pp. 1177-1178, 2000.
[87] R. Virmani, F. D. Kolodgie, A. P. Burke, A. Farb, and S. M. Schwartz, "Lessons
from sudden coronary death: a comprehensive morphological classification
scheme for atherosclerotic lesions," Arterioscler Thromb Vasc Biol, vol. 20, pp.
1262-1275, 2000.
45
[88] H. C. Stary, A. B. Chandler, R. E. Dinsmore, V. Fuster, S. Glagov, W. J. Insull, et
al., "A definition of advanced types of atherosclerotic lesions and a histological
classification of atherosclerosis.," Arterioscler Thromb Vasc Biol, vol. 15, pp.
1512-1531, 1995.
[89] P. Verhamme, R. Quarck, H. Hao, M. Knaapen, S. Dymarkowski, H. Bernar, et
al., "Dietary cholesterol withdrawal reduces vascular inflammation and induces
coronary plaque stabilization in miniature pigs," Cardiovasc Res, vol. 56, pp. 135-
44, Oct 2002.
[90] P. Holvoet, A. Mertens, P. Verhamme, K. Bogaerts, G. Beyens, R. Verhaeghe, et
al., "Circulating oxidized LDL is a useful marker for identifying patients with
coronary artery disease," Arterioscler Thromb Vasc Biol, vol. 21, pp. 844-8, May
2001.
[91] P. Holvoet, J. Vanhaecke, S. Janssens, F. Van de Werf, and D. Collen, "Oxidized
LDL and malondialdehyde-modified LDL in patients with acute coronary
syndromes and stable coronary artery disease," Circulation, vol. 98, pp. 1487-94,
Oct 13 1998.
[92] E. Barsoukov and J. R. Macdonald, Impedance spectroscopy: theory, experiment,
and applications. Malden, MA: Wiley-Interscience, 2005.
[93] M. E. Orazem and B. Tribollet, Electrochemical Impedance Spectroscopy 2008.
[94] L. K. Curtiss, "Reversing atherosclerosis," New England Journal of Medicine, vol.
360, pp. 1144-1146, 2009.
[95] D. D. Heistad, "Unstable coronary-artery plaques," The New England journal of
medicine, vol. 349, pp. 2285-2287, 2003.
[96] Z. S. Galis, "Vulnerable plaque: the devil is in the details," Circulation, vol. 110,
pp. 244-246, 2004.
[97] Y. M. Park, M. Febbraio, and R. L. Silverstein, "CD36 modulates migration of
mouse and human macrophages in response to oxidized LDL and may contribute
46
to macrophage trapping in the arterial intima," The Journal of Clinical
Investigation, vol. 119, pp. 136-145, 2009.
[98] A. Nair, B. D. Kuban, E. M. Tuzcu, P. Schoenhagen, S. E. Nissen, and D. G.
Vince, "Coronary plaque classification with intravascular ultrasound
radiofrequency data analysis," Circulation, vol. 106, pp. 2200-2206, 2002.
[99] N. F. Marrouche, D. O. Martin, O. Wazni, A. M. Gillinov, A. Klein, M. Bhargava,
et al., "Phased-array intracardiac echocardiography monitoring during pulmonary
vein isolation in patients with atrial fibrillation: impact on outcome and
complications," Circulation, vol. 107, pp. 2710-2716, 2003.
[100] I. K. Jang, B. E. Bouma, D. H. Kang, S. J. Park, S. W. Park, K. B. Seung, et al.,
"Visualization of coronary atherosclerotic plaques in patients using optical
coherence tomography: comparison with intravascular ultrasound," Journal of the
American College of Cardiology, vol. 39, pp. 604-609, 2002.
[101] H. Yabushita, B. E. Bouma, S. L. Houser, H. T. Aretz, I. K. Jang, K. H.
Schlendorf, et al., "Characterization of human atherosclerosis by optical
coherence tomography," Circulation, vol. 106, pp. 1640-1645, 2002.
[102] B. T. MacDonald, K. Tamai, and X. He, "Wnt/β-catenin signaling: components,
mechanisms, and diseases," Developmental cell, vol. 17, pp. 9-26, 2009.
[103] F. D. Kolodgie, A. S. J. Katocs, E. E. Largis, S. M. Wrenn, J. F. Cornhill, E. E.
Herderick, et al., "Hypercholesterolemia in the rabbit induced by feeding graded
amounts of low-level cholesterol. Methodological considerations regarding
individual variability in response to dietary cholesterol and development of lesion
type," Arterioscler Thromb Vasc Biol., vol. 16, pp. 1454-1464, 1996.
[104] D. Tang, C. Yang, S. Kobayashi, J. Zheng, and R. P. Vito, "Effect of stenosis
asymmetry on blood flow and artery compression: a three-dimensional fluid-
structure interaction model," Ann Biomed Eng, vol. 31, pp. 1182-93, 2003.
[105] S. M. Schwartz, Z. S. Galis, M. E. Rosenfeld, and E. Falk, "Plaque rupture in
humans and mice," Arterioscler Thromb Vasc Biol, vol. 27, pp. 705-13, 2007.
47
[106] J. A. Berliner and J. W. Heinecke, "The role of oxidized lipoprotein in
atherogenesis," Free Radical Biology and Medicine, vol. 20, pp. 707-727, 1996.
[107] L. Asatryan, R. Hamilton, J. Mario Isas, J. Hwang, R. Kayed, and A. Sevanian,
"LDL phospholipid hydrolysis produces modified electronegative particles with
an unfolded apoB-100 protein," Journal of Lipid Research, vol. 46, pp. 115-122,
January 2005 2005.
[108] Y. Yamaguchi, J. Haginaka, S. Morimoto, Y. Fujioka, and M. Kunitomo,
"Facilitated nitration and oxidation of LDL in cigarette smokers," Eur J Clin
Invest., vol. 35, pp. 186-193, 2005 Mar 2005.
[109] M. Navab, J. A. Berliner, A. D. Watson, S. Y. Hama, M. C. Territo, A. J. Lusis, et
al., "The Yin and Yang of oxidation in the development of the fatty streak. A
review based on the 1994 George Lyman Duff Memorial Lecture," Arterioscler
Thromb Vasc Biol, vol. 16, pp. 831-42, 1996.
[110] S. Ehara, M. Ueda, T. Naruko, K. Haze, A. Itoh, M. Otsuka, et al., "Elevated
levels of oxidized low density lipoprotein show a positive relationship with the
severity of acute coronary syndromes," Circulation, vol. 103, pp. 1955-60, 2001.
[111] C. J. Slager, A. C. Phaff, C. E. Essed, N. Bom, J. C. H. Schuurbiers, and P. W.
Serruys, "Electrical impedance of layered atherosclerotic plaques on human
aortas," IEEE Transac Biomed Eng, vol. 39, pp. 411-419, 1992.

48

Chapter Two: Characterization of Inflammatory
Atherosclerotic Lesions via Electrochemical Impedance
Spectroscopy
1

1
Disclaimer: the content of this chapter has been published as "Characterization of
Inflammatory Atherosclerotic Lesions via Electrochemical Impedance Spectroscopy",
Biosensors and Bioelectronics 30(1): 165-173 (2011)
Acknowledgement: Co-authors of this manuscript include: Fei. Yu, Xiaohu Dai, Tyler
Beebe, Tzung. K. Hsiai. The author of this thesis credits other co-authors for their
significant input in the process of preparing the manuscript for publication.
49

2.1 Chapter Two Introduction
Detection of atherosclerotic lesions prone to rupture is of utmost importance in the
management of patients with acute coronary syndromes or stroke [112, 113]. Despite the
advent of computed tomographic (CT) angiography, high resolution MRI [114],
intravascular ultrasound (IVUS), near-infrared fluorescence (NIRF) [115], time-resolved
laser-induced fluorescence spectroscopy [116] and other techniques, predicting
metabolically active atherosclerotic lesions has remained an unmet clinical need.
Mechanically unstable atherosclerotic plaque is often characterized by a thin-cap fibrous
atheroma (< 65 µ m) and a metabolically active lipid core [117, 118]. Rupture of these
plaques is clinically manifested as acute coronary syndromes or stroke [119-121].
Emerging imaging modalities such as an integrated intravascular ultrasound (IVUS) and
optical coherence tomography (OCT) system have enabled the colocalization of thin-cap
fibroatheroma with intimal hyperplasia and calcification [122, 123]. However, assessing
metabolic states in the inflammatory, albeit non-obtrusive, lesions has remained a
diagnostic challenge.
Atherosclerotic lesions can display distinct electrochemical properties [124, 125].
Biological tissues store charges, and electric impedance (Z) develops as a function of
frequency in response to the applied alternating current (AC). Active lipids and
macrophages cause distinct electrochemical properties in the vessel wall that can be
measured by electrochemical impedance spectroscopy (EIS). Catheter-based linear 4-point
microelectrodes were reported to measure frequency-dependent tissue impedance in New
Zealand White (NZW) rabbits [124-126]. To provide reliable endoluminal EIS
measurements, we applied the concentric bipolar microelectrodes to address non-
homogeneous tissue composition, uneven endoluminal topography, and non-uniform
current distribution in explants of rabbit arteries, and demonstrated elevated impedance
spectrum in the pre-atherosclerotic tissues [127]. These lesions with oxidized Low Density
Lipoprotein (oxLDL) and foam cell infiltrates were considered clinically silent, and were
previously reported to have no significantly different impedance compared with the healthy
aorta [124]. However, the application of concentric bipolar electrodes detected changes in
electrochemical impedance signals in the pre-atherosclerotic lesions [127].
Here, we demonstrate a sensitive and specific approach to characterize metabolically active
lesions via EIS measurements in explants of human aorta. We developed equivalent circuit
models to assess electric circuit parameters in the context of simulating endoluminal EIS
50

measurements. EIS measurements performed on 15 coronary, carotid, and femoral arteries
at various Stary stages of atherosclerotic lesions revealed distinct electrochemical
impedance spectroscopic signals [128, 129]. Endoluminal impedance was significantly
higher in the active lipid-rich lesions as validated by positive anti-oxLDL staining. To
corroborate the specificity of EIS measurements, we demonstrate significant differences in
frequency-dependent impedance signals among fatty streaks (Stary Type II lesions), thin
fibrous cap oxLDL-rich (Type III or IV), oxLDL absent fibroatheroma (Type V), and
calcified lesions (type VII). Hence, we provide new electrochemical insights into the
applications of EIS measurements to detect active metabolic conditions in the mechanically
unstable atherosclerotic lesions.

51

2.2 Chapter Two Material and Methods
2.2.1 Electrochemical Impedance Spectroscopic Measurements
Fresh-frozen human artery specimens were obtained from National Disease Research
Interchange (NDRI) in accordance with the University of Southern California Institutional
Review Board guideline. A total of 15 human coronary, carotid and femoral arterial
segments from 9 donors were analyzed for endoluminal EIS measurements. The arterial
samples were immersed in phosphate buffered saline (PBS) solution (Invitrogen, CA,
USA), and sectioned longitudinally to unfold the endoluminal sides. The gross pathology
of individual specimens revealed various degrees of atherosclerosis as classified by Stary
stages from type I to VII [128, 129]. A total of 147 points of interest with gross lesion-free
condition or various types of atherosclerotic lesions were selected for endoluminal EIS
measurements.
EIS measurements were conducted using the concentric bipolar microelectrodes with a
flat-tip profile (FHC Co., ME, USA) as previously described [127]. Briefly, the concentric
bipolar microelectrode was mounted vertically on a micro-manipulator (World Precision
Instruments, FL, USA), and made in contact with tested tissue at selected measuring point.
An Ag/AgCl electrode (World Precision Instruments, FL, and USA) was used as the
reference electrode. Frequency-dependent impedance was measured from 100Hz to 300
kHz (Gamry Series G 300 potentiostat, PA, USA). The magnitudes and phases of the EIS
measurements were recorded at 20 data points per frequency decade, and the measured
impedance spectrums were analyzed (Gamry Echem Analyst software, PA).
2.2.2 Equivalent circuit model and simulation
To simulate equivalent circuit model for the concentric bipolar electrode-endoluminal
tissue interface, we constructed three models describing both working and counter
electrode interface as well as the tissue impedance. Equivalent Circuit model 1 (EC1) was
comprised of 6 electric elements (Fig. 12a). Both counter electrode (CE) and working
electrode (WE) were denoted as a constant phase elements in parallel with a charge transfer
resistance. The impedance of constant phase element ZCPE can be expressed as:
ZCPE=
1
Y(jω)
a
, (2)
52

Where ‘Y’ represents the capacitance and ‘a’ is the empirical constant describing the
surface property of electrode. When a = 1, the CPE operates as an ideal capacitor. In most
of the cases, a falls between 0 and 1 (0 < a <1), and ZCPE indicates the non-ideal behavior
of electrode-tissue interface capacitance. The charge transfer resistance, RCT, is
predominantly dependent on the chemical and physical properties of electrolyte solution
and electrode material. Blood vessel is considered to harbor both resistive (RB) and
capacitive (CB) properties; thus, both contribute to the overall tissue impedance. The RB
and CB values were mainly dependent on the composition and structure of the tissue,
particularly its water, lipid, ion and charged molecule content. With the assumption of an
extremely large charge transfer resistance (RCT1) at large-area counter electrode and an
ideal double layer capacitance (CDL2) replacing the CPE at working electrode, we
implemented Equivalent Circuit model 2 (EC2) to avoid potential over-fitting to simplify
the simulation (Fig. 12b). In this context, the total number of parameters was reduced to 6.
In addition, by assuming that CPE at counter electrode also acts as an ideal double layer
capacitance (CDL1), Equivalent Circuit model 3 (EC3) further reduced total parameters to
5 (Fig. 12c).
The best-fitting model parameters (RB, CB, Y, a, RCT and CDL) for the 3 equivalent circuits
were simulated, using a simplex algorithm in the Gamry Echem Analyst software. The
difference between the simulated and experimental impedance was represented as a single
“Goodness of Fit” value, equivalent to the square of the overall percentage error. Using
this approach, we were able to compare the agreement between the simulated and
experimental spectra, and to verify the equivalent circuits as the best suited model for
concentric bipolar endoluminal EIS applications.
2.2.3 Histology and Immunohistochemistry
Human specimens were sectioned and immersed in 4% paraformaldehyde for paraffin
fixation immediately following the impedance measurements. Multiple slides with
thickness of 5µ m were cut for histological evaluation. Standard hematoxylin and eosin
(H&E) staining was performed to visualize initima, media, smooth muscle cells, adventitia,
and foam cells. The metabolic states of atherosclerotic lesions were assessed by anti-
oxLDL antibody (mAb4E6) to active lipids, Oil-red-O to lipid content, and von Kossa to
calcification. All histological sections were visualized under Olympus IX70 microscopes
(Olympus, Japan) and captured with a CCD digital camera (ProGres® C3, Jenoptic,
Germany). Metabolically active conditions of the fibroatheromas were classified by the
53

Stary stages in terms of intimal hyperplasia, thin-cap atheroma, active lipids, and
calcification [128, 129].
2.2.4 Statistical analysis
Atherosclerotic lesions were categorized into five types (lesion free/fatty streak/thin cap
oxLDL-rich atheroma/ oxLDL-absent fibroatheroma/calcified lesions) based on
histological evidence. Due to variations in specimen size, thickness, and possible changes
in electrode surface chemistry after multiple applications, inter-specimen variations in
baseline EIS measurements could develop. To standardize comparisons, we normalized all
of the parameter values obtained from simulation to the respective mean parameter values
obtained from the lesion-free sites of the same specimens. Next, one-way analysis of
variance (ANOVA) and two-tailed T-test were used for multi-group comparison and
comparison between lesion and lesion-free groups, respectively. P values < 0.05 were
considered statistically significant.

54

2.3 Chapter Two Results
2.3.1 An optimal equivalent circuits to simulate endoluminal EIS measurements
Both Equivalent Circuits 1 and 2 (EC1 and EC2) predicted frequency-dependent changes
in impedance ( ) and phase ( 𝜃 ), and were in agreement with the endoluminal EIS
measurements in the human carotid arteries accompanied by approximately 2.5% error
(Fig. 12d). Equivalent Circuit 3 (EC3) predicted frequency-dependent changes in
impedance accompanied by approximately 14.8% error and by a significant deviation in
the phase values ( θ). We further compared the individual circuit parameters among the
three equivalent circuits (Fig. 12e), and revealed that RB, CB and Goodness of Fit values
were nearly identical between equivalent circuits 1 and 2. Given that the estimated value
of charge transfer resistance (RCT1) in the counter electrode of EC1 exceeded the
computational limit (10
38
Ω) of the software (Gamry Echem Analyst), we were able to
remove the high RCT1 from EC2. However, the assumption that CPE in the counter
electrode functioned as a double layer capacitor (C DL1) resulted in a decrease in Goodness
of Fit and a deviation in phase ( θ) values in EC3. In this context, EC2 provided the optimal
model to simulate EIS results in the concentric bipolar electrode-endoluminal tissue
interface, and established the basis for the ensuing analysis of endoluminal EIS
measurements.
2.3.2 Endoluminal EIS measurements in the fatty streaks
Endoluminal EIS measurements were compared between fatty streak-rich and fatty streak-
absent sites, followed by immunohistochemistry analysis for anti-oxLDL and Oil-red-O
staining. The fatty streak-absent site was stained negative for anti-oxLDL (Fig. 13a).
Adjacent to this site was the fatty streak–rich site that was stained positive for both anti-
oxLDL and Oil-Red-O (Fig. 13b). The EIS signals revealed the frequency-dependent
differences between fatty streak-rich and fatty streak-free sites from 10 kHz to 300 kHz.
The maximum difference in phase between the two measurements was at ~10 kHz (Fig.
13c). The bar graph provided the statistically significant difference in impedance ranging
from 10 kHz to 100 kHz (p < 0.001, n = 10 for fatty streak-rich, and n = 8 for fatty streak-
free endoluminal surface) (Fig. 13d). Hence, we demonstrate a significant frequency-
dependent increase in impedance in the fatty streaks (Stary Type II lesions) compared to
lesion-free sites.
55

2.3.3 Sensitive endoluminal EIS measurements in the thin-cap atheromas
To address the inflammatory states underneath the fibrous caps, we demonstrate the
application of EIS in en face human carotid arteries. Endoluminal EIS measurements were
compared between intimal hyperplasia and early stage atheroma. Immunohistochemistry
revealed a negative anti-oxLDL staining in region of intimal hyperplasia (Fig. 14a), but
positive anti-oxLDL staining in the thin-capped (50 to 150 µ m in cap thickness) atheroma
that harbored a lipid core (Fig. 14b). Endoluminal EIS measurements revealed an increase
in impedance from 10 kHz to 300 kHz in the thin-cap atheroma (Fig. 14c). The maximal
phase differences between lesion-free and atheroma regions was also at 10 kHz. The bar
graph further provided statistically significant differences in impedance ranging from 10
kHz to 100 kHz (p < 0.01, n = 6 for both thin-cap atheroma and lesion-free) (Fig. 14d).
Thus, we demonstrate a significant frequency-dependent increase in impedance in the
oxLDL-rich atheroma (Type III or IV) compared to the oxLDL-absent lesion-free regions.
2.3.4 Specificity of endoluminal EIS measurements
To assess the specificity for inflammatory states, we performed frequency-dependent EIS
measurements to compare oxLDL-rich and -absent fibrous atheroma in en face human
carotid arteries. Endoluminal EIS measurements compared between intimal hyperplasia
and fibrous atheromas, followed by immunohistochemistry staining to reveal negative anti-
oxLDL staining in both the lesion-free site (Fig. 15a) and the fibrous structure (Fig. 15b).
Endoluminal EIS measurements showed a statistically insignificant difference in
frequency-dependant impedance from 1 kHz to 300 kHz between the lesion-free sites and
fibrous structures (Fig. 15c). The bar graph further supported statistically insignificant
differences in impedances ranging from 10 kHz to 100 kHz (p > 0.05, n = 4 for lesion-free
and n = 6 for fibrous atheromas) (Fig. 15d). Despite the presence of fibrous structure,
insignificant changes in EIS measurements were consistent with the oxLDL-absent lesions
(Type V).
2.3.5 Elevated endoluminal EIS readings in the calcific atheromas
To further assess frequency-dependent EIS measurements in the presence of calcification,
we performed EIS measurements in en face calcific atherosclerotic plaque from explants
of human carotid arteries. H&E and immunohistochemistry revealed negative anti-oxLDL
and von Kossa staining in the lesion-free sites (Fig. 16a), and positive von Kossa staining
56

in the calcified lesions (Fig. 16b). The calcified core was dislodged during subsequent
fixation, resulting in a void in the core. Endoluminal EIS measurements revealed an
increase in impedance from 10 kHz to 100 kHz in the calcified lesions (Fig. 16c). The
maximal phase difference between lesion-free and calcified regions was at ~6 kHz. The
bar graph demonstrated a statistically significant difference in impedance ranging from 10
kHz to 100 kHz (p < 0.01, n = 4 for calcification and n = 6 for calcification-free regions)
(Fig. 16d). Therefore, we demonstrate that calcific lesions (type VII) also engendered a
significant increase in tissue impedance.
2.3.6 Sensitivity and Specificity of endoluminal EIS measurements
To provide sensitivity and specificity of EIS signals, we analyzed the RB (biological
component resistance) values derived from the aforementioned Equivalent Circuit 2 (Fig.
12b). We normalized RB values from lesion sites to the mean RB values from the lesion-
free sites in the identical specimen. We provided statistically significant differences in RB
values among Type II lesions (fatty streaks), Type III or IV lesions (thin-cap oxLDL-rich
atheroma) and Type VII lesions (calcification) (*p < 0.001) compared to the lesion-free
sites. We further corroborated statistically significant differences between ox-LDL-rich
and ox-LDL-absent fibrous atheromas (
#
p < 0.001), and between oxLDL-rich and calcific
lesions (p < 0.05) (Fig. 17). In sum, our electrochemical strategy establishes sensitive and
specific application of EIS measurements to detect inflammatory states of the
atherosclerotic lesions.

57

2.4. Chapter Two Discussion
In this study, electrochemical characterization of fibrous atheromas in terms of impedance
spectroscopy and bioactive lipids offers a novel entry point to identify the high-risk and
rupture-prone plaques. We established an Equivalent Circuit model to analyze our EIS
measurements. We demonstrated that oxLDL within the fibrous atheroma engendered
distinct frequency-dependent electrochemical impedance (EIS) measurements. To
corroborate the specificity of tissue resistance values (RB), we provided statistically
significant differences in the frequency-dependent EIS signals among fatty streaks (Stary
Type II lesions), thin fibrous cap oxLDL-rich (Type III or IV), oxLDL absent
fibroatheroma (Type V), and calcified lesions (type VII). Thus, we demonstrated a
sensitive and specific electrochemical strategy to detect the inflammatory states of
atherosclerotic lesions.
Electrochemical impedance spectroscopy (EIS) is an emerging approach for biosensing
applications [130]. The commonly accepted model focuses on the electrical elements
involved in the working electrode and electrolyte interface; namely, a charge transfer
resistance (RCT), a Warburg impedance (W) and a double layer capacitance (CDL) or
constant phase element (CPE) [130, 131]. For in vitro biosensing applications, a large
surface area provided by the inert platinum or carbon electrode is commonly used as the
counter electrode, providing both high charge transfer resistance and double-layer
capacitance. For this reason, the overall impedance contributed by counter electrode is
considered negligible. For intravascular EIS applications requiring high spatial resolution,
the confined space in the catheters warrants close packaging of both the counter and
working electrodes. For this reason, EIS measurements must account for the
electrochemical interference at both the counter and working electrode interfaces.
In light of the paucity of literature for modeling equivalent circuits for the concentric
bipolar electrode-tissue interface, we constructed three models to include both working and
counter electrode interface as well as tissue impedance. As early as 1970s, the principles
of bio-impedance have been assessed for clinical applications. For example, bioelectrical
impedance analysis (BIA) provided the entire corporeal impedance as an estimation of
percentage total body fat [132-134]. The principle of BIA is based on the premises that
gross tissue impedance is closely related to tissue water content and electrolyte
concentration. Fat-free tissue is known as a good electrical conductor for its high water
(approximately 73%) and electrolytes content (ions and proteins). However, fat tissue is
anhydrous and thus, a poor conductor. In corollary, atherosclerotic lesions display distinct
58

electrochemical properties [124, 125]. The high lipid content, including the negatively
charged active lipids such as oxidized Low-Density Lipoprotein (oxLDL) present in the
pro-inflammatory lesions [135], engenders distinct electrochemical properties in the vessel
wall that can be measured by electrochemical impedance spectroscopy (EIS).
The sensitivity and specificity of intravascular EIS measurements depend on various
biochemical and physical parameters. Surface roughness and electrochemical properties of
the electrodes, artery wall thickness and geometry contribute to accurate modeling of the
Equivalent Circuits. In the current study, we demonstrate that Equivalent Circuit 2 (Fig.
1b) is an optimal model to simulate endoluminal EIS measurements for modeling the
electrical resistive and capacitive properties of the tissue in the vicinity of concentric
bipolar electrodes.
Oxidized LDL induces the transformation of macrophages into lipid-laden foam cells [136],
and activated macrophages secrete factors such as matrix metalloproteinases (MMPs) to
mechanically destabilize plaques [137, 138]. Growing evidence suggests that oxLDL and
thin-cap fibroatheromas (TCFA) rich in macrophage/foam cells are more prone to
mechanical stress and destabilization [139-141]. Hence, electrochemical impedance
spectroscopy (EIS) allows for characterization of the electrical properties at the electrode
and biological interfaces [130]. Intravascular deployment of EIS-based 4-point electrode
probe was reported for detecting atherosclerotic lesions [124, 125, 142-144]. We further
revealed that active lipids and macrophages engendered distinct electrochemical properties
that can be measured by EIS via the concentric bipolar microelectrodes [127].
To establish sensitive and specific strategy for inflammatory states underneath the fibrous
caps, we demonstrate distinct differences in tissue resistance (RB) among fatty streaks
(Stary Type II lesions), thin fibrous cap oxLDL-rich (Type III or IV), and calcified lesions
(type VII) in en face human carotid arteries. Furthermore, RB value was significantly
elevated in the oxLDL-rich atheromas and fatty streaks compared to oxLDL-absent
fibroatheromas, and the difference in RB values were statistically insignificant between
oxLDL-absent fibroatheromas and the lesion-free regions (Fig. 17). It is recognized that
tissue resistance to electrical current is dependent on its intrinsic property in terms of water
content and free-moving electrolytes. We postulate that despite intimal hyperplasia and
smooth muscle cell migration to the endoluminal surface, oxLDL-absent fibroatheromas
harbor comparable water and ionic contents like the rest of the vessel wall, thus rendering
a good electric conductor and a low resistivity path for current flow. In contrast, the lipid
core beneath the fibrous atheroma harbors low water content, resulting in a poor electrical
59

conductor and a high resistivity path for current flow, thus confining the electrical current
flow to the thin fibrous layer of atheroma. In corollary, the calcified core in Type VII
lesions is analogous to a salt crystal and an insulator, thus, rendering a high resistivity path.
In this context, endoluminal EIS signals were elevated in the presence of both active lipids
and calcification, while the normalized RB was significantly higher in the calcific
atheromas (Fig. 17).
In addition to the normalized RB values, intravascular ultrasound (IVUS) can be applied to
distinguish lipid-rich atheroma from calcification. Fibrous cap oxLDL-rich atheroma
generates echolucency while the calcified lesions engenders high echogenicity [145]. Thus,
integration of EIS signals with IVUS images holds promise to co-register non-obstructive
lesions (< 20% stenosis) harboring active lipids and inflammatory foam cells that were
otherwise considered clinically silent, and were previously reported to have no
significantly different impedance compared with the healthy aorta [124].
Currently, the advent of near-infrared fluorescence (NIRF) provides cystein protease
activity as an indicator of inflammation [115]. However, contrast dye injection is indicated.
The use of glucose analogue [
18
F]-fluorodeoxyglucose

(
18
FDG) also provides metabolic
activity by Positron Emission Tomography (PET) [146]. However, radioisotope is required
and the spatial resolution remains inadequate. Encouraging findings from the high-
frequency dual ultrasound (US) and optical coherence tomographic (OCT) probe
demonstrate the feasibility to detect both high resolution thin-cap fibroatheroma (TCFA)
and intimal hyperplasia [123]. Integrating the aforementioned US-OCT system with our
novel electrochemical approach to detect active lipids and macrophages in the vessel wall
will further establish the capacity to characterize intimal thickening, calcification, thin
fibrous cap, calcification and metabolic states.
We hereby assessed endoluminal impedance in explants of human arteries via a concentric
bipolar electrode, and provided a sensitive and specific electrochemical strategy to
characterize fibrous cap atheromas in terms of impedance spectroscopy and active lipids
content. We demonstrate that high content of bioactive metabolic factors within the fibrous
atheroma engendered distinct frequency-dependent electrochemical impedance spectra.
Histology and immunohistochemistry for active lipids and calcification further validated
specificity of the EIS measurements for active metabolic states in en face human arteries.

60

Figure 12. Equivalent circuit models to simulate concentric bipolar electrochemical
impedance spectrum (a) Equivalent Circuit 1 (EC1) encompassed electric circuit
parameters for working and counter electrodes interface as well as arterial wall impedance.
The electrode-endoluminal interface was modeled by a constant phase element (CPE)
having an impedance ZCPE (𝑍 CPE
=
1
Y(jω)
a
) in parallel with a charge transfer resistance
(RCT). The vessel wall harbors both resistive (RB) and capacitive properties (CB). Hence, a
total of 8 electric circuit parameters were included. CE: Counter electrode; WE: working
electrode. (b) Equivalent Circuit 2 (EC2) was a simplified version of EC1. By assuming a
very large charge transfer resistance at the counter electrode interface, RCT1 was removed.
CPE in the working electrode was replaced with a double layer capacitor (CDL2). The total
number of electric circuit components was reduced to 6. (c) Equivalent Circuit 3 (EC3)
was a further simplified version of EC2. By assuming that CPE in the counter electrode
also acted as an ideal double layer capacitor (C DL1), the total number of parameters was
reduced to 5. (d) Both EC1 and EC2 simulated experimental EIS measurements
accompanied by approximately 2.5% error, whereas EC3 was accompanied by 14.8% error
and a deviated phase ( ) values from the endoluminal EIS signals. (e) Selected circuit
parameter values for endoluminal EIS data shown in Fig. 1d. The values were derived from
simulations using EC1, EC2 and EC3, respectively. Y1, a1, RB and CB values derived from
EC1 and EC2 were almost identical. The best fit EC1 and EC2 models had comparable
Goodness of Fit (6.34E-4 and 6.8E-4, respectively), whereas the best fit EC3 model had
significantly higher Goodness of Fit (2.2E-2), indicating larger deviation from
experimental data.
61

Figure 13. Endoluminal EIS measurements of fatty streaks. (a) Anti-oxLDL staining
was negative in the fatty streaks-free region. The right upper insert showed the luminal
wall and right lower insert showed the corresponding negative Oil-red-O staining. (b) Both
anti-oxLDL and Oil-Red-O were positive in the fatty streaks. (c) Endoluminal tissue
impedance increased from 10 kHz to 300 kHz in the presence of fatty streaks. The
maximum phase difference between lesion-free and fatty streak regions developed at ~10
kHz. Impedance measured from fatty streak sites appeared to have greater variance. (d)
Statistically significant difference in impedance of lesion-free sites and fatty streak sites
was observed from 10 kHz to 100 kHz (p <0.01, n = 10 for fatty streaks and n=8 for lesion-
free).
62

Figure 14. Endoluminal EIS measurements of oxLDL-rich fibrous atheroma. (a)
Despite the presence of intimal hyperplasia, anti-oxLDL staining was negative. The left
lower insert showed endoluminal intimal hyperplasia. (b) Anti-oxLDL staining was
positive, and the fibrous cap thickness ranged from 50 to 150µ m. The right lower insert
showed the intra-plaque atheroma. (c) Endoluminal tissue impedance increased from 10
kHz to 300 kHz in the presence of the anti-oxLDL-positive fibrous atheroma. The
maximum phase difference between lesion-free and atheroma sites also developed at ~10
kHz. (d) Maximum difference in impedance was observed from 10 kHz to 100 kHz (p
<0.01, n = 6 for both atheroma and lesion-free sites).
63

Figure 15. Endoluminal EIS measurements of oxLDL-absent fibrous atheroma. (a)
Anti-oxLDL staining was negative in the lesion-free site. (b) Anti-oxLDL staining was
also negative in the fibrous atheroma. (c) Frequency-dependent endoluminal tissue
impedance from 1 kHz to 300 kHz between lesion-free regions and fibrous structures were
statistically insignificant. (d) Bar graph also revealed insignificant difference in frequency-
dependent impedance from 10 kHz to 100 kHz (p > 0.05, n = 4 for lesion-free and n = 6
for oxLDL-absent fibrous atheroma).

64

Figure 16. Endoluminal EIS measurements of calcification. (a) Both anti-oxLDL and
von Kossa staining was negative in the lesion-free regions. (b) Both H&E and von Kossa
staining supported the presence of calcification. (c) Endoluminal EIS measurements
revealed an increase in impedance from 10 kHz to 100 kHz in the presence of calcified
lesions. The maximum phase difference between lesion-free region and calcified lesions
developed at ~6 kHz. (d) Maximum difference in impedance was observed from 10 kHz
to 100 kHz (p < 0.01, n = 4 for calcification and n = 6 for calcification-free sites).

65

Figure 17. Sensitivity and specificity of Endoluminal EIS measurements. The RB
(biological component resistance) values were calculated using Equivalent Circuit 2 (Fig.
1) and normalized to the mean RB values from lesion-free sites on the same specimens.
Arterial specimens with Type II lesions (fatty streaks), Type III or IV lesions (thin cap
oxLDL-rich atheroma) and Type VII lesions (calcification) demonstrated significantly
higher RB values (*p < 0.001 compared to lesion-free sites). However, RB values in oxLDL-
absent fibrous atheroma (Type V or Type VIII) were similar to lesion-free sites (p > 0.05).
In addition, statistically significant difference in RB values were observed between oxLDL-
rich and oxLDL-absent fibrous atheromas (p < 0.001), as well as between oxLDL-rich
fibrous atheromas and calcified lesions (p < 0.05).

66

2.5 References
[112] R. Ross, "Atherosclerosis is an inflammatory disease," American Heart Journal,
vol. 138, pp. S419-20, 1999.
[113] J. L. Anderson, C. D. Adams, E. M. Antman, C. R. Bridges, R. M. Califf, D. E.
Casey Jr, et al., "ACC/AHA 2007 Guidelines for the Management of Patients
With Unstable Angina/Non-ST-Elevation Myocardial Infarction--Executive
Summary," Journal of the American College of Cardiology, vol. 50, pp. 652-726,
2007.
[114] S. G. Worthley, G. Helft, V. Fuster, Z. A. Fayad, M. Shinnar, L. A. Minkoff, et
al., "A novel nonobstructive intravascular MRI coil: in vivo imaging of
experimental atherosclerosis," Arteriosclerosis, Thrombosis, and Vascular
Biology, vol. 23, pp. 346-350, 2003.
[115] F. A. Jaffer, C. Vinegoni, M. C. John, E. Aikawa, H. K. Gold, A. V. Finn, et al.,
"Real-time catheter molecular sensing of inflammation in proteolytically active
atherosclerosis," Circulation, vol. 118, pp. 1802-9, Oct 28 2008.
[116] L. Marcu, M. C. Fishbein, J. M. I. Maarek, and W. S. Grundfest, "Discrimination
of human coronary artery atherosclerotic lipid-rich lesions by time-resolved laser-
induced fluorescence spectroscopy," Arteriosclerosis, Thrombosis, and Vascular
Biology, vol. 21, pp. 1244-1250, 2001.
[117] Y. Vengrenyuk, S. Carlier, S. Xanthos, L. Cardoso, P. Ganatos, R. Virmani, et al.,
"A hypothesis for vulnerable plaque rupture due to stress-induced debonding
around cellular microcalcifications in thin fibrous caps," Proc Natl Acad Sci U S
A, vol. 103, pp. 14678-83, Oct 3 2006.
[118] A. V. Finn, M. Nakano, J. Narula, F. D. Kolodgie, and R. Virmani, "Concept of
vulnerable/unstable plaque," Arteriosclerosis, Thrombosis, and Vascular Biology,
vol. 30, pp. 1282-1292, 2010.
[119] J. A. Ambrose, M. A. Tannenbaum, D. Alexopoulos, C. E. Hjemdahl-Monsen, J.
Leavy, M. Weiss, et al., "Angiographic progression of coronary artery disease and
the development of myocardial infarction," Lead Article, vol. 12, pp. 184-202,
1988.
67

[120] V. Fuster, L. Badimon, J. J. Badimon, and J. H. Chesebro, "The pathogenesis of
coronary artery disease and the acute coronary syndromes," The New England
journal of medicine, vol. 326, pp. 242-250, 1992.
[121] W. C. Little, M. Constantinescu, R. J. Applegate, M. A. Kutcher, M. T. Burrows,
F. R. Kahl, et al., "Can coronary angiography predict the site of a subsequent
myocardial infarction in patients with mild-to-moderate coronary artery disease?,"
Circulation, vol. 78, pp. 1157-1166, 1988.
[122] J. Sanz and Z. A. Fayad, "Imaging of atherosclerotic cardiovascular disease,"
Nature, vol. 451, pp. 953-957, 2008.
[123] X. Li, J. Yin, C. Hu, Q. Zhou, K. K. Shung, and Z. Chen, "High-resolution
coregistered intravascular imaging with integrated ultrasound and optical
coherence tomography probe," Applied Physics Letters, vol. 97, pp. 133702-
133704, 2010.
[124] T. Suselbeck, H. Thielecke, J. Kochlin, S. Cho, I. Weinschenk, J. Metz, et al.,
"Intravascular electric impedance spectroscopy of atherosclerotic lesions using a
new impedance catheter system," Basic research in cardiology, vol. 100, pp. 446-
452, 2005.
[125] I. Streitner, M. Goldhofer, S. Cho, H. Thielecke, R. Kinscherf, F. Streitner, et al.,
"Electric impedance spectroscopy of human atherosclerotic lesions,"
Atherosclerosis, vol. 206, pp. 464-468, 2009.
[126] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an intravascular
impedance catheter for detection of fatty lesions in arteries," IEEE Transactions
on Medical Imaging, vol. 16, pp. 439-446, 1997.
[127] F. Yu, R. Li, L. Ai, C. Edington, H. Yu, M. L. Barr, et al., "Electrochemical
Impedance Spectroscopy to Assess Vascular Oxidative Stress," Annals of
Biomedical Engineering, vol. 39, pp. 287-296, 2011.
[128] H. C. Stary, "Natural history and histological classification of atherosclerotic
lesions: an update," Arterioscler Thromb Vasc Biol, vol. 20, pp. 1177-1178, 2000.
68

[129] H. C. Stary, A. B. Chandler, R. E. Dinsmore, V. Fuster, S. Glagov, W. J. Insull, et
al., "A definition of advanced types of atherosclerotic lesions and a histological
classification of atherosclerosis.," Arterioscler Thromb Vasc Biol, vol. 15, pp.
1512-1531, 1995.
[130] F. Lisdat and D. Schä fer, "The use of electrochemical impedance spectroscopy for
biosensing," Analytical and Bioanalytical Chemistry, vol. 391, pp. 1555-1567,
2008.
[131] W. Franks, I. Schenker, P. Schmutz, and A. Hierlemann, "Impedance
characterization and modeling of electrodes for biomedical applications,"
Biomedical Engineering, IEEE Transactions on, vol. 52, pp. 1295-1302, 2005.
[132] D. Heber, S. Ingles, J. M. Ashley, M. H. Maxwell, R. F. Lyons, and R. M.
Elashoff, "Clinical detection of sarcopenic obesity by bioelectrical impedance
analysis," The American journal of clinical nutrition, vol. 64, pp. 472S-477S,
1996.
[133] U. G. Kyle, I. Bosaeus, A. D. De Lorenzo, P. Deurenberg, M. Elia, J. È . M.
GÛmez, et al., "Bioelectrical impedance analysis--part I: review of principles and
methods," Clinical Nutrition, vol. 23, pp. 1226-1243, 2004.
[134] U. G. Kyle, I. Bosaeus, A. D. De Lorenzo, P. Deurenberg, M. Elia, J. Manuel
Gó mez, et al., "Bioelectrical impedance analysis--part II: utilization in clinical
practice," Clinical Nutrition, vol. 23, pp. 1430-1453, 2004.
[135] A. Sevanian, J. Hwang, H. Hodis, G. Cazzolato, P. Avogaro, and G. Bittolo-Bon,
"Contribution of an in vivo oxidized LDL to LDL oxidation and its association
with dense LDL subpopulations," Arteriosclerosis, Thrombosis, and Vascular
Biology, vol. 16, pp. 784-793, 1996.
[136] M. S. Brown and J. L. Goldstein, "Lipoprotein metabolism in the macrophage:
implications for cholesterol deposition in atherosclerosis," Annual Review of
Biochemistry, vol. 52, pp. 223-261, 1983.
[137] G. C. Cheng, W. H. Briggs, D. S. Gerson, P. Libby, A. J. Grodzinsky, M. L. Gray,
et al., "Mechanical strain tightly controls fibroblast growth factor-2 release from
69

cultured human vascular smooth muscle cells," Circulation Research, vol. 80, pp.
28-36, 1997.
[138] Y. S. Kim, Z. S. Galis, A. Rachev, H. C. Han, and R. P. Vito, "Matrix
metalloproteinase-2 and-9 are associated with high stresses predicted using a
nonlinear heterogeneous model of arteries," Journal of Biomechanical
Engineering, vol. 131, pp. 011009-011018, 2009.
[139] S. Ehara, M. Ueda, T. Naruko, K. Haze, A. Itoh, M. Otsuka, et al., "Elevated
levels of oxidized low density lipoprotein show a positive relationship with the
severity of acute coronary syndromes," Circulation, vol. 103, pp. 1955-60, Apr 17
2001.
[140] S. Zeibig, Z. Li, S. Wagner, H. P. Holthoff, M. Ungerer, A. Bultmann, et al.,
"Effect of the oxLDL Binding Protein Fc-CD68 on Plaque Extension and
Vulnerability in Atherosclerosis," Circulation Research, vol. 108, pp. 695-703,
2011.
[141] G. Chinetti-Gbaguidi, M. Baron, M. A. Bouhlel, J. Vanhoutte, C. Copin, Y. Sebti,
et al., "Human Atherosclerotic Plaque Alternative Macrophages Display Low
Cholesterol Handling but High Phagocytosis Because of Distinct Activities of the
PPAR and LXR  Pathways," Circulation Research, vol. 108, pp. 985-995, 2011.
[142] S. Cho and H. Thielecke, "Influence of electrode position on the characterization
of artery stenotic plaques by using impedance catheter," Biomedical Engineering,
IEEE Transactions on, vol. 53, pp. 2401-2404, 2006.
[143] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an intravascular
impedance catheter for detection of fatty lesions in arteries," IEEE Transaction on
Medical Imaging, vol. 16, pp. 439-446, 1997.
[144] D. K. Stiles and B. Oakley, "Simulated characterization of atherosclerotic lesions
in the coronary arteries by measurement of bioimpedance," Biomedical
Engineering, IEEE Transactions on, vol. 50, pp. 916-921, 2003.
[145] R. Yamada, H. Okura, Y. Miyamoto, T. Kawamoto, Y. Neishi, A. Hayashida, et
al., "A newly developed radio frequency signal-based intravascular ultrasound
tissue charagerization: a comparison between IMAP and integrated backscatter
70

intravascular ultrasound," Journal of the American College of Cardiology, vol.
57, pp. E1882-E1882, 2011.
[146] J. Rudd, E. Warburton, T. Fryer, H. Jones, J. Clark, N. Antoun, et al., "Imaging
atherosclerotic plaque inflammation with [18F]-fluorodeoxyglucose positron
emission tomography," Circulation, vol. 105, p. 2708, 2002.

71

Chapter Three: Elevated Electrochemical Impedance in the
Endoluminal Regions with High Shear Stress: Implication for
Assessing Lipid-Rich Atherosclerotic Lesions
1

1
Disclaimer: the content of this chapter has been published as “Elevated Electrochemical
Impedance in the Endoluminal Regions with High Shear Stress: Implication for Assessing
Lipid-Rich Atherosclerotic Lesions", Biosensors and Bioelectronics 43: 237-244 (2013)
Acknowledgement: Co-authors of this manuscript include: Fei. Yu, Juhyun Lee, Nelson
Jen, Xiang Li, Qian Zhang, Eun S. Kim, Kirk K. Shung, and Tzung. K. Hsiai. The author
of this thesis credits other co-authors for their significant input in the process of preparing
the manuscript for publication.
72

3.1 Chapter Three Introduction
Metabolically active lesions is of clinical significant in the development of mechanically
unstable plaque.[147] Tissue impedance spectroscopy is an emerging electrochemical
strategy to characterize atherosclerotic lesions.[148-150] Elevated electrochemical
impedance spectroscopy (EIS) signals are associated with metabolically active lesions in
both ex vivo [151, 152] and in vivo models.[148-150, 153-155] When the microelectrodes
are in contact with endoluminal surface, EIS signals are reproducible and independent of
lumen diameters, blood viscosity, and the flow rate.[148] For this reason, we integrated
EIS with high-frequency ultrasound and shear stress to assess lipid-laden atherosclerotic
lesions
Currently, gray-scale intravascular ultrasound (IVUS) is deemed as the gold standard for
in vivo imaging of the vessel walls.[156] While IVUS has enabled quantitative assessment
of coronary artery and peripheral vascular disease, assessing thin-capped fibrous atheromas
has been hampered by its gray-scale representation of the artery wall and its limited spatial
resolution.[156] By integrating EIS with high-frequency IVUS at a sampling rate of 400
MHz, we proposed to distinguish thick- versus thin-capped fibrous atheromas that harbor
active lipids; namely, oxidized low density lipoprotein (oxLDL).
Fluid shear stress imparts mechano-signal transduction that is intimately linked with the
initiation and development of atherosclerosis.[151, 157-161] Both the spatial (  / x) and
temporal (  / t) components of shear stress modulate the focal nature of vascular oxidative
stress in the promotion of pro-inflammatory states and post-translational LDL oxidative
modification. [151, 162, 163] In the athero-prone regions, LDL particles transmigrate into
the subendothelial layers, [162] where post-translational oxidative modifications of LDL
particles induce activation of matrix metalloproteinases [164, 165] and up-regulation of
NF- B-mediated adhesion molecules to destabilize the plaque. [152, 165-168]
In this context, we sought to demonstrate oxLDL-laden lesions by integrating
hemodynamic, imaging, and electrochemical approach. We established increased time-
averaged intravascular shear stress (ISSave) in response to high fat diets in the NZW rabbit
model. After 9 weeks, IVUS enabled visualization of endoluminal regions that harbored
atherosclerotic lesions for the assessment of EIS signals by the concentric bipolar
microelectrodes. Histology analysis for prominent anti-oxLDL lesions provided a
validation for the elevated EIS signals. Hence, we introduced an integrated approach to
enhance the characterization of oxLDL-laden atherosclerotic lesions with a translational
implication for mechanically unstable plaque.
73

3.2 Chapter Three Materials and Methods
3.2.1 Microfabrication and Calibration of Catheter-Based Flexible Polymer Sensors
The intravascular thermal sensor was fabricated using surface micromachining techniques
as previously described [169]. The Ti/Pt sensing element (240 μm in length and 80 μm in
width) was encapsulated in parylene C polymer for direct contact with the blood flow. All
materials used, including Parylene C, Ti, and Pt, offered a high level of biocompatibility
for in vivo investigations.
The concentric bipolar microelectrodes for electrochemical impedance spectroscopy (EIS)
measurements sensor were fabricated using a similar surface micromachining technique;
chromium deposition was used instead of the Ti/Pt for the sensing electrodes. The
concentric bipolar microelectrodes consisted of a working and a counter electrode; the
former was the inner pole with a diameter of 100 µ m, and the latter was the outer ring-
shaped electrode with an outer diameter of 500 µ m, and a width of 100 µ m. The spacing
between the inner and outer electrodes was 100 µ m (Figures 18a and 18b).
Both the intravascular thermal sensors and concentric bipolar microelectrodes were
integrated to an electrical coaxial wire of 0.4 mm in diameter (Tyco Electronics, Berwyn,
PA) as a guide wire for intravascular deployment and interrogation. The bonding sites of
the sensors were connected to the terminal end of the coaxial wire leads using conductive
epoxy (EPO-TEK H20E; Epoxy Technology, Billerica, MA). Biocompatible epoxy (EPO-
TEK 301; Epoxy Technology, Billerica, MA) anchored the sensor body onto the coaxial
wire surface (Figure 24).
3.2.2 In vivo Assessment of Intravascular Thermal Profiles in the New Zealand
White Rabbit Model
We acquired intravascular thermal profiles in the descending, thoracic, and peri-renal
abdominal aortas of NZW rabbits, and the data were calibrated to ISS as an approximation
of wall shear stress [170, 171] (Figure 25). All in vivo animal experiments were performed
at the Heart Institute of the Good Samaritan Hospital (Los Angeles, CA) with approval
from its Institutional Animal Care and Use Committee (IACUC). Deployment of the
flexible MEMS sensors into the rabbit’s aorta was performed in compliance with the
74

IACUC approved protocol. Eight age-matched male NZW rabbits (ten weeks, mean body
weight 2442 ± 210 g) were acquired from a local breeder (Irish Farms, Norco, CA) and
maintained in the Good Samaritan Hospital Vivarium. After a seven-day quarantine period,
the rabbits were anesthetized through an intramuscular injection of 50 mg/kg ketamine
(JHP Pharmaceuticals, LLC) combined with 10 mg/kg xylazine (IVX Animal Health, Inc.).
The animals were anticoagulated with heparin (100 units/kg) prior to the sensor
deployment.
To obtain artery dimensions and blood flow rates in the rabbit aortas, we positioned the
ultrasound transducer (Philips SONOS 5500) over the abdomen to interrogate the arterial
blood flow prior to sensor deployment. Continuous blood pressure measurements were
recorded with an automated tail cuff (IITC/Life Science Instruments). In the animal
angiographic laboratory, fluoroscope (Phillips BV-22HQ C-arm) and contrast dye injection
enabled us to localize the position of the sensors in relation to the inner aortic diameter,
allowing for steering the catheter-based sensors.
Catheter-based thermal sensor was deployed into rabbit aorta via left femoral artery cut-
down and advanced into abdominal, thoracic and descending aorta for respective thermal
profile assessment with the aid of fluoroscopy to guide the positioning of the catheter[171].
Constant temperature (CT) circuit was used to drive the thermal sensors for real-time
voltage signal acquisition [172]. The voltage across the sensing element was monitored at
a sampling rate of 2000 Hz by a LabVIEW-based data acquisition system, including a data
acquisition board (USB-6216 DAQ device, National Instruments, Austin, TX) and a laptop
computer (ThinkPad T61, Lenovo, China). Signal processing, wavelet decomposition and
low-pass filters were applied to minimize the background noise[173]. After the baseline
intravascular thermal profile assessment, the femoral artery was re-sutured and skin was
closed with staples to allow recovery. Buprenorphine, an analgesic was administered at
0.02 mg/kg during the first week as needed after baseline measurements. The rabbits were
then randomly divided into 2 groups: 1) normal standard chow diet (ND) (n = 4); and 2)
hypercholesterolemic diet (HD) (n = 4) containing 1.5% cholesterol & 6% peanut oil
(Newco®, CA). After 9 weeks, ISS measurements were repeated with the identical
experiment protocols. The rabbits were then sacrificed and their aortas were isolated for ex
vivo assessments and histological evaluations.

75

3.2.3 Computational Fluid Dynamics (CFD) Simulation
CFD code was developed to compare between analytical and experimental data. Three-
dimensional modeling of rabbit aortic geometries (aortic arch, thoracic, abdominal, renal
aorta) was reconstructed by Solidworks (Concord, Massachusetts, USA). The size of
individual aortic segments was obtained from angiographic and ultrasound imaging. The
inlet velocity profiles were acquired by the pulsed-wave Doppler velocity measurements.
The outlet boundary condition was determined from the mean arterial pressure obtained
during ISS experiments. Geometries were meshed from Solidworks flow simulation. After
defining the boundary conditions and performing geometry meshing, meshed models were
solved. The governing equations were solved by assuming laminar, incompressible, and
unsteady flow under the non-slip condition.
3.2.4 High-Frequency Intravascular Ultrasound Imaging (IVUS) of NZW Rabbit
Aortas
IVUS imaging of the rabbit aorta explants was performed using a custom-built ultrasound
imaging system within 4 hours after rabbits were sacrificed and the aortas were isolated
[174]. Segments approximately 2 cm in length were cut sequentially along the aorta and
maintained in Dulbecco's Modified Eagle Medium (DMEM) for cell viability. Individual
segments were then vertically positioned in a container placed on top of a rotating platform.
The ultrasound transducer was introduced into the aortic lumen from top along the central
axis of the rotating platform to achieve rotational scanning. Two-way pulse-echo
measurement was performed using a single pulser/receiver unit (Olympus NDT, Inc.,
Kennewick, WA). The detected ultrasound echo signals were digitized by a 12 bit data
acquisition board (Gage Applied Technologies, Lockport, IL) operating at the sampling
rate of 400 MHz. A function generator provided a 2.5 KHz trigger signal to the
pulser/receiver and the data acquisition board. Gross histology of atherosclerotic lesions
was identified both visually and from the IVUS images, and the positions of the lesions
with respect to the aorta segment geometry were labeled for the corresponding
electrochemical impedance and immunohistochemistry.
3.2.5 Electrochemical Impedance Spectroscopy Assessment of NZW Rabbit Aortas
Immediately after IVUS assessment, the rabbit aorta segments were flushed with and
maintained in phosphate buffered saline (PBS) for EIS assessment. Endoluminal EIS
76

measurements were performed by using the concentric bipolar microelectrodes at multiple
sites associated with the atherosclerotic lesions and compared with the adjacent healthy
endoluminal regions [148]. To ensure contact between the EIS sensor and the endoluminal
measurement sites on the arterial wall, we mounted the microelectrodes to a steerable guide
wire and made contact with the atherosclerotic lesions by steering the terminal end of guide
wire. An Ag/AgCl electrode immersed in the PBS solution was used as a reference
electrode. EIS measurements were performed by using a Gamry Series G 300 potentiostat
(Gamry Instruments, PA) installed in a desktop computer. An input of 10 mV peak-to-peak
AC voltage with a frequency decay ranging from 300 kHz to 100 Hz was delivered to the
sites. The magnitudes and phases of the impedance were acquired at 20 data points per
frequency decade. After the measurements, the electrical resistance properties of the tissue
were calculated on the basis of equivalent circuit model by using the Gamry Echem Analyst
software suite (Gamry Instruments, PA) as previously described. [148]
3.2.6 Immunohistochemistry
Vascular rings corresponding to the EIS measurement sites were cut from the aorta
segments, and immersed in 4% paraformaldehyde. After 24 hours, they were embedded in
paraffin and cut into serial 5-µ m sections. Immunostaining was performed with standard

techniques in paraffin embedded vascular tissue using biotinylated secondary

antibodies
and streptavidin-conjugated horse radish peroxidase (HRP). OxLDL was stained with
mAb4E6 antibody. [175] Tissue sections were imaged (Olympus IX70 microscope, Japan),
and were captured with a CCD digital camera (ProgRes C3, Jenoptik, Germany).
3.2.7 Statistical Analysis
Data were expressed as means ± SD where it was applicable. A student’s t-test was
performed for statistical comparisons between two groups of values. One-way analysis of
variance (ANOVA) was performed for comparisons of multiple groups of values. The
Tukey procedure was performed to determine the statistical significance among multiple
groups. A P value of < 0.05 was considered statistically significant.

77

3.3 Chapter Three Results
3.3.1 OxLDL-Laden Lesions in the Endoluminal Regions Exposed to Augmented ISS
Representative ISS profiles were validated with the CFD simulations for wall shear stress
(WSS) (Fig. 19), the time-averaged ISS (ISSave) and peak ISS were comparable with the
computed WSS values consistent with the established 23% and 14% experimental
errors[157, 170], respectively. The acquired ISS profiles were then compared between
baseline and after 9 weeks of high-fat diet (Fig. 20, 21) (Figure 27 and 28 for the entire
aorta).
In the normal diet-fed arm, ISSave values increased from distal aortic arch to infra-renal
aorta in relation to the tapering in the diameters of downstream vessels. However, there
was no significant changes in ISS profiles and the ISSave values between the baseline and
after 9 weeks (n=4, P > 0.05) (Fig. 20c). The corresponding cross-section of the thoracic
aorta visualized by the high-frequency IVUS revealed no gross endoluminal lesions in
agreement with the negative immunostaining for anti-oxLDL throughout the entire aorta
(Fig. 20d).
In the fat-fed arm, the magnitude of ISS profiles increased after 9 weeks compared to the
baseline in the representative peri-renal aorta (Fig. 21). Similar to the trend in normal diet-
fed rabbits, ISSave values increased from the distal aortic arch to the infra-renal aortas (Fig.
21c). However, there was a significant augmentation in ISS throughout the entire regions
after 9 weeks (n=4, P < 0.05), accompanied by a greater standard deviations among
individual measurements than those of the baseline. ISSave values increased by 24%, 20%,
30%, and 38% in the distal aortic arch, thoracic, abdominal, and infra-renal aorta,
respectively (n=4, P < 0.05) (Fig. 21c and Table 1). Similarly, the peak ISS values
increased by 28%, 16%, 32%, and 26%, respectively (n=4, P < 0.05) (Table 1). IVUS
imaging of the thoracic aorta section revealed endoluminal lesions that were prominent for
anti-oxLDL staining (Fig. 21d). In parallel, serum LDL increased by 140-fold (n=4, P <
0.05), accompanied by an increase in kinematic viscosity by 1.5-fold (n=4, P < 0.05) after
9 weeks (Fig. 22). Hence, augmented ISSave values in response to high-fat diets were
associated with endoluminal evidence of atherosclerotic lesions as supported by IVUS and
immunohistochemistry.

78

3.3.2 Increased Electrochemical Impedance in Association with OxLDL-Laden
Lesions
Under the IVUS guidance, concentric bipolar microelectrodes were deployed to assess EIS
signals. The frequency-dependent EIS spectra were significantly elevated from 30 kHz to
100 kHz in the oxLDL-laden regions, as evidenced by immuno-staining (Fig. 23a, e and
f) (Figure 29). Significant differences in phase spectra also developed from 10 kHz to ~30
kHz (Fig. 23b). The impedance spectra were subsequently input into the equivalent circuit
model to simulate tissue resistance between the lesion-free and ox-LDL-rich lesions as
previously reported (Fig. 23c).[148] Both simulated impedance spectra and experimental
data were in close agreement associated with a minimal deviation (1% to 5%).
In the equivalent circuit model, the constant phase element (CPE) is associated non-ideal
double layer capacitance at electrode/tissue interface and is defined to have impedance of
Z
C PE
=
1
Y ω
a
(3)
where Y is the empirical admittance and a is an empirical constant phase value between 0
and 1 [176]. Our fitted CPE constants Y and a for healthy rabbit aorta (n = 8) and plaques
(n = 12) are 104± 49 nS, 83± 31 nS, and 0.727± 0.089, 0.781± 0.095, respectively. We
observed no significant difference in Y and a values between healthy rabbit aorta wall and
lesion sites (p > 0.05). The calculated tissue resistance for the oxLDL-rich lesion (679 ±
125 , n = 12) was significantly higher in comparison with that of lesion-free regions (497
± 55 , n = 8) (Fig. 23f).

79

3.4 Chapter Three Discussion
The key findings in the current study are to demonstrate an integrated strategy with a
combination of three micro-sensors to assess endoluminal tissue impedance in the regions
of augmented shear stress as visualized by the high-frequency ultrasound imaging.
Identification of the distinct frequency-dependent EIS signals in the oxLDL-rich lesions
were made possible by the newly developed flexible concentric bipolar microelectrodes
(Figure 18a). In our current study, despite of having a limitation of small sample size, we
demonstrated augmented ISS values in response to high-fat diet by deploying the flexible
micro-thermal sensors to the aorta of NZW rabbits. Interrogation of the regions harboring
augmented shear stress by IVUS revealed atherosclerotic lesions. Elevated EIS signals in
these lesions were associated with prominent anti-oxLDL staining. Hence, our approach
provides an experimental basis to further integrate the three sensors for simultaneous
characterization of mechanically unstable plaque.
Intravascular electrochemical impedance spectroscopy is an emerging technology capable
of differentiating cellular composition in the atherosclerotic plaques, thus offering a
feasible strategy to identify metabolically active and mechanically unstable lesions.[148-
150, 155] We and others have established a quantitative correlation between tissue
impedance and atherosclerotic lesions in terms of active lipid content (oxLDL). Streiners
et al. deployed impedance sensor by using a linear 4-point electrode configuration into ex
vivo human coronary arteries, and demonstrated that inflammatory process in advanced
vulnerable plaques (Type V) engendered an elevated tissue impedance.[150] However, the
linear 4-electrode configuration poses two main constraints: 1) the large size of 4-electode
array (1.4mm in the entire length) limits its capability to assess large lesions; and 2) the
linear-arrays entails a limited spatial resolution to assess small and non-homogeneous
lesions. For these reasons, we proposed the concentric bipolar microelectrodes to provide
high spatial resolution (0.5mm diameter) and symmetric tissue impedance. Using our
concentric bipolar microelectrodes, we distinguished pre-atherosclerotic lesions that
harbored oxLDL and foam cell infiltrates in the descending aorta immediately distal the
aortic arch.[148] Unlike the linear 4-point configuration, we proposed the use of concentric
bipolar microelectrodes to address uneven lesion topography, heterogeneous tissue
composition and non-uniform current distribution; thus, allowing for impedance
assessment independent of sensor orientation at a high spatial resolution. Furthermore, the
compact size of the concentric configuration allows for packaging of an array of concentric
bipolar electrodes onto a balloon catheter for mapping the endoluminal impedance; thus,
enhancing specificity and sensitivity of identifying active lipid-rich lesions.
80

In parallel, we developed equivalent circuit model and performed simulation to isolate the
impedance contribution by the lesion tissue RB (Fig. 23c). We utilized a modified Randle’s
circuit to account for the non-Faraday impedance at tissue-electrode interface and tissue
non-homogeneities.[176, 177]. In our model, the CPE component is primarily associated
with electrode surface properties and surface/solute interactions. We observed no
significant difference in CPE values between healthy rabbit aorta wall and lesion sites
measured with the same concentric bipolar electrodes setup, indicating that the
electrode/tissue interface impedance did not change significantly due to lesion
development. The value of tissue resistance component RB is largely dependent on its water
content, electrolyte concentration, as well as the presence of oxidative/inflammatory
substances and calcification. Applying computational simulation, we were able to
demonstrate a significantly higher tissue resistance in the oxLDL-rich lesions. Hence,
elevated EIS signals in the regions of augmented ISSave helped identify active lipid-rich
lesions in which oxidized LDL and macrophage-derived foam cells are the important pro-
inflammatory components.[178]
By the integrated micro-sensor approach, we further demonstrated augmented
intravascular shear stress (ISS) in response to high-fat diet. Multiple factors were
implicated in the ISS augmentation; namely, changes in the compliance of vasculature,
endoluminal remodeling, increased blood viscosity, and presence of atherosclerotic lesions.
Hypercholesterolemia is well-recognized to reduce arterial compliance.[179] Reduction in
coronary artery compliance in patients with early atherosclerosis is associated with
endothelial dysfunction and an increase in wall shear stress.[180] While compensatory
remodeling of coronary vasculature initially maintains shear stress, [181-183] remodeling-
mediated neointimal hyperplasia is conducive to the development of high shear stress in
response to reduced vessel lumen diameters.[184] The presence of pre-atherosclerotic
lesions further engenders disturbed flow downstream to the stenosis, [185, 186] recruiting
monocyte and LDL particles transmigration into the subendothelial layers [170, 187] as
well as promoting atherothrombosis.[158] Using the ApoE knockout mouse model with
the constrictive carotid artery collars, Ding et al. reported elevated shear stress
within the constrictive regions and low shear stress at proximal regions coupled with
endothelial disfunction[188]. In corollary, we demonstrated augmented
intravascular shear stress after 9 weeks of high-fat diet due to both presence of
endoluminal lesions and a significant increase in blood viscosity. Using the MEMS
thermal sensors, we have also reported that low shear stress developed at the
proximal region or upstream from the stenotic lesions.[157] Our current protocol did
not result in high-degree stenotic lesions. However, we also observed that low time-
81

averaged shear stress developed in the arterial branching points or curvatures where
atherosclerosis preferentially occurred. Finally, augmented in ISS in fat-fed rabbits was
also influenced by blood viscosity. In the current study, high-fat diet resulted in a 30.5%
higher in blood viscosity that that of normal diet (4.23cP vs. 2.94cP), accompanied by a
138.67-fold increase in serum LDL concentration after 9 weeks (26.2 mg/dL to 3659.4
mg/dL). In contrast, LDL concentrations in the normal diet decreased by 67% after 9 weeks
(from 15.8 mg/dL to 5.2 mg/dL).
In conjunction with intravascular ultrasound (IVUS), electrochemical impedance approach
further holds promises to distinguish between thick- and thin-capped fibrous plaques
associated with a large lipid pool. Although other imaging modalities, including X-ray
angiography, are able to identify the plaque morphology, characterization of lipid-rich and
mechanically vulnerable plaque remains a clinical challenge. Thus, we demonstrated that
integrating IVUS and EIS signals afforded real-time and in vivo strategy to identify
mechanically unstable plaque, and additional investigations with larger sample size are
needed to provide a robust statistical power for the pre-clinical studies.
In this study, elevated tissue impedance in the endoluminal regions of augmented shear
stress were demonstrated in the fat-fed NZW rabbit model. Our integrated approach
revealed two new findings: 1) time-averaged ISS increased in the regions of atherosclerotic
lesions as visualized by high-frequency IVUS, and 2) the elevated EIS signals in these
lesions were associated with active lipid content. In this context, integrating intravascular
ultrasound imaging, hemodynamics and tissue impedance offer a translational basis for
combining the three micro-sensors for diagnostic applications.

82

Figure 18. Catheter-based concentric bipolar microelectrodes. (a) Magnification of
concentric bipolar microelectrodes. (b) Packaging of the concentric bipolar
microelectrodes to the coaxial wire.

83

Figure 19. Comparison of intravascular shear stress profiles with Computational
Fluid Dynamics (CFD) simulations. Fluoroscopic guidance allowed for positioning the
catheter at the (a)-(d) distal aortic arch, (e)-(h) thoracic aorta, (i)-(l) peri-renal abdominal
aorta, and (m)-(p) infra-renal aorta. The thermal sensor was able to capture the
characteristic pulsatile profiles along the aorta with a high spatial and temporal resolution.
The red, solid curves denoted ISS obtained by the sensors, whereas the blue dashed curves
denoted the CFD simulations.

84

Figure 20. ISS profiles in response of normal diet. (a) Representative baseline ISS
profiles obtained from the abdominal aorta of rabbits. (b) Representative ISS profiles
obtained from the same region after 9 weeks of normal chow diet (ND). The solid lines
represented averaged shear stress over 10 cardiac cycles; dashed lines represented average
ISS ± standard deviation. (c) Time-average ISS profiles in the distal aortic arch, thoracic
aorta, abdominal aorta and infra-renal aorta regions were compared between baseline and
after 9 weeks of ND. No statistically significant differences in ISS profiles were observed.
(d) Representative cross-section of the thoracic aorta by IVUS imaging and histology
revealed absence of endoluminal lesions on ND.

85

Figure 21. ISS profiles in response to high-fat diet. (a) Representative baseline ISS
profiles obtained from the abdominal aorta of rabbits. (b) Representative ISS measured
obtained from the abdominal aorta of rabbits on high-fat diet (HD) for 9 weeks. The solid
lines represented averaged shear stress over 10 cardiac cycles; dashed lines represented
average ISS ± standard deviation. (c) Time-average ISS profiles in the distal aortic arch,
thoracic aorta, abdominal aorta and renal aorta regions were compared between baseline
and after 9 weeks of HD. * denoted statistically significant differences in time-averaged
ISS signals at baseline and after 9 weeks of HD (P < 0.05). (d) Representative cross-section
by IVUS imaging and histology of the thoracic aorta segment. Positive staining (reddish-
brown) indicated presence of oxLDL. The discontinuations of the endoluminal lesions at
10 and 12 o’clock were due to branching.

86

Figure 22. LDL and blood viscosity at baseline versus after 9 weeks. (a) LDL levels
significantly increased after 9 weeks in rabbits on high-fat diet. (b) Kinematic viscosity
was significantly higher in rabbits on high-fat than on normal diets after 9 weeks.

87

88

Figure 23. Endoluminal EIS assessment of oxLDL-rich lesions. (a) Frequency-
dependent impedance was measured from 10kHz to 100kHz (Logarithm scale). (b) Phase
angles were measured from the corresponding frequency range (Logarithm scale). (c)
Equivalent circuit model to simulate tissue resistance provided validation of the
experimental EIS signals as previously described [148]. CPE: constant phase element. (d)
The representative IVUS image revealed the endoluminal lesions at 4 o’clock. The
corresponding histology confirmed the pre-atherosclerotic lesions. (e)
Immunohistochemistry revealed prominent anti-oxLDL staining (reddish-brown). (f) The
bar graph compared the simulated tissue resistance between normal (n = 8) and oxLDL-
rich lesions (n = 12) (* p < 0.05).

89

Figure 24. The catheter-based MEMS shear stress sensors and concentric bipolar
microelectrodes. (a) The dotted circle at the terminal end of sensor is magnified to reveal
the thermal sensor. (b) Details of the sequential alignment of the thermal sensor and the
concentric bipolar microelectrodes to the coaxial wire revealed connection to the electrical
coaxial wire using the conductive epoxy. (c) A photograph reveals the packaged thermal
sensor and concentric bipolar microelectrodes to the coaxial wire. (d) Angiogram shows
the deployment of catheter.

90

Figure 25. Hemodynamic response was measured as voltage output changes in aorta.
(a) Rabbit aorta was visualized by fluoroscopic angiography with contrast for
measurements of aortic diameter. (b) Fluoroscopy further guided deployment of flexible
intravascular sensor to the ascending aorta and the pacing lead to the right atrium. (c) Real-
time intravascular measurements (blue line) captured the pulsatile voltage profiles. The
signal was filtered by Hilbert wavelet transform (red line). Voltage measurements were
then converted to the corresponding shear stress values as described in the methods.

91

Figure 26. Time frame of the experimental protocol.

92

Figure 27. Intravascular shear stress (ISS) profiles in ND-fed rabbits. (a) Baseline ISS
from the distal aortic arch. (b) Corresponding ISS after 9 weeks. (c) Baseline ISS from the
thoracic aorta. (d) Corresponding ISS after 9 weeks. (e) Baseline ISS from the abdominal
aorta. (f) Corresponding ISS after 9 weeks. (g) Baseline ISS from peri-renal aorta. (h)
Corresponding ISS after 9 weeks. Center solid lines represent the mean shear stress from
10 samples. Upper envelope and lower envelope represent one standard deviation from
mean shear stress. (i) Comparison of time-averaged ISS between baseline and after 9 weeks
of ND. (j) Comparison of peak ISS between baseline and after 9 weeks of ND.
93

Figure 28. Intravascular shear stress (ISS) profiles in HD-fed rabbits. (a) Baseline ISS
from distal aortic arch. (b) Shear stress after 9 weeks from the same distal aortic arch. (c)
Baseline ISS from the thoracic aorta. (d) ISS after 8 weeks from the same thoracic aorta.
(e) Baseline ISS from the abdominal aorta. (f) ISS after 8 weeks from the same abdominal
aorta. (g) Baseline shear stress from peri-renal aorta. (h) ISS after 9 weeks from t he same
peri-renal aorta. The thick solid lines represented the mean shear stress from 10 samples.
Upper envelope and lower envelope represented one standard deviation from the mean ISS.
(i) Comparison of the time-averaged ISS between the baseline and after 9 weeks of HD. (j)
Comparison of peak ISS between the baseline and after 9 weeks of HD. * mark represents
the P values < 0.05.
94

Figure 29. IVUS and histology images to determine endoluminal atherosclerotic
lesions. After 9 weeks of ND, endoluminal lesions were absent (a, b). After 9 weeks of
HD, endoluminal lesions were present (c, d).

95

Table 1. Summary of ISS in Aortas of Rabbits Fed with Normal or High-fat Diet for
9 Weeks
Peak ISS (dyne/cm
2
) Baseline ND 9 Weeks ND Baseline HD 9 Weeks HD
Aortic Arch 39.1± 0.9 40.0± 1.8 28.5± 0.5 39.4± 3.1
Thoracic Aorta 51.6± 2.9 49.60± 2.3 44.4± 0.7 52.8± 2.2
Abdominal Aorta 74.0± 3.3 77.60± 3.3 49.6± 0.9 73.0± 9.0
Renal Aorta 99.6± 4.1 104.3± 4.4 86.0± 0.5 116.3± 15.5
ISSave (dyne/cm
2
)
Aortic Arch 17.0± 0.1 17.9± 0.6 10.2± 0.1 21.2± 1.1
Thoracic Aorta 22.9± 0.5 22.4± 0.6 17.6± 0.2 25.2± 1.0
Abdominal Aorta 34.8± 0.8 36.5± 0.8 19.2± 0.2 31.9± 1.4
Renal Aorta 52.2± 0.6 52.0± 2.3 30.6± 0.3 52.0± 4.6

96

3.5 References
[147] M. Naghavi and E. Falk, "From vulnerable plaque to vulnerable patient,"
Asymptomatic Atherosclerosis, pp. 13-38, 2010.
[148] F. Yu, X. Dai, T. Beebe, and T. Hsiai, "Electrochemical impedance spectroscopy
to characterize inflammatory atherosclerotic plaques," Biosensors and
Bioelectronics, vol. 30, pp. 165-173, 2011.
[149] I. Streitner, M. Goldhofer, S. Cho, H. Thielecke, R. Kinscherf, F. Streitner, et al.,
"Electric impedance spectroscopy of human atherosclerotic lesions,"
Atherosclerosis, vol. 206, pp. 464-468, 2009.
[150] I. Streitner, M. Goldhofer, S. Cho, R. Kinscherf, H. Thielecke, M. Borggrefe, et al.,
"Cellular Imaging of Human Atherosclerotic Lesions by Intravascular Electric
Impedance Spectroscopy," PLoS ONE, vol. 7, p. e35405, 2012.
[151] T. K. Hsiai, S. K. Cho, H. M. Honda, S. Hama, M. Navab, L. L. Demer, et al.,
"Endothelial Cell Dynamics under Pulsating Flows: Significance of High Versus
Low Shear Stress Slew Rates (∂τ/∂t)," Annals of biomedical engineering, vol. 30,
pp. 646-656, 2002.
[152] J. J. Li, X. Meng, H. P. Si, C. Zhang, H. X. Lv, Y. X. Zhao, et al., "Hepcidin
Destabilizes Atherosclerotic Plaque via Overactivating Macrophages After
Erythrophagocytosis," Arteriosclerosis, Thrombosis, and Vascular Biology, vol.
32, pp. 1158-1166, 2012.
[153] T. Suselbeck, H. Thielecke, J. Kochlin, S. Cho, I. Weinschenk, J. Metz, et al.,
"Intravascular electric impedance spectroscopy of atherosclerotic lesions using a
new impedance catheter system," Basic research in cardiology, vol. 100, pp. 446-
452, 2005.
[154] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an intravascular
impedance catheter for detection of fatty lesions in arteries," IEEE transactions on
medical imaging, vol. 16, pp. 439-446, 1997.
97

[155] F. Yu, R. Li, L. Ai, C. Edington, H. Yu, M. Barr, et al., "Electrochemical Impedance
Spectroscopy to Assess Vascular Oxidative Stress," Annals of biomedical
engineering, vol. 39, pp. 287-296, 2011.
[156] H. M. Garcì a-Garcì a, B. D. Gogas, P. W. Serruys, and N. Bruining, "IVUS-based
imaging modalities for tissue characterization: similarities and differences," The
International Journal of Cardiovascular Imaging (formerly Cardiac Imaging), vol.
27, pp. 215-224, 2011.
[157] L. Ai, L. Zhang, W. Dai, C. Hu, K. Kirk Shung, and T. K. Hsiai, "Real-time
assessment of flow reversal in an eccentric arterial stenotic model," Journal of
biomechanics, vol. 43, pp. 2678-2683, 2010.
[158] D. L. Bark Jr and D. N. Ku, "Wall shear over high degree stenoses pertinent to
atherothrombosis," Journal of biomechanics, vol. 43, pp. 2970-2977, 2010.
[159] Y. S. Chatzizisis, A. U. Coskun, M. Jonas, E. R. Edelman, C. L. Feldman, and P.
H. Stone, "Role of Endothelial Shear Stress in the Natural History of Coronary
Atherosclerosis and Vascular Remodeling:: Molecular, Cellular, and Vascular
Behavior," Journal of the American College of Cardiology, vol. 49, pp. 2379-2393,
2007.
[160] C. Cheng, D. Tempel, R. van Haperen, H. C. de Boer, D. Segers, M. Huisman, et
al., "Shear stress-induced changes in atherosclerotic plaque composition are
modulated by chemokines," Journal of Clinical Investigation, vol. 117, pp. 616-26,
Mar 2007.
[161] P. H. Stone, A. U. Coskun, S. Kinlay, M. E. Clark, M. Sonka, A. Wahle, et al.,
"Effect of Endothelial Shear Stress on the Progression of Coronary Artery Disease,
Vascular Remodeling, and In-Stent Restenosis in Humans In Vivo 6-Month
Follow-Up Study," Circulation, vol. 108, pp. 438-444, 2003.
[162] N. Sun, N. B. Wood, A. D. Hughes, S. A. M. Thom, and X. Y. Xu, "Influence of
pulsatile flow on LDL transport in the arterial wall," Annals of biomedical
engineering, vol. 35, pp. 1782-1790, 2007.
98

[163] R. M. Nerem, R. W. Alexander, D. C. Chappell, R. M. Medford, S. E. Varner, and
W. R. TAYLOR, "The study of the influence of flow on vascular endothelial
biology," The American journal of the medical sciences, vol. 316, p. 169, 1998.
[164] R. Li, D. Mittelstein, J. Lee, K. Fang, R. Majumdar, Y. Tintut, et al., "A dynamic
model of calcific nodule destabilization in response to monocyte-and oxidized
lipid-induced matrix metalloproteinases," American Journal of Physiology-Cell
Physiology, vol. 302, pp. C658-C665, 2012.
[165] N. R. Madamanchi, A. Vendrov, and M. S. Runge, "Oxidative stress and vascular
disease," Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 25, pp. 29-38,
2005.
[166] J. Hwang, H. Michael, A. Salazar, B. Lassegue, K. Griendling, M. Navab, et al.,
"Pulsatile Versus Oscillatory Shear Stress Regulates NADPH Oxidase Subunit
Expression," Circulation research, vol. 93, pp. 1225-1232, 2003.
[167] S. Parathath, S. L. Mick, J. E. Feig, V. Joaquin, L. Grauer, D. M. Habiel, et al.,
"Hypoxia Is Present in Murine Atherosclerotic Plaques and Has Multiple Adverse
Effects on Macrophage Lipid MetabolismNovelty and Significance," Circulation
research, vol. 109, pp. 1141-1152, 2011.
[168] S. Lee, J. R. Springstead, B. Parks, C. E. Romanoski, R. Palvolgyi, T. Ho, et al.,
"Metalloproteinase Processing of HBEGF is a Proximal Event in the Response of
Human Aortic Endothelial Cells to Oxidized Phospholipids," Arteriosclerosis,
Thrombosis, and Vascular Biology, vol. 32, pp. 1246-1254, 2012.
[169] H. Yu, L. Ai, M. Rouhanizadeh, D. Patel, E. S. Kim, and T. K. Hsiai, "Flexible
polymer sensors for in vivo intravascular shear stress analysis,"
Microelectromechanical Systems, Journal of, vol. 17, pp. 1178-1186, 2008.
[170] L. Ai, H. Yu, W. Takabe, A. Paraboschi, F. Yu, E. Kim, et al., "Optimization of
intravascular shear stress assessment in vivo," Journal of biomechanics, vol. 42,
pp. 1429-1437, 2009.
[171] F. Yu, L. Ai, W. Dai, N. Rozengurt, H. Yu, and T. K. Hsiai, "MEMS Thermal
Sensors to Detect Changes in Heat Transfer in the Pre-Atherosclerotic Regions of
99

Fat-Fed New Zealand White Rabbits," Annals of biomedical engineering, vol. 39,
pp. 1736-1744, 2011.
[172] M. Rouhanizadeh, T. C. Lin, D. Arcas, J. Hwang, and T. H. Hsiai, "Spatial
variations in shear stress in a 3-D bifurcation model at low Reynolds numbers,"
Annals of Biomedical Engineering, vol. 33, pp. 1360-1374, Oct 2005.
[173] P. Sun, Y. Zhang, F. Yu, E. Parks, A. Lyman, Q. Wu, et al., "Micro-
electrocardiograms to study post-ventricular amputation of zebrafish heart," Ann
Biomed Eng, vol. 37, pp. 890-901, May 2009.
[174] W. Wei, X. Li, Q. Zhou, K. K. Shung, and Z. Chen, "Integrated ultrasound and
photoacoustic probe for co-registered intravascular imaging," Journal of
biomedical optics, vol. 16, p. 106001, 2011.
[175] P. Holvoet, J. Vanhaecke, S. Janssens, F. Van de Werf, and D. Collen, "Oxidized
LDL and malondialdehyde-modified LDL in patients with acute coronary
syndromes and stable coronary artery disease," Circulation, vol. 98, pp. 1487-1494,
1998.
[176] S. Hleli, C. Martelet, A. Abdelghani, F. Bessueille, A. Errachid, J. Samitier, et al.,
"An immunosensor for haemoglobin based on impedimetric properties of a new
mixed self-assembled monolayer," Materials Science and Engineering: C, vol. 26,
pp. 322-327, 2006.
[177] W. Franks, I. Schenker, P. Schmutz, and A. Hierlemann, "Impedance
characterization and modeling of electrodes for biomedical applications," IEEE
Transac Biomed Eng, vol. 52, pp. 1295-1302, 2005.
[178] S. M. Schwartz, Z. S. Galis, M. E. Rosenfeld, and E. Falk, "Plaque rupture in
humans and mice," Arterioscler Thromb Vasc Biol, vol. 27, pp. 705-13, Apr 2007.
[179] C. Giannattasio, M. Failla, G. Emanuelli, A. Grappiolo, L. Boffi, D. Corsi, et al.,
"Local effects of atherosclerotic plaque on arterial distensibility," Hypertension,
vol. 38, pp. 1177-1180, 2001.
100

[180] T. Takumi, E. H. Yang, V. Mathew, C. S. Rihal, R. Gulati, L. O. Lerman, et al.,
"Coronary endothelial dysfunction is associated with a reduction in coronary artery
compliance and an increase in wall shear stress," Heart, vol. 96, pp. 773-778, 2010.
[181] Y. Huo and G. S. Kassab, "Compensatory remodeling of coronary microvasculature
maintains shear stress in porcine left-ventricular hypertrophy," Journal of
Hypertension, vol. 30, p. 608, 2012.
[182] J. M. Dolan, F. J. Sim, H. Meng, and J. Kolega, "Endothelial Cells Express a Unique
Transcriptional Profile under Very High Wall Shear Stress Known to Induce
Expansive Arterial Remodeling," American Journal of Physiology-Cell
Physiology, vol. 302, pp. C1109-C1118, 2012.
[183] H. Samady, P. Eshtehardi, M. C. McDaniel, J. Suo, S. S. Dhawan, C. Maynard, et
al., "Coronary Artery Wall Shear Stress Is Associated With Progression and
Transformation of Atherosclerotic Plaque and Arterial Remodeling in Patients
With Coronary Artery DiseaseClinical Perspective," Circulation, vol. 124, pp. 779-
788, 2011.
[184] P. Zilla, L. Moodley, J. Scherman, H. Krynauw, J. Kortsmit, P. Human, et al.,
"Remodeling leads to distinctly more intimal hyperplasia in coronary than in
infrainguinal vein grafts," Journal of Vascular Surgery, vol. 55, pp. 1734-1741,
2012.
[185] S. S. Varghese and S. H. Frankel, "Numerical modeling of pulsatile turbulent flow
in stenotic vessels," Journal of biomechanical engineering, vol. 125, p. 445, 2003.
[186] B. X. Zhang, "Numerical simulation of pulsatile turbulent flow in tapering stenosed
arteries," International Journal of Numerical Methods for Heat &# 38; Fluid Flow,
vol. 19, pp. 561-573, 2009.
[187] P. F. Davies, A. Remuzzi, E. J. Gordon, C. F. Dewey, and M. A. Gimbrone,
"Turbulent fluid shear stress induces vascular endothelial cell turnover in vitro,"
Proceedings of the National Academy of Sciences, vol. 83, p. 2114, 1986.
[188] S. F. Ding, M. Ni, X. L. Liu, L. H. Qi, M. Zhang, C. X. Liu, et al., "A causal
relationship between shear stress and atherosclerotic lesions in apolipoprotein E
101

knockout mice assessed by ultrasound biomicroscopy," American Journal of
Physiology-Heart and Circulatory Physiology, vol. 298, pp. H2121-H2129, 2010.

102

Chapter Four: Micro-Electrocardiograms to Study Post-
Ventricular Amputation of Zebrafish Heart
1

1
Disclaimer: the content of this chapter has been published as “Micro-Electrocardiograms
to Study Post-Ventricular Amputation of Zebrafish Heart”, Annals of Biomedical
Engineering, 37(5): 890-901 (2009)
Acknowledgement: Co-authors of this manuscript include: Fei. Yu, Juhyun. Lee, Nelson
Jen, Xiang Li, Qian Zhang, Eun S. Kim, Kirk K. Shung, Tzung. K. Hsiai. The author of
this thesis credits other co-authors for their significant input in the process of preparing
the manuscript for publication.
103

4.1 Chapter Four Introduction
The zebrafish (Danio rerio) has emerged as an excellent cardiovascular model, yet
assessing physiological functions of the adult zebrafish heart has been hampered by its
small size and the difficulty of performing studies in an aqueous environment. The majority
of biological research concerning the zebrafish has focused on its developmental stages;
the transparency of the embryo has made the zebrafish a viable model for angiogenesis and
organogenesis. However, limited efforts have been developed to monitor the structures and
electric activities of the adult zebrafish heart in real-time.
Humans lack the regenerative capacity of organisms such as newts and hydra [189, 190].
Injured human hearts can scar and remodel, predisposing the individuals to cardiac
arrhythmia and sudden death[191]. However, zebrafish fully regenerate their myocardium
after up to 20% ventricular resection within two months without evidence of scar tissues
[192]. In response to amputation, thrombosis immediately develops to achieve hemeostasis
in the ventricle. The thrombus is replaced by a fibrin clot 2-4 days post amputation (dpa).
Nascent cardiomyocytes replace the vast majority of the lost ventricular tissue by 30 dpa,
and the structure of the ventricle is fully restored on 60 dpa without scar tissue [192].
Molecular and histological methods have been performed to assess heart regeneration.
However, research tools to study zebrafish cardiac conduction system and mechanics
during the regeneration process have remained under-developed.
Despite a two-chambered heart and a lack of pulmonary vasculature[193], the zebrafish
heart parallels that of humans in terms of QT interval and heart rate[194]. The atrium is
medially dorsal and posterior to the ventricle. Similar to humans, the zebrafish heart is
encased by a pericardial sac in the thoracic cavity, and is situated below the pectoral bone
of the pectoral fins. The bulbous arteriosus is analogous to the human aortic arch with thick
contractile smooth muscle. Blood returns into the sinus venosus, which is analogous to the
vena cava[193].The fundamental electrical properties are remarkably similar to those of
humans[195], and the critical pathways in cardiovascular development parallel higher
vertebrates[196-198]. Hence, the zebrafish is also an emerging model for studying human
inherited cardiac arrhythmia, specifically, long QT syndromes[199].
In 2004, Forouhar et al. reported an electrocardiogram (ECG) of an embryonic zebrafish,
revealing similar atrial and ventricular electrical signals as found in a human ECG, while
encountering challenges in detecting ventricular repolarization (the T wave)[200]. Through
104

the use of oral perfusion and muscle paralytics, MacRae et al. obtained a consistent ECG
of an adult zebrafish with distinct P waves and QRS complexes resembling those of
humans[201]. For the recording, adult fish were paralyzed while perfusion needles were
inserted into the oral cavity[201]. In our study, the surface ECG was we acquired in the
mildly sedated fish. We applied signal processing and wavelet transform to enhance signal-
to-noise ratios
We performed a short-term follow-up of zebrafish ECG signals prior to ventricular
amputation and 2 and 4 dpa. We demonstrated distinct atrial contraction (P), ventricular
depolarization (QRS) and repolarization (QT). PR and QRS intervals remained unchanged
in response to ventricular amputation (n=6, P > 0.05). Corrected QT intervals (QTc)
shortened on 4 dpa (n=6, P<0.05). In parallel, histological findings revealed replacement
of thrombus with fibrin clots. While the specific intervals from the individual fish remained
identical, the voltage (amplitudes) ratios between P waves and QRS complexes were
sensitive to the positioning of electrodes. Ventricular tachycardia or fibrillation was not
observed on 2 and 4 dpa. Overall, these studies have provided a basis to monitor zebrafish
heart function, electrical activities, regeneration, and QT intervals in real-time.

105

4.2 Chapter Four Materials and Methods
4.2.1 Animals
The animal experiments were performed according to protocol approved by the
Institutional Animal Care and Use Committee (IACUC) at the University of Southern
California. Adult zebrafish, 3-4 cm in length, were acquired from Aquatica Tropical
(Florida) and maintained under standard laboratory conditions at 24
o
C. The individual fish
were fed with brime shrimp (hatched from eggs in 10ml in 2 L salt water) daily, kept in
constantly circulating water, and isolated from other fish for ECG follow-up post
amputation. The range of the fish size was from 31 to 38mm.
4.2.2 Heart Resection
Zebrafish were sedated in 5% Tricaine methanesulfonate (4 g Tricaine, 979 ml H2O, 21 ml
tris-HCl) until they floated ventral side up and ceased to move for 5 seconds. Tweezers
were used to remove 3-4 scale layers above the thoracic cavity. A midline incision of 0.25
cm in length was created posterior to the ventricle. The pericardial sac was isolated and
punctured to expose the ventricle. The ventricle was gently raised to expose the apex, and
approximately 20% of the ventricle was excised by scissors. Thrombosis developed
immediately [192], and the zebrafish were returned to freshwater with continuous
oxygenator.
4.2.3 ECG Recording
Baseline ECG signals were recorded one day prior to ventricular amputation, and repeated
ECG interrogations were performed on 2 and 4 days post-amputation. In parallel, ECG
signals were recorded from the control fish without heart resection. All of the studies were
performed in triplicates at 26
°
C. The exact length of the fish was recorded prior to ECG
measurement each time. Fish were sedated as described above and placed in a damp sponge
with the ventral side exposed for electrode placement. Two 29-gauge micro-electrodes (AD
Instrument, Colorado Springs, CO) were positioned at 90
o
to the animal’s ventral epidermis
adjusted by positional walkers (AMI-USC Machine Shop). The positive electrode was
positioned directly above the ventricle, or 2-3 mm below the end of the gills along the
midline where the chest excursion in response to myocardial contraction could be observed
106

(approximately one -fifth of the body length from the head). The negative or reference
electrode was positioned along the midline immediately in front to the anal fin. The relative
distance of the two electrodes was about 10-11 mm, or one-third of the total length such
that the negative electrode was sufficiently away from the heart and could be safely
regarded as ground. The precise placement of both electrodes was established using
calipers to measure the relative distance along the ventral line from mouth to tail. The
scales were removed to facilitate sufficient contact pressure and increase signal to noise
ratio. Both electrodes were inserted into the skin to approximately 1 mm in depth for
stabilizing the electrode perpendicular to ventral epidermis (Fig. 30a). The exact position
of the positive electrode may be adjusted slightly using the micromanipulator to obtain
maximum voltage signals. The individual fish remained stationary in the sponge for less
than three minutes, with a mean ECG recording of one minute. Next, the fish was allowed
to recover from sedation within 5 minutes in an oxygenated water bath free of Tricane
4.2.4 Data Acquisition and Processing
The two electrodes were connected to a high-gain differential amplifier (A-M Systems Inc.
1700 Differential Amplifier, Carlsborg, WA) and the ECG signals were amplified by
1,000-fold and filtered at a cut-off frequency between 1 and 500 Hz as well as at 60 Hz
(notch). The signals were acquired and digitized at a sampling rate of 1,000 Hz (National
Instruments USB-6251 DAQ device, Austin TX, and LabVIEW 8.2.). The recorded signals
were digitally processed using the wavelet transform and threshold with a Matlab 7.5
software (Mathworks inc, Natick, MA) (Fig.30b).
Majority of energy of the Zebrafish ECG signals fell within the range of 2-45Hz frequency.
Within this frequency range, QRS intervals fell in the range of 10-45Hz while T- and P-
waves were in the range of 3-10Hz. The ambient power supply and wiring generated high
frequency harmonic interference at 60Hz. The baseline drifting was below 2Hz, and the
noise from fish gill motion was 2-2kHz. In addition to signal processing, wavelet transform
allowed for filtering various sources of noise.
A wavelet is a mathematical function used to divide a given function or continuous-time
signal into different frequency components. Wavelet transform represents a function by
scaled wavelets in the time domain. [202]. After ECG signals were digitized at 1000Hz,
the digital signals were divided into 10 frequency components or scales by using the “coif5”
wavelet, a 5
th
order coiflet (a discrete wavelet) function designed by Ingrid
107

Daubechies[203, 204]. Coif5 wavelet is symmetrical, useful in preventing de-phasing
image processing. Coif wavelet also allows for a high speed of convergence to reduce
computation[205]. “Adaptive Thresholding”, a process to eliminate with sub-threshold
value noises while retaining the super-threshold signals within different frequency range,
was used to suppress noise from various sources. Finally, the remaining signals were
recomposed by inverse wavelet transform to the final de-noised ECG signals.
4.2.5 Heart Histology
After ECG recording, the fish were sacrificed at 4 dpa and the hearts were processed for
paraffin section (10 m). The sections were de-paraffined, and processed for acid fuchsin-
orange G (AFOG) staining, staining the fibrin and collagen red and blue, respectively.
4.2.6 Statistical Analysis
ECG recordings were identified for PR, QRS, QT, and RR intervals using the
MATLAB® program. ECG signals were selected from rhythm strips containing a
minimum of 20 seconds of distinct P waves, QRS complexes, and T waves. QT intervals
were corrected for heart rate variability by using the following equation [206]:
RR
QT
QT
c
 (4)
All values are expressed as means ± SD. For statistical comparisons of wave intervals, we
used a paired t-test with values of p < 0.05 considered to be statistically significant. In light
of possible non-normal distribution of ECG signals, we compared multiple mean values by
non-parametric one-way Kruskal-Wallis tests [207] and assessed the statistical significance
of change in PR, QRS and QTc among different measurements.

108

4.3 Chapter Four Results
4.3.1 Signal Processing and Filtering
Power supply, wiring, and motion artifacts constituted the main sources of noise in the
recorded ECG signals. Different threshold values were used to de-noise the ECG signals
in various frequency ranges. The main zebrafish ECG signals were excluded from scales
1, 2, 9 and 10 as indicated by the individual frequency components or scales (Fig. 31).
While high threshold values used in these scales allowed for complete elimination of high
frequency noise and the wondering baseline, low threshold values allowed for elimination
of the low amplitude noise (Figs. 31a and b). The P waves, QRS complexes, and T waves
were not affected by the wavelet transform. The zebrafish ECG signals in scales 3, 4, 5, 6
were stronger than those of power supply, wiring, and ambient noise. The signals of low
frequency noise were comparable to those of scales 8 and 9, and were selected for
conversation to ECG signals, particularly the T waves.
After data processing and filtering, ECG signals were obtained (Fig. 32). The wondering
baseline was noticeable prior to wavelet transform (Fig. 32a). The de-noised ECG signals
were obtained by using the appropriate threshold values in the individual frequency
components (scales). Application of wavelet transform at threshold values between 1.9 Hz
and 62.5 Hz generated the distinct P waves, QRS complexes, and ventricular repolarization
patterns or T waves (Fig. 32b). However, filtering frequency at 1.9-7.8Hz resulted in
removal of the T wave pattern (Fig. 32c).
Similar to the human ECG, electrode positions from the heart influenced the amplitude of
the voltage signals. We noted that minute changes in positioning the positive electrode
affected the amplitudes of P waves, QRS complexes, and T waves. Three independent trials
of ECG readings were performed on each zebrafish on the same day post amputation (Fig.
33). A moderate displacement between the positive and negative electrodes, the lateral
distance from the ventral line of fish, the position of the positive electrode with respect to
the heart, and the angle of the electrode needles to the surface of the zebrafish, influenced
the amplitudes of P waves, QRS complexes, and T waves. The P and peaked T waves are
prominent in Figure 33a, whereas the QRS complexes are prominent in Figures 33b and
33c. However, the position of the negative electrode did not significantly affect the ECG
signals. A displacement of the negative electrode at 0.5mm from the midline produced
nearly identical results, indicating that the placement of negative electrode was not an issue
109

affecting ECG recording. Despite the variations in P, R, and T waves, the PR, QRS, and
QT intervals remained unchanged, indicating that signal processing and wavelet transform
provided a reliable means to monitor for changes in ventricular repolarization intervals in
zebrafish heart regeneration.
4.3.2 Zebrafish ECG Signals Following Ventricular Amputation
Ventricular amputation was performed for repeated ECG recordings. The mean heart rate
was 149± 18 beats/min prior to Tricane sedation, and was reduced to 90± 17 beats/min after
sedation (n=9 and 22, respectively) (Table 2). Prior to ventricular amputation, QTc was
used to account for heart rate variability. Representative ECG signals on day 0 (prior to
amputation), 1, 4, 9, and 13 dpa are shown (Fig. 34). The T waves were more prominent
after ventricular amputation, reflecting changes in ventricular repolarization. Repeated
ECG signals were obtained from 4 different zebrafish, 4 dpa (Fig. 35). Distinct P waves,
QRS complexes, and T waves were present among the four individual fish. The histology
of an uncut zebrafish heart revealed an intact ventricle and atrium with prominent
ventricular trabeculae. The amputated ventricles revealed the presence of fibrin and
collagen as evidenced by red and blue staining.
4.3.3 Analysis of PR, QRS and QTc Intervals Analyses of the mean PR, QRS, QT and
QTc intervals were performed on 6 zebrafish prior to and post-amputation (Fig. 36). The
mean values of these intervals and their corresponding standard deviations were obtained
in triplicates for the individual fish. Variations in intervals existed among different fish.
The mean PR and QRS intervals remained unchanged prior to and post amputation (n= 6
fish, P > 0.5). However, the QT intervals were shortened on 4 dpa (n = 6, P = 0.07), and
correction for the heart rate variability supported that the QTc intervals were significantly
shortened on 4 dpa (n = 6, P < 0.05). The PR and QRS intervals reflected the intact atrial
and ventricular conduction, and the shortened QTc implicated changes in ventricular
repolarization in response to myocardial injury. Thus, monitoring ECG signals in zebrafish
provided a means to study cardiac conduction during zebrafish heart generation.

110

4.4 Discussion
The zebrafish heart is an emergent vertebrate model for myocardial regeneration and drug
screening. We demonstrated that real-time zebrafish cardiac function is visible by distinct
P waves for atrial contraction, QRS complexes for ventricular depolarization, and QT
intervals for ventricular repolarization prior to, and 2 and 4 days post amputation. Despite
a two-chamber heart, zebrafish demonstrate heart conduction patterns that mirror those of
humans. In response to ventricular amputation, PR and QRS intervals remained intact and
unchanged in 6 fish that were analyzed. Corrected QT intervals (QTc) shortened on 4 dpa,
suggesting that ventricular resection alters myocardial repolarization. In parallel, histology
revealed that apical thrombi were replaced with fibrin clots and collagen fibers in response
to amputation. While atrial arrhythmia was recorded after prolonged sedation, ventricular
tachycardia or fibrillation was not observed in this study. Human myocardium scars and
remodels in response to acute coronary syndromes; zebrafish heart muscle regenerates in
the absence of scar formation and ventricular remodeling [192]. Thus, we have developed
a reliable technique to assess changes in QT intervals for a longitudinal study.
We reported that ventricular amputation led to a shortened QTc interval without affecting
the PR and QRS intervals from non-anesthetized and non-paralyzed adult zebrafish. In vivo
recording of an adult zebrafish electrocardiogram and assessment of drug-induced QT
prolongation were first made possible by eliminating the motion artifacts with a paralytic

dose of µ -conotoxin GIIIB [194]. In our study, ECG signals were recorded from the mildly
sedated fish without oral perfusion and muscle paralysis. Despite gill motion, reproducible
ECG signals were generated by signal processing and wavelet transform. Milan et al.
reported that agents known to induce QT prolongation in humans led to QT prolongation
in zebrafish [194]. Using the zebrafish model for long QT syndromes in humans, Arnaout
et al. demonstrated that mutation in the kcnh2 gene, which encodes the channel responsible
for the rapidly activating delayed rectifier current (IKr) and accounts for `45% of long QT
syndrome [199], resulted in the inability of zebrafish to generate action potentials and
disruption of calcium release. While kcnh2 mutation was lethal in homozygous embryonic
zebrafish, heterozygous kcnh2 mutation manifested delayed ventricular repolarization and
prolonged QTc intervals in anesthetized, paralyzed adult zebrafish [199]. While normal
function

of channels, receptors, and cytoskeletal proteins contributed to repolarization of
the cardiac myocytes, the shortened QTc interval suggests an alteration in repolarization in
response to ventricular resection.
111

Zebrafish hearts share common structures with mammalian hearts, serving as a model for
vertebrate animal studies. Histological studies show that the ventricle of an adult zebrafish
heart is composed of trabecular and compact myocardium, and surrounded by epicardium
and endocardium (ref-textbook). Sedmera et al. reported that the spread of excitation wave
that occurred through the atrium was uniform. Furthermore, the apex-to-base ventricular

activation pattern was also observed in higher vertebrates in the apparent

absence of
conduction fascicles or a functional equivalent of

the His-Purkinje

system [195]. Thus, our
ECG signals support the notion of atrial excitation, followed by ventricular contraction and
repolarization.
Unlike mammals, zebrafish hearts have remarkable regenerative abilities. Zebrafish heart
regeneration occurs over a period of two months [192]. In response to amputation,
thrombosis immediately develops and nascent cardiomyocytes replace the vast majority of
the lost ventricular tissue by 30 dpa and the structure of the ventricle is fully restored at 60
dpa without scar tissue [192]. Activation of the regenerative potential of human heart tissue
implicates a novel therapeutic approach to supplement or replace conventional
pharmacotherapy and mechanical intervention. Despite the lack of scar tissue, whether
ventricular arrhythmia occurs during the regeneration process has posed an interesting
question. In this context, investigating the cardiac electrophysiology and mechanics of the
regenerating zebrafish heart by the use of micro-electrodes (ECG) translates our tools to
the zebrafish research community and small animal research.
Adult zebrafish are typically one inch in length. In comparison to other vertebrate models
such as mice, the small size of zebrafish has both advantages and disadvantages. One of
the advantages is that large numbers of fish can be housed in limited space for high
throughput experiments. However, the relatively small size of zebrafish renders it
challenging to perform physiological studies. Our micro-ECG approach aims to assist the
zebrafish communities in academics, national labs or pharmaceutical industry. The
equipment can be applied to physiological studies of adult hearts as well as
genetic/molecular characterization and drug screening. We believe that the cost-to-benefit
ratio will favor the application of user-friendly micro-ECG to study a large number of
zebrafish hearts.
Understanding heart regeneration in a vertebrate model system is highly relevant to public
health concern. Coronary heart disease is among the leading causes of morbidity and
mortality in United States and worldwide. Myocardial infarction (MI) results in irreversible
loss of cardiomyocytes in the heart[208]. Injured human hearts heal by scarring, which
112

leads to remodeling and heart failure[209]. In contrast to mammals, zebrafish fully
regenerate myocardium after 20% ventricular resection without scarring[192, 210], thereby
providing a genetically tractable model system to investigate molecular mechanisms of
heart regeneration.
Encouraging results from our labs and others [201] have demonstrated the feasibility of
monitoring the zebrafish heart by the use of micro-ECG. Despite variations in amplitudes,
PR, QRS, and QT intervals remain unchanged in each individual fish. In addition to
zebrafish regeneration, our methodology provides an essential tool to assess the
electrophysiological outcome of genetic, mechanical, or pharmacological perturbation to
the heart. In fact, our ultimate goal is to disseminate the use of micro-ECG technology to
the zebrafish research community and small animal research laboratories.
113

Table 2 Heart Rates In the Presence and Absence of Tricane

Sample
(n)
R-R Interval
(ms)
Heart Rate (beat/min)
No Tricaine 9 403± 49.4 149± 18
With Tricaine
22 667± 131 90 ± 17

114

Figure 30. Zebrafish ECG acquisition and processing. (a) Electrode placement.
Anesthetized zebrafish were placed in a damp sponge, the positive electrode was inserted
in the ventral midline about 2mm posterior to the gill, and the negative electrode was
introduced along the midline immediately in front to the anal fin. (b) Block diagram
illustrates the experiment setup for signal processing and wavelet amplification.

(b)
Positive needle
electrode
Negative needle
electrode
1 cm
(a)
115

Figure 31. Signal processing using Wavelet transform. The ECG signals were sampled
at a rate of 1,000Hz and were divided into 10 scales by using coif5 wavelets. In the
individual scales, the signals were composed of a general component (left column), and
detailed components (right column). The original ECG signals can be reconstructed by
recomposing the last general component (left bottom) and the entire detailed components
(right column).
116

Figure 32. Comparison of ECG signals via two difference filter algorithm. (a) ECG
signals prior to wavelet transform. (b) Application of wavelet transform at threshold values
between 1.9 Hz and 62.5 Hz resulted in distinct P waves, QRS complexes, and ventricular
repolarization patterns consistent with T waves (c) Excluding frequency between 1.9Hz
and 7.8Hz resulted in an elimination of ECG signals for T waves after wavelet transform.

117

Figure 33. Three ECG signals of an amputated zebrafish. Changes in positive electrode
placement resulted in different ECG waveforms, the relative magnitude and polarity of P,
QRS and T waves. Determination of PR, QRS and QT intervals were not affected by the
variability in magnitudes.

0 500 1000 1500 2000 2500 3000 3500 4000
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
Time(ms)
Magnitude
ECG of zebrafish at 4 dpa (trial 1)
T wave
P wave
QRS complex
(a)
0 500 1000 1500 2000 2500 3000 3500 4000
-0.4
-0.2
0
0.2
0.4
0.6
0.8
Time(ms)
Magnitude
ECG of zebrafish at 4 dpa (trial 2)
QRS complex
T wave
P wave
(b)
0 500 1000 1500 2000 2500 3000 3500 4000
-0.4
-0.2
0
0.2
0.4
0.6
0.8
Time(ms)
Magnitude
ECG of zebrafish at 4 dpa (trial 3)
QRS complex
T wave
P wave
(c)
118

Figure 34. Representative ECG signals prior to and post amputation (a) Prior to
amputation. (b) 4 dpa. (c) 9 dpa. (d) 13 dpa. T waves became gradually prominent,
suggesting changes in ventricular repolarization after ventricular amputation.

119

Figure 35. ECG signals from 4 different zebrafish at 4 dpa. (a) The histology of an
uncut zebrafish heart revealed an intact ventricle and atrium. Ventricular trabeculae were
more prominent in the zebrafish heart than those of humans. (b)(c)(d)(e) Comparison of
ECG signals from 4 individual fish with the corresponding histology at 4dpa. Distinct P
waves, QRS complexes, and T waves were present among the four individual fish. The
paraffin sections were stained with acid fuchsin-orange G (ADOG), revealing the fibrin in
red and collagen in blue. Both fibrin and collagen were absent in the intact ventricle. BA
denoted bulbus arteriosus; A, atrium; and V, ventricle.
120

Figure 36. Statistical analyses of the mean PR, QRS, QT and QTc intervals from 6
zebrafish prior to and post-amputation. (a) PR intervals remained unchanged prior to and
post amputation (n=6, P > 0.05). (b) QRS intervals also remained unchanged (n=6, P >
0.05). (c) QT intervals appeared to shorten 4 dpa (n=6, P=0.07). (d) QTc intervals were
significantly shortened 4 dpa (n=6, P < 0.05). The body lengths of fish #1-6 at ECG
recording were measured to be 35, 31, 32, 34, 38 and 34 mm, respectively.

121

4.5 References

[189] R. O. Becker, S. Chapin, and R. Sherry, "Regeneration of the ventricular
myocardium in amphibians," Nature, vol. 248, pp. 145-7, Mar 8 1974.
[190] J. O. Oberpriller and J. C. Oberpriller, "Response of the adult newt ventricle to
injury," J Exp Zool, vol. 187, pp. 249-53, Feb 1974.
[191] E. Braunwald, D. P. Zipes, P. Libby, and R. Bonow, Braunwald's Heart Disease:
A Textbook of Cardiovascular Medicine, 7th ed. Philadelphia: Saunders
Company, 2004.
[192] K. D. Poss, L. G. Wilson, and M. T. Keating, "Heart regeneration in zebrafish,"
Science, vol. 298, pp. 2188-90, Dec 13 2002.
[193] C. Nusslein-Volhard, (Editor), Dahm,R, (Editor), Zebrafish. New York: Oxford
University Press, 2002.
[194] D. J. Milan, I. L. Jones, P. T. Ellinor, and C. A. MacRae, "In vivo recording of
adult zebrafish electrocardiogram and assessment of drug-induced QT
prolongation," Am J Physiol Heart Circ Physiol, vol. 291, pp. H269-73, Jul 2006.
[195] D. Sedmera, M. Reckova, A. deAlmeida, M. Sedmerova, M. Biermann, J.
Volejnik, et al., "Functional and morphological evidence for a ventricular
conduction system in zebrafish and Xenopus hearts," Am J Physiol Heart Circ
Physiol, vol. 284, pp. H1152-60, Apr 2003.
[196] D. Y. Stainier, "Zebrafish genetics and vertebrate heart formation," Nat Rev
Genet, vol. 2, pp. 39-48, Jan 2001.
[197] A. S. Forouhar, A. Hickerson, M. Liebling, A. Moghaddam, J. Lu, H.-J. Tsai, et
al., "The embryonic vertebrate heart tube is a dynamic suction pump," Science,
vol. 312, pp. 751-753, 2006.
[198] J. R. Hove, R. W. Kö ster, A. S. Forouhar, G. Acevedo-Bolton, S. E. Fraser, and
M. Gharib, "Intracardiac fluid-forces are an essential epigenetic factor for
embryonic cardiogenesis," Nature, pp. 172-177, 2003.

Scale 2
122

[199] R. Arnaout, T. Ferrer, J. Huisken, K. Spitzer, D. Y. Stainier, M. Tristani-Firouzi,
et al., "Zebrafish model for human long QT syndrome," Proc Natl Acad Sci U S
A, vol. 104, pp. 11316-21, Jul 3 2007.
[200] A. S. Forouhar, J. R. Hove, C. Calvert, J. Flores, H. Jadvar, and M. Gharib,
"Electrocardiographic characterization of embryonic zebrafish," Conf Proc IEEE
Eng Med Biol Soc, vol. 5, pp. 3615-7, 2004.
[201] D. J. Milan and C. A. MacRae, "Animal models for arrhythmias," Cardiovasc
Res, vol. 67, pp. 426-37, Aug 15 2005.
[202] S. M. Szilagyi and L. Szilagyi, "Wavelet transform and neural-network-based
adaptive filtering forQRS detection," Proceedings of the 22nd Annual
International Conference of the IEEE, vol. 2, pp. 1267-1270, 2000.
[203] C. S. Burrus, R. A. Gopinath, and H. Guo, Introduction to Wavelets and Wavelet
Transforms: A Primer. Upper Saddle River, NJ: Prentice-Hall, 1997.
[204] I. Daubechies, Ten Lectures on Wavelets. Philadelphia, PA: Soc. Indus. Appl.
Math., 1992.
[205] D. L. Donoho, "De-noising by soft-thresholding," IEEE Trans. Inform. Theory,
vol. 41, pp. 613-627, 1995.
[206] E. E. Braunwald, D. P. E. Zipes, P. E. Libby, and R. E. Bonow, "Braunwald's
Heart Disease: A Textbook of Cardiovascular Medicine," 5 edition ed
Philadelphia: Saunders Company, 2004, p. 114.
[207] S. A. Glantz, "Primer of Biostatistics," 6th ed: McGraw-Hill, 2005, pp. 386-388.
[208] J. L. Reeve, A. M. Duffy, T. O'Brien, and A. Samali, "Don't lose heart--
therapeutic value of apoptosis prevention in the treatment of cardiovascular
disease.," J Cell Mol Med., vol. 9, pp. 609-22, 2005.
[209] C. Hahn and M. A. Schuwartz, "The Role of Cellular Adaptation to Mechanical
Forces in Atherosclerosis," Arterioscler Thromb Vasc Biol, 2008.
123

[210] A. Raya, A. Consiglio, Y. Kawakami, C. Rodriguez-Esteban, and J. C. Izpisua-
Belmonte, "The zebrafish as a model of heart regeneration," Cloning Stem Cells,
vol. 6, pp. 345-51, 2004.

124

Chapter Five: Electrocardiogram Signals to Assess Zebrafish
Heart Regeneration: Implication of Long QT Intervals
1

1
Disclaimer: the content of this chapter has been published as “Electrocardiogram Signals
to Assess Zebrafish Heart Regeneration: Implication of Long QT Intervals”, Annals of
Biomedical Engineering, 38(7): 2346-2357 (2010)
Acknowledgement: Co-authors of this manuscript include: Fei. Yu, Rongsong Li,
Elizabeth Parks, Wakako Tababe, Tzung. K. Hsiai. The author of this thesis credits other
co-authors for their significant input in the process of preparing the manuscript for
publication.
125
5.1 Chapter Five Introduction
Aberrant electrical activity of the heart, otherwise known as cardiac arrhythmia, is a leading
cause of sudden cardiac death. Approximately 450,000 individuals develop acute loss of
consciousness and sudden cardiac death in the United States [211]. Zebrafish (Danio rerio)
represents an emerging vertebrate model for cardiovascular research and regenerative
medicine, in part, due to the relatively ease of maintenance and breeding and, in part, due
to its significant promise for high throughput drug-screening [212]. Recent data supports
stem cell based cardiac regeneration in the mammalian systems [213], and zebrafish
myocardium has been shown to regenerate after 20% ventricular resection within 60 days
[214]. Whether structurally regenerated zebrafish hearts displayed functionally normal
conduction phenotype remained undefined.
Despite a two-chambered heart and a lack of pulmonary vasculature [215], the zebrafish
heart electrocardiogram (ECG) is fundamentally similar to that of humans in terms of P
waves, QRS complexes, and T waves [216, 217]. The critical conduction pathways of the
zebrafish in cardiovascular development also parallel that of higher vertebrates [218]. The
zebrafish heart is encased by a pericardial sac in the thoracic cavity below the pectoral bone,
and the atrium is medially dorsal and posterior to the ventricle. The bulbous arteriosus (BA)
is analogous to the human aortic arch with thick contractile smooth muscle. Deoxygenated
blood returns to the sinus venosus (SA), a structure analogous to the vena cava in humans.
Thus, zebrafish is a viable model for developmental biology, cardiac arrhythmia, and drug
discovery [219].
Unlike mammals, zebrafish myocardium fully regenerates over a period of 60 days, as
evidenced by histology [214, 220]. In response to resection, thrombosis immediately
develops to achieve hemostasis in the ventricle. The thrombus is replaced by a fibrin clot
at 2-4 days post resection (dpr). Nascent cardiomyocytes replace the vast majority of the
lost ventricular tissue by 30 dpr and the structure of the ventricle is fully restored at 60
dpr[214]. Zebrafish heart regeneration has been characterized using molecular, genetic,
genomic and immunohistochemical approaches [221]; however, the cardiac conduction
phenotype in the early-staged cardiomyocytes had remained unknown.
Zebrafish cardiac propagation in the regenerated cardiac tissue is a complex process
governed

by the excitable properties of the tissue and its macroscopic

and microscopic
architecture. Encouraging results from our laboratory [222] and others [223] showed the
feasibility of monitoring zebrafish heart regeneration by the use of microelectrodes. In this
126
context, we assessed the histology-conduction relationship in response to ventricular
resection. Zebrafish were periodically sedated for ECG monitoring. Signal processing and
wavelet transform were applied to enhance signal-to-noise ratios [222]. Dynamic ECG
changes developed post ventricular resection, including J point depression and QTc
prolongation. Despite histological evidence of cardiomyocyte regeneration and a gradual
normalization of J point depression at 60 days post resection, QTc intervals remained
prolonged. Our findings suggest that early regenerated cardiomyocytes lacked the
conduction phenotypes of the sham fish. Our in vivo regeneration model provides a non-
invasive approach to assess cardiac conduction with relevance to future assessment of
genetically [219], epigenetically [212], or pharmacologically [216] induced cardiac
phenotypes.

127
5.2 Chapter Five Experimental Designs and Methods
5.2.1 Animals
The animal experiments were performed in compliance with the protocol approved by the
Institutional Animal Care and Use Committee (IACUC) at the University of Southern
California. Adult zebrafish, 3-5 cm in length, were acquired from Tong's Tropical Fish and
Supplies (CA) and maintained under standard laboratory conditions at 24 ° C. The
individual fish were fed daily with brime shrimp (hatched from eggs in 10ml in 2 L salt
water), kept in constantly circulating water, and isolated from other fish for ECG follow-
up post resection.
5.2.2 Heart Resection or Injury
Eighteen fish were divided into two arms: 6 sham operation and 12 apical ventricular
resection. Twelve zebrafish underwent apical ventricular resection according to the
previously described method [222]. Zebrafish were sedated in 5% Tricaine
methanesulfonate (Tricaine). A midline incision of 0.25 cm in length was created posterior
to the ventricle and approximately 20% of the apical ventricle was excised by scissors. The
control fish underwent sham operation; that is, ventral midline incision was performed
without ventricular resection. The zebrafish were returned to freshwater in the presence of
a continuous oxygenator. Five resected fish and one sham fish died. Results from the 5
sham operated fish and 7 surviving fish (#1, #2, #3, #6, #7, #11, #12) with ventricular
resection were analyzed for serial ECG recordings, histology, and immunostaining for gap
junction protein connexin 43.
5.2.3 ECG Recordings and Signal Processing
Baseline ECG signals were recorded one day prior to ventricular resection and repeated
ECG’s were performed twice every week for 59 days following resection. In parallel, ECG
signals were recorded from 5 control sham fish (midline incision without heart resection)
over the identical period.
The ECG measurements were performed using a modified technique [222]. The entire
recording processes were performed in a Faraday cage to shield interference from
128
electromagnetic radiation. Two 29-gauge stainless steel micro-electrodes (AD Instrument,
Colorado Springs, CO) were positioned at 90
o
to the animal’s ventral epidermis. The
recording electrode was positioned directly above the ventricle and the reference electrode
was positioned along the midline immediately in front of the anal fin. Both electrodes were
inserted into the skin to approximately 1 mm in depth. The fish was allowed to recover
from sedation within 5 minutes in an oxygenated water bath free of Tricaine.
Longitudinal ECG signals prior to and 59 days post resection (dpr) were recorded from 12
zebrafish. The ECG signals were amplified 10,000-fold (A-M Systems Inc. 1700
Differential Amplifier, Carlsborg, WA) and filtered at a cut-off frequency of 60 Hz (notch)
and between 0.1 and 500 Hz. The signals were acquired and digitized at a sampling rate of
1,000 Hz (National Instruments USB-6251 DAQ device, Austin TX, and LabVIEW 8.2.).
To enhance signal-to-noise ratios (SNR), we digitally processed the signals using the
wavelet transform and thresholding Matlab algorithm (Matlab: Mathworks inc, Natick,
MA) developed in our laboratory [222]. The parameters used in the algorithm allowed for
systematic recordings for QRS and QTc intervals regardless of electrode placements or the
cardiac vector orientations [216]. J point depression (J) was computed as the difference
between J-point and baseline (signal level prior to P wave) normalized to the QRS voltage
signals. J values for the sham and regenerated ventricles with and without residual
resection/scar tissue were compared. QTc intervals were computed by normalizing to the
heart rates using the standard Bazett formula [222].
5.2.4 Histology
At 60 days, the zebrafish hearts were isolated and fixed with paraffin for 5µ m slides. The
slides were stained with Acid fuchsin orange G (AFOG) and mouse polyclonal connexin-
43 (CX43) antibody (1: 4,000) (Sigma-Aldrich, MO, USA). Fibrin and collagen were
stained orange/red and blue, respectively, (Fig. 37) using the AFOG staining. Using the
connexin-43 antibody staining, connexin-43 positive tissue was stained light brown and
negative tissue was stained light blue. The atria were used as the positive control and the
bulbus arteriosus as the negative. Photos were acquired by the microscope (Leica DM LB2,
Leica Microsystems, Germany) coupled with a CCD digital camera (Spot RT-KE,
Diagnostic Instruments, MI, USA).
The specificity of the mouse connexin-43 primary antibody was verified by using the same
antibody for western blotting on ventricle myocardium tissue collected from intact fish.
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Isolated tissue were lysed in proper volume of RIPA buffer (50 mM Tris-HCl, pH 8.0,
150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate supplemented with protease
inhibitor (PI) cocktail) and phosphotase inhibitor cocktail at 4 ° C for 30 min. After
centrifugation for 5 min at 4 ° C, supernatant was collected as whole cell lysates. Protein
concentration was determined with DCP protein assay kit from Bio-Rad inc. (USA).
Samples with equal protein amount of lysates were run on a 4-20% gradient SDS-PAGE
gel. The proteins were then transferred to PVDF membrane and blotted with primary and
secondary antibodies. Signal was developed with Supersignal Western Pico (Pierce) and
recorded with FluorChem FC2 (Alpha Innotech Inc., CA, USA). Densitometry scan of
western blot were with the software come with FluorChem FC2 machine.
5.2.5 Statistical Analysis
Numerical data will be expressed as mean ± standard deviation. Statistical analysis was
performed for comparisons between sham and resected fish as well as among separate
experiments. For comparisons between two groups, two-sample independent-groups t-
tests were used. Comparisons of multiple mean values are made by one-way analysis of
variance (ANOVA), and statistical significance among multiple groups is determined using
the Tukey procedure (for pairwise comparisons of means between sham and ventricular
resection). P-values of less than 0.05 are considered statistically significant.

130
5.3 Chapter Five Results
5.3.1 Adult zebrafish heart histology
The histological evidence of cardiac regeneration provided a basis to evaluate the status of
the heart regeneration at 60 days. While the Acid fuchsin orange G staining showed
regenerated fibrin tissue (orange) within ventricles (Fig. 37), immunohistochemistry
revealed the regenerated cardiomyocytes and fibroblasts (Fig. 38). In light of the paucity
of available zebrafish antibodies and their lack of specificity, we verified the polyclonal
rabbit CX43 antibody by performing western blotting on zebrafish ventricular myocardium
(Fig. 38a). The protein band corresponded to the 40kD reference band confirming the
molecular weight of CX43. However, the presence of three other bands suggested non-
specific bindings. Nonetheless, the brown staining represented presence of cardiomyocytes
in the ventricles at 60 dpr (Fig. 38b and38c) compared to the blue negative staining for
bulbus arteriosus and nucleated erythrocytes. Moreover, cardiomyocytes displayed the
characteristic muscle striations in reddish-brown.
Histological evaluation revealed that three fish developed incomplete heart regeneration
(#2, #6 and #12). Compared to the sham-operated fish with intact cardiac boundary (Fig.
2b), the fish heart associated with incomplete regeneration exhibited ill-defined boarder or
injured sites associated with a negative CX43-staining (Fig. 38c). In contrast, four fish
developed complete regeneration (#1, #3, #7, #11) associated with a positive CX43-
staining resembling to that of the sham fish (Fig. 37b, Fig. 41d and 41e). There was no
histological difference in CX43-staining density between the regenerated and sham
myocardium.
5.3.2 ECG recordings and signal processing
The initial ECG signals were recorded at 1000 Hz (Fig. 39a). Wavelet transform was
performed by breaking down the signals into 10 frequency segments ranging from 0 Hz to
the Nyquist frequency (½ of sampling frequency, i.e. 500 Hz) (Fig. 39b) [222]. The
individual frequency ranges contained different levels of signal components amidst the
noise, which was suppressed by the set threshold value (Fig 39b). Generally the signals
from frequency range 0.98 Hz to 7.8 Hz were kept entirely to preserve T wave [222]. The
final processed ECG signals were re-constructed by inverse wavelet transform after noise
131
reduction (Fig. 39c). The major features of the ECG were retained while signal-to-noise
ratios were significantly improved.
5.3.3 ECG of the sham fish
In the sham fish that underwent ventral midline incision without ventricular resection,
specific ECG features and intervals remained statically unchanged over the period 59 days
post resection (dpr). Distinct P waves, followed by QRS complexes were noted. T waves
were visible but not prominent at day 0 (Fig. 40). The QTc intervals were similar to the
baseline (P > 0.05, n=5), suggesting preserved ventricular depolarization and
repolarization. The corresponding immunohistochemistry at 60 dpr revealed intact heart
associated with brown staining for CX43 in both atrium and ventricle, light blue for bulbus
arteriosus (as a negative control), and dark blue for nucleated erythrocytes (Figs 40d and
40e).
5.3.4 ECG of fish with scar tissues
Representative ECG recording (fish #12) revealed scar tissues at 59 dpr. P waves and QRS
complexes remained unchanged compared to ECG recordings prior to resection (Fig. 41a
versus Fig. 41c), suggesting preserved atrial and ventricular depolarization. Dynamic
changes in ECG depolarization and repolarization patterns, particularly, J point depression
and QTc prolongation developed post ventricular resection (Fig. 41b). QTc intervals
remained prolonged at 59 dpr (Fig. 41c). J point depression accompanied with ST-T
changes remained statistically significant compared to the sham at 52 dpr (P < 0.01, n=3).
However, J point depression was no longer statistically significant at 59 dpr (P > 0.05,
n=3). Overall ECG patterns displayed similar features commonly observed in human ECG
in response to myocardial ischemia, particularly, the changes in T wave and ST segments
[224]. The corresponding immunohistochemistry showed scar tissues at the apex (Fig.
41d). Dark blue staining over the epicardium and around the scar tissues indicated absence
of CX43 staining (Fig. 38c, Fig. 41e). Thus, our findings suggest that ventricular resection
and incomplete regeneration engendered delayed J-point normalization and prolonged QTc
intervals.

132
5.3.5 ECG of fish undergoing ventricular regeneration
Representative ECG recordings (fish #7) revealed dynamic changes despite histological
finding for complete ventricular regeneration at 60 dpr. ECGs prior to resection showed
distinct P waves, followed by QRS complexes, and non-prominent T waves (Fig. 42a). At
31 dpr, T waves became prominent, accompanied with prolonged QTc intervals; and J
points were depressed, accompanied with notable ST-T changes (Fig. 42b). At 59 dpr, QTc
intervals remained prolonged (P < 0.01, n=4) while J point depression appeared to
normalize to the baseline (P > 0.05, n=4) (Fig. 42c). Immunohistochemistry at 59 dpr
revealed brown staining for CX43 in the ventricles, and the regenerated cardiomyocytes
were indistinguishable from the surrounding tissue (Figs. 42d and e). Hence, our findings
suggest that ECG repolarization remained prolonged in the early-staged cardiomyocytes.
5.3.6 Cardiac resection and heart rate variability
The recorded fish heart rates ranged from 500ms to 1500ms over the period of 60 days.
The heart rates could be influenced by the level of sedation [225]. To account for individual
variability in response to tricaine, we normalized the standard deviation of RR intervals by
the mean RR intervals for all of the recorded ECGs. The calculated values, Var (0 < Var <
1), indicated the degree of variability for a particular ECG recording. Higher Var values
associated with a greater variability. The Var values for the individual ECG recording
intervals for sham (n = 5) and resected fish (n = 7) were grouped together and were
presented as mean ± standard deviation versus days post resection (Fig.43). During the first
17 days, the heart rate variability of sham and resected fish were similar. After 20 dpr, the
resected fish group showed higher variability compared to the control. However, the
variability was statistically significant at merely 3 time points; specifically, 27, 52, and 55
dpr. Given other confounding variables such as hypoxia and sedation, ventricular resection
alone may not be an independent predictor for heart rate variability.
5.3.7 Dynamic changes in J point depression
Next, we assessed changes in J point depression between the sham and ventricle resection.
Mean J points for the sham (n=5), regenerated ventricles (n=4), and scar tissues (n=3) were
compared (Fig. 44). At 10 dpr, J point depression became statistically significant for both
the regeneration group (P < 0.01, n=4) and scar tissue group (P < 0.01, n=3). At 52 dpr, J
133
point depression remained for the scar tissue group. At 59 dpr, J point became statistically
insignificant for both the regeneration and scar tissue group.
5.3.8 Dynamic changes in QTc intervals
Periodic mean QTc intervals were compared over 59 days (Table 1). Starting at 3dpr, the
QTc intervals were significantly different between the sham and fish undergoing
ventricular resection (P < 0.01, n=7). The QTc intervals were not significantly different
between fish undergoing regeneration and those with scar tissues (P > 0.05, n=4 and 3,
respectively). However, distinct QTc prolongation was observed for fish undergoing
ventricular resection compared to the sham (Fig. 45). While the QTc intervals of sham
(blue diamond) remained statistically unchanged, ranging from 275 ms to 350 ms, the mean
QTc intervals remained prolonged for both the regeneration (fish #1, 3, 7, and 11) and scar
tissue groups (fish #2, 6, and 12) at 59 dpr (P < 0.01, n=7). Taken together, fish that
underwent ventricular resection developed prolonged QTc intervals despite positive
cardiomyocyte staining for connexin-43 after 59 dpr (Fig. 3); those that developed scar
tissues developed a more accentuated J-point depression between 10 dpr and 45 dpr (Fig.
44).

134
5.4 Chapter Five Discussion
Zebrafish cardiac conduction in the regenerated cardiac tissue involves the excitable
properties of the tissue and its macroscopic

and microscopic structure. In this study, we
established an in vivo

model for the study of conduction phenotypes in the regenerating
zebrafish heart. By using the microelectrodes, long-term assessment of PR, QRS, and QTc
intervals revealed dynamic changes in response to ventricular resection. Histological
analysis also revealed presence of gap junctions such as CX43 in the regenerated
cardiomyocytes. Despite a gradual normalization of J point depression, ECG repolarization
was not normalized to baseline as compared to the sham. Moreover, early regenerated
cardiomyocytes lacked the conduction phenotypes of the sham fish. Our findings pave the
way for a non-invasive platform to assess cardiac regeneration and conduction phenotypes
for small animal research with relevance to understand cardiac arrhythmias.
Using zebrafish as a vertebrate model system holds promise for understanding
cardiomyocyte conduction. Diverging from the mammalian ancestry 450 million years ago,
zebrafish possess the essential common anatomy of humans [215]. Understanding heart
regeneration in a vertebrate model system is highly relevant to public health concern.
Myocardial infarction results in irreversible loss of cardiomyocytes in the heart [226].
Injured human hearts heal by scarring, which leads to remodeling and heart failure [227].
In contrast to mammals, zebrafish myocardium fully regenerates after 20% ventricular
resection [214, 220]; thereby providing a genetically tractable model to investigate
molecular mechanisms of cardiac myocyte regeneration.
Our longitudinal study demonstrated that zebrafish displayed two gross histological
findings ensuing ventricular resection: (1) cardiomyocyte regeneration (Fish #1, #3, #7and
#11) and (2) regeneration with scar tissue (Fish #2, #6 and #12). All of the fish that
underwent ventricular resection developed QTc prolongation and prominent T waves,
accompanied with J point depression. Furthermore, J point depression was more
accentuated in fish displaying scar tissues from between10 dpr and 45 dpr.
Long QT phenomenon has an important clinical implication for sudden cardiac death [228].
The long QT syndromes (LQTS) are due to delayed repolarization of cardiac myocyte
action potential; thereby, predisposing individuals for torsade de pointes, a lethal form of
ventricular tachycardia, leading to fainting and death [229]. The common in-born etiology
of LQTS is associated with a host of mutations in the rectifier potassium channels. The
135
common exogenous etiology is linked with drug toxicities (class III potassium channel
blockers or psychoactive drugs) [230]. Prolonged QTc reflects delayed repolarization of
action potential. Dysfunctional inward rectifier potassium channels such as IKr may be
implicated [231]. Myocardial ischemia and stem cell therapy also constitute the substrates
for LQTS [232]. J-point depression is implicated in myocardial ischemia. A decrease in
coronary blood perfusion predisposes an individual to develop myocardial injury, leading
to heart attack otherwise known as coronary syndromes. Hence, prolong QTc intervals and
J-point depressions represent myocardial phenotypes of tissue injury in response to
ventricular resection.
The dynamic changes in zebrafish ECG recordings provided a non-invasive means to
assess conduction otherwise hampered by the lack of a suitable human model for heart
regeneration. While normal function of ion channels, receptors, and cytoskeletal proteins
contributed to repolarization of the cardiac myocytes, prolonged QTc intervals suggest an
alteration in repolarization. Milan et al. reported that agents known to induce QT
prolongation in humans also led to QT prolongation in zebrafish [216]. The authors
identified 15 repolarization genes implicating in a network of transmembrane and
cytoplasmic proteins that modulate ion channel function, underscoring zebrafish as a
faithful model for human cardiac repolarization [233]. Using the zebrafish model for long
QT syndromes in humans, Arnaout et al. demonstrated that mutation in the kcnh2 gene,
which encodes the channel responsible for the rapidly activating delayed rectifier current
(IKr), accounts for 45% of long QT syndrome [219] and results in the inability of zebrafish
to generate action potentials and calcium release. While kcnh2 mutation was lethal in
homozygous embryonic zebrafish, heterozygous kcnh2 mutation manifested delayed
ventricular repolarization and prolonged QTc intervals in anesthetized, paralyzed adult
zebrafish [219].
In the era of stem cell and regenerative medicine, there is a considerable interest to assess
the phenotypes of early regenerated cardiomyocytes. Albeit the advances of molecular
imaging and ultrasound modalities to follow stem cell fate, gene therapy and cardiac
function [234], the emerging concern in cardiac stem cell therapy and tissue engineering is
cardiac toxicities that can manifest clinically as atrial or ventricular arrhythmia. Hence,
assessing zebrafish QT intervals in response to ventricular injuries provides a novel
approach to address cardiac arrhythmia or long QT syndromes in the early regenerated
tissues [219, 228].
136
Analogous to human myocardial injury, in our study, QT prolongation in zebrafish was
induced by surgically resecting ~ 20% of the ventricular myocardium. We observed that
prolonged QT intervals persisted despite histological evidence of complete ventricular
regeneration. Antibody staining for connexin-43 was positive in the regenerated
cardiomyocytes; however, early-staged cardiomyocytes might not have fully expressed
potassium channels necessary for excitation and contraction coupling. Thus, the resected
fish could be at risk for cardiac arrhythmia in response to pharmacological perturbation.
Overall, our in vivo model provided a basis for the assessment of cardiac conduction
phenotypes in relation heart regeneration, allowing for a non-invasive and longitudinal
approach to assess gene regulation and ultra-structural

functional properties of regenerated
cardiomyocytes [235]. Recently, the use of microelectrode array (MEA) mapping
technique opened new avenue to assess the functional syncytium [236] and to perform high
throughput phenotyping of genetic deletion or over-expression, as well as lethal mutations
otherwise infeasible in transgenic

animals. Zebrafish regeneration model provides limitless
opportunities to assess conduction phenotypes with relevance to genetic, epigenetic, and
pharmacologic perturbation.

137
Table 3. Periodic QTc Assessment. Periodic mean QTc intervals of fish undergoing
ventricular resection were compared with the sham over 59 days. The differences in QTc
intervals between the sham and resected ventricles were statistically significant starting at
3 dpr (P < 0.05, n=4 and 3, respectively). However, the QTc values between regeneration
and scar tissues were statistically insignificant (P > 0.05, n=4 and 3, respectively).
Dpr (days) 0 3 10 17 24 31 38 45 52 59
QTc Control (ms) 347± 41 320± 32 337± 47 289± 32 326± 43 325± 60 299± 14 348± 64 311± 24 325± 42
QTc
No Scar (ms)
306± 17 495± 57 541± 18 521± 54 554± 11 535± 27 545± 15 528± 53 530± 48 496± 31
QTc
Scarred (ms)
322± 18 450± 65 472± 67 577± 37 499± 45 502± 10 531± 18 527± 41 543± 55 534± 51
P for One-way
ANOVA test
0.158 0.001 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
P value for T-test
(Control vs.
Scarred)
0.655 0.034 0.070 0.000 0.007 0.000 0.000 0.002 0.013 0.015
P value of T-test
(Control vs. No
Scar)
0.200 0.007 0.000 0.000 0.000 0.000 0.000 0.002 0.001 0.000
P value of T-test
(No Scar vs.
Scarred)
0.431 0.429 0.211 0.160 0.163 0.081 0.344 0.992 0.752 0.339

138

Figure 37. Histological study of sham versus ventricular resection at 60 dpr. (a) Acid
fuchsin orange G (AFOG) staining of heart from the sham. Inset showed the ventricle at
100x. (b) AFOG staining of the regenerated ventricle. Dotted line indicates the region of
ventricular resection. Inset highlighted regenerated ventricle at 100x. The ventricle
revealed cardiomyocyte regeneration as reported by Poss et al [3]. Although endo-, mid-
and epicardial layers seemed indistinct, trabeculation was notable in zebrafish heart [8].
The variations in cardiomyocyte “density” or otherwise known as wall thickness were
mainly due the different cardiac contractile stage between systole and diastole at which the
heart was arrested and fixed for histology preparation. A: atrium. V: ventricle. BA: Bulbus
Arteriosus.

139

Figure 38. Immunohistochemistry for cardiomyocyte gap junction protein, connexin-
43. (a) Western blots indicated a band corresponding to the molecular weight of connexin-
140
43. However, the antibody for zebrafish connext-43 is not specific as notable for other
bands. (b) Atrium and ventricle were surrounded by light brown staining whereas bulbus
arteriosus was in light blue staining. Magnification (400X) revealed light born staining in
the individual cardiomyocytes and striation was prevalent. Zebrafish erythrocytes are
nucleated and stained in dark blue. (c) Immunohistochemistry on incompletely regenerated
heart on 60 dpr. Low magnification (40 X) presented bulbus arteriosus (BA) as well as
pericardial tissue in blue, atrium and ventricle in brown; Magnification (400 X) revealed
ventricular cavity and incomplete ventricle boundary in details. CX43 negative scar tissue
with minimal brown staining with prominent nuclei was present near the injured site.

141

Figure 39. Example of signal processing for Fish #11 ECG recorded at 3 dpa. (a) Raw
signal directly recorded from sedated zebrafish using Labview at sampling rate of 1000 Hz.
(b) Breakdown of raw signals into frequency segments via wavelet transform. Low
frequency signals (DC to 0.49Hz) were completely filtered. Thresholds were applied to
each of the individual frequency ranges to suppress corresponding noise levels. Sub-
threshold values were set to zero. Signals within frequency range from 0.98 Hz to 7.81 Hz
were reserved to ensure fidelity of T-waves. (c) Reconstructed ECG signals from processed
frequency segments were shown after applying inverse wavelet transform.

0 1000 2000 3000 4000 5000
-1
0
1
2
3
4
0 1000 2000 3000 4000 5000
-1
0
1
2
3
4
Time (msec)
Voltage(V)
Adaptive Threshold Value
(a) (b)
(c)
142

Figure 40. Representative ECG recording from sham operation fish. (a) ECG
recording at 0 dpr revealed P waves and QRS complexes. T waves were visible but not
prominent. (b) ECG recording at 31 dpr revealed that P waves, QRS intervals, and T waves
were statistically similar to the baseline ECG recording. (c) ECG recordings at 59 dpr
revealed that P wave, QRS complexes and QTc intervals remained statistically unchanged.
(d) Immunohistochemistry for cardiomyocyte gap junction protein, connexin-43, at 60 dpr
disclosed light brown staining in both atrium and ventricle at 40X. Bulbus arteriosus was
in light blue staining. (e) The ventricle remained intact post the sham operation at 100X.
The nucleated erythrocytes were stained dark blue.

143

Figure 41. Representative ECG of fish with incomplete regeneration or residual
resection. (a) ECG prior to resection showed distinct P waves, QRS complexes, and T
waves. (b) ECG at 31 dpr revealed QTc, prolongation, J point and ST depression. (c) ECG
at 59 dpr demonstrated persistent QTc prolongation, J point and ST depression, as well as
prominent T waves. (d) Immunohistochemistry revealed scar tissues at ill-defined boarder
at 40X. (e) At 100X CX43 negative scar tissue with minimal brown staining with
prominent nuclei was present near the injured site. Red arrows point to the blue staining
suggesting scar tissues in the resected region.

144

Figure 42. Representative ECG from fish with ventricular regeneration. (a) ECG
before ventricular resection displayed distinct P waves, followed by QRS complexes. T
waves were not distinguishable. (b) ECG at 31 dpr showed that P waves and QRS
complexes remained unchanged, suggesting intact atrial and ventricular depolarization.
However, prominent T waves developed and QTc intervals became prolonged. ST segment
depression was evidenced by the J point depression. (c) ECG at 59 dpr showed that QTc
intervals remained prolonged, and that ST segment appeared to normalize to baseline. Tall
P wave amplitudes were likely due to electrode placement proximal to the source of sinus
node pacemaker cells. Note that variations in voltage amplitude at various days post
resection were likely due to lead placements. (d) Immunohistochemistry at 60 dpr revealed
light brown staining for connexin-43 in both atrium and ventricle in contrast to light blue
staining for bulbus arteriosus at 40X. (e) Complete cardiomyocyte regeneration was noted
despite persistent prolonged QTc intervals at 100X.

145

Figure 43. Effect of resection on the variability of RR intervals. Variability, Var, was
calculated as the standard deviation of RR interval divided by the mean RR interval. Higher
Var value indicates greater variability. Mean and standard deviation of Var values for
sham-operated and resected fish were presented against the numbers of day post-resection
(dpr). RR variability of sham (n = 5) and resected fish (n = 7) were similar from day 0 to
17 dpr. After 20 dpr, the resected group showed greater Var value. Only 3 time points (27
dpr, 52 dpr and 55 dpr) revealed a statistically significant difference.

146

Figure 44. Dynamic changes in J point depression values between sham and resected
ventricles. J point depression (J) was calculated as the difference between J-point and
baseline (signal level prior to P wave) and normalized with voltage amplitude of QPS
complex. Periodic J values for the sham operation (Control), resected ventricles with
regeneration, resected ventricles with scar tissues (Scarred) were plotted over 59 days. “*”
indicates statistical significant difference between the sham and regeneration (No Scar) (P
< 0.01); “#” indicates statistical significant difference between the sham and scar tissues
(P < 0.01).

147

Figure 45. QTc values between sham and resected ventricles. Statistically significant
differences developed at 3 dpr (P < 0.01, n=7). While the QTc of sham (blue diamond)
remained statistically unchanged ranging from 275 ms to 350 ms, the mean QTc values for
the resected ventricles showed QTc prolongation ranging from 375 ms to 400-600 ms
starting at 3 dpr despite cardiomyocyte regeneration at 59 dpr (P < 0.01, n=7).

148
5.5 References
[211] Z. J. Zheng, J. B. Croft, W. H. Giles, and G. A. Mensah, "Sudden cardiac death in
the United States, 1989 to 1998," Circulation, vol. 104, p. 2158, 2001.
[212] N. C. Chi, R. M. Shaw, B. Jungblut, J. Huisken, T. Ferrer, R. Arnaout, et al.,
"Genetic and physiologic dissection of the vertebrate cardiac conduction system,"
Public Library of Science - Biology, vol. 6, pp. 1006 - 1019, 2008.
[213] O. Bergmann, R. D. Bhardwaj, S. Bernard, S. Zdunek, F. Barnabe-Heider, S.
Walsh, et al., "Evidence for cardiomyocyte renewal in humans," Science, vol.
324, pp. 98-102, Apr 3 2009.
[214] K. D. Poss, L. G. Wilson, and M. T. Keating, "Heart regeneration in zebrafish,"
Science, vol. 298, pp. 2188-90, Dec 13 2002.
[215] C. Nusslein-Volhard (Editor) and R. Dahm (Editor), Zebrafish. New York:
Oxford University Press, 2002.
[216] D. J. Milan, I. L. Jones, P. T. Ellinor, and C. A. MacRae, "In vivo recording of
adult zebrafish electrocardiogram and assessment of drug-induced QT
prolongation," Am. J.Physiol. Heart Circ. Physiol., pp. H269-H273, 2006.
[217] D. Sedmera, M. Reckova, A. deAlmeida, M. Sedmerova, M. Biermann, J.
Volejnik, et al., "Functional and morphological evidence for a ventricular
conduction system in zebrafish and Xenopus hearts," Am J Physiol Heart Circ
Physiol, vol. 284, pp. H1152-60, Apr 2003.
[218] D. Y. Stainier, "Zebrafish genetics and vertebrate heart formation," Nat Rev
Genet, vol. 2, pp. 39-48, Jan 2001.
[219] R. Arnaout, T. Ferrer, J. Huisken, K. Spitzer, D. Y. Stainier, M. Tristani-Firouzi,
et al., "Zebrafish model for human long QT syndrome," Proc Natl Acad Sci U S
A, vol. 104, pp. 11316-21, Jul 3 2007.
149
[220] A. Raya, A. Consiglio, Y. Kawakami, C. Rodriguez-Esteban, and J. C. Izpisua-
Belmonte, "The zebrafish as a model of heart regeneration," Cloning Stem Cells,
vol. 6, pp. 345-51, 2004.
[221] C. L. Lien, M. Schebesta, S. Makino, G. J. Weber, and M. T. Keating, "Gene
expression analysis of zebrafish heart regeneration," PLoS Biol, vol. 4, p. e260,
Aug 2006.
[222] P. Sun, Y. Zhang, F. Yu, E. Parks, A. Lyman, Q. Wu, et al., "Micro-
electrocardiograms to study post-ventricular amputation of zebrafish heart," Ann
Biomed Eng, vol. 37, pp. 890-901, May 2009.
[223] D. J. Milan and C. A. MacRae, "Animal models for arrhythmias," Cardiovasc
Res, vol. 67, pp. 426-37, Aug 15 2005.
[224] B. Surawicz, T. K. Knilans, and T. C. Chou, Chou's electrocardiography in
clinical practice: adult and pediatric, 5th ed.: WB Saunders Company, 2001.
[225] D. J. Y. Hsieh and C. F. Liao, "Zebrafish M2 muscarinic acetylcholine receptor:
cloning, pharmacological characterization, expression patterns and roles in
embryonic bradycardia," British journal of pharmacology, vol. 137, p. 782, 2002.
[226] J. L. Reeve, A. M. Duffy, T. O'Brien, and A. Samali, "Don't lose heart--
therapeutic value of apoptosis prevention in the treatment of cardiovascular
disease," J Cell Mol Med, vol. 9, pp. 609-22, Jul-Sep 2005.
[227] C. Hahn and M. A. Schwartz, "The role of cellular adaptation to mechanical
forces in atherosclerosis," Arterioscler Thromb Vasc Biol, vol. 28, pp. 2101-7,
Dec 2008.
[228] S. G. Priori, P. J. Schwartz, C. Napolitano, R. Bloise, E. Ronchetti, M. Grillo, et
al., "Risk stratification in the long-QT syndrome," New England Journal of
Medicine, vol. 348, p. 1866, 2003.
[229] E. Braunwald, D. P. Zipes, and P. Libby, Heart disease: a textbook of
cardiovascular medicine, 8th ed.: WB Saunders Company, 2001.
150
[230] M. T. Keating, "The long QT syndrome: a review of recent molecular genetic and
physiologic discoveries," Medicine, vol. 75, p. 1, 1996.
[231] M. R. Rosen, M. J. Janse, and A. L. Wit, Cardiac electrophysiology: a textbook:
Futura Publ. Comp., 1990.
[232] R. R. Makkar and P. S. Chen, "Stem Cell Therapy for Myocardial Repair,"
Journal of the American College of Cardiology, vol. 42, pp. 2070-2072, 2003.
[233] D. J. Milan, A. M. Kim, J. R. Winterfield, I. L. Jones, A. Pfeufer, S. Sanna, et al.,
"Drug-Sensitized Zebrafish Screen Identifies Multiple Genes, Including GINS3,
as Regulators of Myocardial Repolarization.," Circulation, vol. 120, pp. 553-559,
2009.
[234] G. Y. Chang, F. Cao, M. Krishnan, M. Huang, Z. Li, X. Xie, et al., "Positron
emission tomography imaging of conditional gene activation in the heart,"
Journal of molecular and cellular cardiology, vol. 43, pp. 18-26, 2007.
[235] I. Kehat, D. Kenyagin-Karsenti, M. Snir, H. Segev, M. Amit, A. Gepstein, et al.,
"Human embryonic stem cells can differentiate into myocytes with structural and
functional properties of cardiomyocytes," Journal of Clinical Investigation, vol.
108, pp. 407-414, 2001.
[236] I. Kehat, A. Gepstein, A. Spira, J. Itskovitz-Eldor, and L. Gepstein, "High-
Resolution Electrophysiological Assessment of Human Embryonic Stem Cell-
Derived Cardiomyocytes. A Novel In Vitro Model for the Study of Conduction,"
Circulation Research, vol. 91, pp. 659-663, 2002.

151

Chapter Six: Flexible Microelectrode Arrays to Interface
Epicardial Electrical Signals with Intracardial Calcium
Transients in Zebrafish Hearts
1

1
Disclaimer: the content of this chapter has been published as “Flexible microelectrode
arrays to interface epicardial electrical signals with intracardial calcium transients in
zebrafish hearts”, Biomedical Microdevices 14(2): 357-366 (2012)
Acknowledgement: Co-authors of this manuscript include: Fei. Yu, Yu Zhao, Jie Gu,
Katherine L. Quigley, Neil C. Chi, Yu-Chong Tai, Tzung. K. Hsiai. The author of this
thesis credits other co-authors for their significant input in the process of preparing the
manuscript for publication.
152
6.1 Chapter Six Introduction
Real-time monitoring of heart regeneration in a vertebrate model system is highly
relevant to tissue engineering and stem cell therapy. Myocardial infarction results in
irreversible loss of cardiac tissue [237]. Injured human hearts heal by scarring, which
leads to remodeling, arrhythmia, and heart failure [238]. In contrast, Zebrafish (Danio
rerio) fully regenerate their myocardium after 20% ventricular amputation [239, 240],
thus providing a genetically tractable model system for high-throughput research,
including antiarrhythmic [241] and psychoactive drug discoveries [242, 243], as well as
human inherited cardiac arrhythmias, blood, and sleep disorders [244, 245].
Zebrafish hearts harbor remarkably similar cardiac electrical properties as those of
humans [246]. In addition to their similar surface electrical cardiograms (ECG) [247],
the critical pathways in cardiovascular development parallel to higher vertebrates [248].
Histological studies show that the ventricle of adult zebrafish hearts is composed of
trabecular myocardium and compact myocardium, surrounded by epicardium and
endocardium. More importantly, genes involved in zebrafish heart development are
highly conserved with higher vertebrates. In terms of size, the average length of an adult
fish is at 2 to 4 cm. The mean heart rate is at 151 ± 30 beats/min, and the pericardial sac
is encased in the thoracic cavity below the pectoral fin at 1 mm beneath the skin. While
the small size is conducive to high-throughput research, the small heart size (1-2 mm in
length) renders it challenging to perform functional physiological analyses. In this
context, the advent of flexible micro-electronics allows for interrogation of small animal
systems in real-time.
Here, we demonstrated electrical and optical coupling of cardiac conduction in zebrafish
hearts via flexible microelectrodes with high spatial resolution otherwise difficult with
the existing external needle electrodes [247, 249, 250]. We implanted one counter and
three detecting microelectrodes to the chest cavity of adult zebrafish and the reference
electrodes to the tails. Application of signal processing and wavelet transform revealed
three distinct epicardial ECG recordings at 10 µ m apart. In corollary, we demonstrated
that intracellular Calcium transients (Ca
2+
) in the single cell and tissue levels were in
synchrony with the epicardial ECG recordings. Thus, interfacing flexible microelectrodes
provides enhanced spatial resolution at ~ 5 m for real-time longitudinal monitoring of
electrical signals from the non-planar and dynamic cardiac surface.
153
6.2 Chapter Six Materials and Methods
6.2.1 Animals
The animal experiments were performed in compliance with the protocol approved by the
Institutional Animal Care and Use Committee (IACUC) at the University of Southern
California. Adult zebrafish, 3-5 cm in length, were acquired from Tong's Tropical Fish
and Supplies (CA) and maintained under standard laboratory conditions at 24 ° C. The
individual fish were fed daily with brime shrimp (hatched from eggs in 10ml in 2 L salt
water), and were kept in constantly circulating water.
6.2.2 Microfabrication of flexible electrode arrays
Approximately 10 μm of parylene C was deposited on a silicon wafer with a photoresist
sacrificial layer to the underside (Fig. 46). A second layer of photoresist was deposited,
followed by selective exposure and development to a diamond-shape pattern. Next, a
gold layer was deposited via Physical Vapor Deposition (PVD) via a titanium adhesion
layer. Gold metal liftoff process was used to define pitches with a thickness from 200
nm to 300 nm. A final parylene coating at approximately 2 μm in thickness formed the
top insulation layer. The electrodes and connection pads were exposed and the overall
geometry of the array was defined by oxygen plasma reactive-ion etching via a thick
photoresist etch mask. Finally, the arrays were peeled from the wafer in a water bath or
released through removal of the sacrificial photoresist in acetone (Fig. 47a).
6.2.3 Microelectrode array implantation
The fish were sedated using tricaine methanesulfonate (MS-222), and placed in a damp
sponge to expose the ventral side for visualization under the microscope as previously
described [250, 251]. Two parallel incisions of approximately 1.5 mm in length and 1.5
mm apart were performed on the chest (Fig. 47b), and two triangular structures of the
chest stripe were inserted into the incision sites for direct microelectrode contact with the
epicardium. The tail stripe was placed on the abdominal region with the reference
electrode in contact with the abdominal skin. Both parylene-coated stripes were sutured
to the fish body.
154
6.2.4 Data processing of epicardial ECG signals
The ECG measurements were performed using a modified technique [250]. The entire
recording processes were performed in a Faraday cage to shield interference from
electromagnetic radiation. The ECG signals were amplified by 10,000-fold (A-M
Systems Inc. 1700 Differential Amplifier, Carlsborg, WA), and were filtered at a cut-off
frequency of 60 Hz (notch) between 0.1 and 500 Hz. The signals were acquired and
digitized at a sampling rate of 1,000 Hz (National Instruments USB-6216 DAQ device,
Austin TX, and LabVIEW 8.2). Wavelet transform and thresholding analysis were
applied to enhance signal-to-noise ratios (Matlab 2007a software, MathWorks, Inc.) for
the individual ECG signals recording as previously reported [250].
Wavelet transform was performed to filter various sources of noise. A wavelet was a
mathematical function used to divide a given function or continuous-time signal into
different frequency components. The individual components were investigated with a
resolution corresponding to their scales. Wavelet transform represented a function by
scaled wavelets in time domain [252]. After ECG signals were digitized at a rate of
1,000 Hz, the digital signals were divided into 10 scales by using “coif5” wavelet. The
coif5 is a 5th order coiflet (a discrete wavelet) function designed (by Ingrid Daubechies)
[253] to enable the best performance for de-noising ECG signals [254]. Wavelet
transform further allowed for a high speed convergence to reduce computation time
[255]. Thresholding analysis was applied to suppress the gill motion noise that was
merged within ECG signals. The remaining signals were recomposed by the inverse
wavelet transform, resulting in the de-noised ECG signals. P wave (atrial contraction),
QRS complexes (ventricular contraction) and ST segment (ventricular repolarization)
were distinguishable for the epicardial ECG recordings, and QRS intervals were
measured from the beginning of upstroke (Q) to the troughs (S). The signal-to-noise ratio
(SNR) in decibel was calculated before and after processing using the following formula:
S NR = 20 log
10
A
sign al
A
n o ise
(1)
Where A is the root mean square (RMS) voltage for signal and noise, respectively. SNR
for the epicardial ECG recordings and surface ECG recordings were compared to
evaluate the feasibility of electrode array-based epicardial ECG applications.
155
6.2.5 Optical Calcium transients in the entire and apical region of the adult
zebrafish hearts
Intracellular cardiomyocyte calcium concentrations allowed for monitoring the
propagation of electrical signal across the heart with a 320 x 240 pixels resolution. The
experimental setup and methods for simultaneous optical imaging of Calcium were
described previously [256]. In brief, adult zebrafish were sedated in a 0.02% solution of
Tricaine Methanesulfonate in water until they stop moving completely. Hearts were
removed from the fish via midline incision and were then incubated in incubation solution
(IS) containing 5 M Fluo-4-acetoxymethyl ester dissolved in dimethyl sulfoxide and
pluronic F-127 (0.1%) (Invitrogen) for 40 minutes. Hearts were then washed in Tyrode's
solution (in mM, NaCl 136, KCL 5.4, NaH2PO4 0.3, MgCl2 1.0, Glucose 5.0, HEPES 10
and CaCl2 1.8, pH adjusted to 7.4 with NaOH ) before transferring to an experimental
chamber (T=23
o
C) on a inverted microscope (Olympus). Whole heart Calcium imaging
was performed with a resolution of 320 x 240 pixels at 30 frames/second using a CCD
camera (Model LCL 811K, Watec America, Las Vegas, NV)[257] and apical heart
imaging was recorded by a digital camera (Canon EOS 550D) at 50 frames per second.
Optical mapping and myocardial calcium dynamics were superimposed to provide direct
comparison.
6.2.6 Optical Calcium transients in a single cell
After heart was removed from fish as described above, ventricle was isolated under
microscope. Ventricles then will be digested in the calcium-free salt solution (SS)
containing the following (in mM): NaCl 136, KCl 5.4, NaH2PO4 0.33, HEPES 10 (pH
7.4) supplemented with 0.1mg/mL trypsin and 1mg/mL collagenase. Digestion was
allowed at 37˚C for 20mins. The tissue was then transferred into SS supplemented with
0.1mM CaCl2 and 1mg/mL BSA, and cells were isolated by gentle trituration using fire-
polished pastor pipette of different pore sizes.
Next, isolated cardiomyocytes were centrifuged and resuspended with the IS for 30mins.
After incubation cells were washed and transferred into standard Tyrode’s solution for
imaging. ImageJ (NIH) and Matlab (Mathworks) software were used for data processing.

156
6.3 Chapter Six Results
6.3.1 Eipcardial ECG signal processing
The initial ECG signals were recorded at 1,000 Hz (Fig. 48a). Wavelet transform was
performed by breaking down the signals into 10 frequency segments ranging from 0 Hz
to the Nyquist frequency (½ of sampling frequency, i.e. 500 Hz) (Fig. 48b) [258]. The
individual frequency ranges contained different levels of signal components in the
presence of noise, which was suppressed by the pre-set threshold value (Fig. 48b).
Signals in the frequency range between 0.98 Hz and 7.8 Hz were not filtered to preserve
T wave [250]. The final ECG signals were re-constructed by inverse wavelet transform
after noise reduction (Fig. 48c). The P waves, QRS complexes and T waves were
retained, allowing for significantly improved signal-to-noise ratios from approximately
2dB for the raw data to 7dB after processing.
6.3.2 Epicardial ECG versus surface ECG signals
Implantation of flexible microelectrode arrays in the zebrafish myocardium revealed
distinct P waves, QRS complexes and T waves (Fig. 49). While ECG signals recorded
from Electrodes A and B revealed high amplitude QRS complexes (Fig. 49a and 4b),
electrode C revealed low amplitude QRS complexes and inverted ST segments (Fig.
49c). In comparison with the surface ECG signals (Fig. 49d), epicardial microelectrode
array demonstrated a higher signal strength (1.1 volts) compared to surface needle
microelectrodes (0.8 volts) with comparable SNR (7.1dB vs. 7.4dB for epicardial ECG
and surface ECG, respectively).
6.3.3 Interfacing epicardial ECG signals with propagation of Calcium transient in
the entire hearts
Excitation conduction across the myocardium revealed propagation of electrical signals
beginning from the atrium (A), through AV ring (AVR), to the ventricle (V) (Fig. 50). In
response to cardiac contraction, a sequence of intracellular Calcium transients was
captured in an area measured at 1870 x 1400 µ m
2
, accompanied by an image resolution
of 320 x 240 pixels. As the Calcium transients propagated from the atrium, through the
AV ring, to the ventricle (Fig. 50b), instantaneous changes in Ca
2+
as a function of time
157
were consistent with the sequence of atrial contraction (P waves) and ventricular
contraction (QRS complexes). Isochronal map revealed that fast conduction rate
developed in both atrium and ventricle as evidenced by the longer distance between the
white dash lines, whereas slow conduction rate occurred in the AV ring region as
evidenced by a shorter distance between the dash lines (Fig. 50c). The sequence of
pseudo-colored snapshots of Ca
2+
fluorescence corresponded to atrial contraction from 0
ms to 167 ms (P waves); ventricular contraction was initiated within the first 167
ms(QRS complexes) and propagated up to 267 ms, followed by ventricular repolarization
until 400 ms (ST segments) (Fig. 50d). Thus, application of Calcium mapping provided
a basis to validate conduction phenotypes from epicardial ECG signals.
6.3.4 Interfacing epicardial ECG signals with intracardiac calcium transients in a
single cell and an apical region
In corollary, Calcium transients were captured in the apical region of a contracting
ventricle (Fig. 51a). The mean grey value intensity plots of the five regions of interests
(ROIs) revealed Calcium transients, d[Ca
2+
]/dt, in synchrony with the epicardial ECG
signals (Fig. 51b). Intracellular Calcium transients were also captured in three ROIs from
a single contracting cardiomyocyte (Fig. 51c). Corresponding mean fluorescence
intensity plots demonstrated propagation of Calcium transients at single cell level (Fig.
51d). Thus, epicardial ECG recordings provide a reliable means of label-free monitoring
of conduction phenotypes.

158
6.4 Chapter Six Discussion
Heart failure inflicts nearly 5 million people in the US, and an additional 550,000 new
cases are diagnosed each year. Despite current regimens, heart failure remains the
leading cause of morbidity and mortality in the United States and developed world
mainly from inadequate replacement of infracted myocardium. Despite limited capacity
for cardiomyocytes to divide, this regeneration is insufficient to overcome the significant
loss of myocardium [259-261]. However, zebrafish (Danio rerio) possess a remarkable
capacity to regenerate a significant amount of myocardium in injured hearts, and thus,
represent a viable vertebrate model for regeneration [239].
In this study, we demonstrated real-time recordings of electrical conduction phenotypes
in small vertebral animal such as zebrafish via flexible microelectrode arrays. Distinct P
waves, QRS complexes, and ST segments were comparable to those of surface ECG.
Furthermore, the dynamic intracellular Calcium transients were in synchrony with the
epicardial ECG signals, and propagation of Calcium from the atrium to ventricle was
coupled with one cycle of ECG signals. Thus, interfacing flexible microelectrodes with
the epicardium was conducive to real-time recording of electrical signals from the
contracting hearts with high spatial resolution at ~ 20 m.
The advent of flexible microelectrodes allows for physiological interrogation of the small
animal systems at the interface between electronics and living tissue [262, 263]. Flexible
intravascular thermal sensors have been deployed to the aortas of New Zealand White
rabbits model on hypercholeterolemic diet to asses arterial regions prone to
atherosclerotic plaques [264]. Novel flexible parylene-based high-density electrode
arrays have also been applied for electrical stimulation in the retinas and spinal cords
[265]. These electrode arrays were microfabricated according to single-metal-layer and,
most recently, by the dual-metal-layer processes. Electrode arrays have also been
implanted and tested in the spinal cords of murine models, with the ultimate goal of
restoring locomotion post spinal cord injury. These arrays provide a high density and
precise spatial control of stimulation and recording otherwise impossible with the
traditional fine-wire electrodes [266]. Furthermore, with the recent advancement of
epidermal electronic technologies [267], long-term epicardial ECG monitoring for small
vertebrates in aqueous environment are made possible with wireless powered,
implantable multielectrode microelectronics.
159
Dipole, the polarity of the cardiac pacemaker, the distance of the electrode from the
dipole, and the strength of the electrical field influence ECG recording [268]. For
example, the amplitude of ventricular contraction (QRS) signals varies depending on the
distance and direction from the dipole (Fig. 52). The strength of electric potential (Ep) is
proportional to the solid angle ( ) and charge surface (  = voltage/unit of the solid
angle), and is inversely proportional to the square of the distance from the source.
Consistent with the 12-lead surface ECG signals in humans, the amplitude and shape of
QRS complexes and ST segments varied depending on the solid angle ( ) and the
strength of charge surface (  = voltage/unit of the solid angle) from the pacemaker
source or dipole (Fig. 49). Compared to previously reported surface ECG recording
[251], the epicardial multielectrode ECG system was able to provide information about
the pacemaker dipole as well as the asymmetricity of the ventricle repolarization, and will
be particularly useful in monitoring ECG changes during heart regeneration.
In this study, we also demonstrated that epicardial ECG signals correlated with optical
Calcium transients in the whole hearts. Optical Calcium transients required resection of
the entire hearts or isolation of cadiomyocytes from the fish, whereas epicardial ECG
recordings were performed in real-time without sacrificing the animals. While Calcium
transients revealed contraction of individual cardiomyocyte and atrioventricular
activation in the entire hearts, flexible microelectrodes allow for a global assessment of
electric conduction from atrial contraction to ventricular contraction and repolarization.
In addition, Calcium dye is cytotoxic, undergoing photobleaching during prolonged
recording. Thus, interfacing flexible microelectrodes with contracting epicardium offered
a non-invasive and long-term strategy to assess tissue regeneration.
Overall, flexible microelectrodes provided an entry point to identify the specific electrical
responses to tissue injury, drug-screening, and regenerating hearts. These phenotypic
effects are otherwise difficult with optical mapping alone. By allowing for direct
electrode contact with the non-planar surface of the pericardium, epicardial ECG signals
will further address the inter-observer and inter-lab variations, as well as changes in solid
angles between trials with the use of a pair of microelectrodes. Furthermore, application
of flexible microelectrodes allows for addressing a higher pacing threshold in the
regenerating part of the heart where the regenerated myocardium is not fully conductive.
In the era of regenerative medicine, flexible microelectrodes are conducive to maintain a
connection with the living tissues without damaging the host cells; thus, enabling
longitudinal monitoring of the conduction phenotypes in the specific regions of injured
and regenerating myocardium.
160

Figure 46. Microfabrication steps for flexible microelectrode arrays. The
microelectrode array is embedded in a sandwich structure as parylene-metal-parylene.
Steps 1-4: Approximately 8 μm of parylene C was deposited on a silicon wafer, followed
by spin-coating with a photoresist sacrificial layer. Steps 5-6: A gold metal liftoff process
was used to define a 16 μm pitch with a thickness from 200 nm to 300 nm. Step 7: A
second parylene deposition (~1 μm) formed the insulation. Steps 8-10: A small via at 6
μm x 6 μm was patterned in the insulation layer by oxygen plasma reactive-ion etching.
Step 11: The top layer of photoresist was striped to expose the microelectrode arrays.
Step 12: The arrays were peeled from the wafer in a water bath or released through
removal of the sacrificial photoresist in acetone.

161

Figure 47 (a) Flexible microelectrode arrays for zebrafish ECG recording. The chest strip
(upper electrodes) consisted of detecting electrodes (A, C, and D) and the tail strip (lower
electrode) consisted of a reference electrode. (b) Microelectrode array implantation
scheme. The chest strip was inseted into the chest cavity above the pericardium. The tail
strip was secured to the tail.

162

Figure 48 Wavelet transform and noise-reduction algorithm for zebrafish ECG
recordings. (a) Raw signal directly recorded from sedated zebrafish at sampling rate of
1000 Hz. (b) Breakdown of raw signals into frequency segments via coif5 wavelet
transform. Low frequency signals (DC to 0.98 Hz) were completely filtered. Thresholds
were applied to the individual frequency ranges for suppression of corresponding noise
levels. Sub-threshold values were set to zero. Signals within frequency range from 0.98
Hz to 7.81 Hz were reserved to ensure fidelity of T-waves. (c) Filtered ECG signals were
reconstructed by performing inverse wavelet transform from processed frequency
segments.

163

Figure 49. ECG signals acquired from multi-lead array. (a) ECG signals recorded
from Electrode A with respect to (wrt) the reference electrode. (b) Electrode B with
respect to the reference. (c) Electrode C with respect to Electrode D. (d) ECG signals
acquired by the sue of surface needle microelectrodes. Signals after noise-reduction from
epicardial microelectrode array demonstrated comparable signal strength (0.6 - 1.1 V olts
vs. 0.8 V olts, respectively) as well as signal to noise ratio (7.1dB vs. 7.4dB, respectively)
compared to surface needle ECG.

164

Figure 50. Calcium transients in the entire zebrafish heart. (a) Raw optical Ca
fluorescence image. A, atrium; A VR, A V ring; V , ventricle. Red arrow indicates the
conduction direction. (b) Upper panel shows Ca
i
transients traces from representative
sites in the A, A VR and V . The lower panel shows line scans of Ca fluorescence along the
red line in A, showing AV conduction velocity (slope of leading edge) is delayed through
A VR compared to A and V . (c) Isochronal map showing the fast conduction velocity in
both A (greater separation between dashed white isochrones) and slow conduction
velocity through A V ring area (crowded isochrones). The dashed green square indicates
the mapped area in panel D. (d) Pseudo-colored snapshots of Ca fluorescence at the
various times indicated at the lower left of the each snapshot, Red = high calcium; yellow
and green = intermediate calcium, and blue-black = low calcium. The white arrow shows
the direction of conduction. Scale bars: 200 m (A, C); 1 sec (B).

165

Figure 51. (a) Apical region of a contracting ventricle was captured. Five ROIs were
represented by boxes and the apex (edge not shown) was indicated by an arrow. Scale
bar, 200μm. (b) Corresponding mean fluorescence intensity plots of ROIs 1-5 revealed
166
propagation of Calcium transients, normalized d[Ca
2+
]/dt of rising slope were calculated,
d[Ca
2+
]/dt=11.45± 1.11 (n=5), dotted lines represent linear fitting of slopes. From box 1 to
box 5 that were superimposed in one epicardial ECG cycle (1.2 sec). Arrows indicated
peak amplitudes that were normalized to 1 with arbitrary unit (AU). (c) Three regions of
interests (ROIs) denoted by white boxes 1, 2 and 3 were captured from a single
contracting cardiomyocyte. Scale bar, 20μm. (d) Corresponding mean grey value
intensity plots of 3 ROIs illustrated Calcium transients. Second rising calcium transients
were used to calculated normalized d[Ca
2+
]/dt (d[Ca
2+
]/dt=8.13± 0.93, n=3) as indicated
by arrows, dotted lines show linear fitting of three rising slope. Amplitude of calcium
transients were normalized to 1, with arbitrary unit (AU).

167

Figure 52 (a) Comparison between human and zebrafish ECG signals. Due to a smaller
heart size and higher mean heart rates, the PR interval for zebrafish is shorter than that of
humans. T waves usually display a biphasic pattern acquired from the pseudo-unipolar
electrodes. (b) Lead placements in relation to the vector direction. QRS amplitudes in P1
and P2 are dependent on the electrode lead position. E
p
denotes electric potential,  the
solid angle, and  the strength of charge surface (  = voltage/unit of the solid angle).

168
6.5 Chapter Six References
[237] J. L. Reeve, A. M. Duffy, T. O'Brien, and A. Samali, "Don't lose heart--
therapeutic value of apoptosis prevention in the treatment of cardiovascular
disease," J Cell Mol Med, vol. 9, pp. 609-22, Jul-Sep 2005.
[238] C. Hahn and M. A. Schwartz, "The role of cellular adaptation to mechanical
forces in atherosclerosis," Arterioscler Thromb Vasc Biol, vol. 28, pp. 2101-7,
Dec 2008.
[239] K. D. Poss, L. G. Wilson, and M. T. Keating, "Heart regeneration in zebrafish,"
Science, vol. 298, pp. 2188-90, Dec 13 2002.
[240] A. Raya, A. Consiglio, Y. Kawakami, C. Rodriguez-Esteban, and J. C. Izpisua-
Belmonte, "The zebrafish as a model of heart regeneration," Cloning Stem Cells,
vol. 6, pp. 345-51, 2004.
[241] L. I. Zon and R. T. Peterson, "In vivo drug discovery in the zebrafish," Nature
Reviews Drug Discovery, vol. 4, pp. 35-44, 2005.
[242] J. Rihel, D. A. Prober, A. Arvanites, K. Lam, S. Zimmerman, S. Jang, et al.,
"Zebrafish behavioral profiling links drugs to biological targets and rest/wake
regulation," Science, vol. 327, pp. 348-351, 2010.
[243] J. S. Mitcheson, J. Chen, M. Lin, C. Culberson, and M. C. Sanguinetti, "A
structural basis for drug-induced long QT syndrome," Proceedings of the National
Academy of Sciences of the United States of America, vol. 97, pp. 12329-12333,
2000.
[244] K. Stoletov, L. Fang, S. H. Choi, K. Hartvigsen, L. F. Hansen, C. Hall, et al.,
"Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake
in hypercholesterolemic zebrafish," Circulation research, vol. 104, pp. 952-960,
2009.
[245] T. Yokogawa, W. Marin, J. Faraco, G. Pé zeron, L. Appelbaum, J. Zhang, et al.,
"Characterization of Sleep in Zebrafish and Insomnia in Hypocretin Receptor
Mutants," Public Library of Science - Biology, vol. 5, pp. 2379-2397, 2007.
169
[246] D. Sedmera, M. Reckova, A. deAlmeida, M. Sedmerova, M. Biermann, J.
Volejnik, et al., "Functional and morphological evidence for a ventricular
conduction system in zebrafish and Xenopus hearts," Am J Physiol Heart Circ
Physiol, vol. 284, pp. H1152-60, Apr 2003.
[247] D. J. Milan and C. A. MacRae, "Animal models for arrhythmias," Cardiovasc
Res, vol. 67, pp. 426-37, Aug 15 2005.
[248] D. Y. Stainier, "Zebrafish genetics and vertebrate heart formation," Nat Rev
Genet, vol. 2, pp. 39-48, Jan 2001.
[249] A. S. Forouhar, J. R. Hove, C. Calvert, J. Flores, H. Jadvar, and M. Gharib,
"Electrocardiographic characterization of embryonic zebrafish," Conf Proc IEEE
Eng Med Biol Soc, vol. 5, pp. 3615-7, 2004.
[250] P. Sun, Y. Zhang, F. Yu, E. Parks, A. Lyman, Q. Wu, et al., "Micro-
electrocardiograms to study post-ventricular amputation of zebrafish heart,"
Annals of Biomedical Engineering, vol. 37, pp. 890-901, 2009.
[251] F. Yu, R. Li, E. Parks, W. Takabe, and T. K. Hsiai, "Electrocardiogram signals to
assess zebrafish heart regeneration: implication of long QT intervals," Annals of
Biomedical Engineering, vol. 38, pp. 2346-2357, 2010.
[252] S. M. Szilagyi and L. Szilagyi, "Wavelet transform and neural-network-based
adaptive filtering for QRS detection," Proceedings: 22nd Annual IEEE - EMBS
International Conference, vol. 2, pp. 1267-1270, 2000.
[253] C. S. Burrus, R. A. Gopinath, and H. Guo, Introduction to Wavelets and Wavelet
Transforms: A Primer. Upper Saddle River, NJ: Prentice-Hall, 1997.
[254] H. G. R. Tan, A. Tan, P. Khong, and V. Mok, "Best wavelet function
identification system for ecg signal denoise applications," in International
Conference on Intelligent and Advanced Systems, Kuala Lumpur, 2007, pp. 631-
634.
[255] D. L. Donoho, "De-noising by soft-thresholding," IEEE Trans. Inform. Theory,
vol. 41, pp. 613-627, 1995.
170
[256] M. Valderrabano, F. Chen, A. S. Dave, S. T. Lamp, T. S. Klitzner, and J. N.
Weiss, "Atrioventricular ring reentry in embryonic mouse hearts," Circulation,
vol. 114, pp. 543-9, Aug 8 2006.
[257] L. M. Galli, T. Barnes, T. Cheng, L. Acosta, A. Anglade, K. Willert, et al.,
"Differential inhibition of Wnt-3a by Sfrp-1, Sfrp-2, and Sfrp-3," Dev Dyn, vol.
235, pp. 681-90, Mar 2006.
[258] P. Sun, Y. Zhang, F. Yu, E. Parks, A. Lyman, Q. Wu, et al., "Micro-
electrocardiograms to study post-ventricular amputation of zebrafish heart," Ann
Biomed Eng, vol. 37, pp. 890-901, May 2009.
[259] P. C. Hsieh, V. F. Segers, M. E. Davis, C. MacGillivray, J. Gannon, J. D.
Molkentin, et al., "Evidence from a genetic fate-mapping study that stem cells
refresh adult mammalian cardiomyocytes after injury," Nat Med, vol. 13, pp. 970-
4, Aug 2007.
[260] O. Bergmann, R. D. Bhardwaj, S. Bernard, S. Zdunek, F. Barnabe-Heider, S.
Walsh, et al., "Evidence for cardiomyocyte renewal in humans," Science, vol.
324, pp. 98-102, Apr 3 2009.
[261] K. Bersell, S. Arab, B. Haring, and B. Kuhn, "Neuregulin1/ErbB4 signaling
induces cardiomyocyte proliferation and repair of heart injury," Cell, vol. 138, pp.
257-70, Jul 23 2009.
[262] H. Yu, L. Ai, M. Rouhanizadeh, D. Patel, E. S. Kim, and T. K. Hsiai, "Flexible
polymer sensors for in vivo intravascular shear stress analysis," Journal of
Microelectromechanical Systems, vol. 17, pp. 1178-1186, 2008.
[263] D. C. Rodger, J. D. Weiland, M. S. Humayun, and Y. C. Tai, "Scalable high lead-
count parylene package for retinal prostheses," Sensors and Actuators B:
Chemical, vol. 117, pp. 107-114, 2006.
[264] F. Yu, L. Ai, W. Dai, H. Yu, and T. K. Hsiai, "MEMS Thermal Sensors to Detect
Changes in Heat Transfer in the Pre-Atherosclerotic Regions of Fat-Fed New
Zealand White Rabbits," Annals of Biomedical Engineering, vol. 39, pp. 1736-
1744, 2011.
171
[265] D. C. Rodger, A. J. Fong, L. W., H. Ameri, I. Lavrov, H. Zhong, et al., "High-
density Flexible Parylene-based Multielectrode Arrays for Retinal and Spinal
Cord Stimulation, Transducers '07, Lyon, France," 2007.
[266] D. C. Rodger, W. Li, H. Fong, A. J. Ameri, E. Meng, J. Burdick, et al., "Flexible
microfabricated parylene multielectrode arrays for retinal stimulation and spinal
cord field modulation," in Proc. 4th International IEEE-EMBS Special Topic
Conference on Microtechnologies in Medicine and Biology (MMB’06), Okinawa,
Japan, 2006, pp. 31-34.
[267] D. H. Kim, N. Lu, R. Ma, Y. S. Kim, R. H. Kim, S. Wang, et al., "Epidermal
electronics," Science, vol. 333, pp. 838-843, 2011.
[268] E. Braunwald, (Editor), Zipes, DP, (Editor), Libby, P, (Editor), Bonow, R,
(Editor) Braunwald's Heart Disease: A Textbook of Cardiovascular Medicine, 7
edition ed. Philadelphia: Saunders Company, 2004.

172

CHAPTER SEVEN: IMPLANTABLE
MICROELECTRONIC MEMBRANES TO INVESTIGATE
CARDIAC ELECTRICAL PHENOTYPES IN SMALL
ANIMAL MODELS OF HEART REGENERATION
1

1
Disclaimer: the content of this chapter are being prepared as a manuscript for
publication under the title “Implantable Microelectronic Membranes to investigate
cardiac electrical phenotypes in small animal model of heart regeneration”.
Acknowledgement: Co-authors of this manuscript include: Hung Cao, Yu Zhao, Fei Yu,
Michael Harrison, Juhyun Lee, Ali Darehzereshki, Ching-Ling Lien, Neil C. Chi, Yu-
Chong Tai, Tzung. K. Hsiai. The author of this thesis credits other co-authors for their
significant input in the process of preparing the manuscript for publication.
173
7.1 Chapter Seven Introduction
In the era of stem cell and regenerating medicine, flexible micro sensors enable
implantable electronics to monitor changes in electrical conduction in injured and
regenerating organ systems. Epidermal electronics was demonstrated to acquire
electroencephalography (EEG) signals from the brain of the rat model. Implantable
electronics revealed the feasibility to assess traumatic spinal injuries [269], and to
stimulate photoreceptors to restore visual impairment [270]. Development of flexible
microelectrode arrays (MEA) further synchronized epicardial electrical signals with
myocardial Calcium transients in zebrafish hearts.
Myocardial infarction results in irreversible loss of heart tissues or cardiomyocytes [271].
Injured human hearts heal by scarring, which leads to remodeling and subsequently, heart
failure [272]. Unlike adult mammalian tissues, certain fish and amphibians maintain a
regenerative capacity throughout adult life. The conventional view of mammalian hearts
as having virtually no regenerative capacity is now questioned by the recent animal and
human studies, in which new cardiomyocytes may arise from existing cardiomyocytes
and progenitor or stem cells. Zebrafish (Danio rerio) hearts fully regenerate after 20% of
ventricular resection [273, 274], thereby providing a genetically tractable system for drug
discoveries and inherited cardiac arrhythmias. Through a genetic fate mapping, Porrello
et al. discovered the transient regenerating capacity of 1-day-old neonatal mice after birth,
but this capacity is lost by 7 days of age. Thus, recognition of the limited regenerative
capacity in the mammalian heart is engendering enthusiasm about the body's plasticity
for its therapeutic potential.
Atrial and ventricular ECG signals in adult zebrafish were first revealed to be akin to
those of humans in 2004. Later, distinguishable P wave and QRS complex were
demonstrated by using oral perfusion to maintain oxygenation and muscle paralytics to
reduce mechanical noise [275]. Evolving ECG signals from larvae to adult zebrafish were
further made possible via micro-electrode pipette to uncover P waves and QRS
complexes at 14 days after fertilization (dfp) [276]. In the small animal models of heart
regeneration; namely, zebrafish and neonatal mice, ECG signals were acquired by using a
pair of micro-needle electrodes connected to a differential amplifier in the Faraday cage.
However, long-term monitoring of changes in electric conduction phenotypes required
repeated ECG acquisitions, rendering the micro-needle approach stressful to the animals
and inadequate to provide spatial resolution.
174
In this context, we developed a flexible electronic membrane with high spatial and
temporal resolution to elucidate changes in conduction phenotypes in response to injured
myocardium in small animal model of heart regeneration. The electronic membrane was
based on parylene C with four gold electrodes forming an implantable micro-electrode
array (MEA) membranes. The MEA detected injured currents; namely, ST-depression
with high spatial resolution, akin to myocardial ischemia in humans. Furthermore, the
flexible membrane remained functional despite being in the aquatic environment and in
the freely swimming zebrafish. Thus, we provided a novel approach to advance our
understanding of conduction mechanisms underlying injured myocardium with a
translation implication for assessing small animal models of tissue regeneration.

175
7.2 Chapter Seven Designs and Methods
7.2.1. Microelectrode array (MEA) fabrication
MEAs were micro-fabricated to enable biocompatible and flexible membranes that could
be conformed to the non-planar anatomic surface. A 5 µ m-thick parylene C layer was
first deposited onto a hexamethyldisilazane-treated (HMDS) silicon wafer. Next, a double
layer of Au (0.2 µ m) on Ti (0.02 µ m) was deposited by thermal evaporation and
patterned by a regular gold etchant. Another 5-µ m thick parylene C was deposited, and
the recording sites and connecting pads were defined by oxygen plasma etching. Four
working electrodes were positioned to the caudal region of the MEA while the reference
electrode was in the mid-section (Fig. 53). The connecting pads were electrically
connected to a customized flexible flat cable (FFC) via conductive epoxy, and
polydimethylsiloxane (PDMS) was applied to establish electrical insulation and
mechanical strength.
In the case of zebrafish, additional wing components were implemented to facilitate long-
term implantation (Fig. 53). The electrodes in the caudal region of MEA were thermally
annealed to allow for implantation into the epicardial region of zebrafish. A pair of wings
were circumferentially enclosed around the fish, and were dorsally connected via epoxy
(Vetbond, 3M Animal Care Products, St. Paul, MN, USA) (Fig. 54). The relative
dimension of the reference electrode to the working electrodes (50, 100, 200 and 300 µ m
in diameter) were easily modified to optimize contact area with the non-planar anatomy.
7.2.2. Characterization of implantable membrane electronics
A pair of Au planar electrodes formed a transducer for measuring the ionic potential
difference between working and reference points (Fig. 55). In light of the specific
dimensions and the frequency range of ECG (2 to 125 Hz), the metal-electrolyte interface
was modeled as a double-layer capacitor in parallel with a resistor. The impedances of
electrodes with different diameters were characterized in the 0.7% saline solution with
sufficient conductivity (300-series Impedance Analyzer, Gamry Instruments, Warminster,
PA, USA). The impedance of the electrode with a diameter of 300 µ m was less than 1
MΩ at 100 Hz (Fig. 55d).
176
The equivalent circuits were composed of the front-end planar metal electrodes in direct
contact with signal source and the back-end instrumentation amplifier with high input
impedance. The conductive nature of living organisms was modeled as a network of
resistors. The individual ECG recording (working) and the reference electrodes probed
into two different points of this resistor network. One of the working electrodes (A, B, C
or D) and the reference electrode were positioned proximally to the hearts and towards
the tail, respectively (Fig. 55a). The shunt capacitors between the recording cables
remained small and negligible as compared to the electrode impedances and amplifier
input impedance. The bandwidth of input impedance was amplified to as high as a
parallel 1000-G resistor that was in parallel with a 5-pF capacitor. The impedances of a
working electrode with a diameter of 300 µ m versus the reference electrode remained
below 1 MΩ at 100 Hz, enabling a significant voltage drop across the instrumentation
amplifier. To minimize decoupling through the low-impedance path formed by fish body,
we positioned the electrodes proximal to the source of cardiac conduction.
7.2.3. Real-time acquisition of electrical conduction in zebrafish and neonatal mice
a. Electrical phenotypes in injured zebrafish
Sedated adult zebrafish (in 0.04% Tricaine methane sulfonate) was placed on a damp
sponge for microscopic procedures in compliance with the Institutional Animal Care and
Use Committee (IACUC) as previously described [277]. A 2-mm-long horizontal
incision was created at 0.5 mm caudal to the heart. The MEA head containing four
working electrodes (WE) was inserted into the epicardium while the reference electrode
(RE) was anchored towards the fish tail. The pair of wings was dorsally adhered as
described above. The fish was allowed to recover in water free of Tricane (Fig. 56a and
56b).
During electrical signal acquisition, the sedated fish implanted with the MEA was placed
in a Faraday cage (Fig. 56c). The signals were amplified by 10,000-fold (A-M Systems
Inc. 1700 Differential Amplifier, Carlsborg, WA), and filtered between 0.1 and 500 Hz
and at a cut-off frequency of 60 Hz (notch). The filtered signals were digitized at a
sampling rate of 1,000 Hz (National Instruments USB-6251 DAQ device, Austin TX, and
LabVIEW 8.2), and the signal-to-noise ratio (SNR) was enhanced by using the both
wavelet analysis and noise-reduction techniques as previously described.
177
b. ECG acquisition in neonatal mice
The mouse was anesthetized by a subcutaneous injection of 80 µ g/gr body weight
Ketamine/xylazine, and the caudal portion of MEA membrane was adhered to the chest
(Fig. 56d) in compliance with the Children’s Hospital Los Angeles (CHLA) IACUC
committee. To enhance SNR, we employed a micro-needle electrode for reference to
reduce the interfacing impedance. The signal processing was performed as described for
the zebrafish.
c. Heart injury procedures
Wild type adult zebrafish (Tong’s Tropical Fish and Supplies, Los Angeles, CA, USA)
and one-day old neonatal mice (ICR/CD-1 strain, Charles River Laboratories, MA)
underwent ventricular cryo-injury via liquid nitrogen-chilled metal probes. The sedated
zebrafish was mounted on a stereo microscope for an open-chest procedure. Ventricular
injury was induced via a liquid-nitrogen-chilled metal probe (diameter 0.8 mm) for 24
seconds. The fish was returned to the fresh water for recovery. After 3-7 days, the fish
underwent MEA implantation. Neonatal mice were cryo-injured by employing the similar
procedure. After 7 days, the mice underwent ECG signal acquisition.

178
7.3 Chapter Seven Results
7.3.1. Cryo-injury induced aberrant electrical phenotypes in both neonatal and
zebrafish
Using MEA membrane, we demonstrated ECG data obtained in a cryo-injured neonatal
mouse heart prior to and after signal-processing (Figs. 57a and b). Using the micro-
needle electrodes, we were able to validated the MEA approach (Fig. 57c). Akin to
human single lead recording, a single electrode placement failed to detect the ST-
depression in response to cryo-injury, which was uncovered by the use of MEA
membrane (Figs. 57a and b). The signals were enhanced after signal processing via
wavelet transform (Fig. 57b). The signals to noise ratios (SNRs) were calculated to be
12.6 dB for the raw and 20.8 dB for the processed data, respectively. While the needle
electrodes revealed distinct P waves and QRS complexes, the MEA membranes
uncovered distinct T waves and ST-depression, otherwise under-detected by the single
micro-needle approach.
7.3.2. Electrical signal acquisition in aquatic environment via MEA membranes
We further demonstrated the feasibility for prolonged ECG monitoring in freely
swimming zebrafish via the MEA membrane on Day 1, Day 2 and Day 3, respectively
(Fig. 58). There were no significant drifts in ECG signals in terms of P waves and QRS
complexes. Modifications in MEA design and implantation technique would further
improve the SNR and fish survival for long-term investigations.
Injury currents, namely ST-depression, persisted on day 3 (Fig. 59b). In this case, we
acquired the baseline ECG signals prior to cryo-injury (Fig. 59a), and repeated the
measurements on day 3 by inserting the MEA membrane into the epicardium. Unlike
human ECG, T wave became more distinct in response to cryo-injury in zebraifish. Thus,
the use of MEA membrane highlighted the capability of high-density electrodes to
uncover small regions of myocardial ischemia as evidenced by ST-depression, otherwise
technically challenging with the micro-needle approach. [278]

179
7.3.3. Electrical signal acquisition for injury currents via multi-channels
For the 4-chamber human hearts, 12-lead ECG has routinely been used to identify the
specific regions of myocardial injury. For the 2-chamber zebrafish heart, four-lead ECG
was applied to detect changes in ST segments in response to cryo-injury (Fig. 60a). Four
working electrodes enabled 4-lead ECG signals with improved spatial and temporal
resolution. Variations in the voltage amplitude among the four ECG signals reflected the
different points of reference in electrode placement with respect to the cardiac conduction
vector. In this example, the T wave signals were more prominent in electrode leads B, C
and D than that of A. Also notable were ST depression in electrode lead D and less
significant ST depression in leads B and C, which were absent in electrode lead A (Fig.
60b). Similar to the 12-lead ECG in humans, our 4-channel signal acquisition allowed for
identification of specific regions of myocardial injury for small animal models of heart
regeneration.

180
7.4 Chapter Seven Discussions
The novelties of the current study are the minimally invasive approach to uncover
aberrant electrical phenotypes in injured myocardium for small animal models of
regeneration. The design and development of flexible MEA membranes enabled
detection of injured myocardial currents with high spatial and temporal resolution,
otherwise challenging with the single micro-electrode approach. The electronic
membranes remained functional in the freely swimming zebrafish in real-time, offering
feasibility for prolonged period of signal acquisition. Thus, arrays of micro-electrodes
afford us the potential to scale up to large vertebral models of tissue regeneration.
Understanding heart regeneration in a vertebrate model system is highly essential to
public health. Heart failure remains the leading cause of morbidity and mortality in the
US and developed world due to failure to adequately replace lost ventricular myocardium
from ischemia-induced infarct. Thus, studies of electrical phenotypes in small vertebral
and mammalian heart regeneration using intimately implanted ECG microelectrode
arrays would provide new insights into electromechanical coupling of regenerating cells
in host hearts [279]. The advent of flexible microelectronic membrane provides an
intimate application to interrogate electrical depolarization and repolarization. By
interfacing microelectrode arrays with the injured and regenerating hearts, we will be
able to analyze electrical phenotypes with high spatial and temporal resolution.
The MEA membrane provided the first ECG evidence of injured current in the cro-
injured myocardium of neonatal mouse and zebrafish. While the MEA membrane clearly
revealed distinct ST-depression (Figs. 57 and 59), the different probing positions of
micro-needle electrodes might fail to uncover the injured current. In this experiment, a
needle electrode was used for reference along with working electrodes on the MEA
device. If the built-in planar reference electrode were applied, the high impedance of the
mouse skin would render low amplitudes in the acquired ECG signals. However,
application of conductive epoxy coupled with an increase in the surface area of reference
electrode will reduce the high impedance issue.
The MEA implantation were able to remained operational as long as the fish were alive,
allowing for acquiring ECG over 3 days (Fig. 58). Despite the swift movement of the
animals in the aquatic environment, the head portion of the MEA membrane remained
implanted in the epicardium, allowing for reproducible P wave, QRS complex and T
181
waves over 3 days. In response to cryo-injury, there was a significant change prior to and
3 days post injury in the ECG signals (Fig. 59), revealing aberrant repolarization in the
injured heart. The histological heart slides further provided site of injury, complementing
the ECG finding.
The inevitable inflammatory responses at the incision site precluded the fish survival for
over 4 days. The application of epoxy to the dorsal part of the fish further engendered
tissue irritation and edema (Fig. 56b). To address fish discomfort and stress, we propose
to reduce the width of MEA at the incision site and to implement a smart locking
mechanism to secure the MEA for the future investigation.
Simultaneous 4-channel recording further uncovered specific regions of injury current
with high spatial and temporal resolution. ECG data from all four electrodes revealed
synchronization in ECG intervals (Fig. 60). The individual electrodes in the MEA
membrane were capable of identifying site-specific conduction signals
4
. Further
improvement in developing high-density MEA membranes would enable unequivocal
detection of myocardial injury in the anterior, posterior and lateral walls, reminiscent of
12-lead ECG in humans.
The current MEA membranes also afforded the possibility of long-term electrical signal
acquisition. In comparison with the conventional needle micro-electrodes, the
biocompatible and flexible polymer-based MEA membranes demonstrated advantages in
signal stability (Fig. 58), spatial resolution (Fig. 60), and minimally-invasive approach
for implantation with sustained MEA function. The micro-fabrication processes are also
able to integrate with additional sensors (i.e. strain sensor for electromechanical
coupling) and/or technologies (i.e. CMOS technology). Thus, the MEA membranes
provide a test-bed for a compact and multi-modality system for elucidating biophysical
properties of injured and regenerating tissues.
In summary, we addressed the bioengineering challenges with innovative design to
uncover conduction phenotypes of injured tissues of small animal models of heart
regeneration. This integration-enabled design paves the foundation for wireless
technology for long-term acquisition of ECG signals in real-time. The success of this
approach will enable investigations of myocardial regeneration in the Tg(fli-1:GFP),
Tg(hsp70:Wnt3a) and Tg(hsp70:DDK-1) transgenic zebrafish lines to elucidate the
molecular mechanisms underlying heart regeneration. The development of high-density
182
MEA membranes will further afford the opportunity to uncover aberrant
electroencephalography (EEG) signals from the injured brain of small animal models
with translational significance to regenerative medicine.

183

Figure 53. Micro-fabrication of the flexible MEA membrane. (a) Step-by-step
fabrication processes resulted in a 4-lead ECG. (b) Schematic diagram of the MEA
membrane highlighted 4 working electrodes A, B, C and D, respectively, in the MEA
head, the reference electrode and contact pads in the rear.

184

Figure 54. Flexible MEA membranes for non-planar anatomy. (a) MEA membranes
were dovetailed to the zebrafish anatomy. (b) The photo shows the EMA membrane
franked by flexible wings. (c) The MEA head was annealed to curve upwards to the
ventricular contour. (d) The photo reveled a flexible MEA membrane that could be
conformed to the chest of neonatal mice. (e) The connector was used to link the MEA
membrane with the recording system. (f) The MEA was connected to the connector
(soldered to the red cable).

185

Figure 55. Characterization of the MEA impedance. (a) The flexible MEA membrane
was placed on the abdomen of the zebrafish. (b) Equivalent circuit model consisted of 1)
the front-end planar metal electrodes in contact with contracting heart (signal source),
and 2) the rear-end instrumentation amplifier associated with high input impedance. (c)
Frequency-dependent impedance increased with the decrease in surface area. (d)
Impedance increased in response to a decrease in the diameter of the electrode.

186

Figure 56. Real-time ECG acquisition in zebrafish and neonatal mice. (a) The micro-
electrode portion of the MEA membrane was implanted in the epicardium of zebrafish. A
pair of transparent wings circumferentially enclosed the fish body. (b) The MEA
membrane was implanted in a freely swimming zebrafish. (c) ECG acquisition in the
Faraday cage illustrated the sedated zebrafish lying a damped sponge. (d) Neonatal
mouse ECG recording highlighted the MEA membrane adhered to the chest of the mouse
and the external reference electrode positioned to the lower abdomen.

187

Figure 57. Aberrant electric phenotypes acquired from a cryo-injured neonatal
mouse. (a) Raw data was acquired via a MEA membrane on the chest. (b) Signal
processing enhanced the SNR for ST-depression. (c) Validation was performed with
micro-needle electrodes. Analogous to human rhythm recording with one lead, a single
needle placement on the chest missed the ST-depression. (d) Corresponding histology of
neonatal mice at 7 day after birth with sham operation (top) and cryoinjury (bottom) at
birth. Mice heart after cryoinjury displayed significant necrotic scar at anterior side of left
ventricle (black arrow). Vertical bar: 1mm. Image obtained from courtesy of Dr. Ching-
Ling Lien at Children’s Hospital Los Angeles.

(d)
Sham Operation
7 days post severe cryoinjury
188

Figure 58. Zebrafish ECG signals acquired via an implanted MEA membrane over
3 days. Electrical signal acquisition revealed P wave, and QRS complexes and T waves
from the same animal on (a) Day 1, (b) Day 2, and (c) Day 3, respectively.

189

Figure 59. ST depression in response to cryo-injury. ECG signals acquired from the
implanted MEA membrane in the same zebrafish (a) prior to, and (b) post cryo-injury on
day 3. Myocardial ischemia remained on Day 3 as evidenced by ST depression. (c)
Corresponding histology of the zebrafish heart 3 days post cryoinjury displayed a small
scar at the posterior-ventral side of the epicardium.

(c)
190

Figure 60. Simultaneous four-channel ECG signal acquisition. The implanted MEA
membrane captured a region of myocardial injury otherwise undetected via the single
electrode lead. (a) 4 channels were recorded simultaneously showing the synchronization
in ECG intervals. (b) Electrodes A revealed P, QRS, and T morphology without evidence
of myocardial injury currents. Electrodes B, C and D (most obvious) uncovered a region
of myocardium where cryo-injury induced ST depression.

191
7.5 References
[269] Z. Yu, C. Tsay, S. P. Lacour, S. Wagner, and B. Morrison, "Stretchable
microelectrode arrays--a tool for discovering mechanisms of functional deficits
underlying traumatic brain injury and interfacing neurons with neuroprosthetics,"
Conf Proc IEEE Eng Med Biol Soc, vol. Suppl, pp. 6732-5, 2006.
[270] H. Ameri, G. J. Chader, J. G. Kim, S. R. Sadda, N. A. Rao, and M. S. Humayun,
"The effects of intravitreous bevacizumab on retinal neovascular membrane and
normal capillaries in rabbits," Invest Ophthalmol Vis Sci, vol. 48, pp. 5708-15,
Dec 2007.
[271] J. L. Reeve, A. M. Duffy, T. O'Brien, and A. Samali, "Don't lose heart--
therapeutic value of apoptosis prevention in the treatment of cardiovascular
disease," J Cell Mol Med, vol. 9, pp. 609-22, Jul-Sep 2005.
[272] C. Hahn and M. A. Schwartz, "The role of cellular adaptation to mechanical
forces in atherosclerosis," Arterioscler Thromb Vasc Biol, vol. 28, pp. 2101-7,
Dec 2008.
[273] K. D. Poss, L. G. Wilson, and M. T. Keating, "Heart regeneration in zebrafish,"
Science, vol. 298, pp. 2188-90, Dec 13 2002.
[274] A. Raya, A. Consiglio, Y. Kawakami, C. Rodriguez-Esteban, and J. C. Izpisua-
Belmonte, "The zebrafish as a model of heart regeneration," Cloning Stem Cells,
vol. 6, pp. 345-51, 2004.
[275] D. J. Milan, I. L. Jones, P. T. Ellinor, and C. A. MacRae, "In vivo recording of
adult zebrafish electrocardiogram and assessment of drug-induced QT
prolongation," Am J Physiol Heart Circ Physiol, vol. 291, pp. H269 - H273, 2006.
[276] F. Yu, J. Huang, K. Adlerz, H. Jadvar, M. Hamdan, J. Chen, et al., "Evolving
Cardiac Conduction Phenotypes in Developing Zebrafish Larvae: Implications to
Drug Sensitivity," Zebrafish, vol. In Revision, 2010.
[277] F. Yu, R. Li, E. Parks, W. Takabe, and T. K. Hsiai, "Electrocardiogram Signals to
Assess Zebrafish Heart Regeneration," Annals of Biomedical Engineering, vol.
38, pp. 2346-2357, 2010.
192
[278] R. O. Bonow, D. L. Mann, D. P. Zipes, and P. Libby, Braunwald's Heart Disease:
A Textbook of Cardiovascular Medicine, 2-Volume Set, 9th ed.: Saunders, 2011.
[279] D. Y. Stainier, "Zebrafish genetics and vertebrate heart formation," Nat Rev
Genet, vol. 2, pp. 39-48, Jan 2001.

193
Conclusion
In this thesis, we have demonstrated the application of electrical signals to address
clinically relevant issues, namely, detection of mechanically unstable atherosclerotic
plaques and investigation of conduction phenotype and functional heart regeneration in
animal models. We developed novel technologies in both areas and paved the way for
translational researches and contributed to the unmet clinical need of diagnosis and
treatment of acute coronary syndrome.
Atherosclerotic lesions harbor pro-inflammatory substrates; namely, oxLDL and
macrophage-derived foam cells infiltrates, which engender distinct electrochemical
properties. We were able to determine the lesions’ frequency-dependent electrical and
dielectrical behavior by recording the EIS of the vascular tissue. In our study, we
introduced concentric bipolar microelectrodes to address the non-uniform and complex
tissue current distribution, uneven endoluminal topography and non-uniform current
distribution, in both rabbit aortas and human arteries with high spatial resolution. We
provided a sensitive and specific electrochemical strategy to characterize fibrous cap
atheromas in terms of impedance spectroscopy and active lipids content. We demonstrate
that high content of bioactive metabolic factors within the fibrous atheroma engendered
distinct frequency-dependent electrochemical impedance spectra. Histology and
immunohistochemistry for active lipids and calcification further validated specificity of
the EIS measurements for active metabolic states in en face human arteries. Hence, we
demonstrate that the application of EIS strategy was sensitive to detect fibrous cap
oxLDL-rich lesions and specific to distinguish oxLDL-absent fibroatheroma. We further
attemped to combine intravascular EIS with ISS and IVUS to detect elevated tissue
impedance in the endoluminal regions of augmented shear stress in the fat-fed NZW
rabbit model. Our integrated approach revealed two new findings: 1) time-averaged ISS
increased in the regions of atherosclerotic lesions as visualized by high-frequency IVUS,
and 2) the elevated EIS signals in these lesions were associated with active lipid content.
In this context, integrating intravascular ultrasound imaging, hemodynamics and tissue
impedance offer a translational basis for combining the three micro-sensors for diagnostic
applications.
Zebrafish (Danio rerio) possess the remarkable capacity to regenerate a significant
amount of myocardium in injured hearts, and thus represent an emerging vertebrate
model for regenerative medicine and cardiovascular research. Because cardiac
194
development, structure, and function are relatively conserved between lower vertebrates
and mammals, further mechanistic investigations into this regeneration and integration of
myocardium into damaged hearts may yield future insights into cellular therapies for
human heart failure and myocardial infarctions. Our recent electrophysiology studies in
zebrafish revealed that regenerating myocardium may not electrically couple with
uninjured myocardium, it remains to be investigated as to whether structurally
regenerated zebrafish hearts exhibit functionally normal physiologic phenotypes with
complete integration of regenerated myocardium with host myocardium. Zebrafish
cardiac propagation in the regenerated cardiac tissue is a complex process governed

by
the excitable properties of the tissue and its macroscopic

and microscopic architecture. In
this context, we assessed the histology-conduction relationship in response to ventricular
resection, and developed a multi-model approach to investigate this regeneration model.
Encouraging results from our laboratory and others showed the feasibility of monitoring
zebrafish heart regeneration by the use of microelectrodes, MEA, and Doppler
Ultrasound. Our in vivo regeneration model and technology platform provide a non-
invasive approach to assess cardiac conduction with relevance to future assessment of
genetically, epigenetically, or pharmacologically induced cardiac phenotypes.

195
Bibliography

[1] A. Maton, R. L. J. Hopkins, C. W. McLaughlin, S. Johnson, M. Q. Warner, D.
LaHart, et al., Human Biology and Health, 3 ed. Englewood Cliffs, NJ, USA:
Prentice Hall, 1997.
[2] C. K. Zarins, D. P. Giddens, B. K. Bharadvaj, V. S. Sottiurai, R. F. Mabon, and
S. Glagov, "Carotid bifurcation atherosclerosis. Quantitative correlation of
plaque localization with flow velocity profiles and wall shear stress," Circulation
research, vol. 53, pp. 502-514, 1983.
[3] P. F. Davies, C. F. Dewey Jr, S. R. Bussolari, E. J. Gordon, and M. A. Gimbrone
Jr, "Influence of hemodynamic forces on vascular endothelial function: in vitro
studies of shear stress and pinocytosis in bovine aortic cells," Journal of Clinical
Investigation, vol. 73, pp. 1121-1129, 1984.
[4] N. DePaola, M. A. Gimbrone Jr, P. F. Davies, and C. F. Dewey Jr, "Vascular
endothelium responds to fluid shear stress gradients," Arteriosclerosis and
Thrombosis, vol. 12, pp. 1254-1257, 1992.
[5] C. F. Dewey Jr, S. R. Bussolari, M. A. Gimbrone Jr, and P. F. Davies, "The
dynamic response of vascular endothelial cells to fluid shear stress," Journal of
biomechanical engineering, vol. 103, pp. 177-185, 1981.
[6] J. A. Frangos, T. Y. Huang, and C. B. Clark, "Steady shear and step changes in
shear stimulate endothelium via independent mechanisms--superposition of
transient and sustained nitric oxide production," Biochemical and Biophysical
Research Communications, vol. 224, pp. 660-665, 1996.
[7] J. J. Chiu, D. L. Wang, S. Chien, J. Skalak, and S. Usami, "Effects of disturbed
flow on endothelial cells," Journal of biomechanical engineering, vol. 120, pp.
2-8, 1998.
[8] J. Hwang, A. Saha, Y. C. Boo, G. P. Sorescu, J. S. McNally, S. M. Holland, et
al., "Oscillatory Shear Stress Stimulates Endothelial Production of O 2- from p
47 phox-dependent NAD (P) H Oxidases, Leading to Monocyte Adhesion,"
Journal of Biological Chemistry, vol. 278, pp. 47291-47298, 2003.
196
[9] J. Hwang, H. Michael, A. Salazar, B. Lassegue, K. Griendling, M. Navab, et al.,
"Pulsatile versus oscillatory shear stress regulates NADPH oxidase subunit
expression: implication for native LDL oxidation," Circulation research, vol. 93,
pp. 1225-1232, 2003.
[10] R. Ross, "Atherosclerosis--an inflammatory disease," N Engl J Med, vol. 340, pp.
115-26, Jan 14 1999.
[11] D. D. Heistad, "Unstable coronary-artery plaques," The New England journal of
medicine, vol. 349, pp. 2285-2287, 2003.
[12] L. K. Curtiss, "Reversing atherosclerosis," N Engl J Med, vol. 360, pp. 1144-6,
Mar 12 2009.
[13] B. C. Berk, R. W. Alexander, T. A. Brock, M. A. Gimbrone Jr, and R. C. Webb,
"Vasoconstriction: a new activity for platelet-derived growth factor," Science,
vol. 232, pp. 87-90, 1986.
[14] M. M. Brooks, S. C. Chung, T. Helmy, W. B. Hillegass, J. Escobedo, K. A.
Melsop, et al., "Health Status After Treatment for Coronary Artery Disease and
Type 2 Diabetes Mellitus in the Bypass Angioplasty Revascularization
Investigation 2 Diabetes Trial," Circulation, vol. 122, pp. 1690-1699, 2010.
[15] N. R. Madamanchi, A. Vendrov, and M. S. Runge, "Oxidative stress and
vascular disease," Arterioscler Thromb Vasc Biol, vol. 25, pp. 29-38, Jan 2005.
[16] Topper JN, Cai J, Falb D, and G. M. Jr., "Identification of vascular endothelial
genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2,
manganese superoxide dismutase, and endothelial cell nitric oxide synthase are
selectively up-regulated by steady laminar shear stress," Proc Natl Acad Sci U S
A., vol. 93, pp. 10417-22, 1996 Sep 17 1996.
[17] R. M. Nerem, R. W. Alexander, D. C. Chappell, R. M. Medford, S. E. Varner,
and W. R. Taylor, "The study of the influence of flow on vascular endothelial
biology," Am J Med Sci, vol. 316, pp. 169-75, 1998.
[18] O. Traub and B. C. Berk, "Laminar shear stress: mechanisms by which
endothelial cells transduce an atheroprotective force," Arterioscler Thromb Vasc
Biol, vol. 18, pp. 677-85, 1998.
197
[19] L. K. Curtiss, "Reversing atherosclerosis?" N Engl J Med, vol. 360, pp. 1144-6,
Mar 12 2009.
[20] R. Ross, "The pathogenesis of atherosclerosis: a perspective for the 1990s,"
Nature, vol. 362, pp. 801-809, 1993.
[21] V. Fuster, "Atherosclerosis, thrombosis, and vascular biology," in Cecil
Medicine, L. Goldman and D. Ausiello, Eds., 23rd ed Philadelphia, PA, USA:
Saunders Elsevier, 2007.
[22] L. Ai, M. Rouhanizadeh, J. C. Wu, W. Takabe, H. Yu, M. Alavi, et al., "Shear
stress influences spatial variations in vascular Mn-SOD expression: implication
for LDL nitration," American Journal of Physiology- Cell Physiology, vol. 294,
pp. C1576-C1585, 2008.
[23] C. Cheng, D. Tempel, R. van Haperen, H. C. de Boer, D. Segers, M. Huisman, et
al., "Shear stress-induced changes in atherosclerotic plaque composition are
modulated by chemokines," Journal of Clinical Investigation, vol. 117, pp. 616-
26, Mar 2007.
[24] H. Watkins and M. Farrall, "Genetic susceptibility to coronary artery disease:
from promise to progress," Nature Reviews Genetics, vol. 7, pp. 163-173, 2006.
[25] A. V. Finn, M. Nakano, J. Narula, F. D. Kolodgie, and R. Virmani, "Concept of
vulnerable/unstable plaque," Arteriosclerosis, Thrombosis, and Vascular
Biology, vol. 30, pp. 1282-1292, 2010.
[26] A. Didangelos, D. Simper, C. Monaco, and M. Mayr, "Proteomics of acute
coronary syndromes," Current atherosclerosis reports, vol. 11, pp. 188-195, 2009.
[27] A. Maseri and V. Fuster, "Is there a vulnerable plaque?," Circulation, vol. 107,
pp. 2068-2071, 2003.
[28] S. Glagov, E. Weisenberg, C. K. Zarins, R. Stankunavicius, and G. J. Kolettis,
"Compensatory enlargement of human atherosclerotic coronary arteries," New
England Journal of Medicine, vol. 316, pp. 1371-1375, 1987.
[29] Z. Y. Li, S. P. S. Howarth, T. Tang, and J. H. Gillard, "How critical is fibrous
cap thickness to carotid plaque stability?: A flow-plaque interaction model,"
Stroke, vol. 37, pp. 1195-1199, 2006.
198
[30] A. M. Varnava, P. G. Mills, and M. J. Davies, "Relationship between coronary
artery remodeling and plaque vulnerability," Circulation, vol. 105, pp. 939-943,
2002.
[31] P. Schoenhagen, K. M. Ziada, D. G. Vince, S. E. Nissen, and E. M. Tuzcu,
"Arterial remodeling and coronary artery disease: the concept of," Journal of the
American College of Cardiology, vol. 38, pp. 297-306, 2001.
[32] K. Thygesen, J. S. Alpert, and H. D. White, "Universal definition of myocardial
infarction," Circulation, vol. 116, pp. 2634-2653, 2007.
[33] J. L. Reeve, A. M. Duffy, T. O'Brien, and A. Samali, "Don't lose heart--
therapeutic value of apoptosis prevention in the treatment of cardiovascular
disease," J Cell Mol Med, vol. 9, pp. 609-22, Jul-Sep 2005.
[34] C. Murray and A. Lopez, "WHO World Health Report " World Health
Organization, Geneva, Switzerland2002.
[35] E. Boersma, N. Mercado, D. Poldermans, M. Gardien, J. Vos, and M. L.
Simoons, "Acute myocardial infarction," The Lancet, vol. 361, pp. 847-858,
2003.
[36] R. Ross, "Atherosclerosis is an inflammatory disease," American Heart Journal,
vol. 138, pp. S419-20, 1999.
[37] J. Sanz and Z. A. Fayad, "Imaging of atherosclerotic cardiovascular disease,"
Nature, vol. 451, pp. 953-957, 2008.
[38] J. L. Anderson, C. D. Adams, E. M. Antman, C. R. Bridges, R. M. Califf, D. E.
Casey Jr, et al., "ACC/AHA 2007 Guidelines for the Management of Patients
With Unstable Angina/Non-ST-Elevation Myocardial Infarction--Executive
Summary," Journal of the American College of Cardiology, vol. 50, pp. 652-726,
2007.
[39] Wikipedia. (Mar 24). "Atherosclerosis":
http://en.wikipedia.org/wiki/Atherosclerosis. Available:
http://en.wikipedia.org/wiki/Atherosclerosis
[40] R. P. Choudhury, V. Fuster, J. J. Badimon, E. A. Fisher, and Z. A. Fayad, "MRI
and characterization of atherosclerotic plaque: emerging applications and
199
molecular imaging," Arterioscler Thromb Vasc Biol, vol. 22, pp. 1065-1074,
2002.
[41] A. Schmermund and R. Erbel, "Unstable coronary plaque and its relation to
coronary calcium," Circulation, vol. 104, pp. 1682-1687, 2001.
[42] "American Heart Association, "Heart Disease and Stroke Statistics - 2006
Update"," American Heart Association2006.
[43] S. Zeibig, Z. Li, S. Wagner, H. P. Holthoff, M. Ungerer, A. Bultmann, et al.,
"Effect of the oxLDL Binding Protein Fc-CD68 on Plaque Extension and
Vulnerability in Atherosclerosis," Circulation research, vol. 108, pp. 695-703,
2011.
[44] K. R. Aroom, M. T. Harting, C. S. Cox, R. S. Radharkrishnan, C. Smith, and B.
S. Gill, "Bioimpedance Analysis: A Guide to Simple Design and
Implementation," Journal of Surgical Research, vol. 153, pp. 23-30, 2009.
[45] L. A. Geddes, "Historical evolution of circuit models for the electrode-
electrolyte interface," Ann Biomed Eng, vol. 25, pp. 1-14, 1997.
[46] I. Streitner, M. Goldhofer, S. Cho, H. Thielecke, R. Kinscherf, F. Streitner, et al.,
"Electric impedance spectroscopy of human atherosclerotic lesions,"
Atherosclerosis, vol. 206, pp. 464-468, 2009.
[47] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an
intravascular impedance catheter for detection of fatty lesions in arteries," IEEE
transactions on medical imaging, vol. 16, pp. 439-446, 1997.
[48] K. R. Foster and H. P. Schwan, "Dielectric properties of tissues," Handbook of
biological effects of electromagnetic fields, pp. 25-102, 1996.
[49] T. Suselbeck, H. Thielecke, J. Kochlin, S. Cho, I. Weinschenk, J. Metz, et al.,
"Intravascular electric impedance spectroscopy of atherosclerotic lesions using a
new impedance catheter system," Basic research in cardiology, vol. 100, pp.
446-452, 2005.
[50] C. Hahn and M. A. Schwartz, "The role of cellular adaptation to mechanical
forces in atherosclerosis," Arterioscler Thromb Vasc Biol, vol. 28, pp. 2101-7,
Dec 2008.
200
[51] E. R. Porrello, A. I. Mahmoud, E. Simpson, J. A. Hill, J. A. Richardson, E. N.
Olson, et al., "Transient Regenerative Potential of the Neonatal Mouse Heart,"
Science, vol. 331, pp. 1078-1080, 2011.
[52] P. C. Hsieh, V. F. Segers, M. E. Davis, C. MacGillivray, J. Gannon, J. D.
Molkentin, et al., "Evidence from a genetic fate-mapping study that stem cells
refresh adult mammalian cardiomyocytes after injury," Nature Medicine, vol. 13,
pp. 970-4, Aug 2007.
[53] O. Bergmann, R. D. Bhardwaj, S. Bernard, S. Zdunek, F. Barnabe-Heider, S.
Walsh, et al., "Evidence for cardiomyocyte renewal in humans," Science, vol.
324, pp. 98-102, Apr 3 2009.
[54] K. Bersell, S. Arab, B. Haring, and B. Kuhn, "Neuregulin1/ErbB4 signaling
induces cardiomyocyte proliferation and repair of heart injury," Cell, vol. 138,
pp. 257-70, Jul 23 2009.
[55] C. Mason and P. Dunnill, "A brief definition of regenerative medicine,"
Regenerative Medicine, vol. 3, pp. 1-6, 2008.
[56] "Regenerative medicine glossary," Regenerative Medicine, vol. 4 (4 Suppl), pp.
S1 - 88, 2009.
[57] C. E. Murry, H. Reinecke, and L. M. Pabon, "Regeneration gaps: observations
on stem cells and cardiac repair," Journal of the American College of Cardiology,
vol. 47, pp. 1777-1785, 2006.
[58] W. Roell, T. Lewalter, P. Sasse, Y. N. Tallini, B. R. Choi, M. Breitbach, et al.,
"Engraftment of connexin 43-expressing cells prevents post-infarct arrhythmia,"
Nature, vol. 450, pp. 819-824, 2007.
[59] K. D. Poss, L. G. Wilson, and M. T. Keating, "Heart regeneration in zebrafish,"
Science, vol. 298, pp. 2188-90, Dec 13 2002.
[60] N. C. Chi, R. M. Shaw, B. Jungblut, J. Huisken, T. Ferrer, R. Arnaout, et al.,
"Genetic and physiologic dissection of the vertebrate cardiac conduction
system," Public Library of Science - Biology, vol. 6, pp. 1006 - 1019, 2008.
[61] D. Sedmera, M. Reckova, A. deAlmeida, M. Sedmerova, M. Biermann, J.
Volejnik, et al., "Functional and morphological evidence for a ventricular
201
conduction system in zebrafish and Xenopus hearts," Am J Physiol Heart Circ
Physiol, vol. 284, pp. H1152-60, Apr 2003.
[62] R. Arnaout, T. Ferrer, J. Huisken, K. Spitzer, D. Y. R. Stainier, M. Tristani-
Firouzi, et al., "Zebrafish model for human long QT syndrome," Proceedings of
the National Academy of Sciences of the United State of America, vol. 104, pp.
11316 - 11321, 2007.
[63] R. Kopp, T. Schwerte, and B. Pelster, "Cardiac performance in the zebrafish
breakdance mutant," Journal of Experimental Biology, vol. 208, pp. 2123-2134,
2005.
[64] D. J. Milan, I. L. Jones, P. T. Ellinor, and C. A. MacRae, "In vivo recording of
adult zebrafish electrocardiogram and assessment of drug-induced QT
prolongation," Am J Physiol Heart Circ Physiol, vol. 291, pp. H269 - H273,
2006.
[65] C. Nusslein-Volhard (Editor) and R. Dahm (Editor), Zebrafish. New York:
Oxford University Press, 2002.
[66] D. Y. Stainier, "Zebrafish genetics and vertebrate heart formation," Nat Rev
Genet, vol. 2, pp. 39-48, Jan 2001.
[67] K. Kikuchi, J. E. Holdway, A. A. Werdich, R. M. Anderson, Y. Fang, G. F.
Egnaczyk, et al., "Primary contribution to zebrafish heart regeneration by gata4+
cardiomyocytes," Nature, vol. 464, pp. 601-605, 2010.
[68] P. Sun, Y. Zhang, F. Yu, E. Parks, A. Lyman, Q. Wu, et al., "Micro-
electrocardiograms to study post-ventricular amputation of zebrafish heart," Ann
Biomed Eng, vol. 37, pp. 890-901, May 2009.
[69] D. J. Milan and C. A. MacRae, "Animal models for arrhythmias," Cardiovasc
Res, vol. 67, pp. 426-37, Aug 15 2005.
[70] Y. Vengrenyuk, S. Carlier, S. Xanthos, L. Cardoso, P. Ganatos, R. Virmani, et
al., "A hypothesis for vulnerable plaque rupture due to stress-induced debonding
around cellular microcalcifications in thin fibrous caps," Proc Natl Acad Sci U S
A, vol. 103, pp. 14678-83, Oct 3 2006.
[71] J. A. Ambrose, M. A. Tannenbaum, D. Alexopoulos, C. E. Hjemdahl-Monsen, J.
Leavy, M. Weiss, et al., "Angiographic progression of coronary artery disease
202
and the development of myocardial infarction," Lead Article, vol. 12, pp. 184-
202, 1988.
[72] V. Fuster, L. Badimon, J. J. Badimon, and J. H. Chesebro, "The pathogenesis of
coronary artery disease and the acute coronary syndromes," The New England
Journal of Medicine, vol. 326, pp. 242-250, 1992.
[73] W. C. Little, M. Constantinescu, R. J. Applegate, M. A. Kutcher, M. T. Burrows,
F. R. Kahl, et al., "Can coronary angiography predict the site of a subsequent
myocardial infarction in patients with mild-to-moderate coronary artery
disease?," Circulation, vol. 78, pp. 1157-1166, 1988.
[74] R. Ross, "Atherosclerosis is an inflammatory disease," Am Heart J, vol. 138, pp.
S419-420, 1999.
[75] R. P. Choudhury, V. Fuster, J. J. Badimon, E. A. Fisher, and Z. A. Fayad, "MRI
and characterization of atherosclerotic plaque: emerging applications and
molecular imaging," Arterioscler Thromb Vasc Biol, vol. 22, pp. 1065-1074,
2002.
[76] A. Schmermund and R. Erbel, "Unstable coronary plaque and its relation to
coronary calcium," Circulation, vol. 104, pp. 1682-1687, 2001.
[77] T. Suselbeck, H. Thielecke, J. Kochlin, S. Cho, I. Weinschenk, J. Metz, et al.,
"Intravascular electric impedance spectroscopy of atherosclerotic lesions using a
new impedance catheter system," Basic research in cardiology, vol. 100, pp.
446-452, 2005.
[78] I. Streitner, M. Goldhofer, S. Cho, H. Thielecke, R. Kinscherf, F. Streitner, et al.,
"Electric impedance spectroscopy of human atherosclerotic lesions,"
Atherosclerosis, vol. 206, pp. 464-468, 2009.
[79] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an
intravascular impedance catheter for detection of fatty lesions in arteries," IEEE
transactions on medical imaging, vol. 16, pp. 439-446, 1997.
[80] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an
intravascular impedance catheter for detection of fatty lesions in arteries," IEEE
Transac Med Imaging, vol. 16, pp. 439-446, 1997.
203
[81] K. R. Aroom, M. T. Harting, C. S. Cox, R. S. Radharkrishnan, C. Smith, and B.
S. Gill, "Bioimpedance Analysis: A Guide to Simple Design and
Implementation," Journal of Surgical Research, vol. 153, pp. 23-30, 2009.
[82] K. R. Foster and H. P. Schwan, "Dielectric properties of tissues," Handbook of
biological effects of electromagnetic fields, pp. 25-02, 1996.
[83] L. A. Geddes, "Historical evolution of circuit models for the electrode-
electrolyte interface," Ann Biomed Eng, vol. 25, pp. 1-14, 1997.
[84] W. Franks, I. Schenker, P. Schmutz, and A. Hierlemann, "Impedance
characterization and modeling of electrodes for biomedical applications," IEEE
Transac Biomed Eng, vol. 52, pp. 1295-1302, 2005.
[85] E. T. McAdams, A. Lackermeier, J. A. McLaughlin, D. Macken, and J. Jossinet,
"The linear and non-linear electrical properties of the electrode-electrolyte
interface," Biosensors and Bioelectronics, vol. 10, pp. 67-74, 1995.
[86] H. C. Stary, "Natural history and histological classification of atherosclerotic
lesions: an update," Arterioscler Thromb Vasc Biol, vol. 20, pp. 1177-1178,
2000.
[87] R. Virmani, F. D. Kolodgie, A. P. Burke, A. Farb, and S. M. Schwartz, "Lessons
from sudden coronary death: a comprehensive morphological classification
scheme for atherosclerotic lesions," Arterioscler Thromb Vasc Biol, vol. 20, pp.
1262-1275, 2000.
[88] H. C. Stary, A. B. Chandler, R. E. Dinsmore, V. Fuster, S. Glagov, W. J. Insull,
et al., "A definition of advanced types of atherosclerotic lesions and a
histological classification of atherosclerosis.," Arterioscler Thromb Vasc Biol,
vol. 15, pp. 1512-1531, 1995.
[89] P. Verhamme, R. Quarck, H. Hao, M. Knaapen, S. Dymarkowski, H. Bernar, et
al., "Dietary cholesterol withdrawal reduces vascular inflammation and induces
coronary plaque stabilization in miniature pigs," Cardiovasc Res, vol. 56, pp.
135-44, Oct 2002.
[90] P. Holvoet, A. Mertens, P. Verhamme, K. Bogaerts, G. Beyens, R. Verhaeghe, et
al., "Circulating oxidized LDL is a useful marker for identifying patients with
204
coronary artery disease," Arterioscler Thromb Vasc Biol, vol. 21, pp. 844-8,
May 2001.
[91] P. Holvoet, J. Vanhaecke, S. Janssens, F. Van de Werf, and D. Collen,
"Oxidized LDL and malondialdehyde-modified LDL in patients with acute
coronary syndromes and stable coronary artery disease," Circulation, vol. 98, pp.
1487-94, Oct 13 1998.
[92] E. Barsoukov and J. R. Macdonald, Impedance spectroscopy: theory, experiment,
and applications. Malden, MA: Wiley-Interscience, 2005.
[93] M. E. Orazem and B. Tribollet, Electrochemical Impedance Spectroscopy 2008.
[94] L. K. Curtiss, "Reversing atherosclerosis," New England Journal of Medicine,
vol. 360, pp. 1144-1146, 2009.
[95] D. D. Heistad, "Unstable coronary-artery plaques," The New England journal of
medicine, vol. 349, pp. 2285-2287, 2003.
[96] Z. S. Galis, "Vulnerable plaque: the devil is in the details," Circulation, vol. 110,
pp. 244-246, 2004.
[97] Y. M. Park, M. Febbraio, and R. L. Silverstein, "CD36 modulates migration of
mouse and human macrophages in response to oxidized LDL and may
contribute to macrophage trapping in the arterial intima," The Journal of Clinical
Investigation, vol. 119, pp. 136-145, 2009.
[98] A. Nair, B. D. Kuban, E. M. Tuzcu, P. Schoenhagen, S. E. Nissen, and D. G.
Vince, "Coronary plaque classification with intravascular ultrasound
radiofrequency data analysis," Circulation, vol. 106, pp. 2200-2206, 2002.
[99] N. F. Marrouche, D. O. Martin, O. Wazni, A. M. Gillinov, A. Klein, M.
Bhargava, et al., "Phased-array intracardiac echocardiography monitoring during
pulmonary vein isolation in patients with atrial fibrillation: impact on outcome
and complications," Circulation, vol. 107, pp. 2710-2716, 2003.
[100] I. K. Jang, B. E. Bouma, D. H. Kang, S. J. Park, S. W. Park, K. B. Seung, et al.,
"Visualization of coronary atherosclerotic plaques in patients using optical
coherence tomography: comparison with intravascular ultrasound," Journal of
the American College of Cardiology, vol. 39, pp. 604-609, 2002.
205
[101] H. Yabushita, B. E. Bouma, S. L. Houser, H. T. Aretz, I. K. Jang, K. H.
Schlendorf, et al., "Characterization of human atherosclerosis by optical
coherence tomography," Circulation, vol. 106, pp. 1640-1645, 2002.
[102] B. T. MacDonald, K. Tamai, and X. He, "Wnt/β-catenin signaling: components,
mechanisms, and diseases," Developmental cell, vol. 17, pp. 9-26, 2009.
[103] F. D. Kolodgie, A. S. J. Katocs, E. E. Largis, S. M. Wrenn, J. F. Cornhill, E. E.
Herderick, et al., "Hypercholesterolemia in the rabbit induced by feeding graded
amounts of low-level cholesterol. Methodological considerations regarding
individual variability in response to dietary cholesterol and development of
lesion type," Arterioscler Thromb Vasc Biol., vol. 16, pp. 1454-1464, 1996.
[104] D. Tang, C. Yang, S. Kobayashi, J. Zheng, and R. P. Vito, "Effect of stenosis
asymmetry on blood flow and artery compression: a three-dimensional fluid-
structure interaction model," Ann Biomed Eng, vol. 31, pp. 1182-93, 2003.
[105] S. M. Schwartz, Z. S. Galis, M. E. Rosenfeld, and E. Falk, "Plaque rupture in
humans and mice," Arterioscler Thromb Vasc Biol, vol. 27, pp. 705-13, 2007.
[106] J. A. Berliner and J. W. Heinecke, "The role of oxidized lipoprotein in
atherogenesis," Free Radical Biology and Medicine, vol. 20, pp. 707-727, 1996.
[107] L. Asatryan, R. Hamilton, J. Mario Isas, J. Hwang, R. Kayed, and A. Sevanian,
"LDL phospholipid hydrolysis produces modified electronegative particles with
an unfolded apoB-100 protein," Journal of Lipid Research, vol. 46, pp. 115-122,
January 2005 2005.
[108] Y. Yamaguchi, J. Haginaka, S. Morimoto, Y. Fujioka, and M. Kunitomo,
"Facilitated nitration and oxidation of LDL in cigarette smokers," Eur J Clin
Invest., vol. 35, pp. 186-193, 2005 Mar 2005.
[109] M. Navab, J. A. Berliner, A. D. Watson, S. Y. Hama, M. C. Territo, A. J. Lusis,
et al., "The Yin and Yang of oxidation in the development of the fatty streak. A
review based on the 1994 George Lyman Duff Memorial Lecture," Arterioscler
Thromb Vasc Biol, vol. 16, pp. 831-42, 1996.
[110] S. Ehara, M. Ueda, T. Naruko, K. Haze, A. Itoh, M. Otsuka, et al., "Elevated
levels of oxidized low density lipoprotein show a positive relationship with the
severity of acute coronary syndromes," Circulation, vol. 103, pp. 1955-60, 2001.
206
[111] C. J. Slager, A. C. Phaff, C. E. Essed, N. Bom, J. C. H. Schuurbiers, and P. W.
Serruys, "Electrical impedance of layered atherosclerotic plaques on human
aortas," IEEE Transac Biomed Eng, vol. 39, pp. 411-419, 1992.
[112] R. Ross, "Atherosclerosis is an inflammatory disease," American Heart Journal,
vol. 138, pp. S419-20, 1999.
[113] J. L. Anderson, C. D. Adams, E. M. Antman, C. R. Bridges, R. M. Califf, D. E.
Casey Jr, et al., "ACC/AHA 2007 Guidelines for the Management of Patients
With Unstable Angina/Non-ST-Elevation Myocardial Infarction--Executive
Summary," Journal of the American College of Cardiology, vol. 50, pp. 652-726,
2007.
[114] S. G. Worthley, G. Helft, V. Fuster, Z. A. Fayad, M. Shinnar, L. A. Minkoff, et
al., "A novel nonobstructive intravascular MRI coil: in vivo imaging of
experimental atherosclerosis," Arteriosclerosis, Thrombosis, and Vascular
Biology, vol. 23, pp. 346-350, 2003.
[115] F. A. Jaffer, C. Vinegoni, M. C. John, E. Aikawa, H. K. Gold, A. V. Finn, et al.,
"Real-time catheter molecular sensing of inflammation in proteolytically active
atherosclerosis," Circulation, vol. 118, pp. 1802-9, Oct 28 2008.
[116] L. Marcu, M. C. Fishbein, J. M. I. Maarek, and W. S. Grundfest,
"Discrimination of human coronary artery atherosclerotic lipid-rich lesions by
time-resolved laser-induced fluorescence spectroscopy," Arteriosclerosis,
Thrombosis, and Vascular Biology, vol. 21, pp. 1244-1250, 2001.
[117] Y. Vengrenyuk, S. Carlier, S. Xanthos, L. Cardoso, P. Ganatos, R. Virmani, et
al., "A hypothesis for vulnerable plaque rupture due to stress-induced debonding
around cellular microcalcifications in thin fibrous caps," Proc Natl Acad Sci U S
A, vol. 103, pp. 14678-83, Oct 3 2006.
[118] A. V. Finn, M. Nakano, J. Narula, F. D. Kolodgie, and R. Virmani, "Concept of
vulnerable/unstable plaque," Arteriosclerosis, Thrombosis, and Vascular
Biology, vol. 30, pp. 1282-1292, 2010.
[119] J. A. Ambrose, M. A. Tannenbaum, D. Alexopoulos, C. E. Hjemdahl-Monsen, J.
Leavy, M. Weiss, et al., "Angiographic progression of coronary artery disease
and the development of myocardial infarction," Lead Article, vol. 12, pp. 184-
202, 1988.
207
[120] V. Fuster, L. Badimon, J. J. Badimon, and J. H. Chesebro, "The pathogenesis of
coronary artery disease and the acute coronary syndromes," The New England
journal of medicine, vol. 326, pp. 242-250, 1992.
[121] W. C. Little, M. Constantinescu, R. J. Applegate, M. A. Kutcher, M. T. Burrows,
F. R. Kahl, et al., "Can coronary angiography predict the site of a subsequent
myocardial infarction in patients with mild-to-moderate coronary artery
disease?," Circulation, vol. 78, pp. 1157-1166, 1988.
[122] J. Sanz and Z. A. Fayad, "Imaging of atherosclerotic cardiovascular disease,"
Nature, vol. 451, pp. 953-957, 2008.
[123] X. Li, J. Yin, C. Hu, Q. Zhou, K. K. Shung, and Z. Chen, "High-resolution
coregistered intravascular imaging with integrated ultrasound and optical
coherence tomography probe," Applied Physics Letters, vol. 97, pp. 133702-
133704, 2010.
[124] T. Suselbeck, H. Thielecke, J. Kochlin, S. Cho, I. Weinschenk, J. Metz, et al.,
"Intravascular electric impedance spectroscopy of atherosclerotic lesions using a
new impedance catheter system," Basic research in cardiology, vol. 100, pp.
446-452, 2005.
[125] I. Streitner, M. Goldhofer, S. Cho, H. Thielecke, R. Kinscherf, F. Streitner, et al.,
"Electric impedance spectroscopy of human atherosclerotic lesions,"
Atherosclerosis, vol. 206, pp. 464-468, 2009.
[126] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an
intravascular impedance catheter for detection of fatty lesions in arteries," IEEE
Transactions on Medical Imaging, vol. 16, pp. 439-446, 1997.
[127] F. Yu, R. Li, L. Ai, C. Edington, H. Yu, M. L. Barr, et al., "Electrochemical
Impedance Spectroscopy to Assess Vascular Oxidative Stress," Annals of
Biomedical Engineering, vol. 39, pp. 287-296, 2011.
[128] H. C. Stary, "Natural history and histological classification of atherosclerotic
lesions: an update," Arterioscler Thromb Vasc Biol, vol. 20, pp. 1177-1178,
2000.
[129] H. C. Stary, A. B. Chandler, R. E. Dinsmore, V. Fuster, S. Glagov, W. J. Insull,
et al., "A definition of advanced types of atherosclerotic lesions and a
208
histological classification of atherosclerosis.," Arterioscler Thromb Vasc Biol,
vol. 15, pp. 1512-1531, 1995.
[130] F. Lisdat and D. Schä fer, "The use of electrochemical impedance spectroscopy
for biosensing," Analytical and Bioanalytical Chemistry, vol. 391, pp. 1555-
1567, 2008.
[131] W. Franks, I. Schenker, P. Schmutz, and A. Hierlemann, "Impedance
characterization and modeling of electrodes for biomedical applications,"
Biomedical Engineering, IEEE Transactions on, vol. 52, pp. 1295-1302, 2005.
[132] D. Heber, S. Ingles, J. M. Ashley, M. H. Maxwell, R. F. Lyons, and R. M.
Elashoff, "Clinical detection of sarcopenic obesity by bioelectrical impedance
analysis," The American journal of clinical nutrition, vol. 64, pp. 472S-477S,
1996.
[133] U. G. Kyle, I. Bosaeus, A. D. De Lorenzo, P. Deurenberg, M. Elia, J. È . M.
GÛmez, et al., "Bioelectrical impedance analysis--part I: review of principles
and methods," Clinical Nutrition, vol. 23, pp. 1226-1243, 2004.
[134] U. G. Kyle, I. Bosaeus, A. D. De Lorenzo, P. Deurenberg, M. Elia, J. Manuel
Gó mez, et al., "Bioelectrical impedance analysis--part II: utilization in clinical
practice," Clinical Nutrition, vol. 23, pp. 1430-1453, 2004.
[135] A. Sevanian, J. Hwang, H. Hodis, G. Cazzolato, P. Avogaro, and G. Bittolo-Bon,
"Contribution of an in vivo oxidized LDL to LDL oxidation and its association
with dense LDL subpopulations," Arteriosclerosis, Thrombosis, and Vascular
Biology, vol. 16, pp. 784-793, 1996.
[136] M. S. Brown and J. L. Goldstein, "Lipoprotein metabolism in the macrophage:
implications for cholesterol deposition in atherosclerosis," Annual Review of
Biochemistry, vol. 52, pp. 223-261, 1983.
[137] G. C. Cheng, W. H. Briggs, D. S. Gerson, P. Libby, A. J. Grodzinsky, M. L.
Gray, et al., "Mechanical strain tightly controls fibroblast growth factor-2 release
from cultured human vascular smooth muscle cells," Circulation Research, vol.
80, pp. 28-36, 1997.
[138] Y. S. Kim, Z. S. Galis, A. Rachev, H. C. Han, and R. P. Vito, "Matrix
metalloproteinase-2 and-9 are associated with high stresses predicted using a
209
nonlinear heterogeneous model of arteries," Journal of Biomechanical
Engineering, vol. 131, pp. 011009-011018, 2009.
[139] S. Ehara, M. Ueda, T. Naruko, K. Haze, A. Itoh, M. Otsuka, et al., "Elevated
levels of oxidized low density lipoprotein show a positive relationship with the
severity of acute coronary syndromes," Circulation, vol. 103, pp. 1955-60, Apr
17 2001.
[140] S. Zeibig, Z. Li, S. Wagner, H. P. Holthoff, M. Ungerer, A. Bultmann, et al.,
"Effect of the oxLDL Binding Protein Fc-CD68 on Plaque Extension and
Vulnerability in Atherosclerosis," Circulation Research, vol. 108, pp. 695-703,
2011.
[141] G. Chinetti-Gbaguidi, M. Baron, M. A. Bouhlel, J. Vanhoutte, C. Copin, Y.
Sebti, et al., "Human Atherosclerotic Plaque Alternative Macrophages Display
Low Cholesterol Handling but High Phagocytosis Because of Distinct Activities
of the PPARg and LXRa Pathways," Circulation Research, vol. 108, pp. 985-
995, 2011.
[142] S. Cho and H. Thielecke, "Influence of electrode position on the characterization
of artery stenotic plaques by using impedance catheter," Biomedical Engineering,
IEEE Transactions on, vol. 53, pp. 2401-2404, 2006.
[143] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an
intravascular impedance catheter for detection of fatty lesions in arteries," IEEE
Transaction on Medical Imaging, vol. 16, pp. 439-446, 1997.
[144] D. K. Stiles and B. Oakley, "Simulated characterization of atherosclerotic
lesions in the coronary arteries by measurement of bioimpedance," Biomedical
Engineering, IEEE Transactions on, vol. 50, pp. 916-921, 2003.
[145] R. Yamada, H. Okura, Y. Miyamoto, T. Kawamoto, Y. Neishi, A. Hayashida, et
al., "A newly developed radio frequency signal-based intravascular ultrasound
tissue charagerization: a comparison between IMAP and integrated backscatter
intravascular ultrasound," Journal of the American College of Cardiology, vol.
57, pp. E1882-E1882, 2011.
[146] J. Rudd, E. Warburton, T. Fryer, H. Jones, J. Clark, N. Antoun, et al., "Imaging
atherosclerotic plaque inflammation with [18F]-fluorodeoxyglucose positron
emission tomography," Circulation, vol. 105, p. 2708, 2002.
210
[147] M. Naghavi and E. Falk, "From vulnerable plaque to vulnerable patient,"
Asymptomatic Atherosclerosis, pp. 13-38, 2010.
[148] F. Yu, X. Dai, T. Beebe, and T. Hsiai, "Electrochemical impedance spectroscopy
to characterize inflammatory atherosclerotic plaques," Biosensors and
Bioelectronics, vol. 30, pp. 165-173, 2011.
[149] I. Streitner, M. Goldhofer, S. Cho, H. Thielecke, R. Kinscherf, F. Streitner, et al.,
"Electric impedance spectroscopy of human atherosclerotic lesions,"
Atherosclerosis, vol. 206, pp. 464-468, 2009.
[150] I. Streitner, M. Goldhofer, S. Cho, R. Kinscherf, H. Thielecke, M. Borggrefe, et
al., "Cellular Imaging of Human Atherosclerotic Lesions by Intravascular
Electric Impedance Spectroscopy," PLoS ONE, vol. 7, p. e35405, 2012.
[151] T. K. Hsiai, S. K. Cho, H. M. Honda, S. Hama, M. Navab, L. L. Demer, et al.,
"Endothelial Cell Dynamics under Pulsating Flows: Significance of High Versus
Low Shear Stress Slew Rates (∂τ/∂t)," Annals of biomedical engineering, vol. 30,
pp. 646-656, 2002.
[152] J. J. Li, X. Meng, H. P. Si, C. Zhang, H. X. Lv, Y. X. Zhao, et al., "Hepcidin
Destabilizes Atherosclerotic Plaque via Overactivating Macrophages After
Erythrophagocytosis," Arteriosclerosis, Thrombosis, and Vascular Biology, vol.
32, pp. 1158-1166, 2012.
[153] T. Suselbeck, H. Thielecke, J. Kochlin, S. Cho, I. Weinschenk, J. Metz, et al.,
"Intravascular electric impedance spectroscopy of atherosclerotic lesions using a
new impedance catheter system," Basic research in cardiology, vol. 100, pp.
446-452, 2005.
[154] M. K. Konings, W. Mali, and M. A. Viergever, "Development of an
intravascular impedance catheter for detection of fatty lesions in arteries," IEEE
transactions on medical imaging, vol. 16, pp. 439-446, 1997.
[155] F. Yu, R. Li, L. Ai, C. Edington, H. Yu, M. Barr, et al., "Electrochemical
Impedance Spectroscopy to Assess Vascular Oxidative Stress," Annals of
biomedical engineering, vol. 39, pp. 287-296, 2011.
[156] H. M. Garcì a-Garcì a, B. D. Gogas, P. W. Serruys, and N. Bruining, "IVUS-
based imaging modalities for tissue characterization: similarities and
211
differences," The International Journal of Cardiovascular Imaging (formerly
Cardiac Imaging), vol. 27, pp. 215-224, 2011.
[157] L. Ai, L. Zhang, W. Dai, C. Hu, K. Kirk Shung, and T. K. Hsiai, "Real-time
assessment of flow reversal in an eccentric arterial stenotic model," Journal of
biomechanics, vol. 43, pp. 2678-2683, 2010.
[158] D. L. Bark Jr and D. N. Ku, "Wall shear over high degree stenoses pertinent to
atherothrombosis," Journal of biomechanics, vol. 43, pp. 2970-2977, 2010.
[159] Y. S. Chatzizisis, A. U. Coskun, M. Jonas, E. R. Edelman, C. L. Feldman, and P.
H. Stone, "Role of Endothelial Shear Stress in the Natural History of Coronary
Atherosclerosis and Vascular Remodeling:: Molecular, Cellular, and Vascular
Behavior," Journal of the American College of Cardiology, vol. 49, pp. 2379-
2393, 2007.
[160] C. Cheng, D. Tempel, R. van Haperen, H. C. de Boer, D. Segers, M. Huisman, et
al., "Shear stress-induced changes in atherosclerotic plaque composition are
modulated by chemokines," Journal of Clinical Investigation, vol. 117, pp. 616-
26, Mar 2007.
[161] P. H. Stone, A. U. Coskun, S. Kinlay, M. E. Clark, M. Sonka, A. Wahle, et al.,
"Effect of Endothelial Shear Stress on the Progression of Coronary Artery
Disease, Vascular Remodeling, and In-Stent Restenosis in Humans In Vivo 6-
Month Follow-Up Study," Circulation, vol. 108, pp. 438-444, 2003.
[162] N. Sun, N. B. Wood, A. D. Hughes, S. A. M. Thom, and X. Y. Xu, "Influence of
pulsatile flow on LDL transport in the arterial wall," Annals of biomedical
engineering, vol. 35, pp. 1782-1790, 2007.
[163] R. M. Nerem, R. W. Alexander, D. C. Chappell, R. M. Medford, S. E. Varner,
and W. R. TAYLOR, "The study of the influence of flow on vascular
endothelial biology," The American journal of the medical sciences, vol. 316, p.
169, 1998.
[164] R. Li, D. Mittelstein, J. Lee, K. Fang, R. Majumdar, Y. Tintut, et al., "A
dynamic model of calcific nodule destabilization in response to monocyte-and
oxidized lipid-induced matrix metalloproteinases," American Journal of
Physiology-Cell Physiology, vol. 302, pp. C658-C665, 2012.
212
[165] N. R. Madamanchi, A. Vendrov, and M. S. Runge, "Oxidative stress and
vascular disease," Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 25,
pp. 29-38, 2005.
[166] J. Hwang, H. Michael, A. Salazar, B. Lassegue, K. Griendling, M. Navab, et al.,
"Pulsatile Versus Oscillatory Shear Stress Regulates NADPH Oxidase Subunit
Expression," Circulation research, vol. 93, pp. 1225-1232, 2003.
[167] S. Parathath, S. L. Mick, J. E. Feig, V. Joaquin, L. Grauer, D. M. Habiel, et al.,
"Hypoxia Is Present in Murine Atherosclerotic Plaques and Has Multiple
Adverse Effects on Macrophage Lipid MetabolismNovelty and Significance,"
Circulation research, vol. 109, pp. 1141-1152, 2011.
[168] S. Lee, J. R. Springstead, B. Parks, C. E. Romanoski, R. Palvolgyi, T. Ho, et al.,
"Metalloproteinase Processing of HBEGF is a Proximal Event in the Response
of Human Aortic Endothelial Cells to Oxidized Phospholipids," Arteriosclerosis,
Thrombosis, and Vascular Biology, vol. 32, pp. 1246-1254, 2012.
[169] H. Yu, L. Ai, M. Rouhanizadeh, D. Patel, E. S. Kim, and T. K. Hsiai, "Flexible
polymer sensors for in vivo intravascular shear stress analysis,"
Microelectromechanical Systems, Journal of, vol. 17, pp. 1178-1186, 2008.
[170] L. Ai, H. Yu, W. Takabe, A. Paraboschi, F. Yu, E. Kim, et al., "Optimization of
intravascular shear stress assessment in vivo," Journal of biomechanics, vol. 42,
pp. 1429-1437, 2009.
[171] F. Yu, L. Ai, W. Dai, N. Rozengurt, H. Yu, and T. K. Hsiai, "MEMS Thermal
Sensors to Detect Changes in Heat Transfer in the Pre-Atherosclerotic Regions
of Fat-Fed New Zealand White Rabbits," Annals of biomedical engineering, vol.
39, pp. 1736-1744, 2011.
[172] M. Rouhanizadeh, T. C. Lin, D. Arcas, J. Hwang, and T. H. Hsiai, "Spatial
variations in shear stress in a 3-D bifurcation model at low Reynolds numbers,"
Annals of Biomedical Engineering, vol. 33, pp. 1360-1374, Oct 2005.
[173] P. Sun, Y. Zhang, F. Yu, E. Parks, A. Lyman, Q. Wu, et al., "Micro-
electrocardiograms to study post-ventricular amputation of zebrafish heart," Ann
Biomed Eng, vol. 37, pp. 890-901, May 2009.
213
[174] W. Wei, X. Li, Q. Zhou, K. K. Shung, and Z. Chen, "Integrated ultrasound and
photoacoustic probe for co-registered intravascular imaging," Journal of
biomedical optics, vol. 16, p. 106001, 2011.
[175] P. Holvoet, J. Vanhaecke, S. Janssens, F. Van de Werf, and D. Collen,
"Oxidized LDL and malondialdehyde-modified LDL in patients with acute
coronary syndromes and stable coronary artery disease," Circulation, vol. 98, pp.
1487-1494, 1998.
[176] S. Hleli, C. Martelet, A. Abdelghani, F. Bessueille, A. Errachid, J. Samitier, et
al., "An immunosensor for haemoglobin based on impedimetric properties of a
new mixed self-assembled monolayer," Materials Science and Engineering: C,
vol. 26, pp. 322-327, 2006.
[177] W. Franks, I. Schenker, P. Schmutz, and A. Hierlemann, "Impedance
characterization and modeling of electrodes for biomedical applications," IEEE
Transac Biomed Eng, vol. 52, pp. 1295-1302, 2005.
[178] S. M. Schwartz, Z. S. Galis, M. E. Rosenfeld, and E. Falk, "Plaque rupture in
humans and mice," Arterioscler Thromb Vasc Biol, vol. 27, pp. 705-13, Apr
2007.
[179] C. Giannattasio, M. Failla, G. Emanuelli, A. Grappiolo, L. Boffi, D. Corsi, et al.,
"Local effects of atherosclerotic plaque on arterial distensibility," Hypertension,
vol. 38, pp. 1177-1180, 2001.
[180] T. Takumi, E. H. Yang, V. Mathew, C. S. Rihal, R. Gulati, L. O. Lerman, et al.,
"Coronary endothelial dysfunction is associated with a reduction in coronary
artery compliance and an increase in wall shear stress," Heart, vol. 96, pp. 773-
778, 2010.
[181] Y. Huo and G. S. Kassab, "Compensatory remodeling of coronary
microvasculature maintains shear stress in porcine left-ventricular hypertrophy,"
Journal of Hypertension, vol. 30, p. 608, 2012.
[182] J. M. Dolan, F. J. Sim, H. Meng, and J. Kolega, "Endothelial Cells Express a
Unique Transcriptional Profile under Very High Wall Shear Stress Known to
Induce Expansive Arterial Remodeling," American Journal of Physiology-Cell
Physiology, vol. 302, pp. C1109-C1118, 2012.
214
[183] H. Samady, P. Eshtehardi, M. C. McDaniel, J. Suo, S. S. Dhawan, C. Maynard,
et al., "Coronary Artery Wall Shear Stress Is Associated With Progression and
Transformation of Atherosclerotic Plaque and Arterial Remodeling in Patients
With Coronary Artery DiseaseClinical Perspective," Circulation, vol. 124, pp.
779-788, 2011.
[184] P. Zilla, L. Moodley, J. Scherman, H. Krynauw, J. Kortsmit, P. Human, et al.,
"Remodeling leads to distinctly more intimal hyperplasia in coronary than in
infrainguinal vein grafts," Journal of Vascular Surgery, vol. 55, pp. 1734-1741,
2012.
[185] S. S. Varghese and S. H. Frankel, "Numerical modeling of pulsatile turbulent
flow in stenotic vessels," Journal of biomechanical engineering, vol. 125, p. 445,
2003.
[186] B. X. Zhang, "Numerical simulation of pulsatile turbulent flow in tapering
stenosed arteries," International Journal of Numerical Methods for Heat &# 38;
Fluid Flow, vol. 19, pp. 561-573, 2009.
[187] P. F. Davies, A. Remuzzi, E. J. Gordon, C. F. Dewey, and M. A. Gimbrone,
"Turbulent fluid shear stress induces vascular endothelial cell turnover in vitro,"
Proceedings of the National Academy of Sciences, vol. 83, p. 2114, 1986.
[188] S. F. Ding, M. Ni, X. L. Liu, L. H. Qi, M. Zhang, C. X. Liu, et al., "A causal
relationship between shear stress and atherosclerotic lesions in apolipoprotein E
knockout mice assessed by ultrasound biomicroscopy," American Journal of
Physiology-Heart and Circulatory Physiology, vol. 298, pp. H2121-H2129, 2010.
[189] R. O. Becker, S. Chapin, and R. Sherry, "Regeneration of the ventricular
myocardium in amphibians," Nature, vol. 248, pp. 145-7, Mar 8 1974.
[190] J. O. Oberpriller and J. C. Oberpriller, "Response of the adult newt ventricle to
injury," J Exp Zool, vol. 187, pp. 249-53, Feb 1974.
[191] E. Braunwald, D. P. Zipes, P. Libby, and R. Bonow, Braunwald's Heart Disease:
A Textbook of Cardiovascular Medicine, 7th ed. Philadelphia: Saunders
Company, 2004.
[192] K. D. Poss, L. G. Wilson, and M. T. Keating, "Heart regeneration in zebrafish,"
Science, vol. 298, pp. 2188-90, Dec 13 2002.
215
[193] C. Nusslein-Volhard, (Editor), Dahm,R, (Editor), Zebrafish. New York: Oxford
University Press, 2002.
[194] D. J. Milan, I. L. Jones, P. T. Ellinor, and C. A. MacRae, "In vivo recording of
adult zebrafish electrocardiogram and assessment of drug-induced QT
prolongation," Am J Physiol Heart Circ Physiol, vol. 291, pp. H269-73, Jul 2006.
[195] D. Sedmera, M. Reckova, A. deAlmeida, M. Sedmerova, M. Biermann, J.
Volejnik, et al., "Functional and morphological evidence for a ventricular
conduction system in zebrafish and Xenopus hearts," Am J Physiol Heart Circ
Physiol, vol. 284, pp. H1152-60, Apr 2003.
[196] D. Y. Stainier, "Zebrafish genetics and vertebrate heart formation," Nat Rev
Genet, vol. 2, pp. 39-48, Jan 2001.
[197] A. S. Forouhar, A. Hickerson, M. Liebling, A. Moghaddam, J. Lu, H.-J. Tsai, et
al., "The embryonic vertebrate heart tube is a dynamic suction pump," Science,
vol. 312, pp. 751-753, 2006.
[198] J. R. Hove, R. W. Kö ster, A. S. Forouhar, G. Acevedo-Bolton, S. E. Fraser, and
M. Gharib, "Intracardiac fluid-forces are an essential epigenetic factor for
embryonic cardiogenesis," Nature, pp. 172-177, 2003.
[199] R. Arnaout, T. Ferrer, J. Huisken, K. Spitzer, D. Y. Stainier, M. Tristani-Firouzi,
et al., "Zebrafish model for human long QT syndrome," Proc Natl Acad Sci U S
A, vol. 104, pp. 11316-21, Jul 3 2007.
[200] A. S. Forouhar, J. R. Hove, C. Calvert, J. Flores, H. Jadvar, and M. Gharib,
"Electrocardiographic characterization of embryonic zebrafish," Conf Proc IEEE
Eng Med Biol Soc, vol. 5, pp. 3615-7, 2004.
[201] D. J. Milan and C. A. MacRae, "Animal models for arrhythmias," Cardiovasc
Res, vol. 67, pp. 426-37, Aug 15 2005.
[202] S. M. Szilagyi and L. Szilagyi, "Wavelet transform and neural-network-based
adaptive filtering forQRS detection," Proceedings of the 22nd Annual
International Conference of the IEEE, vol. 2, pp. 1267-1270, 2000.
[203] C. S. Burrus, R. A. Gopinath, and H. Guo, Introduction to Wavelets and Wavelet
Transforms: A Primer. Upper Saddle River, NJ: Prentice-Hall, 1997.
216
[204] I. Daubechies, Ten Lectures on Wavelets. Philadelphia, PA: Soc. Indus. Appl.
Math., 1992.
[205] D. L. Donoho, "De-noising by soft-thresholding," IEEE Trans. Inform. Theory,
vol. 41, pp. 613-627, 1995.
[206] E. E. Braunwald, D. P. E. Zipes, P. E. Libby, and R. E. Bonow, "Braunwald's
Heart Disease: A Textbook of Cardiovascular Medicine," 5 edition ed
Philadelphia: Saunders Company, 2004, p. 114.
[207] S. A. Glantz, "Primer of Biostatistics," 6th ed: McGraw-Hill, 2005, pp. 386-388.
[208] J. L. Reeve, A. M. Duffy, T. O'Brien, and A. Samali, "Don't lose heart--
therapeutic value of apoptosis prevention in the treatment of cardiovascular
disease." J Cell Mol Med., vol. 9, pp. 609-22, 2005.
[209] C. Hahn and M. A. Schuwartz, "The Role of Cellular Adaptation to Mechanical
Forces in Atherosclerosis," Arterioscler Thromb Vasc Biol, 2008.
[210] A. Raya, A. Consiglio, Y. Kawakami, C. Rodriguez-Esteban, and J. C. Izpisua-
Belmonte, "The zebrafish as a model of heart regeneration," Cloning Stem Cells,
vol. 6, pp. 345-51, 2004.
[211] Z. J. Zheng, J. B. Croft, W. H. Giles, and G. A. Mensah, "Sudden cardiac death
in the United States, 1989 to 1998," Circulation, vol. 104, p. 2158, 2001.
[212] N. C. Chi, R. M. Shaw, B. Jungblut, J. Huisken, T. Ferrer, R. Arnaout, et al.,
"Genetic and physiologic dissection of the vertebrate cardiac conduction
system," Public Library of Science - Biology, vol. 6, pp. 1006 - 1019, 2008.
[213] O. Bergmann, R. D. Bhardwaj, S. Bernard, S. Zdunek, F. Barnabe-Heider, S.
Walsh, et al., "Evidence for cardiomyocyte renewal in humans," Science, vol.
324, pp. 98-102, Apr 3 2009.
[214] K. D. Poss, L. G. Wilson, and M. T. Keating, "Heart regeneration in zebrafish,"
Science, vol. 298, pp. 2188-90, Dec 13 2002.
[215] C. Nusslein-Volhard (Editor) and R. Dahm (Editor), Zebrafish. New York:
Oxford University Press, 2002.
217
[216] D. J. Milan, I. L. Jones, P. T. Ellinor, and C. A. MacRae, "In vivo recording of
adult zebrafish electrocardiogram and assessment of drug-induced QT
prolongation," Am. J.Physiol. Heart Circ. Physiol., pp. H269-H273, 2006.
[217] D. Sedmera, M. Reckova, A. deAlmeida, M. Sedmerova, M. Biermann, J.
Volejnik, et al., "Functional and morphological evidence for a ventricular
conduction system in zebrafish and Xenopus hearts," Am J Physiol Heart Circ
Physiol, vol. 284, pp. H1152-60, Apr 2003.
[218] D. Y. Stainier, "Zebrafish genetics and vertebrate heart formation," Nat Rev
Genet, vol. 2, pp. 39-48, Jan 2001.
[219] R. Arnaout, T. Ferrer, J. Huisken, K. Spitzer, D. Y. Stainier, M. Tristani-Firouzi,
et al., "Zebrafish model for human long QT syndrome," Proc Natl Acad Sci U S
A, vol. 104, pp. 11316-21, Jul 3 2007.
[220] A. Raya, A. Consiglio, Y. Kawakami, C. Rodriguez-Esteban, and J. C. Izpisua-
Belmonte, "The zebrafish as a model of heart regeneration," Cloning Stem Cells,
vol. 6, pp. 345-51, 2004.
[221] C. L. Lien, M. Schebesta, S. Makino, G. J. Weber, and M. T. Keating, "Gene
expression analysis of zebrafish heart regeneration," PLoS Biol, vol. 4, p. e260,
Aug 2006.
[222] P. Sun, Y. Zhang, F. Yu, E. Parks, A. Lyman, Q. Wu, et al., "Micro-
electrocardiograms to study post-ventricular amputation of zebrafish heart," Ann
Biomed Eng, vol. 37, pp. 890-901, May 2009.
[223] D. J. Milan and C. A. MacRae, "Animal models for arrhythmias," Cardiovasc
Res, vol. 67, pp. 426-37, Aug 15 2005.
[224] B. Surawicz, T. K. Knilans, and T. C. Chou, Chou's electrocardiography in
clinical practice: adult and pediatric, 5th ed.: WB Saunders Company, 2001.
[225] D. J. Y. Hsieh and C. F. Liao, "Zebrafish M2 muscarinic acetylcholine receptor:
cloning, pharmacological characterization, expression patterns and roles in
embryonic bradycardia," British journal of pharmacology, vol. 137, p. 782, 2002.
[226] J. L. Reeve, A. M. Duffy, T. O'Brien, and A. Samali, "Don't lose heart--
therapeutic value of apoptosis prevention in the treatment of cardiovascular
disease," J Cell Mol Med, vol. 9, pp. 609-22, Jul-Sep 2005.
218
[227] C. Hahn and M. A. Schwartz, "The role of cellular adaptation to mechanical
forces in atherosclerosis," Arterioscler Thromb Vasc Biol, vol. 28, pp. 2101-7,
Dec 2008.
[228] S. G. Priori, P. J. Schwartz, C. Napolitano, R. Bloise, E. Ronchetti, M. Grillo, et
al., "Risk stratification in the long-QT syndrome," New England Journal of
Medicine, vol. 348, p. 1866, 2003.
[229] E. Braunwald, D. P. Zipes, and P. Libby, Heart disease: a textbook of
cardiovascular medicine, 8th ed.: WB Saunders Company, 2001.
[230] M. T. Keating, "The long QT syndrome: a review of recent molecular genetic
and physiologic discoveries," Medicine, vol. 75, p. 1, 1996.
[231] M. R. Rosen, M. J. Janse, and A. L. Wit, Cardiac electrophysiology: a textbook:
Futura Publ. Comp., 1990.
[232] R. R. Makkar and P. S. Chen, "Stem Cell Therapy for Myocardial Repair,"
Journal of the American College of Cardiology, vol. 42, pp. 2070-2072, 2003.
[233] D. J. Milan, A. M. Kim, J. R. Winterfield, I. L. Jones, A. Pfeufer, S. Sanna, et al.,
"Drug-Sensitized Zebrafish Screen Identifies Multiple Genes, Including GINS3,
as Regulators of Myocardial Repolarization.," Circulation, vol. 120, pp. 553-559,
2009.
[234] G. Y. Chang, F. Cao, M. Krishnan, M. Huang, Z. Li, X. Xie, et al., "Positron
emission tomography imaging of conditional gene activation in the heart,"
Journal of molecular and cellular cardiology, vol. 43, pp. 18-26, 2007.
[235] I. Kehat, D. Kenyagin-Karsenti, M. Snir, H. Segev, M. Amit, A. Gepstein, et al.,
"Human embryonic stem cells can differentiate into myocytes with structural
and functional properties of cardiomyocytes," Journal of Clinical Investigation,
vol. 108, pp. 407-414, 2001.
[236] I. Kehat, A. Gepstein, A. Spira, J. Itskovitz-Eldor, and L. Gepstein, "High-
Resolution Electrophysiological Assessment of Human Embryonic Stem Cell-
Derived Cardiomyocytes. A Novel In Vitro Model for the Study of Conduction,"
Circulation Research, vol. 91, pp. 659-663, 2002.
219
[237] J. L. Reeve, A. M. Duffy, T. O'Brien, and A. Samali, "Don't lose heart--
therapeutic value of apoptosis prevention in the treatment of cardiovascular
disease," J Cell Mol Med, vol. 9, pp. 609-22, Jul-Sep 2005.
[238] C. Hahn and M. A. Schwartz, "The role of cellular adaptation to mechanical
forces in atherosclerosis," Arterioscler Thromb Vasc Biol, vol. 28, pp. 2101-7,
Dec 2008.
[239] K. D. Poss, L. G. Wilson, and M. T. Keating, "Heart regeneration in zebrafish,"
Science, vol. 298, pp. 2188-90, Dec 13 2002.
[240] A. Raya, A. Consiglio, Y. Kawakami, C. Rodriguez-Esteban, and J. C. Izpisua-
Belmonte, "The zebrafish as a model of heart regeneration," Cloning Stem Cells,
vol. 6, pp. 345-51, 2004.
[241] L. I. Zon and R. T. Peterson, "In vivo drug discovery in the zebrafish," Nature
Reviews Drug Discovery, vol. 4, pp. 35-44, 2005.
[242] J. Rihel, D. A. Prober, A. Arvanites, K. Lam, S. Zimmerman, S. Jang, et al.,
"Zebrafish behavioral profiling links drugs to biological targets and rest/wake
regulation," Science, vol. 327, pp. 348-351, 2010.
[243] J. S. Mitcheson, J. Chen, M. Lin, C. Culberson, and M. C. Sanguinetti, "A
structural basis for drug-induced long QT syndrome," Proceedings of the
National Academy of Sciences of the United States of America, vol. 97, pp.
12329-12333, 2000.
[244] K. Stoletov, L. Fang, S. H. Choi, K. Hartvigsen, L. F. Hansen, C. Hall, et al.,
"Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid
uptake in hypercholesterolemic zebrafish," Circulation research, vol. 104, pp.
952-960, 2009.
[245] T. Yokogawa, W. Marin, J. Faraco, G. Pé zeron, L. Appelbaum, J. Zhang, et al.,
"Characterization of Sleep in Zebrafish and Insomnia in Hypocretin Receptor
Mutants," Public Library of Science - Biology, vol. 5, pp. 2379-2397, 2007.
[246] D. Sedmera, M. Reckova, A. deAlmeida, M. Sedmerova, M. Biermann, J.
Volejnik, et al., "Functional and morphological evidence for a ventricular
conduction system in zebrafish and Xenopus hearts," Am J Physiol Heart Circ
Physiol, vol. 284, pp. H1152-60, Apr 2003.
220
[247] D. J. Milan and C. A. MacRae, "Animal models for arrhythmias," Cardiovasc
Res, vol. 67, pp. 426-37, Aug 15 2005.
[248] D. Y. Stainier, "Zebrafish genetics and vertebrate heart formation," Nat Rev
Genet, vol. 2, pp. 39-48, Jan 2001.
[249] A. S. Forouhar, J. R. Hove, C. Calvert, J. Flores, H. Jadvar, and M. Gharib,
"Electrocardiographic characterization of embryonic zebrafish," Conf Proc IEEE
Eng Med Biol Soc, vol. 5, pp. 3615-7, 2004.
[250] P. Sun, Y. Zhang, F. Yu, E. Parks, A. Lyman, Q. Wu, et al., "Micro-
electrocardiograms to study post-ventricular amputation of zebrafish heart,"
Annals of Biomedical Engineering, vol. 37, pp. 890-901, 2009.
[251] F. Yu, R. Li, E. Parks, W. Takabe, and T. K. Hsiai, "Electrocardiogram signals
to assess zebrafish heart regeneration: implication of long QT intervals," Annals
of Biomedical Engineering, vol. 38, pp. 2346-2357, 2010.
[252] S. M. Szilagyi and L. Szilagyi, "Wavelet transform and neural-network-based
adaptive filtering for QRS detection," Proceedings: 22nd Annual IEEE - EMBS
International Conference, vol. 2, pp. 1267-1270, 2000.
[253] C. S. Burrus, R. A. Gopinath, and H. Guo, Introduction to Wavelets and Wavelet
Transforms: A Primer. Upper Saddle River, NJ: Prentice-Hall, 1997.
[254] H. G. R. Tan, A. Tan, P. Khong, and V. Mok, "Best wavelet function
identification system for ecg signal denoise applications," in International
Conference on Intelligent and Advanced Systems, Kuala Lumpur, 2007, pp.
631-634.
[255] D. L. Donoho, "De-noising by soft-thresholding," IEEE Trans. Inform. Theory,
vol. 41, pp. 613-627, 1995.
[256] M. Valderrabano, F. Chen, A. S. Dave, S. T. Lamp, T. S. Klitzner, and J. N.
Weiss, "Atrioventricular ring reentry in embryonic mouse hearts," Circulation,
vol. 114, pp. 543-9, Aug 8 2006.
[257] L. M. Galli, T. Barnes, T. Cheng, L. Acosta, A. Anglade, K. Willert, et al.,
"Differential inhibition of Wnt-3a by Sfrp-1, Sfrp-2, and Sfrp-3," Dev Dyn, vol.
235, pp. 681-90, Mar 2006.
221
[258] P. Sun, Y. Zhang, F. Yu, E. Parks, A. Lyman, Q. Wu, et al., "Micro-
electrocardiograms to study post-ventricular amputation of zebrafish heart," Ann
Biomed Eng, vol. 37, pp. 890-901, May 2009.
[259] P. C. Hsieh, V. F. Segers, M. E. Davis, C. MacGillivray, J. Gannon, J. D.
Molkentin, et al., "Evidence from a genetic fate-mapping study that stem cells
refresh adult mammalian cardiomyocytes after injury," Nat Med, vol. 13, pp.
970-4, Aug 2007.
[260] O. Bergmann, R. D. Bhardwaj, S. Bernard, S. Zdunek, F. Barnabe-Heider, S.
Walsh, et al., "Evidence for cardiomyocyte renewal in humans," Science, vol.
324, pp. 98-102, Apr 3 2009.
[261] K. Bersell, S. Arab, B. Haring, and B. Kuhn, "Neuregulin1/ErbB4 signaling
induces cardiomyocyte proliferation and repair of heart injury," Cell, vol. 138,
pp. 257-70, Jul 23 2009.
[262] H. Yu, L. Ai, M. Rouhanizadeh, D. Patel, E. S. Kim, and T. K. Hsiai, "Flexible
polymer sensors for in vivo intravascular shear stress analysis," Journal of
Microelectromechanical Systems, vol. 17, pp. 1178-1186, 2008.
[263] D. C. Rodger, J. D. Weiland, M. S. Humayun, and Y. C. Tai, "Scalable high
lead-count parylene package for retinal prostheses," Sensors and Actuators B:
Chemical, vol. 117, pp. 107-114, 2006.
[264] F. Yu, L. Ai, W. Dai, H. Yu, and T. K. Hsiai, "MEMS Thermal Sensors to
Detect Changes in Heat Transfer in the Pre-Atherosclerotic Regions of Fat-Fed
New Zealand White Rabbits," Annals of Biomedical Engineering, vol. 39, pp.
1736-1744, 2011.
[265] D. C. Rodger, A. J. Fong, L. W., H. Ameri, I. Lavrov, H. Zhong, et al., "High-
density Flexible Parylene-based Multielectrode Arrays for Retinal and Spinal
Cord Stimulation, Transducers '07, Lyon, France," 2007.
[266] D. C. Rodger, W. Li, H. Fong, A. J. Ameri, E. Meng, J. Burdick, et al., "Flexible
microfabricated parylene multielectrode arrays for retinal stimulation and spinal
cord field modulation," in Proc. 4th International IEEE-EMBS Special Topic
Conference on Microtechnologies in Medicine and Biology (MMB’06),
Okinawa, Japan, 2006, pp. 31-34.
222
[267] D. H. Kim, N. Lu, R. Ma, Y. S. Kim, R. H. Kim, S. Wang, et al., "Epidermal
electronics," Science, vol. 333, pp. 838-843, 2011.
[268] E. Braunwald, (Editor), Zipes, DP, (Editor), Libby, P, (Editor), Bonow, R,
(Editor) Braunwald's Heart Disease: A Textbook of Cardiovascular Medicine, 7
edition ed. Philadelphia: Saunders Company, 2004.
[269] Z. Yu, C. Tsay, S. P. Lacour, S. Wagner, and B. Morrison, "Stretchable
microelectrode arrays--a tool for discovering mechanisms of functional deficits
underlying traumatic brain injury and interfacing neurons with
neuroprosthetics," Conf Proc IEEE Eng Med Biol Soc, vol. Suppl, pp. 6732-5,
2006.
[270] H. Ameri, G. J. Chader, J. G. Kim, S. R. Sadda, N. A. Rao, and M. S. Humayun,
"The effects of intravitreous bevacizumab on retinal neovascular membrane and
normal capillaries in rabbits," Invest Ophthalmol Vis Sci, vol. 48, pp. 5708-15,
Dec 2007.
[271] J. L. Reeve, A. M. Duffy, T. O'Brien, and A. Samali, "Don't lose heart--
therapeutic value of apoptosis prevention in the treatment of cardiovascular
disease," J Cell Mol Med, vol. 9, pp. 609-22, Jul-Sep 2005.
[272] C. Hahn and M. A. Schwartz, "The role of cellular adaptation to mechanical
forces in atherosclerosis," Arterioscler Thromb Vasc Biol, vol. 28, pp. 2101-7,
Dec 2008.
[273] K. D. Poss, L. G. Wilson, and M. T. Keating, "Heart regeneration in zebrafish,"
Science, vol. 298, pp. 2188-90, Dec 13 2002.
[274] A. Raya, A. Consiglio, Y. Kawakami, C. Rodriguez-Esteban, and J. C. Izpisua-
Belmonte, "The zebrafish as a model of heart regeneration," Cloning Stem Cells,
vol. 6, pp. 345-51, 2004.
[275] D. J. Milan, I. L. Jones, P. T. Ellinor, and C. A. MacRae, "In vivo recording of
adult zebrafish electrocardiogram and assessment of drug-induced QT
prolongation," Am J Physiol Heart Circ Physiol, vol. 291, pp. H269 - H273,
2006.
223
[276] F. Yu, J. Huang, K. Adlerz, H. Jadvar, M. Hamdan, J. Chen, et al., "Evolving
Cardiac Conduction Phenotypes in Developing Zebrafish Larvae: Implications
to Drug Sensitivity," Zebrafish, vol. In Revision, 2010.
[277] F. Yu, R. Li, E. Parks, W. Takabe, and T. K. Hsiai, "Electrocardiogram Signals
to Assess Zebrafish Heart Regeneration," Annals of Biomedical Engineering,
vol. 38, pp. 2346-2357, 2010.
[278] R. O. Bonow, D. L. Mann, D. P. Zipes, and P. Libby, Braunwald's Heart Disease:
A Textbook of Cardiovascular Medicine, 2-Volume Set, 9th ed.: Saunders, 2011.
[279] D. Y. Stainier, "Zebrafish genetics and vertebrate heart formation," Nat Rev
Genet, vol. 2, pp. 39-48, Jan 2001.

Abstract (if available)

Abstract Atherosclerosis, particularly mechanically unstable lesions and resulting acute coronary syndromes remain to be one of the leading causes of death and permanent disabilities in the world. We hereby aim to address this clinical issue by applying electrical signals to assess the cardiovascular phenomenon. In particular, we developed novel electrical approaches to two important issues in early diagnosis and treatment of acute coronary syndrome: 1) detection of unstable plaque in vivo for early medical intervention, and 2) assessment of cardiac conduction and mechanical coupling during heart regeneration in small animal models. ❧ Despite advances in diagnosis and therapy, atherosclerotic cardiovascular disease remains the leading cause of morbidity and mortality in the Western world. Predicting metabolically active atherosclerotic lesions has remained an unmet clinical need. We developed an electrochemical strategy to characterize the inflammatory states of high-risk atherosclerotic plaques. Using the concentric bipolar microelectrodes, we sought to demonstrate distinct Electrochemical Impedance Spectroscopic (EIS) measurements for unstable atherosclerotic plaques that harbored active lipids and inflammatory cells. We demonstrated increased impedance in response to oxidized low density lipoprotein (oxLDL)-laden lesions. Using equivalent circuits to simulate vessel impedance at the electrode-endoluminal tissue interface, we demonstrated specific electric elements to model working and counter electrode interfaces as well as the tissue impedance. We hereby assessed the feasibility of integrating EIS with intravascular ultrasound (IVUS) and shear stress (ISS) to provide a new strategy to assess oxLDL-laden lesions in the fat-fed New Zealand White (NZW) rabbits. By applying electrochemical impedance in conjunction with shear stress and high-frequency ultrasound sensors, we provided a new strategy to identify oxLDL-laden lesions. Our study demonstrated the feasibility of integrating EIS, ISS, and IVUS for a catheter-based approach to assess mechanically unstable plaque. ❧ On the other arm, zebrafish (Danio rerio) is an emerging genetic model for regenerative medicine. In humans, myocardial infarction results in the irreversible loss of cardiomyocytes. Zebrafish hearts fully regenerate after resection or cryoinjury, without either scarring or arrhythmias. To study this cardiac regeneration, we developed microelectrode electrocardiograpm (ECG) platform, and subsequently implantable flexible multi-electrode membrane arrays that measure the epicardial electrocardiogram signals of zebrafish in real-time. The microelectrode electrical signals allowed for a high level of both temporal and spatial resolution. We observed delayed electric repolarization in either the regenerated hearts or scar tissues. Early regenerated cardiomyocytes lacked the conduction phenotypes of the sham fish. The electrical signals were in synchrony with optically measured calcium concentration as well as Doppler Echocardiogram. These microelectrode devices therefore provide a real-time analytical tool for monitoring conduction phenotypes of small vertebral animals with a high temporal and spatial resolution.

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Asset Metadata

Creator Yu, Fei (author)

Core Title Electrical signals to assess cardiovascular phenomenon

School Viterbi School of Engineering

Degree Doctor of Philosophy

Degree Program Biomedical Engineering

Publication Date 05/14/2013

Defense Date 03/25/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag atherosclerosis,ECG,electrochemical impedance spectroscopy,OAI-PMH Harvest,unstable plaque,zebrafish

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hsiai, Tzung K. (committee chair), Kim, Eun Sok (committee member), Shung, Kirk Koping (committee member), Yen, Jesse T. (committee member)

Creator Email feiyu@usc.edu,ronaldoyu@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-256202

Unique identifier UC11294464

Identifier etd-YuFei-1691.pdf (filename),usctheses-c3-256202 (legacy record id)

Legacy Identifier etd-YuFei-1691.pdf

Dmrecord 256202

Document Type Dissertation

Format application/pdf (imt)

Rights Yu, Fei

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA