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NEAR INFRARED OPTICAL IMAGING AND MAGNETIC RESONANCE CHARACTERIZATION OF DYSTROPHIC AND DAMAGED MUSCLE

By

STEPHEN MARK CHRZANOWSKI

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2016

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© 2016 Stephen Mark Chrzanowski

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My family, friends and mentors, who carried me through the troughs and lifted me to the

peaks during the PhD - I could not have done this without you

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ACKNOWLEDGMENTS

We are like dwarfs on the shoulders of giants, so that we can see more than they, and things at a greater distance, not by virtue of any sharpness of sign on our part, or any physical distinction, but because we are carried high and raised up by their giant size.

–Benand of Chartres, circa 1130

Dr. Glenn A. Walter’s substantial scientific accolades are internationally

respected, but his accomplishments as a human being far outweigh what he has

achieved in the world of science. Throughout our personal and professional relationship

as mentor-mentee, he was continually my ever-optimistic guiding force throughout my

pre-doctoral training years. I am confident to say that his mentorship will not conclude

following the completion of my training period under his protective shield.

I am a product of the village of individuals that have guided me along this route,

each enlightening our journey in their own unique ways. Dr. Krista Vandenborne

encouraged me to ‘enjoy the journey,’ despite my greatest efforts to only focus on the

results. The remainder of my advisory committee, including Drs. Barry Byrne, Peter

Sayeski, and Huabei Jiang, each contributed vital seeds of knowledge to foster me into

the evolving physician-scientist in training that I’ve become today. I would not be here if

it wasn’t for the renegade Dr. Steve Hsu, who while program director of the University of

Florida’s MD-PhD program, provided the initial opportunity to begin my journey as an

MD-PhD. I must go about thanking Dr. Robbie Regenhardt as well, who asked me one

evening stumbling through midtown to be his replacement as the MD-PhD student

advocate, allowing me a glimpse of how our College of Medicine runs. The current

Executive Committee of Drs. William “Stratford” May, Al Lewin, Lisa Spyrida, and Linda

Bloom have been constant golden examples of exemplary scientists and mentors.

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Drs. Celine Baligand, Ravneet Vohra, Fan Ye, and Rebecca Willcocks are the

“boots on the ground” field generals who have demonstrated exemplary patience,

teaching me all of the applicable skills that are used on a daily basis in the clinical and

preclinical laboratories. My colleagues, Brittany Lee, Abhinandan Batra, Wootaek Lim,

Umar Alabasi, Harneet Arora, Alison Barnard, and Ishu Arpan, have been fantastic

peers to provide humor, scientific inquiry, perspective, and always have been a helping

hand in our many projects. Much gratitude is necessary to the oft unheralded behind

the scenes team of Christa Stout, Jenny Fairfield, Hilary and Renee Cunkle, Cathy

Powers, Seth Panayiotou, and Andres Saagova, as they have consistently kept me on

track to succeed in the lab, without receiving due credit themselves.

Beyond the lab, my journey as a member of the muscular dystrophy family began

long before my time at the University of Florida. The Kapusta and Trevis families have

greatly shaped my life journey, and any contribution of scientific knowledge I may

compose pales in comparison to the contribution their families have made to my life.

Also, the Muscular Dystrophy Association camps in Cleveland, Cincinnati, and

Jacksonville have provided a lifetime of stories through the inappropriate sense of

humor that many of the campers possess.

I’m incredibly lucky to have not one best friend, but rather four in Bryan Trevis

and Drs. Nicholas Peter James Perry, Narayanasarma Singam, and Damon Fu, who

have been my security net of reassurance, humor, perspective, and enlightenment

throughout this PhD. Through our years at Cincinnati together, Sarma, Fu, and Nic

consistently provided positive encouragement in and beyond the classroom. When

Bryan and I journeyed 10,000 miles across the USA, he taught me that it is not the

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destination that matters, but rather the journey to get there. Rosha Poudyal, more than

anyone else has encouraged me through the troughs and celebrated my successes

throughout graduate school, and words do not suffice to tell my appreciation for the

support and love she has provided during this journey.

A special token of appreciation is warranted to Drs. Christy Holland and Kate

Hitchcock, who both saw potential in me, and taught me to believe in myself, more than

I thought possible. Dr. Hitchcock in particular deserves gratitude, for not only showing

me how to be a successful physician-scientist, but for also serendipitously providing

fantastic clinical care halfway across the country to my significant other, as she battled

meningitis.

And finally, I must thank my family, near and far, for instilling in me a sense of

love for each other, an interest in science, and a stubborn tenacious nature.

Funding for this research was provided by the Department of Defense

(MD110050), NIH/NICHD (HD043730), NIH/NHLBI (NL083810), NIH/NIAMS

(AR056973), the Muscular Dystrophy Association (MDA4170), and Parent Project

Muscular Dystrophy (PPMD8509).

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES .......................................................................................................... 13

LIST OF FIGURES ........................................................................................................ 14

LIST OF ABBREVIATIONS ........................................................................................... 17

ABSTRACT ................................................................................................................... 19

CHAPTER

1 MUSCLE AND THE MUSCULAR DYSTROPHIES ................................................ 21

Skeletal Muscle ....................................................................................................... 21

Growth and Repair of Skeletal Muscle ............................................................. 21 Structural Organization ..................................................................................... 23 Force Generation ............................................................................................. 24

Muscle Contraction ........................................................................................... 24 Dystrophin Associated Glycoprotein Complex .................................................. 25

Dystrophin ........................................................................................................ 26 Muscular Dystrophies ............................................................................................. 27

Clinical Features ............................................................................................... 29 Pathophysiology ............................................................................................... 30 Preclinical Models of Muscular Dystrophies ..................................................... 32

Invertebrates .............................................................................................. 32 Murine models ........................................................................................... 33

Rat models ................................................................................................. 35 Porcine models .......................................................................................... 35 Canine models ........................................................................................... 36

Therapies ......................................................................................................... 37 Genetic manipulation ................................................................................. 37 Enhancing muscle growth .......................................................................... 41 Minimizing inflammation ............................................................................. 42

Other strategies ......................................................................................... 43 Challenges of Therapeutic Trials ...................................................................... 45

2 NON-INVASIVE ASSESSMENTS OF MUSCLE HEALTH ..................................... 50

Electrical Impedance Myography ............................................................................ 51 Ultrasound .............................................................................................................. 52

Elastography ........................................................................................................... 53 Computed Tomography .......................................................................................... 56

Positron Emission Tomography .............................................................................. 57

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Magnetic Resonance Imaging and Spectroscopy ................................................... 58

Basics of Nuclear Magnetic Resonance ........................................................... 58 Origin of Magnetization .................................................................................... 59

Spin and Precession Frequency ....................................................................... 60 Manipulation of Signal ...................................................................................... 60 Measurable Parameters of MR ......................................................................... 61

Longitudinal relaxation (T1) and pulse repetition time (TR) ........................ 61 Transverse relaxation (T2) and echo time (TE) .......................................... 63

Image formation ............................................................................................... 66 Slice selection ............................................................................................ 67 Phase and frequency encoding .................................................................. 67

MR contrast ...................................................................................................... 68 Gadolinium based contrast agents ............................................................. 69

Iron oxide based contrast agents ............................................................... 70 Other contrast agents ................................................................................ 70

Magnetic Resonance Spectroscopy ................................................................. 71

Signal acquisition ....................................................................................... 71 Chemical shift ............................................................................................ 71

Applications of MRI and MRS in skeletal muscle ............................................. 72

MRI and MRS Summary ................................................................................... 74 Near Infrared Optical Imaging ................................................................................. 75

Near Infrared Optical Spectroscopy ................................................................. 75 Contrast Enhanced Near Infrared Optical Imaging ........................................... 76 Applications in Skeletal Muscle ........................................................................ 80

Conclusion .............................................................................................................. 80

3 OUTLINE OF EXPERIMENTS ................................................................................ 89

Overview ................................................................................................................. 89 Preclinical Studies: Detection Damaged, Diseased, and Healthy Murine Muscle ... 89

Acutely Induced Damage to Healthy Mouse Muscle ........................................ 90 Hypothesis ................................................................................................. 90 Specific aim ................................................................................................ 90

Exacerbation and Amelioration of Damage in Dystrophic Mouse Muscle ........ 91 Hypothesis ................................................................................................. 91 Specific aim ................................................................................................ 91

Vascular Drug Delivery Capabilities of ICG Enhanced Near Infrared Optical Imaging ................................................................................................................ 92

Hypothesis ........................................................................................................ 92 Specific Aim ...................................................................................................... 92

Clinical Studies ....................................................................................................... 93 Hypothesis ........................................................................................................ 93

Specific Aim ...................................................................................................... 93

4 METHODOLOGY ................................................................................................... 94

Pre-Clinical Work .................................................................................................... 94

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Animal Handling and Care................................................................................ 94

Mouse Strains .................................................................................................. 94 Control mice ............................................................................................... 94 mdx mice.................................................................................................... 94 γsg -/- mice ................................................................................................. 95

Preclinical Interventions.................................................................................... 96 Immobilization-reambulation studies .......................................................... 96 Downhill treadmill running .......................................................................... 97

Recombinant adeno-associated virus administration ................................. 98 Vascular perfusion experiments ................................................................. 98

Delivery of ICG Loaded Nanoparticles to Dystrophic Muscle ........................... 99 Synthesis and optimization of particles ...................................................... 99 In vivo capabilities of ICG loaded nanoparticles ....................................... 100

Methods.......................................................................................................... 101 Near Infrared Optical Imaging ............................................................................... 101

Magnetic Resonance Imaging and Spectroscopy ................................................. 102

Magnetic Resonance Imaging ........................................................................ 102 Magnetic Resonance Spectroscopy ............................................................... 103

Tissue Analysis ..................................................................................................... 104

Histology ......................................................................................................... 104 Spectrophotometry ......................................................................................... 105

Clinical Studies ..................................................................................................... 106 Heterogeneous Muscle Pathology is Revealed in DMD ................................. 106

Study design ............................................................................................ 106

Magnetic resonance acquisition and measures ....................................... 106

MRI and function data evaluation ............................................................. 107

Magnetic Resonance Imaging Identifies Dystrophic Muscle in the Upper Extremity ..................................................................................................... 108

Study design ............................................................................................ 109 Magnetic resonance acquisition and measures ....................................... 109 MRI data analysis .................................................................................... 110

Functional evaluation ............................................................................... 110 Near Infrared Optical Imaging Detects Acute Muscle Damage ...................... 110

Study design ............................................................................................ 111 Exercise testing ........................................................................................ 111 Magnetic resonance imaging and spectroscopy ...................................... 112

Indocyanine green enhanced near infrared optical imaging ..................... 113

Blood draws and questionnaire ................................................................ 113

5 NEAR INFRARED OPTICAL IMAGING IN A PRE-CLINICAL MODEL OF ACUTE MUSCLE DAMAGE ................................................................................. 119

Introduction ........................................................................................................... 119 Techniques to Assess Muscle Damage ......................................................... 119 Near Infrared Imaging and Indocyanine Green .............................................. 120

Results .................................................................................................................. 122 Animal Procedures ......................................................................................... 122

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Near Infrared Imaging of Mouse Hindlimbs .................................................... 122

Magnetic Resonance Imaging and Spectroscopy Confirm Muscle Damage Following Cast Immobilization and Reambulation ....................................... 123

Histology of Healthy and Damaged Muscle .................................................... 124 Spectrophotometric Quantification of ICG and EBD ....................................... 125 Correlation Between MRI and Near Infrared Optical Imaging ......................... 125

Discussion ............................................................................................................ 126 Near Infrared Optical Imaging as a Novel Method to Assess Muscle

Damage....................................................................................................... 127 Limitations to Experiments ............................................................................. 130

Summary .............................................................................................................. 131

6 QUANTIFICATION OF MUSCLE PATHOLOGY IN MDX AND GSG -/- MICE ....... 140

Introduction ........................................................................................................... 140 Muscular Dystrophies Render Muscle More Susceptible to Damage ............. 140 Mdx and Gsg -/- Mouse Models ....................................................................... 140

Techniques to Assess Muscle Damage Due to Dystrophies .......................... 141

Near Infrared Optical Imaging and Indocyanine Green & Current Uses ......... 142 Objectives ....................................................................................................... 143

Results .................................................................................................................. 144

Near Infrared Optical Imaging and Magnetic Resonance Imaging and Spectroscopy of Dystrophic Mice Allows for Identification of Muscle Pathology .................................................................................................... 144

Eccentric Loading by Downhill Treadmill Running Induces Quantifiable Muscle Damage to Older Mdx Mice ............................................................ 144

Restoration of γ-Sarcoglycan in Muscle is Observed by Near Infrared Optical Imaging ........................................................................................... 145

Magnitude of Effect Size is Comparable Between NIR Optical Imaging, MRI, and MRS ............................................................................................. 146

Histological Assessment of Tissue Confirms Restoration of γ-Sarcoglycan ... 146 Discussion ............................................................................................................ 146

Major Findings ................................................................................................ 146

Importance of Non-Invasive Biomarkers of Disease Progression and Regression .................................................................................................. 147

Importance of NIR Optical Imaging’s Contribution as an Outcome Measure Across Animals and Humans ...................................................................... 148

Comparison Between NIR Optical Imaging and MR ....................................... 150 Limitations ...................................................................................................... 150 Summary ........................................................................................................ 151

7 NIR OPTICAL IMAGING CAN DETECT CHANGES IN MAJOR VASCULATURE ................................................................................................... 160

Introduction ........................................................................................................... 160 Results .................................................................................................................. 162

Discussion and Summary ..................................................................................... 163

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8 POTENTIAL OF NEAR INFRARED RESPONSIVE PARTICLES AND QUANTIFICATION OF DRUG DELIVERY ........................................................... 166

Introduction ........................................................................................................... 166

Results .................................................................................................................. 169 Synthesis and Characterization of ICG-PLA Particles .................................... 169 Photostability at Room and Physiological Temperatures ............................... 170 In Vivo Contrast Enhanced NIR Optical Imaging of PLA-ICG via

Subcutaneous Injections: ............................................................................ 170 In Vivo Contrast Enhanced NIR Optical Imaging of PLA-ICG via

Intramuscular Injections .............................................................................. 171 Discussion ............................................................................................................ 171

Particle Synthesis and Characterization ......................................................... 171

Application of Particles to Animal Models ....................................................... 173 Summary of Delivery of Nanoparticles .................................................................. 175

9 DAMAGED AND DYSTROPHIC MUSCLE IN HUMANS ...................................... 181

A Multislice Analysis Reveals Heterogeneity within Lower Limbs of Boys with DMD .................................................................................................................. 181

Introduction ..................................................................................................... 181 Results ........................................................................................................... 184

Involvement of DMD in muscle presents in non-uniform manner ............. 184 Relationship between MRI scores, function and age ............................... 184

Discussion ...................................................................................................... 185

Limitations ...................................................................................................... 188

Summary of a Multislice Assessment of the Lower Leg in DMD .................... 189 Preliminary Assessment of the Upper Extremity in DMD by MRI .......................... 190

Introduction ..................................................................................................... 190

Results ........................................................................................................... 191 Discussion ...................................................................................................... 192

Summary of Upper Extremity Findings ........................................................... 193 Differences Between Concentric and Eccentric Lower Arm Exercises ................. 193

Introduction ..................................................................................................... 193

Results ........................................................................................................... 194 Discussion ...................................................................................................... 195 Summary Concentric and Eccentric Lower Arm Exercises............................. 196

10 CONCLUSION ...................................................................................................... 204

Overview ............................................................................................................... 204 Summary of Experiments ...................................................................................... 205

Capabilities of ICG Enhanced NIR Optical Imaging in Preclinical Models ...... 205 Potential of Near Infrared Responsive Particles ............................................. 206 Clinical Application of MRI and NIR Optical Imaging ...................................... 206

LIST OF REFERENCES ............................................................................................. 208

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BIOGRAPHICAL SKETCH .......................................................................................... 258

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LIST OF TABLES

Table page 5-1 Frequency of long 1H2O-T2 component in damaged hindlimbs of immobilized-

reambulated mice. ............................................................................................ 135

5-2 NIR optical imaging radiant efficiency measures were correlated to 1H2O-T2, MRI-T2, and Optical Density 780 nm / mg tissue, and r2 values (with associated p values in parentheses). ................................................................................. 139

6-1 Effect size magnitude demonstrates comparable differences between NIR optical imaging and MR measures. .................................................................. 158

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LIST OF FIGURES

Figure page 1-1 The sarcolemma and dystrophin associated glycoprotein complex. ................... 49

1-2 Binding sites and protein structure of dystrophin. Numbers refer to the spectrin-like repeats throughout the protein. NTD, N terminal domain; CR, Cysteine Rich region; CTD, C terminal domain. ................................................. 49

2-1 A spin echo sequence showing the initial 90° RF pulse, followed by the generated FID, the refocusing 180° RF pulse, and the additional 90° pulse of the next sequence. ............................................................................................. 81

2-2 Longitudinal (T1) relaxation curves showing the difference in relaxation between fat and muscle, and how different TR acquisitions (along the x-axis) alter the difference in signal generated between tissue types. ........................... 82

2-3 Transverse (T2) relaxation curves showing the difference in relaxation between muscle and edema, and how different TE acquisitions (along the x-axis) alter the difference in signal decay between tissue types. ......................... 83

2-4 Inversion recovery technique to calculate T1 demonstrating representative signal recovery profiles for edema, muscle, and lipid. ........................................ 84

2-5 Progressive saturation technique demonstrating how different acquisition times within the same recovery curve can be used to calculate T1. .................... 85

2-6 A Carr-Purcell sequence showing the initial 90° RF pulse, followed by a train of 180°RF pulses in the X plane, with each refocusing the FID in the opposite direction. ............................................................................................................. 86

2-7 A Carr-Purcell-Meiboom-Gill Pulse sequence showing the initial 90° RF pulse, followed by a train of 180°RF pulses given in the rotating frame, with each refocusing the FID in the same direction. .................................................. 87

2-8 Electromagnetic spectrum, highlighting the location of the near infrared range. ................................................................................................................. 88

4-1 Radiant efficiency reaches a steady state level between 30 minutes to 12 hours following ICG an intravenous injection. ................................................... 114

4-2 Fat suppressed T1 weighted image shows muscles of the lower leg in subjects with and without DMD, with arrows pointing to the TA (solid) and Per (dashed), highlighting intramuscular differences. ............................................. 115

4-3 Schematic representation of slice selections along the length of the lower leg. 116

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4-4 The qualitative MRI grading scale used to assess pathology within DMD muscle. ............................................................................................................. 117

4-5 Schematic study design of clinical study utilizing NIR to detect muscle damage. ........................................................................................................... 118

5-1 Two-dimensional NIR optical imaging shows an increase and recovery of fluorescent signal in muscle during reambulation following immobilization. ..... 132

5-2 MRI-T2 shows damage and recovery of soleus, but not gastrocnemius nor tibialis anterior muscles during reambulation .................................................... 133

5-3 Spectroscopic findings confirm increase in 1H2O-T2 and reveal long T2 components in the soleus of immobilized-reambulated hindlimbs .................... 134

5-4 Histological assessment confirms damage and recovery in the reambulated soleus muscle of the immobilized-reambulated hindlimbs ................................ 136

5-5 Spectrophotometric assessment confirms dye uptake into the soleus muscle at the peak of muscle damage. Absorbance, measuring EBD (5-5A) and ICG (5-5B) throughout the week of reambulation are quantified. ............................. 137

5-6 Increased radiant efficiency correlates to increased markers of damage in the soleus muscle ................................................................................................... 138

6-1 Dystrophy induced muscle pathology can be detected by NIR optical imaging, MRI, and MRS ................................................................................... 152

6-2 Increased radiant efficiency correlates with increased magnetic resonance measures in healthy and dystrophic mice ......................................................... 153

6-3 NIR optical imaging, MRI, and MRS confirm increased damage to muscle following treadmill exercising in mdx mice ........................................................ 154

6-4 Increased total radiant efficiency correlates with increased magnetic resonance measures before and after damage induced by treadmill running .. 155

6-5 gsg -/- mice treated with AAV demonstrate decreased near infrared fluorescence and lower MRI-T2 and 1H2O-T2 relaxation times following treatment .......................................................................................................... 156

6-6 Increased total radiant efficiency correlates with increased magnetic resonance measures in gsg -/- mice with and without restorative AAV therapy. 157

6-7 Representative immunofluorescence images with and without AAV delivery of γ-sarcoglycan ............................................................................................... 159

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7-1 Differences between major vasculature and surrounding muscle are able to be spatially and temporally identified ................................................................ 164

7-2 A hyperemic response is able to be quantified through NIR optical imaging. ... 165

8-1 Representative size distribution (8-1A), aggregation properties (8-1B), and fluorescence characteristics (8-1C) of ICG-PLA particles ................................. 176

8-2 Photostability at room (25°C, 8-2A) and physiologic (37°C, 8-2B) temperature of ICG-PLA particles and ICG alone. ................................................................ 177

8-3 Subcutaneous injections of PLA-ICG show prolonged maintained signal compared to Lactated Ringer’s Solution and ICG alone visually (8-3A) and quantitatively (8-3B). ......................................................................................... 178

8-4 Intramuscularly injected PLA-ICG particles maintain prolonged fluorescent signal (8-4A) at 1 (8-4B) and 28 (8-4C) days following injections. .................... 179

8-5 Ex vivo NIR optical images of excised muscles following intramuscular injections into the gastrocnemius demonstrate in vivo stability of PLA-ICG particles visually (8-5A) and quantitatively (8-5B). ............................................ 180

9-1 Qualitative MRI Scores from two representative DMD patients demonstrating differences in involvement along the length of six lower leg muscle groups. .... 197

9-2 Comprehensive degree of involvement in all slices of all subjects’ muscles .... 198

9-3 Age and function are related to MRIsingle and MRImulti scores. ........................... 199

9-4 Cross sectional analysis of upper extremity muscles in boys with DMD. .......... 200

9-5 Age and PUL function as related to MRI-T2 and MRI qualitative scores. .......... 201

9-6 Fat suppressed axial MR images of concentrically (9-6A) and eccentrically (9-6B) exercised human forearms with quantification (9-6C) of T2 relaxation times taken from the deep flexor muscles of the forearms. .............................. 202

9-7 Three dimensional absorbance reconstructions of human forearms were taken two days following eccentric (9-7A) and concentric (9-7B) exercise. ...... 203

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LIST OF ABBREVIATIONS

AAV adeno-associated virus

B0 Magnitude of static magnetic field

B1 Magnitude of excitatory radiofrequency field

BMD Becker muscular dystrophy. A form of muscular dystrophy with partial expression of the protein dystrophin. Less severe than Duchenne muscular dystrophy.

DAG Complex Dystrophin associated glycoprotein complex. A transmembrane complex of glycoproteins that link the subsarcolemmal cytosolic protein dystrophin to the extracellular matrix. This complex includes several subunits including sarcoglycans, dystroglycans, sarcospan, and syntrophins. Mutations to any of these proteins frequently lead to the limb girdle muscular dystrophies.

DMD Duchenne muscular dystrophy. The most common and severe muscular dystrophy, resulting from a lack of the protein dystrophin.

DWI Diffusion weighted imaging

ECM Extracellular matrix

FID Free induction decay

FOV Field of view

GAS Gastrocnemius muscle

gsg Gamma sarcoglycan. This is the mouse model of limb girdle muscular dystrophy, type 2C. Mice lacking gamma sarcoglycan (gsg-/-) demonstrate a severe phenotype of muscular dystrophy.

LGMD Limb girdle muscular dystrophy. This includes several forms of muscular dystrophy, identified by the dysfunctional protein of the dystrophin associated glycoprotein complex.

mdx Muscular dystrophy X-linked. This is the mouse model of Duchenne muscular dystrophy. The Dmd gene of the mouse has a premature stop codon in exon 23, resulting in an absence of the dystrophin protein.

MRI Magnetic resonance imaging

MRS Magnetic resonance spectroscopy

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NIR Near infrared

NMR Nuclear magnetic resonance

OI Optical Imaging

RF Radio frequency

SNR Signal to noise ratio

Sol Soleus muscle

STEAM Stimulated Echo acquisition mode

T1 Longitudinal relaxation rate constant

T2 Transverse relaxation rate constant

TA Tibialis anterior muscle

TE Echo time

TR Pulse repetition time

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

NEAR INFRARED OPTICAL IMAGING AND MAGNETIC RESONANCE CHARACTERIZATION OF DYSTROPHIC AND DAMAGED MUSCLE

By

Stephen Mark Chrzanowski

May 2016

Chair: Name Glenn A. Walter Co-chair: Barry J. Byrne Major: Medical Sciences – Physiology and Pharmacology The muscular dystrophies are a heterogeneous spectrum of neuromuscular

disorders that lead to rapid wasting of muscle and premature mortality. Duchenne

muscular dystrophy is the most common and one of the most devastating forms of

muscular dystrophy, leading to early loss of ambulation and death by the 3rd decade.

Current means to measure therapeutic efficacy for these diseases remain

inadequate, limited to invasive muscle biopsies and functional testing. Muscle biopsies

are inadequate because they are invasive, provide a limited sampling of this very

heterogeneous disease, and further damage already degenerative tissue. Functional

testing possesses inherent variables that remain difficult to control, such as subject

motivation and compliance. An ideal methodology of assessing therapeutic treatment must

be: highly sensitive and specific to biologic changes, inexpensive, non-invasive, minimally

exposing to harmful radiation, and comfortable for patients. Near infrared (NIR) optical

imaging (OI) and magnetic resonance imaging (MRI) and spectroscopy (MRS) may offer

potential as non-invasive modalities to quantitatively assess muscle pathology in acutely

injured and diseased muscle. Using an FDA approved near infrared fluorophore, we

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tested whether healthy and damaged muscle could be imaged and differentiated with

near NIR-OI, with confirmation provided by MRI, MRS, and histological assessment.

To assess acute muscle damage, healthy mice were cast immobilized in a

plantar flexed position for two weeks after which, mice were allowed to freely ambulate

and data were collected. Further, mdx and gsg mice were cross-sectionally compared to

age-matched unaffected mice. Next, data were collected from additional mdx mice that

were subjected to downhill treadmill running. The missing protein in gsg mice was

restored through an AAV treatment, and mice were imaged following therapy.

In the immobilization-reambulation model, damage was observed in the soleus

muscle of the immobilized leg by MRI-T2, 1H2O-T2, NIR-OI, and histologically compared

to the non-casted contralateral leg, demonstrating a peak of damage followed by

recovery. Both models of dystrophic mice demonstrated significant differences from

their control counterparts. AAV therapy in the gsg mice restored markers of muscle

damage back to baseline levels. This work supports NIR-OI as a feasible, cost effective,

non-invasive, longitudinal means to quantify muscle health.

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CHAPTER 1 MUSCLE AND THE MUSCULAR DYSTROPHIES

Skeletal Muscle

Skeletal muscle has many significant roles to the human body. Its primary role is

to provide force production, but it also plays key roles of normal maintenance of

metabolism. Unique among tissue types, skeletal muscle is impressively adaptive and

plastic, responding to anabolic, sarcopenic, and pathologic factors, allowing for

appropriate remodeling (Adams and McCue, 1998; Baldwin, 1996; Hood, 2001;

Janssen et al., 2002; Kandarian and Jackman, 2006; Kraemer et al., 2000; Roy et al.,

1991).

Growth and Repair of Skeletal Muscle

Skeletal muscle plays several roles, from providing both gross and fine motor

control to maintaining metabolic homeostasis. Importantly, muscle demonstrates

tremendous plasticity, able to either atrophy or hypertrophy, depending on the external

stimuli applied to the muscle (Hood, 2001; Kraemer et al., 2000; Lieber and Fridén,

2000; Roy et al., 1991). Weight bearing activities, even as simple as opposing gravity,

allow muscle to maintain their physiological integrity, whereas stark atrophy begins to

occur upon removal of resistance (Adams and McCue, 1998; Baldwin, 1996; Baldwin

and Haddad, 2001; Carlson et al., 1999; Dunn et al., 1999; Rittweger et al., 2005; Tesch

et al., 2004). Within days of injury, muscle has also demonstrated great capacity for

being able to regenerate itself (Ciciliot and Schiaffino, 2010; Lepper et al., 2011;

Pimorady-Esfahani et al., 1997; Tesch et al., 2004; Turner and Badylak, 2012).

Following damage to muscle, a cascade of cytokines and growth factors are released,

recruiting semi-pluripotent satellite cells and inflammatory cells to the injured muscle

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fibers (Tidball, 1995, 2005, 2011). Maintenance of muscle health remains critical to

homeostatic maintenance of overall health.

Satellite cells are the semi-pluripotent stem cells of muscle, capable of dividing

into new muscle cells as well as self-regenerating their own populations (Aziz et al.,

2012; Ferrari et al., 1998; Schultz, 1985; Schultz et al., 1985). Recruitment of satellite

cells allows for either fusion with already existing populations of existing damaged fibers

or with each other to form new fibers (Schultz et al., 1985). Mature muscle fibers exist

in a post-mitotic stage of development and do not divide further. Growth and repair, by

way of satellite cells, occurs by the introduction of new myonuclei to the already existing

myofibers (Tedesco et al., 2010). Satellite cells primarily reside between the

sarcolemma and the basal lamina, predominantly exist in a quiescent state in healthy

adult muscle (Aziz et al., 2012; Schultz, 1985). Because satellite cells have the unique

capability to create both new myonuclei and replenish their own population, when

satellite cell populations are depleted, muscle is unable to appropriately regenerate

itself (Boldrin et al., 2015; Fry et al., 2015). Satellite cells are biochemically

characterized by being positive for M-cadherin, Pax7, Myf5, and nCAM-1 (Péault et al.,

2007; Relaix et al., 2005). Upon fusion and activation with existing myofibers, embryonic

myosin is expressed, allowing researchers to identify growing and repairing myofibers

(DiMario et al., 1991; Murry et al., 1996). Further, the repaired fibers exhibit centrally

located nuclei, allowing for histological identification of fiber growth and repair.

Traditionally, the ‘exhaustion’ of satellite cells has been though to be through

mechanisms such as a loss of telomeres (Decary et al., 1997, 2000; Heslop et al., 2000;

Mouly et al., 2005; Renault et al., 2000; Sacco et al., 2010), but more recently, it has

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been shown that dystrophin helps organize the nucleic acid content within dividing

satellite cells. The organization of chromosomal alignment during division is lost in

dystrophic satellite cells, leading to an inability to adequately provide sufficient satellite

cell activity (Dumont et al., 2015). This dynamic and responsive process allows for

continual growth, repair, and maintenance of skeletal muscle. In diseases that affect

muscle, such as the muscular dystrophies, damaging insults to muscle are relentless,

eventually leading to an inability for the reparative processes to keep up with the

pathologic damage that occurs to the muscle, analogous to the red queen syndrome.

Structural Organization

At the most gross level of organization, skeletal muscles are bound by the

epimysium, a tight connective tissue sheath. Numerous fascicles exist within the

muscle, bound by the perimysium. Skeletal muscle is composed of numerous

multinucleated myofibers, which are organized in tightly bound bundles of individual

myofibers called fascicles. The distance between myonuclei are very regulated,

allowing for establishment of myonuclear domains (Allen et al., 1999). The active

contractile apparatus of myofibers are contained within myofibrils. Myofibrils are

interconnected by desmin, an intermediate filamentous protein that forms a three-

dimensional scaffolding around z-disks, connecting the entire contractile apparatus to

the subscarolemmal cytoskeleton. Regular repeating structural units, sarcomeres,

organize myofibrils. Sarcomeres are the basal contractile units of muscle, composed of

regularly arranged and overlapping repeating thin actin filaments and thick myosin

filaments. The interdigitated overlapping actin / myosin complexes slide past each other

during contractions, allowing for force generation within muscle. At the end of each

sarcomere is the z-disk, which structurally organizes filaments, and also gives striated

24

muscle its recognized striped appearance. Further, z-disks are tethered to the centers

of each sarcomere by titin, the largest known protein in humans (Labeit and Kolmerer,

1995).

Force Generation

The most recognized purpose of muscle is to generate force for movement. As

an action potential propagates along t-tubules into the interior of myofibers, voltage

gated calcium ion channels on the sarcoplasmic reticulum open, resulting in an increase

of Ca2+ concentration within the sarcoplasm (Berridge, 1993; Spudich and Watt, 1971).

During the non-contraction phase, tropomyosin blocks binding between actin and

myosin. Normally, globular troponin is bound to the tropomyosin and when Ca2+ binds to

Troponin C, the tropomyosin are moved. This exposes myosin binding sites on actin,

allowing myosin heads to form cross bridges with actin. From the previous cycle of

movement, ADP and Pi are attached to the myosin head. Upon binding of the myosin

heads to the actin, following removal of tropomyosin, the Pi is released. The release of

Pi causes triggers the ‘powerstroke,’ allowing actin myofilament to move past the

myosin, which releases ADP from the myosin head. The bond between the myosin

head and actin is broken when ATP binds to the myosin head. Hydrolysis of ATP to

ADP and Pi releases energy, which is used to recock the myosin head. If Ca2+ is

present, the entire series of event repeats.

Muscle Contraction

The three primary types of contraction to occur within muscle are: concentric,

isometric, and eccentric (Jones and Rutherford, 1987). Concentric contractions occur

when sarcomeres and muscle concurrently shorten together, as a load less than that of

maximum tetanic contraction is generated. Isometric exercises are those that allows for

25

activation of muscle while maintaining its length. The force generated during isometric

contractions are dependent on the length of muscle during contraction, which in turn is

determined by the amount of cross bridges formed in the contracting muscles.

Eccentric contractions occur when lengthening of the muscle occurs while

simultaneously contracting. As loads opposing the muscle increase, it reaches a point

where the external load is greater than the force that muscle can generate, causing

lengthening in the muscle. This has the potential to damage muscle by tearing the

sarcolemma. In healthy individuals with adequate repair capabilities, this is not a

problem, but in individuals with pre-existing muscle pathologies, they may be

inadequately able to repair damaged muscle (Weller et al., 1990). Eccentric loading is

an important concept of muscle that will be revisited throughout this dissertation.

Dystrophin Associated Glycoprotein Complex

Costameres, a sophisticated complex of proteins associated with cytoskeletal

proteins, link the contractile apparatus to the extracellular matrix (ECM). The dystrophin

associated glycoprotein (DAG) complex is an important costameric complex that

contains several vital proteins helping to stabilize the myofibers during contraction.

Mutations to any of the DAG complex proteins cause a variety of muscular dystrophies,

resulting from weakened sarcolemmal membranes, leading to increased susceptible to

damage and insult the myofibers. To ensure adequate distribution of stresses to the

muscle, the contractile apparatuses are linked to the ECM (Ervasti and Campbell, 1991,

1993; Ibraghimov-Beskrovnaya et al., 1992). The DAG complex is sophisticated

organization of proteins traversing the sarcolemmal membrane, whose primary purpose

is to provide stability and distribute transmission of intracellular contractile forces to the

ECM (Ervasti and Campbell, 1991, 1993; Ibraghimov-Beskrovnaya et al., 1992). By

26

distributing the stresses formed by the contractile apparatus of actin-myosin, the DAG

complex effectively minimizes focal stress at any single location of the sarcolemma,

broadly distributing the stresses over larger areas, which minimizes stress induced

damage to the sarcolemmal membrane. The complex is composed of a number of

proteins, including dystrophin, dystroglycans (α, β), sarcoglycans (α, β, γ, δ), sarcospan,

syntrophins (α1, β1, β2, γ1, δ1, and δ2), and α-dystrobrevin (Ibraghimov-Beskrovnaya

et al., 1992) as highlighted in Figure 1-1. Dystrophin binds to binds both to cytoplasmic

actin, as well as the transmembrane β-dystroglycan. β-dystroglycan additionally binds to

α-dystroglycan, which in turn binds to lamanin-2 of the extracellular matrix (ECM).

Thus, the DAG complex effectively transmits stresses generated by the actin-myosin

machinery within the myofibers to ECM, protecting muscle fibers from contraction

induced injury. Muscular dystrophies arise when structural or functional proteins of the

DAG complex are rendered less than optimally effective, leading to weakness and

vulnerability of the sarcolemmal membrane (Campbell, 1995; Ervasti et al., 1990; Laval

and Bushby, 2004).

Dystrophin

The discovery of the DMD gene on the X chromosome invigorated a new era of

DMD research, as it was the first gene identified using positional cloning, (Hoffman et

al., 1987; Koenig et al., 1988). The dystrophin gene, located at Xp21 on the human

chromosome, is the largest protein coding gene identified, spanning 2.5 million base

pairs and 79 exons, corresponding to 1.5% of the entire X-chromosome, and 0.1% of

the entire human genome. The DMD gene codes for the protein dystrophin, which

consists of 4 primary domains: an amino terminus that binds to actin, a rod domain, a

cysteine rich domain, and a carboxy terminus (Figure 1-2) (Ahn and Kunkel, 1993;

27

Koenig et al., 1988). Dystrophin binds to a number of subsarcolemmal components

including actin, anionic membrane lipids, Par-1B, nNOS, cholesterol, microtubules, β-

dystroglycan, syntrophin, and dystrobrevin (Figure 1-2). Several isoforms, the results of

splicing variants, exist in different tissues of the body, suggesting multiple roles of

dystrophin (Feener et al., 1989; Muntoni et al., 2003).

Muscular Dystrophies

Phenotypically, the muscular dystrophies are defined a common clinical

presentation of progressive, degenerative, and irreversible muscle weakness. With the

advent of modern sequencing technologies, over 50 different forms of muscular

dystrophy have been identified, based on the genetic mutation causing pathology to the

muscle (Amato and Griggs, 2011; Kang PB and Griggs RC, 2015).

Pathophysiologically, the muscular dystrophies result from perturbations to proteins that

maintain the integrity of the sarcolemmal membrane. While a common feature of the

muscular dystrophies is progressive muscle weakness and wasting, each muscular

dystrophy is uniquely individual, both genetically and phenotypically. Some forms of

muscular dystrophy are seen in infancy and childhood, while others do not present

symptoms until middle age or later. The different forms of muscular dystrophy also vary

in the distribution and extent of disease involvement throughout the body, rate of

progression, and pattern of inheritance. The muscular dystrophies include Duchenne

and Becker muscular dystrophy (DMD and BMD, respectively), the family of Limb Girdle

muscular dystrophies (LGMD), Congenital muscular dystrophy, Distal muscular

dystrophy, Emery-Dreifuss muscular dystrophy, Fascioscapulohumeral muscular

dystrophy, Oculopharyngeal muscular dystrophy, among others (Bönnemann et al.,

28

1996; Emery, 2002; Flanigan, 2012, 2014; Guglieri et al., 2008; Kang PB and Griggs

RC, 2015; Nigro and Piluso).

The most common of the muscular dystrophies is DMD, with an incidence of 1 in

5,000 live male births (Mah et al., 2014; Mendell et al., 2012). DMD is caused by a out

of frame causing mutation to the dystrophin gene, which normally encodes for the

dystrophin protein. Dystrophin, when functional, is a structural and metabolic protein

that connects the intracellular contractile actin to the DAG complex, stabilizing the

sarcolemmal membrane during muscle contractions (Ervasti and Campbell, 1991;

Hoffman et al., 1987). BMD is a less severe variant of DMD, also caused by mutations

to the dystrophin gene, but are in frame mutations, rather than out of frame mutations,

as in DMD, leading to partially truncated and semi-functional dystrophin protein (Koenig

et al., 1989; Monaco et al., 1988).

The limb girdle muscular dystrophies (LGMD) were originally identified by their

phenotypic presentation in the clinic, namely being muscular dystrophies that affect the

pelvic and shoulder girdle muscles (Guglieri et al., 2008; Laval and Bushby, 2004). To

date, there are at least 20 different subtypes of LGMD and it is a constantly evolving

area of research (Guglieri et al., 2008; Laval and Bushby, 2004). LGMD’s can be either

dominantly inherited (Type 1) or recessively inherited (Type 2). For interest of our work,

we focused on studying LGMD-2C, which is inherited through autosomal recessive

fashion, possessing mutations to the γ-sarcoglycan gene (McNally et al., 1996a;

Noguchi et al., 1995). γ-sarcoglycan is a protein of the DAG complex, which helps

stabilize the membrane during contractions, leading to membrane weakness when

dysfunctional (McNally et al., 1996a, 1996b).

29

Clinical Features

The common clinical presentation for the muscular dystrophies are irreversible

and progressive muscle weakness, but each muscular dystrophy has its own unique

phenotypic presentation. Ensuing discussion will focus primarily on DMD and LGMD-

2C, as those are the two muscular dystrophies that we focused on in our studies.

Males with DMD present with a characteristic and progressive muscle wasting

(Bushby et al., 2010a, 2010b). Even though individuals with DMD lack dystrophin from

birth, clinical symptoms of muscle weakness do not present immediately. By the ages of

3 to 5 years, males with DMD have noticeable mobility differences compared to their

peers, and pseudo-hypertrophy of the calf muscles can be observed. Simple activities,

such as running, jumping, and even walking can be difficult for young boys with DMD.

As the disease progresses, boys develop a characteristic ‘toe-walking’ gait. Due to

weakness of the gluteal muscles and abductor muscles of the lower limb, a

trendelenburg gait and lumbar lordosis become apparent. Upon rising up from the floor,

children with DMD exhibit a characteristic “Gower’s sign” (Tyler, 2003). Gower’s sign is

a clinical observation of when individuals roll into a prone-like position, then bring their

hands up their body to raise to a standing position, first pushing off the ground, then use

their hands to ‘walk’ up their knees, then hips, as they propel themselves to a standing

position. With aging, males continue to demonstrate progressive weakening, leading to

further complications. Boys typically lose ambulation by the teens. Cardiac and

respiratory complications inevitably occur, and these are the leading causes of mortality

in the DMD population (Gomez-Merino and Bach, 2002; McNally et al., 2015; Melacini

et al., 1996). Life expectancy for males with DMD used to be in the teens, but due to

symptomatic treatment to manage the disease, individuals are now living into the late

30

20s (Eagle et al., 2002). Because of the heterogeneity of mutations that individuals with

DMD may have, varying ranges of clinical phenotypes are present. Mutations to isoform

Dp71 have been observed to cause non-progressive cognitive impairment in addition to

the typical progressive muscle wasting findings (Moizard et al., 2000). The much less

severe dystrophinopathy, BMD, maintains the reading frame of dystrophin and presents

with similar symptoms, though significantly delayed (Monaco et al., 1988). Individuals

with BMD present much more gradually than DMD, and frequently live into mid to late

adulthood.

The LGMDs are a diverse group of muscular dystrophies, originally identified by

their clinical phenotypes prior to basic molecular and genetic capabilities. In general,

the LGMDs present with weakness in the hip and shoulder girdle muscles. The distal

muscles are usually spared, if affected at all. Additionally, cardiopulmonary

complications are typical in the LGMDs as well. Though less prevalent than BMD/DMD,

LGMD-2C is autosomally inherited, meaning that females and males are equally

affected. Clinically, patients with LGMD-2C present with a great amount of phenotypic

variability and diagnosis usually occurs through genetic testing (El Kerch et al., 2014;

Kefi et al., 2003). LGMD-2C patients present with a childhood onset of proximal to distal

weakness and usually lose ambulation in their mid-teens. Respiratory and cardiac

complications frequently arise in the 3rd decade of life, leading to early mortality.

Pathophysiology

Repeated cycles of degeneration and repair are the trademark of the

pathophysiological damage to myofibers in the muscular dystrophies. Pathological

insult to the myofibers is primarily due to increased susceptibility and vulnerability to

contraction induced injury, due to weakened sarcolemmal membranes (Ervasti and

31

Campbell, 1993; Petrof, 2002; Petrof et al., 1993). The function of the DAG complex is

to distribute contraction generated stresses throughout the sarcolemmal membrane,

which stabilizes and protects the sarcolemmal membrane. During contraction of

dystrophic muscle, the contractile component (actin) does not structurally anchor

properly to the sarcolemmal membrane and ECM, leading to tearing of the sarcolemma

(Ervasti and Campbell, 1991, 1993; Ibraghimov-Beskrovnaya et al., 1992). This

contraction induced tearing is easily repaired in healthy muscle, but due to the ease of

damage infliction into dystrophic muscle, repair mechanisms are unable to keep pace

with damage (Sacco et al., 2010). This leads to ongoing cycles of damage and repair.

This membrane fragility leads to inappropriate influx and efflux of compounds, such as

Ca2+ entering the cells and creatine kinase leaving the cells and being able to be

detected at elevated levels in circulation (Brancaccio et al., 2007; Turner et al., 1988;

Wrogemann and Pena, 1976). Increases in intracellular Ca2+ leads to activation of

various calpains and caspases, which ultimately lead to degeneration of affected

muscle fibers (Chargé and Rudnicki, 2004; Cohn and Campbell, 2000; Sandri et al.,

2001; Wadosky et al., 2011). Upon histological observation, signs of damage and

repair are simultaneously observed in muscle. Pockets of inflammatory cells can be

observed, as macrophages surround necrotic fibers (Acharyya et al., 2007; Tidball and

Wehling-Henricks, 2007). Though membrane permeability can be observed by

measures of dye uptake into muscle, it is difficult to observe this in vivo in real time

(Hamer et al., 2002; Palacio et al., 2002). Also, because of the heterogeneous

distribution of disease, biopsy samples may not be representative of the overall state of

damage to muscle. As the muscular dystrophies heterogeneously affect muscle

32

throughout the body, a pressing need for a non-invasive, real time, and in vivo modality

to assess the state of muscle health is apparent.

Preclinical Models of Muscular Dystrophies

Several preclinical models have been developed to help study disease

progression and therapeutic intervention in the muscular dystrophies. No genetic model

perfectly mimics the human phenotype of the respective diseases, each model with its

own advantages and disadvantages. Preclinical models have been critical in

developing efficacious therapies for the muscular dystrophies. The most frequently

utilized animals are murine models of disease. The animal models utilized in these

studies, as well as several important models are highlighted in the following section.

Invertebrates

Caenorhabiditis elegans (C. elegans) were the first multicellular organism to have

its entire genome sequenced (C. elegans Sequencing Consortium, 1998). Importantly,

approximately 60% of human genetic disorders are ably identifiable in C. elegans

(Shaye and Greenwald, 2011). Dys1, the C. elegan’s homolog of dystrophin, provides

movement through contractions of longitudinal striated muscles (Gieseler et al., 2000;

Grisoni et al., 2002). Advantages of C. elegans are that they are incredibly easy to

genetically modify, and due to a short life cycle, can be studied in a very time efficient

manner. This makes C. elegans ideal for preliminary first line testing of genetic

modifications and pharmacological therapies. Another invertebrate model that is

commonly used is the Drosophila melanogaster, or commonly known as the fruit fly.

Drosophila are a very common model used to study developmental and neurological

disorders, as humans and drosophila share many similar molecular, cellular, and

physiological traits in muscle. Further, approximately 75% of human diseases share

33

homologues in the fruit fly, including the muscular dystrophy related proteins (Greener

and Roberts, 2000). Similar to C. elegans, drosophila provides an elementary animal

model that serves as an early stage to test therapies.

Murine models

Mice are one of the most commonly studied type of laboratory animal because of

their inexpensive cost, ease of genetic modifiability, and relatively time efficient ability to

perform studies. They serve as the lowest form of vertebrates that are commonly

employed in research studies, allowing for rapid translation to clinical studies.

The mdx mouse is the most well recognized and thoroughly studied model of

DMD (Hoffman et al., 1987). These mice are aptly named ‘mdx’ because they are x-

linked muscular dystrophy mice (Bulfield et al., 1984). Phenotypically, mdx mice differ

from the human natural course of disease, though they are similarly missing the

dystrophin protein. Whereas humans with DMD experience premature mortality from the

disease, mdx mice have comparable life spans to unaffected counterparts. Their

muscle undergoes a unique progression of disease, with an initially high inflammatory

component (3-10 weeks), coupled with degeneration and necrosis, followed by eventual

recovery, until they experience precipitous decline in the final weeks of their lives (Lynch

et al., 2001; Vohra et al., 2015). Unlike humans, mdx mice have a unique ability to

highly upregulate utrophin, a dystrophin homolog in their muscle, which is hypothesized

to account for the stabilization and recovery of muscle after the initial bout of

inflammation (Blake et al., 2002; Tinsley et al., 1998). Additionally, a major difference

between the disease in mdx mice and humans is that the mdx mice lack fatty deposition

in their muscles. Several mutations have been induced to the dystrophin gene in mdx

mice, but the most commonly used variant are those with a spontaneous point mutation,

34

causing a premature stop codon mutation in exon 23 of the dystrophin gene, leading to

inappropriate premature termination of translation of the dystrophin protein in these

mice (Sicinski et al., 1989). Other variants include the mdx52 which also lack the

shorter dystrophin isoforms Dp140 and Dp160 and mdx2-5CV which entirely lack

revertant fibers (Araki et al., 1997). Mdx mice are traditionally bread on the C57/BL6

background, but additional studies have led to mdx mice bred with other strains, leading

to varying phenotypes (Fukada et al., 2010; Heydemann et al., 2005). One strain that

mdx mice have been crossed with is the dba strain, which have inherently higher basal

inflammatory states, which in turn, lead to accelerated pathology in these mice, as

compared to mdx mice on C57 backgrounds (Heydemann et al., 2005).

Double knock out (dko) mice lack both dystrophin and utrophin from birth

(Deconinck et al., 1997; Grady et al., 1997). They have an incredibly severe phenotype,

are physically stunted, with profound muscle weakness, severe lumbar-kyphosis,

cardiomyopathy, and exhibit early mortality compared to mdx and unaffected

counterparts (Deconinck et al., 1998). An explanation for the more severe phenotype

that these mice show is because of the concurrent lack of utrophin, which is conversely

upregulated in mdx mice. While this mouse strain more accurately reflects the clinical

phenotype that humans with DMD demonstrate (Willmann et al., 2009), it is a difficult

breed to work with though because of the severity of pathology and fragility of these

mice leads to unanticipated death during experimentation.

As previously elaborated, the LGMDs are a diverse group of muscular

dystrophies, clinically identified by their presentation of proximal to distal muscle

weakness, but more accurately, by the type of genetic mutations that they exhibit.

35

Mouse models of several LGMDs, lacking specific DAG complex proteins, have been

created to study the LGMDs. These include, but are not limited to the α-sarcoglycan

null (αsg -/-) (Duclos et al., 1998), γ-sarcoclygan null (gsg-/-) (McNally et al., 1996a;

Noguchi et al., 1995), and calveolin-3 null (Galbiati et al., 2001) models. Phenotypically,

these mice demonstrate similar pathologies to mdx mice, though their etiologies are

specific to the respective genes that are knocked out.

Rat models

While mice provide adequate models of DMD, they are not entirely

representative of the human version of the disease. Larger animals, as highlighted in

this chapter, provide a more representative model of the human disease, but are

expensive, time consuming, and difficult to handle. Considering the limitations of smaller

animals, the rat maybe a suitable species. Using TALENs targeting exon 23, dystrophin

deficient rats have been generated to solve this problem (Larcher et al., 2014; Le

Guiner et al., 2014). These rats exhibited no measurable levels of dystrophin,

accompanied with all of the characteristic skeletal muscle lesions that are observed in

humans, such as inflammation, lipid deposition, and fibrosis. Additionally, similar to

humans as well, the hearts of dystrophin deficient rats exhibited a dilated

cardiomyopathy pathology with significant functional defects like humans. However,

because working with rats is more costly and time consuming than the well established

mouse models, we decided to not work with rats further for our studies.

Porcine models

Moving up the animal hierarchy, an additional animal used as a model of DMD is

the pig. A dystrophin deficient porcine model has been identified (Hollinger et al., 2014;

Selsby et al., 2015). Skeletal muscles of these pigs demonstrate comparable pathology

36

to the human version of the disease, with abundant focal necrotic lesions.

Phenotypically, they exhibit impaired mobility and elevated serum creatine kinase

levels. However, death in these dystrophin deficient pigs is usually sudden, caused by

cardiac EKG abnormalities, which is not entirely representative of the human model.

For reasons similar to why we chose to not use the rat, we similarly chose to not

perform our investigations on the pig, as it is a quite costly and time consuming animal

model to work with.

Canine models

Several canine models of muscular dystrophy have been identified, such as the

golden retriever (GRMD), beagle, rottweiler, labrador retriever, Tibetan spaniel, German

shorthaired pointer, and cocker spaniel dogs (Allamand and Campbell, 2000; Cooper et

al., 1988; Sharp et al., 1992; Shimatsu et al., 2003; Valentine et al., 1988). The most

commonly studied of all of these is the GRMD model, which demonstrates a very similar

pathogenesis to the human form of DMD (Allamand and Campbell, 2000). GRMD dogs

have a similar progression of concurrent muscle degeneration and regeneration and

inflammation within muscles. This progresses to an abnormal gait, atrophy of muscles,

and ultimately premature mortality. A beagle model (CXMDJ) was developed via artificial

fertilization from a GRMD model (Shimatsu et al., 2003, 2005). The beagle offers a

comparable model to the GRMD model, but with added benefit of working with a smaller

animal. An additional benefit of the CXMDJ line is the reported cardiac involvement,

closely mimicking that of what is observed in humans (Hayashita-Kinoh et al., 2015;

Yugeta et al., 2006). A drawback to working with canines to study the muscular

dystrophies is that trials involving canines are expensive, tedious, and very time

consuming compared to working with mouse models. However, dogs do provide an

37

excellent and necessary translational model from mice to humans in studying muscular

dystrophy.

Therapies

Therapies for the muscular dystrophies can be broadly categorized into two

categories, symptom managing and curative. To date, treatments have remained

symptomatic for human patients affected by the muscular dystrophies, though exciting

and promising clinical trials are underway. Therapies can be further divided by

mechanism, including those that modify genetic information (either DNA or RNA), those

that enhance muscle mass, those that minimize global inflammation, and those that

modify other aspects to muscle health.

Genetic manipulation

Because most muscular dystrophies have been identified by mutations to single

genes, focused corrections of such mutations remain ideal therapies for these diseases.

Several genetic approaches have been investigated, including DNA manipulation and

RNA modification strategies. In DMD, the fundamental cause of disease is an absence

of dystrophin; therefore, the majority of therapies focus on allowing for restoration of the

dystrophin protein.

Restoring the missing DNA is the most direct candidate to replace the mutated

gene of interest. Direct injections of plasmids containing the full length dystrophin gene,

followed by electroporation to facilitate uptake, have demonstrated valuable proof of

concept findings (Bertoni et al., 2006; Ferrer et al., 2004; Gollins et al., 2003). A

concern for this technique is the elicitation of an immune response to the foreign full

length protein that was previously missing from the host, though in mdx mice, a minimal

immune response has been observed, perhaps due to the presence of revertant fibers

38

(Ferrer et al., 2004). While exciting, these results are likely not translatable to humans

due to the inability to systemically deliver the gene throughout the body with long term

expression (Wolff and Budker, 2005). Recently, the CRISPR/Cas9 system has been

developed as a modality of modifying DNA in living system in a specific and sensitive

manner (Cong et al., 2013; Wang et al., 2013). Preclinically, expression of human

dystrophin has been recorded in immunodeficient mice following CRISPR/Cas9 therapy

(Ousterout et al., 2015). In theory, this similarly would be able to be applied to any

patient affected by a genetic disorder, though much controversy exists over the ethics of

genetically modifying human genomes (Bosley et al., 2015).

AAV therapies have demonstrated limited success in DMD. Because the

dystrophin cDNA transcript is too large (14 kb) to fit inside of the AAV virus (which has a

capacity of 4.7 kb), several groups have proposed using truncated dystrophin, removing

non-essential portions of the cDNA, while retaining essential regions (Fabb et al., 2002),

or multiple trans-spliced vectors that ultimately contain the entire dystrophin gene (Lai et

al., 2005). This has been met with modest success in mouse models of muscular

dystrophy. In the LGMDs, our colleagues have demonstrated success in delivery of

human α-sarcoglycan in mice (Pacak et al., 2007). In LGMD-2C, AAV has also shown

restorative effects in by replacing the missing gamma-sarcoglycan with human gamma-

sarcoglycan (Cordier et al., 2000). Typically, to obtain muscle specificity, a

promoter/enhancer specific to muscle (muscle creatine kinase or desmin) are used.

Additionally, these findings are moving towards clinical trials, such as those for LGMD-

2A, which have demonstrated safe and effective vector delivery delivering the α-

sarcoglycan to human muscle (Mendell et al., 2009). Complications that may arise

39

when delivering the AAVs loaded with genes for foreign proteins include immune

tolerance issues and comprehensive delivery of the virus to all muscle (Yuasa et al.,

2002).

Manipulation of the RNA transcript has been another major target of a variety of

therapies. RNA modifying therapies can be broadly classed into either stop codon

readthrough, or exon splicing therapies. Nonsense mutations account for approximately

5-15% of all DMD mutations, caused by point mutations that result in premature stop

codons at inappropriate locations in the dystrophin gene (Aartsma-Rus et al., 2006).

Ultimately, this causes either no protein to be translated, or translation of unstable

protein that is quickly degraded, leading to an insufficiency of functional dystrophin

(Ohlendieck and Campbell, 1991). Stop codon suppression was originally synthesized

through the finding that aminoglycosides, specifically gentamicin, an antibacterial

therapeutic agent, caused translation of dystrophin in mdx mice that lacked it due to

their nonsense mutations (Barton-Davis et al., 1999; Wagner et al., 2001). In theory,

this should make a full size dystrophin protein upon complete translation. PTC

Therapeutics is further investigating these preclinical findings. Through intensive broad

drug screening studies, PTC Therapeutics discovered Ataluren (TranslarnaTM, PTC124),

which has shown potent read through capabilities while minimizing side effects (Finkel,

2010; Welch et al., 2007). Currently, Ataluren (TranslarnaTM) has received approval for

clinical distribution in the European Union, with requirements for follow up data to the

current Phase 3 trial (Ryan, 2014). Phase 3 clinical trials for ataluren are underway in

America, and suggest clinical benefit, but further data collection is still required (Barth et

al., 2013).

40

The other primary strategy of RNA modification is through exon skipping drugs.

The two exon skipping drugs that are currently in clinical trials for DMD are Eteplirsen

(Sarepta Therapeutics, Inc.) and Drisapersen (Prosensa / Biomarin) (Cirak et al., 2012;

van Deutekom et al., 2007; Goemans et al., 2011; Guncay and Yokota, 2015; Kinali et

al., 2009; Kole and Krieg, 2015; Mendell et al., 2013). Both drugs are modified

antisense oligonucleotides (AONs) that restore the translational reading frame by

splicing out the mutation contained regions of the dystrophin mRNA through splice site

steric hindrance. This allows for production of a truncated, yet mostly functional

dystrophin protein, shifting the more severe DMD phenotype to a less severe BMD

phenotype. It has been shown that exons 44-55 of the dystrophin gene are mutated at

higher frequency than other regions, suggesting that this is a high yield region to target

(Aartsma-Rus et al., 2004). Preclinical studies of these therapies have demonstrated

great efficacy in restoring dystrophin production, albeit truncated dystrophin, and

localization to the sarcolemmal surface. Optimization delivery of these drugs has

remained a challenge, but through the addition to co-polymers, (Kim et al., 2009; Nelson

et al., 2009; Rimessi et al., 2009; Sirsi et al., 2009), and through recombinant adeno-

associated virus (rAAV) vectors (van Deutekom et al., 2007), increased efficacy has

been observed. Through preclinical studies, it is estimated that less than 30% of

dystrophin protein restoration is needed in order to demonstrate clinical phenotypic

efficacy of these drugs (Heemskerk et al., 2007). Clinical trials however have

experienced limited success, with questionable demonstration of therapeutic efficacy by

ways of the primary outcome measure (6 minute walk test) (Hoffman, 2014; Mendell et

al., 2013). Phase 3 trials are still underway for both drugs in the United States.

41

Cocktails of multi-exon skipping regiments theoretically can target a greater distribution

of mutations, compared to single focused AON targets, which may help a greater

number of patients in the future (Aartsma-Rus et al., 2004).

Enhancing muscle growth

Ensuring that adequate amounts of muscle are present for one of the

aforementioned therapies remains a goal for the muscular dystrophies. Myostatin, a

member of the transforming growth factor-B (TGF-B) superfamily, is a negative

regulator of muscle growth (McPherron et al., 1997). Decreased activity and levels of

myostatin have demonstrated potent hyperplasia in skeletal muscle (McPherron and

Lee, 1997; Thomas et al., 2000; Williams, 2004). In the muscular dystrophies,

decreased activity of myostatin, either through knockdown or sequestering antibodies,

has demonstrated mitigation of disease burden (Bogdanovich et al., 2002; Nakatani et

al., 2008). Another approach that may enhance muscle growth is through the

upregulation of insulin-like growth factor 1 (IGF-1). IGF-1 has been shown to decrease

necrosis, increase muscle volume, and enhance regenerative capacity in mdx skeletal

muscle (Barton et al., 2002; Shavlakadze et al., 2004). One of the receptors for

myostatin, activin receptor type IIB (ActRIIB), is an additional target to mitigate disease

within muscle. In mdx mice, blocking ligand binding to ActRIIB has improved muscle

strength and function following a 12 week treatment of an inhibitor composed of the

extracellular portion of the ActRIIB fused to the Fc portion of murine IgG (Pistilli et al.,

2011). To be able to ensure adequate muscle is present to receive corrective therapies

would be a major benefit to clinical trials; therefore, these muscle growth promoting

treatments are frequently coupled with other therapies to synergistically enhance the

repair and growth of muscle in the muscular dystrophies (Hoogaars et al., 2012).

42

Minimizing inflammation

Dystrophic muscle exists in a constant state of elevated inflammation, leading to

perpetual insult and eventual degeneration (Porter et al., 2002). The only therapy that

has proven to prolong ambulation and delay the onset of symptoms in DMD remains

corticosteroids (Bushby et al., 2010a; Fenichel et al., 1991). While corticosteroids do not

treat the underlying genetic defect in the muscular dystrophies, they do manage the

symptoms and prolong clinical function in boys with DMD. The standard of care

recommendation in management of DMD is to prescribe prednisone (or deflazacort in

the European Union) to mitigate the symptoms and progression of disease (Angelini,

2007; Balaban et al., 2005; Fisher et al., 2005). However, despite the advantageous

prolongation of ambulation and slowing of damage to the heart, corticosteroids are not

without negative side effects, such as weight gain, weakened immune systems, and

weakened bones. Despite these negative side effects, corticosteroids are

recommended to give to children with DMD, starting at a young age.

Because of the vast side effects from traditional steroids, such as

immunodeficiency, hyperglycemia, fluid retention, osteoporosis, growth delay, and

delayed puberty, several novel investigations are attempting to promote the beneficial

results of steroids without the negative side effects. VBP15 is an orally administered

drug that protects and protects muscle through anti-inflammatory signaling and

membrane stabilizing pathways through the glucocorticoid receptor in mdx mice (Heier

et al., 2013). Importantly, it also demonstrates reduced hormonal transcriptional

activity, minimizing the negative side effects that traditional glucocorticoids cause (Heier

et al., 2013). Through modifications of the 21-aminosteroid compounds, VBP15 retain

NF-kB inhibitory properties without the negative glucocorticoid side effect profiles

43

(Reeves et al., 2013). Currently, a Phase 1/2 clinical trial in boys with DMD is

underway, investigating CAT-1004, the orally administered small molecule that targets

NF-kB, while minimizing the negative side effects of traditional glucocorticoids

(ClinicalTrials.gov Identifier: NCT02439216).

Another approach to minimize inflammation that is currently being investigated is

to target tumor necrosis factor alpha (TNF-α) with neutralizing antibodies to lower the

systemically elevated inflammatory state that exists in the muscular dystrophies. TNF-α

is a pro-inflammatory cytokine that is upregulated in a number of pathological states,

including DMD. Several therapies to lower TNF-α levels had demonstrated positive

effects on dystrophic muscle (Grounds and Torrisi, 2004; Hodgetts et al., 2006).

Other strategies

Many other non-specific approaches to mitigate the pathology induced by the

muscular dystrophies have been investigated, as highlighted in the following section.

Another therapy that has demonstrated positive findings is through the

restoration of nitric oxide (NO) to the vasculature of muscle. Neuronal nitric oxide

synthase (nNOS) has two binding domains to dystrophin and is the enzyme responsible

for generating NO in the endothelium of vasculature (Brenman et al., 1995; Chang et

al., 1996). In DMD, nNOS is mislocalized away from the subsarcolemmal surface

where it normally resides, leading to diminished NO levels in the vascular endothelium

that normally supplies appropriately moderated blood flow, ultimately leading to

functional ischemia (Lai et al., 2009). Substantial preclinical trials, and preliminary

clinical trials studying the restoration of NO have shown promising results (De

Arcangelis et al., 2015; Ennen et al., 2013; Martin et al., 2012; Nelson et al., 2014;

Thomas et al., 2012; Zhang et al., 2013). At the time of writing, Eli Lilly is in the midst of

44

a clinical trial, testing the ability of Tadalifil to slow the decline in ambulation in boys with

DMD (ClinicalTrials.gov identifier: NCT01865084).

Recently, protease inhibitors have demonstrated the ability to enhance delivery

of dystrophin, even with mutations, to the sarcolemmal surface in DMD (Hollinger et al.,

2014; Mázala et al., 2015). In DMD, dystrophin that is incomplete, or partially truncated

is typically quickly degraded prior to reaching the sarcolemmal surface by protease

enzymes, but through inhibition of these enzymes, delivery of some dystrophin to the

sarcolemmal surfaces was able to be accomplished, allowing for protection from the

natural progression of disease in muscle.

One central dogma explaining the irreversible nature of DMD is that the

regenerative capacity of satellite cells, the stem cells of skeletal muscle, eventually

exhausts and are no longer able to create new myofibers (Decary et al., 1997, 2000;

Heslop et al., 2000; Mouly et al., 2005; Renault et al., 2000; Sacco et al., 2010). The

mechanisms causing ‘exhaustion’ of satellite cells have recently been questioned, with

new suggestions that poor intracellular organization leads to an inability to self-

regenerate, rather than the traditionally believed shortening of telomeres (Dumont et al.,

2015). Several strategies have been developed to correct dystrophic tissue, either by

gene correct to indigenous satellite cells, or through delivery of exogenous healthy

satellite cells. The breadth of research in cell mediated restoration of muscle is beyond

the scope of this dissertation, but is briefly discussed below. Implantation of cells within

mdx muscle have suggested promise, though early clinical trials have suggested

otherwise (Skuk, 2004). Other studies have attempted to implant mesangioblasts into

dystrophic muscle within the GRMD dog (Galvez et al., 2006; Sampaolesi et al., 2006).

45

In all, cell therapy has been met with limited success to mitigate the muscular pathology

that the muscular dystrophies cause.

An additional approach to restore muscle health and function in DMD is through

the upregulation of utrophin, a homolog of dystrophin. Unlike the mdx mice, which

substantially upregulate utrophin, humans do not upregulate utrophin significantly

(Tinsley et al., 1998). However, utrophin is present in significant amounts at birth and in

the neuromuscular junction, suggesting that the body may not reject it as a foreign

material, as it already exists in the host body (Perkins and Davies, 2002). Though

initially promising, upregulation of utrophin has been met with limited success thus far

(Hirst et al., 2005).

Challenges of Therapeutic Trials

Following the initial discovery of dystrophin, a great deal of hope and excitement

filled researchers as a cure for DMD and the rest of the muscular dystrophies seemed

to loom in the near future (Hoffman et al., 1987). Unexpected obstacles have provided

many hurdles to overcome. The dystrophin gene is tremendously large, (2.4 Mb),

requiring creative approaches to packaging truncated dystrophin into vectors

(Athanasopoulos et al., 2004). Further, the diversity of mutations makes it difficult to

provide a single ‘cure-all’ genetic therapy, requiring different therapies for each specific

mutation (Bhattacharya et al., 2014). This in turn, creates frustration at the regulatory

levels, as each patient specific therapy is required to undergo the full regimented

reviews that are required from drugs – a difficult task to accomplish in a very small

number of affected subjects. Furthermore, DMD is a rare disease, occurring in

approximately one in 5,000 live male births, making patient recruitment and enrollment

a challenge to execute adequately powered studies. An additional challenge, specific to

46

clinical trials is that of inclusion and exclusion criteria. Adequate mobility and strength

are commonly required for individuals to participate in clinical trials. Because the

muscular dystrophies are degenerative in nature, this makes recruitment and enrollment

significantly challenging, as many individuals become ineligible and are unable to

participate in trials after they become non-ambulatory. Additionally, another major

challenge is to determine the optimal time to enroll subjects into clinical trials. This is a

difficulty because boys with DMD undergo relatively normal growth and development for

the first 5 years of their lives, at which their functional ability begins to plateau, followed

by precipitous decline (Bushby et al., 2010a). This is a challenge for several reasons.

First, the primary outcome measures used in clinical trials remain function and strength

based, so question remains on the optimal time to test boys with DMD. Secondly, with

disease progression, muscle is replaced by fatty tissue, and eventually scar tissue,

making make of these muscle specific acting therapies ineffective as less muscle is

available to receive therapy as individuals’ muscles deteriorate with aging. A major

challenge that is encountered in a variety of therapies are immunotolerance issues.

This is particularly a problem with AAV administration, as the body may ‘reject’ the

foreign mini/micro-dystrophin put into the body, or develop tolerance to the viral capsid,

making future therapies more difficult. Finally, because of the spectrum of interventions

and therapies that subjects are a part of, the definition of what a ‘control’ subject is

remains hard to define and natural history data is elusive to researchers. Clinical trials

remain difficult to design, execute, perform, and analyze for many reasons listed, and

because of this, have not progressed as quickly as the scientific community and public

have hoped (Aartsma-Rus et al., 2014; Ricotti et al., 2015).

47

A pressing issue faced in clinical trails is the development of adequate outcome

measures and biomarkers of disease progression or therapeutic regression. Logical

outcome measures to assess muscle health in the wake of disease and therapy are

biopsy samples (Anthony et al., 2014; Taylor et al., 2012). Tissue samples allow for a

variety of data to be collected, including protein quantification, immunohistochemistry, or

traditional histological techniques. Unfortunately, many of the muscular dystrophies

heterogeneously affect tissue throughout the body, meaning that samples taken from

one geographic location may not be representative of the remainder of the body

(Desguerre et al., 2009; Kinali et al., 2011). It has been shown that at least three biopsy

locations are required to obtain an accurate representation of muscle fiber type

distribution in human muscle (Lexell and Taylor, 1989). Muscle biopsies are inadequate

because they are invasive, provide a limited sampling of this heterogeneous disease, and

further damage already degenerative tissue. Additionally, in DMD, there are many

different types and locations of mutations to dystrophin, and antibodies that may bind

certain portions of the protein may not bind to certain truncated forms of dystrophin that

may still be present in BMD types of dystrophinopathies. Finally, the muscular

dystrophies are irreversibly progressive disorders, and taking a tissue sample of a

terminally optimal tissue is an emotionally and physically traumatic experience, so it is

not ethical to take continual biopsy samples from this population. The other primary

measure of therapeutic intervention is through functional and strength tests. Logically,

increases in strength and functional ability are what therapies strive to increase (Bushby

et al., 2010b; Davidson et al., 2014; Henricson et al., 2013a; McDonald et al., 2013).

Functional tests [six minute walk (6MWT), supine to stand, stair climb, ten meter walk/run,

48

etc.] possess inherent variables that remain difficult to control, such as subject’s motivation

and compliance. Currently, the 6MWT remains the only FDA approved primary outcome

measure for clinical therapies for DMD, but functional testing in general may lack the

sensitivity to detect subtle changes and impacts of such a therapy, as was observed in

recent clinical trials for DMD (Bushby et al., 2014; Flanigan et al., 2014; Mendell et al.,

2013). Additionally, the 6MWT is not appropriate for either very young DMD boys or the

non-ambulatory population. An ideal and robust methodology of assessing therapeutic

treatment must be: highly sensitive and specific to biologic changes, inexpensive, non-

invasive, inclusive of all subjects, provide minimal harmful radiation exposure, and be

comfortable for the patient.

49

Figure 1-1. The sarcolemma and dystrophin associated glycoprotein complex.

Figure 1-2. Binding sites and protein structure of dystrophin. Numbers refer to the

spectrin-like repeats throughout the protein. NTD, N terminal domain; CR, Cysteine Rich region; CTD, C terminal domain.

50

CHAPTER 2 NON-INVASIVE ASSESSMENTS OF MUSCLE HEALTH

Non-invasive imaging techniques to assess muscle health have been developed

as an alternative to more traditional invasive histological studies. Histology has been

considered the gold standard to study muscle for decades for legitimate reasons. It

allows for the most direct observation of tissue and can assess many quantitative

measures, such as protein or gene expression, or architectural integrity and structure of

regions of interest. An unfortunate necessity in utilizing histological outcome measures

is their terminal nature of utilization of the tissue, limiting the ability to perform

longitudinal studies. Furthermore, because pre-clinical histological endpoints are

terminal, greater numbers of animals are required to perform studies in order to perform

pseudo-longitudinal studies. Additionally, the muscular dystrophies involve muscle in a

heterogeneous fashion as previously described, so histological samples may only

provide a snapshot that is not truly representative of the whole state of disease. For

these reasons, great effort has been put forth to develop non-invasive, safe, repeatable,

sensitive, quantitative technologies to assess the health of muscle.

In our studies, the primary technologies that we utilized are magnetic resonance

imaging (MRI) and spectroscopy (MRS) and near infrared (NIR) optical imaging.

Measures obtained by these technologies have several advantages over traditional

histological assessments. Repeated acquisitions of data are able to be obtained,

allowing for longitudinal studies to be performed with fewer subjects. Additionally, data

over large spatial areas of the body are able to be collected, which is of tremendous

benefit in diseases that heterogeneously affect the body, allowing researchers to see

the whole distribution of disease. Thirdly, data collection occurs in a non-destructive

51

manner, a critical component when working with degenerative illnesses that do not

recover to formerly healthy states. Finally, subject involvement in the tests are

minimized, allowing for highly objective and quantifiable data to be obtained. At the

current time, the scientific community is entering a new epoch, moving away from

invasive, subjective, and non-repeatable biomarkers and outcome measures towards

non-invasive imaging technologies that allow for repeatable, quantitative, and sensitive

data collection from subjects, requiring minimal involvement from patients and subjects.

The following chapter discusses a number of different technologies and the advantages

and disadvantages of each.

Electrical Impedance Myography

Electrical impedance myography (EIM) is a non-invasive technique used to

assess muscle health, utilizing physiological bioimpedance properties of muscle to

detect perturbations in muscle during disease states (Rutkove, 2009). This technique

has been highly successful as a biomarker for several diseases, such as amyotrophic

lateral sclerosis (Esper et al., 2006; Rutkove et al., 2007, 2012), inflammatory

myopathies (Tarulli et al., 2005), radiculopathies (Rutkove et al., 2005), DMD (Li et al.,

2014; Rutkove and Darras, 2013; Rutkove et al., 2014; Shklyar et al., 2015), spinal

muscular atrophy (Rutkove et al., 2010), and congenital muscular dystrophies

(Schwartz et al., 2016). In simple terms, EIM models in vivo muscle as a basic

resistor/capacitor circuit, attributing resistance to extracellular and intracellular fluids,

and capacitance to the membranes within muscle. Whether or not muscles have

maintained sarcolemmal membranes, the measured reactance within muscle may be

different. Further, muscle with inflammation or edema will have different measured

signals as more or less extracellular or intracellular fluid may be present. Additional

52

modifiers of the EIM signal include atrophy and disorganization of muscle, as well as

lipid and fibrotic deposition – common to many of the muscular dystrophies. Another

key concept that EIM utilizes is the anisotropic nature of muscle, as they are normally

aligned in a parallel fashion during healthy states. Naturally, current that flows

orthogonal to fiber orientation experiences greater resistance, which can be quantified

through EIM.

Although EIM demonstrates many positive characteristics, several key limitations

do exist in utilizing EIM to assess the state of health. First, spatial resolution is limited

by the location and placement of electrodes. Additionally, subcutaneous fat and skin

may alter the received EIM signals, although this can be overcome through utilizing

multiple electrodes and multiple frequencies to assess fat and muscle. Overall, EIM is a

very exciting technology that offers great potential as a biomarker in various

neuromuscular disorders.

Ultrasound

Ultrasound is a safe, non-invasive imaging technology that utilizes high

frequency sound waves to tomographically detect contrast in tissues of interest.

Ultrasonic imaging utilizes transducers, which both send and receive high frequency

sound waves into and from the body. As receivers, they acquire the reflected signal off

tissue in the body, returning to the receiver at different times based on the tissue

composition that was imaged, creating contrast in the imaged region. Due to its ease of

use, inexpensiveness, and mobility, ultrasound is used for a variety of neuromuscular

disorders (Heckmatt et al., 1982; Pillen et al., 2008). In muscle, ultrasound has been

used to look at disruptions to normal architecture, orientation of fibers, fasciculation’s,

increases in edema and inflammation, as well as atrophy and hypertrophy (Heckmatt et

53

al., 1980; Jansen et al., 2012; Maurits et al., 2004; Pillen et al., 2006, 2009; Reimers et

al., 1996). Specifically in DMD, ultrasound has been used with accuracy to quantify

pathology within muscle, demonstrating the ability to differentiate the amount of

pathology and muscle from fat (Rutkove et al., 2014; Shklyar et al., 2015; Zaidman et

al., 2014, 2015). Comparisons between quantitative backscatter analysis and the gray

scale level values have correlated strongly with functional measures in boys with DMD,

demonstrating the ability of ultrasound to track disease progression and muscle

pathology.

While having many advantages, ultrasound does possess certain limitations

however, including limited penetration of signal (especially in obese patients), limited

spatial sensitivity, and lack of metabolic information. Though very easy to use, because

of these limitations, ultrasound is not considered the gold standard to assess and

quantify muscle pathology in the muscular dystrophies.

Elastography

Elastography is another medical imaging modality, specifically utilized to assess

elastic properties of muscle (Drakonaki et al., 2012). The most archaic form of

elastography is taught to health professionals in training as manual palpation.

Increased stiffness in tissues such as the thyroid, breast, and prostate raise suspicion of

cancerous pathology if felt. Certainly, more technical and quantitative methods of

measuring stiffness are preferred, and through technology advances, the field of

elastography has developed. Elastography is most commonly performed utilizing

magnetic resonance or ultrasound, as described below.

Elastography can similarly be performed using ultrasound, based on the general

principle that stress applied to tissue causes unique deformations to it, dependent on

54

the intrinsic elastic properties of the tissue. Diseases to the musculoskeletal system

alter the biomechanical properties of tissue, creating measurable differences between

healthy and pathologic states of tissue. Several types of ultrasound elastography are

available for use, unique in the type of stress application, detection of tissue

displacement, acquisition of data, and reconstruction of images. The most commonly

applied method of performing MRE is the sonoelasticity method, developed by Parker et

al (Lerner et al., 1990; Parker et al., 1990). In this technique, a vibrational mechanical

stress is applied low frequency ultrasound waves are applied to compress tissue,

usually applied by handheld ultrasound transducers, followed by reception of signals

through the same probes. In principle, the compressive force causes displacement that

can be calculated through comparing data before and after the compressive pulse

sequences are applied.

In the musculoskeletal system, ultrasound elastography is particularly useful

when studying tendons and muscle. The Achilles tendon is the most well studied

tendon, and abnormally stiff and soft tendons have been able to be identified,

suggesting increased susceptibility to injury in abnormal tendons (Yamamoto et al.,

2016). In muscle, inflammatory myositis has been able to be assessed, demonstrating

increased stiffness due to fibrosis, and decreased stiffness as a result of fatty infiltration

(Botar-Jid et al., 2010). In congenital muscular dystrophy, ultrasound elastography

demonstrated strong correlation between traditional ultrasound and MRI findings

(Drakonaki and Allen, 2010). In an interesting study in cerebral palsy, ultrasound

elastography was used to identify the stiffest regions of muscle that would benefit

greatest from botulism toxin injection therapy (Vasilescu et al., 2010).

55

Like the other imaging modalities discussed, ultrasound elastography has both

advantages and disadvantages. It is very inexpensive, fast, and non-invasive, with a

broad range of potential applications. However, applications to humans thus far have

been limited to the research developmental stage, and application for clinical uses is still

limited. Technical difficulties include a lack of quantification methods, inter-user

variability, and limited reproducibility.

Magnetic resonance elastography (MRE), similar to ultrasound elastography,

measures changes in strain in tissue, though through different mechanisms. Following

mechanical application of strain, MRE can directly visualize and quantitatively measure

propagating acoustic strain waves within tissue (Dresner et al., 2001; Manduca et al.,

2001, 2001; Muthupillai and Ehman, 1996; Muthupillai et al., 1995, 1996). In principle,

three steps are required to perform MRE (Mariappan et al., 2010). First, shear waves

must be generated within tissue. Second, MR images are acquired, depicting the

propagation of the induced shear waves. Thirdly, images of the shear waves must be

processed to generate quantitative maps of tissue stiffness. Phase contrast MRI

techniques are utilized to spatially map and measure shear wave displacement

patterns. Shear waves are applied from the surface of the area of interest at the same

frequency of the motion sensitizing gradients, causing a measurable phase shift in the

received gradients. This makes it possible to spatially ‘tag’ tissue directly, allowing for

displacement calculations, and thus, strain measures. This allows for images to be

composed, and localized shear moduli to be calculated, demonstrating the spatial

differences in elastic properties within tissue. Currently, the primary use for MRE in the

clinic is to assess liver diseases, and is an adequate non-invasive alternative to

56

biopsies. For research purposes, elastic properties of muscle has been well

characterized as changes in stiffness are a well characterized result of disease in

muscle (Dresner et al., 2001; Ringleb et al., 2007; Sack et al., 2002). Upon

comparison of healthy subjects to those with a variety of neuromuscular disorders,

differences in stiffness of muscle were able to be recorded via MRE (Basford et al.,

2002). To date, MRE in muscle is still strictly a research modality in muscle, but cans

still provide us with valuable information.

Similar to the other technologies, MRE has both advantages and disadvantages.

An advantage of MRE is that a high resolution spatial map is able to be composed,

encompassing entire organs. Recent advances in technology have significantly cut

down on the time that it takes to perform MRE. In comparison to ultrasound

elastography, MRE reigns superior in depth of penetration of signal, as ultrasound

encounters difficulties penetrating tissue in obese patients. A drawback to using MRE is

that it is still relatively unexplored in muscle, and that it is not yet ready for clinical

application.

Computed Tomography

Computed Tomography (CT) is another non-invasive imaging technology that

has been used to assess muscle in neuromuscular disorders. CT utilizes ionizing

radiation to create images of the body, based on the tissue’s intrinsic ability to block and

reflect the X-ray beam. CT has much higher resolution than ultrasound, and can

distinguish between muscle, bone, tendons, ligaments, and fluid with good sensitivity.

CT has previously been used to assess the state of muscle pathology in DMD, but few

studies have been performed due to inherent risks of CT (Jones et al., 1983; King et al.,

2005; Liu et al., 1993b; Stern et al., 1984). Because CT utilizes ionizing radiation, it is

57

contraindicated to use in pediatric populations, and therefore, not frequently used to

study muscular dystrophies.

Positron Emission Tomography

Positron emission tomography (PET) is a form of imaging that produces high

resolution three dimensional images of functional processes within the body. Following

administration of a positron emitting radionucleotide contrast agent, PET systems detect

gamma rays emitted from the metabolized positron emitting substrate. A brief waiting

period is required for the radioactive tracer isotopes to be metabolized in the desired

tissue of interest. As the radioisotope undergoes beta decay, it emits a positron. When

a positron and electron collide, gamma photons are created and travel in opposite

directions. The pair of gamma photos are detected by a photomultiplier camera on the

PET scanner, are able to be spatially located, and image reconstruction is able to be

performed. PET imaging is most frequently utilized in the oncology field, where cancer

tumors demonstrate erroneous metabolism, which is able to be measured and identified

by PET imaging. In muscle, PET imaging is infrequently utilized. In one study, using

15O labeled water, muscle blood flow was accurately able to be measured (Ruotsalainen

et al., 1997). In another study, the commonly used 18F-deoxyglucose measured the

exercise tolerance of skeletal muscle following either rosiglitazone or metformin

treatment (Hallsten et al., 2002). Overall, there are more technically simple methods of

assessment to assess the metabolic states of muscle; therefore, PET imaging is not

very widely used in muscle. An advantage to PET imaging is that it can detect deeper

laying tissue groups. Similar to CT imaging through, a primary disadvantage is that

PET imaging requires the use of ionizing radiation, limiting its use in pediatric

populations.

58

Magnetic Resonance Imaging and Spectroscopy

Changes in muscle health, assessed by magnetic resonance imaging (MRI) and

spectroscopy (MRS) are fundamental portions of the work discussed in this

dissertation. MRI has the capability to produce high quality three dimensional images,

with excellent contrast of soft tissues as well as high spatial resolution in real time within

living animals. MRS provides high spectral resolution, revealing insight into metabolic

processes and biochemical composition within muscle. Because of the breadth of

quantitative information that MR provides in a longitudinal fashion with minimal risk, it is

frequently used to assess natural disease progression and therapeutic intervention in

the muscular dystrophies (Hollingsworth, 2014; Mercuri et al., 2007). Because MR is a

fundamental technique employed throughout this research, a more in depth description

will be provided to better understand the remainder of the work.

Basics of Nuclear Magnetic Resonance

Several fundamental components are required to perform MR experiments,

including a static magnetic field (B0), a radio frequency (RF) coil used to excite spins

and receive signal, electromagnetic gradient coils used for spatial encoding of signal,

RF amplifiers used to receive, amplify, and transmit the signal, and a computer station

used to manage the system. The static magnetic field strength (B0) is measured by the

magnetic field strength unit Tesla (T), which is equivalent to 10,000 Gauss (G). As

comparison, the atmosphere on Earth has a magnetic field strength of approximately

0.5 G. Pre-clinical research magnets operate between 0.1 - 21.1T and human magnets

have static field strengths between 0.1T and 11.1T. Currently, clinical MR scanners

greater than 4T are required to have an investigational device exemption (IDE), and

most clinical MRI scanners operate at 2T or less. Working at higher field strengths allow

59

for larger induced nuclear polarization, which in turn allows for larger signal strength. In

this work, all animal experiments were performed at 4.7T, and human experiments at

3.0T.

Origin of Magnetization

A fundamental principle of nuclear magnetic resonance (NMR) is that atoms

containing an odd number of protons and/or neutrons possess intrinsic angular

momentum (ρ). This leads the atoms to having a non-zero spin, which is able to be

manipulated by external magnetic fields. In turn, this allows for measurements of time

dependent signals as they return to equilibrium following the external magnetic

manipulation. Though the work presented here uses 1H nuclei as the signal source,

other elements can also be used, such as 13C, 15N, 17O, 19F, 23Na, and 31P.

Simplistically stated, the axis of rotation of nuclear spins aligns either parallel or

anti-parallel to the static magnetic field. Equation 2-1, the Boltzmann equation, helps

describe the relative distribution of spins in both orientations.

𝑁−

𝑁+⁄ = 𝑒−𝛥𝐸 𝑘𝑇⁄ (2-1)

where N+ spins in the lower quantum energy state are more frequent than N- in the

higher energy state, ΔE is the energy difference between spin states, k is the Boltzmann

constant (1.3805 x 10-23 J/K), and T is temperature. Because the relative distribution of

spins in parallel and anti-parallel directions are very close, there is a small net surplus of

lower energy parallel (N-) spins aligned in the z-axis, creating a magnetic moment (µ).

The difference in energy (ΔE) is equal to twice the product of the field strength (B) and

magnetic moment (µ) of the nuclei.

ΔE = μB (2-2)

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When fully relaxed, this net magnetization vector (on the arbitrarily designated z-axis) is

referred to as MZ.

Spin and Precession Frequency

Equation 2-3, the Larmor equation describes the nuclear spin frequency about an

axis.

𝜔 = 𝛾𝐵 (2-3)

where ω is the precession frequency (MHz), γ is the gyromagnetic ratio the atomic

nuclei (MHz/T), and B0 is the magnetic field (T). For the purpose of our work, we

exclusively work with hydrogen nucleus, which has a gyromagnetic ratio of 42.576

MHz/T. Because all of our preclinical studies were performed on a 4.7T scanner, the

calculated precessional frequency of the 1H nucleus is 200.107 MHz around the z-axis.

Manipulation of Signal

To generate a measurable signal, a second magnetic field (B1) is generated in

the transverse (X-Y) plane, perpendicular to the B0 z-axis of precession. To accomplish

this, an RF pulse at the resonant frequency, transmitted orthogonally to the z-axis is

applied, tipping the net magnetization vector into the X-Y plane. The nuclei continue to

precess as they are tipped out of the z-axis and into the X-Y plane. Magnetization

signal acquisition only occurs in the X-Y plane and is referred to as MXY. Immediately

following the 90° pulse, the MXY is greatest, as MZ is approximately zero because of the

90° pulse. As MXY begins to freely precess around the z-axis, an electromagnetic field

(EMF) induces a current in an RF receiver coil. With time, the signal dampens, resulting

in an exponentially decaying sinusoidal free induction decay (FID), containing frequency

and amplitude information of the signal, making MRI and MRS possible, as shown by

the initial FID in Figure 2-1.

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Measurable Parameters of MR

Two primary measurable phenomena result following the initial 90° pulse: T1

(longitudinal) relaxation (Figure 2-2), and T2 (transverse) relaxation (Figure 2-3), and are

described in the following section.

Longitudinal relaxation (T1) and pulse repetition time (TR)

Immediately following the 90° pulse, the MZ is approximately zero and the

magnetization is entirely in the transverse plane. With time, the net magnetization

returns to equilibrium in alignment with the B0 longitudinal axis, until MZ is equal to M0.

The longitudinal relaxation time (T1) is the time constant that describes this return to

equilibrium of MZ, as quantitatively described in Equation 2-4.

𝑀𝑧(𝑡) = 𝑀0(1 − 𝑒−𝑡

𝑇1) (2-4)

where Mz is the longitudinal magnetization component, t is time, M0 is the initial

magnetization, and T1 is the exponential time constant required to recover 63% of

equilibrium. Through using several techniques, two of which are highlighted in the

upcoming paragraph, and Equation 2-4, a recovery curve can be generated. The T1 time

constant is also referred to as spin-lattice relaxation, in reference to the time it takes to

transfer energy from the spins to the lattice environment as it relaxes to lower energy

states. With this understanding, a general rule is that more solid like tissues have

shorter T1 times than fluid like samples.

Two fundamental techniques to measure T1 are inversion recovery and

progressive saturation. Advantages of doing T1 weighted imaging are several fold.

First, certain tissue can be “suppressed” based on its T1 values, frequently seen in fat-

suppression type imaging. Additionally, based on the discrimination of T1 relaxation

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times, strong contrast can be generated, to better distinguish tissue types, as shown in

Figure 2-2.

Inversion recovery is a technique that utilizes traditional spin-echo sequences

(Figure 2-1, described in greater depth the next section), preceded by a 180° inverting

pulse. The inversion time (TI) is the time between the initial 180° inverting pulse and

the 90° pulse. The purpose of this initial pulse is to flip the initial magnetization (M0) into

the opposite direction of the static B0 field. During the TI interval, these tissues undergo

longitudinal relaxation, as they return to their basal magnetization along the z-axis. At

the initiation of the spin echo sequence (beginning with the 90° pulse), tissues can be

differentiated by their different intrinsic T1 relaxation times. By varying TI, image

contrast can be enhanced and certain tissues’ signals can be suppressed. In the

muscular dystrophies, contributions from lipid signals are frequently suppressed to

highlight anatomical differences. Another important feature that benefits using inversion

recovery is because the initial 180° flips all spins opposite of the B0, field there is twice

the dynamic range for distinguishing tissue types as they must return to M0 from –M0

rather than from 0 to M0. However, despite all of this, there are several disadvantages

to using inversion recovery, such as increased scan times, increased flow related

artifacts, and diminished signal to noise as tissues are suppressed.

Another fundamental T1 technique frequently utilized is called progressive

saturation. In progressive saturation experiments, the data is acquired at multiple

acquisition times throughout the acquisition of data, allowing for varied Mz amplitudes at

each respective TR acquisition to be obtained (Figure 2-5). Eventually, at very long TR

times, the Mz returns to its baseline value of M0. From these data acquired, one is able

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to obtain a T1 relaxation curve, from the varied acquisition times utilized, with each

respective Mz. An advantage of using progressive saturation is that NMR spectra can

be acquired much quicker than conventional studies. In conventional relaxation

experiments, sufficient time is allowed for the spins to fully recover (usually five or more

times TR), but in progressive saturation, all of the data is acquired within a single

recovery of signal, making these scans much quicker. In progressive saturation

experiments, one does not wait until the magnetization has fully recovered before re-

running the excitation and data acquisition sequences. Because of this, each data

capture allows for creation of a recovery profile as a function of multiple TRs.

An important consideration regarding T1 is the strength of the B0 magnetic field.

The practical significance of higher magnetic fields is that tissues have longer T1 times,

and that greater time intervals are required between data collection to allow MZ to

adequately return to equilibrium. This allows the next sequence of data samples to start

at the same initial magnetization, allowing MZ = M0. The time interval between the

application of two 90° RF pulses is called the repetition time (TR) (Figure 2-1).

Typically, to ensure full relaxation to baseline conditions, the TR exceeds the T1 of

samples approximately five to six times to allow for sufficient recovery of MZ to the B0

axis. If T1 > TR, then the MZ will not relax entirely to M0 and subsequent MXY values will

have less magnitude than former signal acquisitions. In these cases, signal (MXY) will

be reduced because the TR is too short to completely longitudinally relax the signal, and

is then said to be T1 weighted.

Transverse relaxation (T2) and echo time (TE)

To understand T2 relaxation (Figure 2-3), it is appropriate to utilize the spin echo

sequence (Figure 2-1) to understand how to calculate T2. Immediately after the 90°

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pulse, a second measurable phenomena, an exponential decay of the MXY signal

occurs. As soon as the 90° pulse is turned off, the transverse magnetization is at its

maximum. In most situations, the time constant T2* (described later) contributes to the

decay of transverse magnetization greater than T2, causing significant signal loss. This

can be corrected allowing for measurement of T2 by utilizing an echo of the FID. To

accomplish this, a 180° pulse in the transverse plane is applied, ‘flipping’ and refocusing

the FID. The sequence run using a 180° pulse to refocus the FID is called a spin-echo

sequence (Figure 2-1). The period allotted to refocus the FID echo is defined as the

echo time (TE).

In the most elementary of spin-echo experiments, the TE is equal to twice the

time interval between the initial 90° pulse and the refocusing 180° pulse (Figure 2-1).

Multiple sequences run with variable TEs allow for several points along a decay curve to

be obtained, allowing for calculation of T2 using Equation 2-5.

𝑀𝑥𝑦(𝑡) = 𝑀0𝑒−𝑡

𝑇2 (2-5)

The decay of the transverse magnetization is due to spin-spin interactions,

gradually decaying the net magnetization signal in the transverse plane. For this

reason, T2 relaxation is frequently referred to as spin-spin decay or transverse

relaxation.

There are two primary contributors to loss of the MXY: spin-spin interactions, as

previously mentioned, and local magnetic field inhomogeneties that cause acceleration

of the FID decay quicker than the theoretical T2. Recalling Equation 2-3, the precession

frequency is directly proportional to the external magnetic field, suggesting that local

magnetic field inhomogeneties may accelerate or slow down precession, leading to

65

dephasing of local nuclei, and a more rapidly decaying FID. This observed T2 is referred

to as T2* (“T2 star”).

Building upon the fundamental spin-echo sequence, additional sequences that

are frequently utilized include the Carr-Purcell (CP) and Carr-Purcell-Meiboom-Gill

(CMPG) pulse sequences, as described below. Similar to elementary SE sequences,

the CP sequence uses a 90° pulse to initially tip magnetization into the transverse

plane, but rather than using a single 180° pulse to flip the spins, a train of evenly spaced

180° RF pulses are applied along the same axis (Figure 2-6). This in turn causes for

the generation of echoes with alternating magnitudes (e.g., the first negative, second

positive, third negative, etc..). Each echo generated has a measurable amplitude, and

each subsequent echo has a smaller amplitude compared to former echoes. The decay

in echo amplitude is measurable, and reflects the T2 of the samples. Importantly, the

alternating series of 180° pulses are used to prevent the buildup of phase accumulation

during the signal decay. The CMPG pulse sequence is a modification of the CP

technique. While the CP pulse sequence applies its train of 180° pulses along the static

x-axis, the CPMG pulse sequence applies the 180° RF pulses along the y-axis of the

rotating frame (Figure 2-7). In the CP sequence, the 180° pulses given along the same

axis, leading to non-exact 180° pulses given and reduced transverse magnetization

after each subsequent 180° pulse. The Meiboom-Gill improvement upon the CP pulse

is to provide the 180° pulses along the rotating frame axis following the initial 90° RF

pulse, leading to a mitigation of lost transverse magnetization after each 180° pulse.

Additional information that is revealed from the CPMG pulse sequence is that the T2 *

can also be revealed in addition to the T2 of the sample. Also, because the 180° pulses

66

are in the rotating frame, the magnitude of each echo is positive, rather than alternating

as in the CP pulse sequences. The initial decay of signal following the 90° pulse is due

to inhomogeneties of the magnetic field (i.e., the B0 field) and allows us to calculate T2 *.

In CPMG sequences however, this is reversible through the 180° pulses in the rotating

frame. T2 relaxation on the other hand, is irreversible as each echoes amplitude decays

with time.

Image formation

MR data collected contains all of the information necessary to create images,

through the ability to decompress spatially unique MR signals. This is accomplished

through using gradients. Prior to data acquisition, inhomogeneties within the B0

magnetic field are corrected. Due to many factors, such as machine error in making

coils, shifting of the wires from magnetic or passive forces, magnetic impurities within

the wires, mechanical stresses from transporting and installing the magnets, or any

additional number of disturbances, the B0 field is inherently inhomogeneous. This is

corrected through measuring and observing the B0 field, applying small currents to coils

that are slightly larger than the primary coils to appropriately modify the field to attempt

to make the B0 field as homogenous as possible, which is called shimming. Along

orthogonal axes, spatially controlled local magnetic fields are created using gradient

coils, which create a gradient of magnetic fields to spatially encode spins according to

their frequency and phase, depending on their location in the local magnetic field.

Through further manipulation of applied and received signals, we are able to spatially

reconstruct images based on principles outlined in the following section.

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Slice selection

Slice selection is performed to isolate a single plane within a region of interest, by

exciting only the spins in that plane or a range of frequencies. The slice selection

gradient can be implemented in any directional gradient. To accomplish slice selection,

a perpendicular RF pulse is applied simultaneously to a linear field gradient along the

direction of axis of the object. The result is selective excitation of spins whose Larmor

frequency matches that of the RF pulse. Importantly, spins that were not excited remain

in the z axis and are not measured. The slice thickness is determined by the

bandwidth of the gradient applied, determining the range of frequencies that are excited.

Though signal is obtained from the entire slice, the image is not yet able to be formed

as additional information is required. In order to gather the additional dimensions of

information, phase and frequency encoding are performed.

Phase and frequency encoding

An additional technique used to spatially encode MR signal is performed by

encoding phase in a spatially dependent manner. Following slice selection, an

orthogonal gradient is applied, causing spins to precess at frequencies dependent on

their position along that axis. The rate and magnitude of precession are spatially

dependent, giving information about their position in a single axis of the slice selected.

The last part of data acquisition uses the read gradient. The read gradient is

turned on as data is acquired, spatially encoding the desired axis with varied

frequencies. The ultimate result of using phase and frequency encoding is that signal of

all locations are able to be spatially identified by their unique phase and frequency in

data space. This data exists in a time domain called k-space. K-space data is then

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Fourier transformed, transforming data from the time to spatial domain, allowing for

processing of the information as a recognizable MR image.

MR contrast

Contrast can be obtained through several different manners. T1 and T2 contrast

can be generated by varying the parameters of different pulse sequences. For

instance, T1 contrast can be generated through using shorter or longer TR times. With

shorter TR times, magnetization does not fully recover to the initial M0, and tissues with

shorter T1 relaxation appear brighter as they are more recovered. Because of this,

tissues with long T1 times, such as edema, appear darker than those with shorter T1

times, such as lipid (Figure 2-4). Similar to T1 weighting, T2 weighting can also be

utilized to generate contrast based on tissues inherent magnetization properties.

Biological samples with high fluid content or high lipid density tend to increase T2

relaxation times due to their high proton density. This causes those areas to appear

brighter compared to surrounding areas. Solid tissues, such as fibrotic tissue, bone,

and muscle, conversely have shorter T2 relaxation times, and appear darker as their

signal has already relaxed and is lost quicker from the transverse plane (Figure 2-3).

MR contrast agents are substances that increase visibility of target structures by

increasing contrast in tissue. Broadly stated, MR contrast agents work by modifying

local magnetic fields, altering the T1 and T2 relaxation times, creating increased contrast

in target tissues. An important concept to consider is that all MR contrast agents do not

penetrate all tissue equally, leading to spatial segregation of contrast, further enhancing

contrast. Several primary fluid compartments that contrast agents can be contained

within include the intracellular compartment, the interstitial compartment, and the

intravascular compartment. The intracellular compartment contains all fluid that is

69

contained within cells, and contrast agents that preferentially accumulate within cells

include certain magnetic nanoparticles, manganese derivatives and ultrasmall

paramagnetic iron oxide particles (Mornet et al., 2004; Pankhurst et al., 2003; Wilhelm

et al., 2003). Next, the interstitial compartment, frequently described as tissue space

that surrounds cells, is the immediate microenvironment that allows nutrients, ions, and

other particles across the cell barrier. Lymphatic vasculature, important to many

oncologic processes, is considered to be interstitial space. MR contrast agents that are

contained within the interstitial compartment include superparamagnetic nanoparticles,

Gd-DPTA conjugates, gadofluorine 8 (Schering AG, Berlin, Germany), and gadorterate

meglumine (Donahue et al., 1994; Harisinghani et al., 2003; Misselwitz et al., 1999;

Ruehm et al., 2001). Lastly, the intravascular compartment is described as the space

that blood naturally exists within. Contrast agents that remain in the intravascular

include free Gd-DPTA, albumin conjugated to Gd-DPTA, and other gadolinium based

agents (Flacke et al., 2001; Ogan et al., 1987; Weinmann et al., 2003). Overall, the

most commonly used contrast agents are gadolinium (Gd) based products, but several

other types include iron oxide, iron platinum, manganese, and biologically based

contrast agents (Caravan et al., 1999; Gupta and Gupta, 2005). Importantly, there are

no blood pooling MR contrast agents that are approved by the Food and Drug

Administration for use in pediatric populations.

Gadolinium based contrast agents

Gd based contrast agents the most commonly used MR contrast agents, and

work by facilitating shortening both T1 and T2 relaxation times in the tissues that it is

accumulated. At lower concentrations, T1 shortening effects are dominant in Gd, but at

higher concentrations, T2 weighting effects begin to be observed (Bleicher and Kanal,

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2008). Common applications of Gd T1 enhanced imaging is often seen through studies

interested in perturbations in vasculature, such as stroke (Chauveau et al., 2010; Saleh

et al., 2004) or cancers (Knopp et al., 1999; Padhani et al., 2000). Frequently,

gadolinium based contrast agents are utilized to assess neurological pathologies, such

as brain tumors (Brada et al., 2001; Grosu et al., 2005; Nelson et al., 1999; Warnke et

al., 1995) or multiple sclerosis (Barkhof et al., 1997; Beck et al., 2002; Kappos et al.,

2010; van Oosten et al., 1996; Polman et al., 2006).

Iron oxide based contrast agents

Another type of MR contrast agent are iron oxide based agents. Iron oxide is

frequently conjugated with a biocompatible medium, such as dextran or carboxydextran,

to increase delivery and efficacy of contrast (Gupta and Gupta, 2005; Laurent et al.,

2008; Sun et al., 2008). Iron oxide based contrast agents function by reducing T2*

relaxation times in tissue with close proximity to the iron oxide. Clinically, one of the

more common iron oxide based contrast imaging purposes has been to detect cancer

metastases (Harisinghani et al., 2003).

Other contrast agents

Other modalities have been employed to increase MR contrast. Reporter genes,

such as ferritin or arginine kinase, have been able to provide endogenous contrast of

tissue iron. (Bengtsson et al., 2010; Forbes et al., 2014a; Ziv et al., 2010). Manganese

is another element that has been used for MR contrast, specifically to enhance T1

contrast. Manganese ions (Mn2+) demonstrate similar properties to Ca2+ and are able to

be enter through the same calcium channels, thus can serve as a proxy to measure

changes in calcium flux (Liu et al., 2004).

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Magnetic Resonance Spectroscopy

Magnetic resonance spectroscopy (MRS) or nuclear magnetic resonance (NMR)

spectroscopy, uses many similar principles to MRI, providing information regarding the

chemical makeup, rather than spatial information as in MRI. Whereas contributions to

MRI-T2 signal can be from anything in the region of interest, (such as T2 shortening

fibrotic tissue, or T2 lengthening lipid-infiltrated or edematous tissue), spectroscopy

specifically analyzes a particular molecule (e.g. 1H, 13C, or 31P) to determine the specific

spectral makeup of that nuclei. Frequently, spectroscopy can be used to identify the

chemical makeup of a sample, due to the unique chemical shift ‘fingerprint’ of different

functional groups. In our experiments, we specifically worked with 1H to assess the state

of health of muscle.

Signal acquisition

NMR reactive nuclei resonate at particular frequencies, depending on the field

strength and nuclei of interest. A 90° RF pulse is applied, tipping the spins into the

transverse receiving plane, as the signal decays as an damping sinusoid in the X-Y

plane, a free induction decay (FID) is obtained as an electrical signal in the receive coil.

The individual frequencies (resonances in ppm) contained with the FID are obtained

using a fast Fourier Transform (FFT).

Chemical shift

Spin generating magnetic fields produce measurable magnetic moments. In the

presence of an external magnetic field (i.e., B0), two spin states exist, in alignment and

opposition of the external magnetic field. The difference in energy (∆E) between the

two states increases as field strength increases and is proportional to the magnetic

moment, as described in Equation 2-2. Further, different localized environments affect

72

other local magnetic environments, leading to shielding or deshielding of protons from

the magnetic fields. This ultimately causes differential RF energy absorptions by the

nuclei, based on localized shielding. The differences in energy are measured in units

parts per million (ppm). PPM is a relative frequency shift, which allows the comparison

of chemical shifts from compounds measured at different magnetic fields with absolute

frequencies related to B0 (2-2).

Applications of MRI and MRS in skeletal muscle

Due to the excellent spatial resolution of soft tissue in MRI, and superior spectral

resolution available in MRS, MRI and MRS have revealed a wealth of information about

healthy and pathologic muscle over the past several decades (Baudin et al., 2015;

Bongers et al., 1992; Cole et al., 1993; Damon et al., 2002; Frimel et al., 2005a; Fulford

et al., 2014; Hollingsworth, 2014; Hsieh et al., 2007; Lamminen, 1990; Mercuri et al.,

2007; Triplett et al., 2014; Vohra et al., 2015; Wang et al., 2009).

A plethora of MRI scan sequences can be run to reveal different information

regarding the state of health of muscle. T1-weighted imaging can reveal contrast

between skeletal muscle and lipid, providing valuable anatomic information regarding

the state of lipid infiltration into dystrophic muscle. In T1-weighted scans, lipid and fat

have a higher signal and appear bright white, while edema and water (the primary

contributors to muscle signal) decay quicker, appearing dark and black. In the muscular

dystrophies, T1-weighted imaging is used most frequently to reveal anatomic

information about muscle, including information such as muscle volume, cross sectional

area, and muscle thickness and length. These measures indicate information about

muscle in various states of health and disease, such as revealing amounts of muscle

atrophy in cachectic states or growth following exercise and training (Sorichter et al.,

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1995). In DMD, proximal muscles are first affected, and this is able to be observed by

T1-weighted images, showing abnormal measures in the gluteus maximus and adductor

magnus, followed by the thigh muscles, and eventually, the lower leg muscles (Akima et

al., 2012; Liu et al., 1993b).

T2-weighted scans are used to differentiate tissue based on the intrinsic T2

relaxation times of tissue. Because water has higher T2 relaxation times than healthy

tissue (darker gray), edematous and inflamed tissue (increased contrast on T2 weighted

scans) is able to be differentiated from otherwise healthy tissue. In the muscular

dystrophies, T2 weighted imaging provides valuable information, as T1 weighting fails to

differentiate acute dystrophic lesions, due to prior fatty involvement, from healthy

muscle at higher field strengths (Chang et al., 1981; Misra et al., 1980). In both clinical

and preclinical studies, T2-weighted MRI has demonstrated to be able to detect

damaged and edematous muscle by quantification of the transverse (T2) relaxation time

constant (Ababneh et al., 2005, 2005; Bendszus et al., 2002; Clarkson and Hubal, 2002;

Foley et al., 1999; Mathur et al., 2011; Shellock et al., 1991). The first study to measure

observable differences in T2 as in an exercise study, revealing that exercised muscle

had longer T2 relaxation times (Evans et al., 1998). Since then, elevated T2 times have

been observed in a number of states that reflect increased water content in muscle,

both through healthy mechanisms such as that found after eccentric exercising, and in

pathologic states, reflecting increased inflammation and edema within muscle. In

healthy individuals, increases in T2 are found to be transient elevated, but chronically

elevated T2 values may indicate underlying pathology within muscle. In healthy

individuals, eccentric exercise has been shown to elevate T2 values within muscle,

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peaking at 2 days after exercises at the peak of muscle damage (Cermak et al., 2012;

Foley et al., 1999; Shellock et al., 1991; Sorichter et al., 1995). Elevated T2 has also

been measured in other eccentric loading protocols in preclinical models (Caron et al.,

2009; Frimel et al., 2005b).

Spectroscopy in muscle has also proved helpful to differentiate healthy,

damaged, and diseased muscle (Bongers et al., 1992; Forbes et al., 2014b; Hsieh et al.,

2007; Lund et al., 2003; Martins-Bach et al., 2012; Park et al., 1995). Different than

MRI-T2 measurements, whose signal are contributed to by a number of factors, 1H2O- T2

looks specifically at the water content within muscle, eliminating other contributing

factors such as lipid deposition. This allows researchers to better assess inflammation

and edema within muscle than MRI-T2 (Bongers et al., 1992; Brizidine et al., 2013;

Fayad et al., 2014; Hsieh et al., 2007, 2009).

Other spectroscopic studies within muscle have used other elements, such as

13C and 31P to assess different properties of muscle. 31P spectroscopy can measure

both ATP energetics and pH within a muscle, providing valuable information about

muscle through these data. Through measuring the amounts of phosphocreatine (PCr)

and phosphate groups of ATP, one can assess energy and pH status within muscle

(Haseler et al., 2004; Lanza et al., 2006; Lund et al., 2003; Weidman et al., 1991).

MRI and MRS Summary

MRI and MRS have demonstrated the ability to non-invasively determine the

state of health of muscle, providing quantitation of how injured or diseased muscle is

when compared to healthy muscle. Due to the safe, repeatable, and quantitative nature

of MRI and MRS, information about the state of health of muscle, natural progression of

disease, and response to therapeutic intervention are studied in this dissertation.

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Near Infrared Optical Imaging

Near Infrared (NIR) Optical Imaging is a non-invasive imaging modality that

utilizes photons in the NIR range to view specimens of interest (Figure 2-8). The

advantages of operating in the NIR range relate to the concept of photon propagation

through tissue and the optical signal to noise ratio (SNR). The NIR range (700-900 nm)

allows for deep propagation of photons through thick biological tissue, including skin,

fat, bone, and muscle in vivo compared to smaller wavelength ranges. Propagation of

photons through tissue is determined by scattering experienced by photons, leading to

diffusion of light. Diffusion of light causes a decrease in resolution, so by allowing for

minimal diffusion because of greater propagation, NIR light has increased resolvability

due to decreased scattering. Additionally, tissue autofluorescence, which is a problem

for most visible range optical imaging, is minimized in the NIR range. Through

applications of NIR filter sets, autofluorescence is nearly entirely eliminated by reducing

background fluorescence, and increasing SNR. The ability to increase photon

propagation and SNR give NIR optical imaging advantages that other optical techniques

are able to utilize.

Near Infrared Optical Spectroscopy

Currently, NIR spectroscopy (NIRS) is commonly utilized to measure perfusion

status, oxygenation, and blood flow within muscles (Boushel and Piantadosi, 2000;

Boushel et al., 2000; Brizidine et al., 2013; Ferrante et al., 2009; Ferrari et al., 2004;

Guenette et al., 2008, 2011; Hamaoka et al., 2007; Mancini et al., 1994; McCully and

Hamaoka, 2000; Messere and Roatta, 2015; Olivier et al.; Torricelli et al., 2004;

Vogiatzis et al., 2008; Wolf et al., 2007). Valuable information can be elucidated from

NIRS, such as increased fluid retention in muscle after eccentric exercises, or

76

decreased perfusion in dystrophic muscle. Beyond muscle, NIRS has been used to

detect intracranial bleeding (Gopinath et al., 1993, 1995; Kirkpatrick et al., 1995).

However, despite the valuable information that is able to be collected, NIRS lacks to

provide sensitive spatial resolution.

Contrast Enhanced Near Infrared Optical Imaging

Through the use of exogenous contrast agents, several imaging models have

been developed for NIR optical imaging. The most widely used NIR chromophore is

indocyanine green (ICG), an FDA approved NIR fluorescent contrast dye (Cherrick et

al., 1960; Frangioni, 2003; Weissleder, 2001). ICG absorbs in a broad range, from 600

to 900 nm and emits fluorescence between 750 and 950 nm. For purpose of ensuing

discussion, our studies found that the maximum absorption occurs at 780 nm, and the

maximum excitation occurs at 820 nm in vivo. ICG is hepatically metabolized, and is

not absorbed by the intestinal mucous membrane, rendering its toxicity as low

compared to other organic fluorophores (Alford et al., 2009). ICG is a blood pooling

agent, binding tightly to blood serum proteins such as albumin, thus, serves to highlight

vasculature (Chen et al., 1999; Desmettre et al., 2000; Kobayashi et al., 2014; Raabe et

al., 2003). Similarly, through the enhanced permeation and retention effect, ICG can

passively accumulate in a manner similar to Evans blue dye (EBD) in histological

studies or gadolinium in contrast enhanced MR (Corlu et al., 2007; Hamer et al., 2002;

Ntziachristos et al., 2000). ICG has been used in a variety of clinical applications, such

as such as imaging of the vasculature of the retina (Chen et al., 1999; Desmettre et al.,

2000; Herbort et al., 1998; Mueller et al., 2002), breast cancer tumors (Gurfinkel et al.,

2000; Ntziachristos et al., 2000; Troyan et al., 2009; Verbeek et al., 2014; Zelken and

Tufaro, 2015), cerebral vasculature and tumors (Haglund et al., 1996; Raabe et al.,

77

2003), gastrointestinal vessels (Borotto et al., 1999), and cardiac vasculature and

myocardial perfusion (Nakayama et al., 2002; Taggart et al., 2003). To date, the FDA

approved version of ICG has been only utilized for spectroscopic purposes, and not yet

been used to spatially image muscle.

While ICG is certainly the most established NIR fluorescent contrast agent, other

NIR fluorescent contrast agents include the general class of cyanine dyes and several

photodynamic therapeutic agents (Ebert et al., 2011; Sevick-Muraca et al., 2002).

Cyanine dyes have served as contrast agents that have been conjugated to monoclonal

antibodies targeting tumor associated antigens to demonstrate tumor specific targeting

(Ballou et al., 1995; Folli et al., 1992, 1994; Pèlegrin et al., 1991; Soukos et al., 2001).

The cyanine dyes Cy3, Cy5, and Cy5.5 have served to bind anti-SSEA-1, and

importantly showed that tumors in deep tissues, normally not visible to fluorescent

marker that operate at smaller wavelengths, was able to be quantitatively visualized

(Ballou et al., 1995). Other interesting conjugates to cyanine dyes include pamidronate,

a bisphosphonate derivative, to visualize bone structure, to visualize osteoblast activity

(Zaheer et al., 2001). While potentially able to hone in on target tissue better, several

disadvantages to antibody and peptide conjugation of cyanine dyes exist. These

disadvantages include adverse immune reactions, prolonged circulation, and elevated

background fluorescence (due to the lengthy stay in circulation (Goldsmith, 1997).

Other interesting avenues of research include enzyme cleaving activatable dyes.

The concept behind these are that they are inert, unable to be visualized, until cleaved

by a particular enzyme present in certain locations of the body. Weissleder and

colleagues and others performed several experiments developing Cy5.5 loaded polymer

78

particles that were inactive until the polymers eroded away (Mahmood et al., 1999;

Tung et al., 1999; Weissleder et al., 1999). When concentrated within the polymer at a

high local concentration, fluorescence auto-quenched, but upon disintegration of the

polymer by cathepsins, the auto-quenching properties were lifted as the dye now

existed in lower concentrations. Similarly, matrix metalloproteinases-2 substrates were

bound to Cy5.5 by Bremer et al, and upon activation of the proteinase, the Cy5.5 is

observable, serving as a proxy to the proteinase activity (Bremer et al., 2001). While

many of these studies were performed with the intention of imaging cancerous targets,

fewer studies have been performed in muscle. Baudy et al elegantly demonstrated the

use of a cathepsin cleavable substrate that highlighted muscle damage and repair in a

mouse model (Baudy et al., 2011). Overall, there are many exciting NIR responsive

fluorescent contrast agents in development, but ICG remains the primary dye of interest

investigated because of its long demonstration of safety in the clinic.

Several applications of contrast enhanced NIR optical imaging include diffuse

optical tomography (DOT), fluorescence reflectance imaging (FRI), optical coherence

tomography (OCT), and fluorescence mediated molecular tomography (FMT). DOT is

based on the deliverance of low energy electromagnetic radiation to one or more

locations on the body, measuring both tissue’s transmission and reflection properties of

the delivered light (Hielscher et al., 2002). Based on tissues’ inherently different

scattering and absorption properties, reconstructions of the spatial distribution of the

optical properties within the sample are composed, providing valuable insight to the

tomographical makeup of samples of interest. FRI, also known as epi-illumination, is

utilized to capture surface and subsurface fluorescent activity from samples of interest.

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Light, at a pre-filtered wavelength, shines onto samples, and fluorescence is collected

from the same side of tissue. When light passes through samples and is collected on

the opposite side of the tissue sample, this is known as trans-illumination. OCT is

another optical imaging technique that measures optical scattering to capture high

resolution three dimensional images (Huang et al., 1991). OCT is analogous to

ultrasound, though using optics rather than sound waves to generate images. Optical

beams are directed at the sample of interest. As the photons enter the tissue sample,

most scatter at large angles rather than being directly reflected back. Through use of an

interferometer, the distance travelled by received photons is calculated, rejecting most

photons that have scattered multiple times before detection. This allows for

reconstruction tissue samples with resolution at the submicron level. A limitation to

OCT is that it is limited to several millimeters below the sample surface, because at

greater depths, the amount of light that escapes without scattering is limited. Lastly,

FMT provides quantitative three dimensional images based on the distribution of

fluorescent probes within a sample (Ntziachristos et al., 2003) . As photons are

propagated through tissue from multiple projection sources, data are collected and

reconstructed to provide tomographic distribution of the fluorochrome within deep

tissue. FMT expands on the principles of diffraction tomography, which is recording

photon reflection to measure shapes of objects, but additionally incorporates absorption

and fluorescent measurements to accurately reconstruct fluorescent reporters. Based

on emission and excitation, data are reconstructed to determine quantitative fluorophore

concentration within tissue.

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Applications in Skeletal Muscle

Though NIRS has been used extensively to study muscle, contrast enhanced

NIR optical imaging has limited demonstration in muscle. A demonstration of contrast

enhanced NIR optical imaging in muscle was performed by Baudy et al, where caged

NIR cathepsin B substrates were utilized to visualized damaged, dystrophic, and treated

muscle in mice (Baudy et al., 2011). However, the use of FDA approved fluorophores

has not yet been demonstrated to image muscle pathology, though it has been used to

assess pathology in a number of different organ systems. For our research, we utilize

ICG contrast enhanced NIR optical imaging to quantify and observe healthy, damaged,

diseased, and treated muscle.

Conclusion

The muscular dystrophies are devastating diseases, relentlessly and

progressively deteriorating muscle. Though therapies continue to rapidly progress to

treat the diseases, adequate measures to longitudinally assess therapeutic efficacy lag

behind. Traditional measures of muscle health, including muscle biopsy, are invasive,

traumatic, and provide a limited field of view of the state of muscle health. Non-invasive

measures, such as MR and NIR optical imaging may provide longitudinal measures of

muscle health, in a safe and quantitative manner. While MR has shown that dystrophic

muscle, and inducible damage to muscle can be reliably quantified, contrast enhance

NIR optical imaging has not yet demonstrated the ability to detect muscle damage. We

hope to demonstrate the ability of contrast enhanced NIR optical imaging to detect

inducible damage, disease, and recovery of disease by therapeutic intervention in the

remainder of these studies.

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Figure 2-1. A spin echo sequence showing the initial 90° RF pulse, followed by the

generated FID, the refocusing 180° RF pulse, and the additional 90° pulse of the next sequence.

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Figure 2-2 Longitudinal (T1) relaxation curves showing the difference in relaxation

between fat and muscle, and how different TR acquisitions (along the x-axis) alter the difference in signal generated between tissue types.

0.0 0.5 1.00.0

0.5

1.0

Time (ms)

Rela

tive S

ign

al (a

.u.)

Fat

Muscle

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Figure 2-3. Transverse (T2) relaxation curves showing the difference in relaxation

between muscle and edema, and how different TE acquisitions (along the x-axis) alter the difference in signal decay between tissue types.

0.0 0.5 1.00.0

0.5

1.0

Time (ms)

Rela

tive S

ign

al (a

.u.)

Muscle

Edema

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Figure 2-4. Inversion recovery technique to calculate T1 demonstrating representative

signal recovery profiles for edema, muscle, and lipid.

0.5 1.0

-1.0

-0.5

0.0

0.5

1.0

Repetition Time (ms)

Mag

neti

zati

on

Z (a

.u.)

Edema

Muscle

Lipid

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Figure 2-5. Progressive saturation technique demonstrating how different acquisition

times within the same recovery curve can be used to calculate T1.

0.0 0.5 1.00.0

0.5

1.0

Acquisition Time (ms)

Ma

gn

eti

za

tio

n Z

(a

.u.)

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Figure 2-6. A Carr-Purcell sequence showing the initial 90° RF pulse, followed by a train

of 180°RF pulses in the X plane, with each refocusing the FID in the opposite direction.

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Figure 2-7. A Carr-Purcell-Meiboom-Gill Pulse sequence showing the initial 90° RF

pulse, followed by a train of 180°RF pulses given in the rotating frame, with each refocusing the FID in the same direction.

88

Figure 2-8. Electromagnetic spectrum, highlighting the location of the near infrared

range.

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CHAPTER 3 OUTLINE OF EXPERIMENTS

Overview

The ultimate objective of this work focuses on the development of near infrared

optical imaging to quantitatively assess the state of health in muscle. Experiments were

conducted at both the preclinical and clinical levels. Preclinically, muscle was assessed

in several states of health: acutely damaged non-diseased muscle, chronically damaged

dystrophic muscle, acutely damaged dystrophic muscle, and therapeutically treated

dystrophic muscle. Additional preclinical experiments were performed to assess

additional abilities of contrast enhanced near infrared optical imaging. Specifically,

experiments to test the capabilities of contrast enhanced near infrared (NIR) optical

imaging to assess vascular drug delivery utilizing biocompatible nanoparticles were also

performed. Clinically, acutely damaged non-dystrophic muscle was studied by NIR

optical imaging. Throughout all studies, additional data was collected to support the

near infrared optical imaging findings, including MRI and MRS findings at preclinical and

clinical levels, as well as histological and spectrophotometric data at the pre-clinical

level.

Overall Hypothesis: Contrast enhanced near infrared optical imaging can

monitor and quantify cellular muscle damage and changes in perfusion in healthy,

damaged, and diseased muscle in a safe, repeatable, noninvasive manner.

Preclinical Studies: Detection Damaged, Diseased, and Healthy Murine Muscle

We hypothesize that indocyanine green (ICG), as a fluorescent NIR contrast

agent, will preferentially accumulate in damaged muscle cells, and can be monitored in

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vivo. This section of the dissertation will provide proof of concept data via several

experiments in multiple mouse models of muscle damage.

Acutely Induced Damage to Healthy Mouse Muscle

This set of experiments tests the concept that ICG will accumulate in acutely

damaged muscle cells of healthy mice, and can be monitored in vivo. Using a well

established model of immobilization followed by reambulation (Frimel et al., 2005b), the

deep soleus of the mouse hindlimb experiences damage and recovery in a time

dependent manner. This intervention causes a well-defined time course of acute

damage, followed by recovery, which we are able to non-invasively monitor through

several modalities. We initially chose to use healthy rather than dystrophic mice to

conduct these experiments to minimize the variable amounts of muscle damage that

contributions of muscle damage from the natural progression of muscular dystrophies in

mice. ICG enhanced NIR optical imaging was performed at various time points during

reambulation following cast immobilization, allowing for assessment of acute muscle at

different states of damage and recovery. The ability to use contrast enhanced NIR

optical imaging to measure muscle health was further confirmed by magnetic resonance

imaging, proton spectroscopy, histological, and spectrophotometric measures.

Hypothesis

ICG enhanced NIR imaging will detect muscle damage and recovery in an acute

model of muscle damage in healthy mice and correlate with other measures of muscle

damage.

Specific aim

The primary aim of this experiment was to determine if ICG enhanced NIR optical

imaging is capable to detect muscle damage in healthy mice following acute insult to the

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muscle. Specifically, we sought to determine if (A) changes in NIR optical imaging

fluorescent signal could detect inducible muscle damage and if (B) NIR optical imaging

fluorescence correlated with other measures of muscle damage.

Exacerbation and Amelioration of Damage in Dystrophic Mouse Muscle

Following detection of pathology to otherwise healthy muscle in a well controlled

model of muscle damage, we sought to detect damage as a result of disease to muscle,

specifically in Duchenne (mdx) and limb girdle 2C (gsg -/-) muscular dystrophy mouse

models. Following baseline detection of disease, we then exacerbated and mitigated

the muscle damage, through eccentric loading by downhill treadmill running to mdx

mice and delivery of the missing γ-sarcoglycan gene by way of AAV delivery in gsg -/-

mice, respectively. Parallel to the prior experiments, a cohort of additional data,

including MRI-T2, 1H2O-T2, histological, and spectrophotometric measures were

collected to confirm the contrast enhanced near infrared optical imaging findings.

Hypothesis

ICG enhanced near infrared optical imaging is able to assess damage to muscle

caused by natural progression of disease, as well as exacerbation of muscle damage

from eccentric exercises and mitigation of pathology through therapeutic intervention.

Specific aim

The primary aim of this experiment was to determine if ICG enhanced NIR optical

imaging is capable to cross sectionally detect muscle damage in dystrophic mice, as

well as in dystrophic mice that underwent additional insult or amelioration of muscle

damage. In exploring this aim, we sought to (A) differentiate healthy from dystrophic

muscle by contrast enhanced near infrared optical imaging, (B) measure additional

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burden of muscle damage following an eccentric exercise protocol, and (C) assess

disease mitigation through a corrective AAV therapy.

Vascular Drug Delivery Capabilities of ICG Enhanced Near Infrared Optical Imaging

Indocyanine green (ICG, Cardio green, Fox-green, or IC-Green), to date, has

been applied for many different utilizations besides imaging muscle damage, including

imaging vasculature as highlighted in Chapter 2. As muscle compromises a significant

portion of the human body, system delivery of therapeutic agents is necessary to obtain

adequate therapeutic treatment. This is a challenging obstacle to overcome in the

muscular dystrophies due to perfusion defects, tissue damage, and fibrosis, limiting the

ability to deliver needed agents to the required areas. We propose that dually loaded

biocompatible nanoparticles may be able to accomplish this through providing stable

optical contrast to visualize the destination of particles, while concurrently delivering a

payload of interest.

Hypothesis

ICG can be used to quantitatively study changes in vascular perfusion and

biocompatible nanoparticles can be loaded with indocyanine green and uptake can be

visualized in an in vivo environment.

Specific Aim

Biocompatible nanoparticles, optimized to contain indocyanine green, will be

delivered to the body and quantitatively tracked. Specifically, we hope to accomplish

several sub-aims, including (A) demonstration of vascular perturbation measurements

using unencapsulated indocyanine green (B) synthesis of ICG loaded biocompatible

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nanoparticles, and (C) demonstration of maintained stability using ICG loaded

biocompatible nanoparticles in an in vivo model.

Clinical Studies

The ultimate destination of any translational research study is in the clinic. The

final experiments of our work are a culmination of all of the aforementioned preclinical

studies. Temporary muscle damage has been shown to be able to be induced into

healthy muscle through eccentric exercises, and this has been shown to be measurable

through MRI-T2 and 1H2O-T2 (Clarkson and Hubal, 2002; Clarkson et al., 1986; Foley et

al., 1999; Fulford et al., 2014; Sorichter et al., 1995). Further, MR measures have

demonstrated the ability to assess the disease in dystrophic muscle in humans.

Building off of the pre-clinical findings, I aimed to demonstrate the abilities of NIR optical

imaging and MR imaging and spectroscopy to detect damaged muscle in humans.

Hypothesis

I hypothesize that near infrared optical imaging and magnetic resonance imaging

and spectroscopy can identify damaged and dystrophic muscle in humans.

Specific Aim

The primary aim of these clinical studies is to identify damaged muscle in

humans by NIR optical imaging and MR imaging and spectroscopy. Specifically, I

intend to show that (A) damage to muscle of healthy humans, as a result of an eccentric

exercise protocol and (B) dystrophic versus unaffected muscle can be measured in the

upper and lower extremities.

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CHAPTER 4 METHODOLOGY

Pre-Clinical Work

Animal Handling and Care

All pre-clinical studies were approved by the University of Florida’s Institutional

Animal care and use committee and the Department of Defense’s Animal Care and Use

Review Office. Mice were housed in an AALAC regulated facility (12 hour light/dark,

72°F, 42% humidity) and provided food ad libetum. Mouse strains included healthy

control C57BL/6ScNJ and C57BL/10ScNJ mice, a dystrophin null model for DMD (mdx,

C57BL/10ScSn-Dmdmdx/J), and a γ-sarcoglycan null model for LGMD2C (gsg -/-,

Sgcgtm1Mcn) as described in the upcoming section.

Mouse Strains

Control mice

Healthy control mice used in these studies were either of the C57BL/6 or

C57BL/10 strains and were procured through breeding colonies maintained by

University of Florida Animal Care Services. These mice are some of the most widely

used strains and are regularly used as healthy control comparisons in studies. The

C57BL/10J strain is phenotypically and genotypically similar to the C57BL/6J strain, with

minor allelic differences. Both strains have lifespans of approximately two years, unless

sacrificed earlier and serve as healthy controls for all muscle related experiments

(Finch, 1994).

mdx mice

The Duchenne muscular dystrophy mouse model, the mdx (C57BL/10ScSn-

Dmdmdx/J ) mouse, is one of the most widely studied mouse models (Aartsma-Rus and

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van Putten, 2014; Anderson et al., 1988; Barton et al., 2002; Barton-Davis et al., 1999;

Blain et al., 2015; Bobet et al., 1998; Brussee et al., 1997; Burns et al., 2015; Gillis,

1996; Griffin and Rosiers, 2009; Hodgetts et al., 2006; Hoffman et al., 1987; Lynch et

al., 2001; Mathur et al., 2011; Pastoret and Sebille, 1995; Porter et al., 2002; Sicinski et

al., 1989; Weller et al., 1990). Initially, mice were obtained from The Jackson

Laboratory (Bar Harbor, ME, USA), and were subsequently bred through University of

Florida Animal Care Services. The mdx strain is a result of a spontaneous mutation that

occurred in a C57BL/10ScSn colony, first described as a model for DMD and BMD in

1984 (Bulfield et al., 1984). In 1987, a premature stop codon mutation on exon 23 of

the dystrophin gene was identified through reverse positional cloning (Hoffman et al.,

1987). The phenotypic presentation of these mice is relatively minor compared to the

human disease, frequently living up to two years of age. These mice demonstrate a

characteristic timeline characterized as early and widespread inflammation and necrosis

within muscle, followed by hypertrophy and recovery of muscle, and do not demonstrate

a severe dystrophic phenotype until the last few months of life (Bulfield et al., 1984;

Vohra et al., 2015).

γsg -/- mice

The γ-sarcoglycan null mouse (gsg -/-) is the genetic model of the human limb

girdle muscular dystrophy 2C (LGMD-2C), lacking a functional γ-sarcoglycan in the

dystrophin associated glycoprotein complex (Barton, 2010; Hack et al., 1998; McNally et

al., 1996a, 1996b). As described in Chapter 1, the limb girdle muscular dystrophies

arise from mutations to various sarcoglycan components of the dystrophin associated

glycoprotein complex (Guglieri et al., 2008; Laval and Bushby, 2004; Pegoraro and

Hoffman, 1993). Eventhough dystrophin is still present, absence of one of these

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subunits renders the sarcolemma weak and susceptible to damage. Phenotypically,

these mice present with a moderately severe disease, having widespread lesions within

muscle, cardiomyopathy, and extensive tissue fibrosis (Hack et al., 1998). The gsg -/-

mice in this study were a generous gift from Elisabeth Barton, and the colony was

maintained at the University of Florida’s Animal Care Services.

Preclinical Interventions

To support of the greater direction of our labs studying muscle pathology, several

interventions were performed on healthy and dystrophic mice, providing insult and

therapeutic protection against muscle damage.

Immobilization-reambulation studies

In our attempt to demonstrate the ability of contrast enhanced NIR optical

imaging to detect muscle damage, we first sought to use a well controlled model of

muscle damage to demonstrate proof of principle data. Cast immobilization and

reambulation in mice has previously demonstrated a characteristic timecourse of

inducible damage, followed by recovery from the muscle damage (Frimel et al., 2005b).

Building off of the work that had previously been performed, we modified the cast

immobilization protocol, allowing for internal controls within each mouse, as described

below.

C57/BL6J mice (n = 60 males) were bred in-house through the University of

Florida’s Animal Care Services, and were 6-8 weeks of age during experimentation. In

addition to the regular chow diet, a dough diet with elevated protein and fat levels

(BioServ, Flemington, NJ) was provided for the mice at the base of the cages during the

entire procedure to ensure dietary needs were met during and after hindlimb

immobilization. Right hindlimbs were immobilized (IMM) in a plantar flexed position, first

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by medical grade paper tape, followed by plaster of paris (OrthoTape, Blufton, SC), and

finally an encompassing single layer of casting material (Patterson Medical, Warrenville,

IL), as previously described (Frimel et al., 2005a, 2005b). The contralateral leg (non-

IMM) served as each individual’s own control. Mice were checked daily for abrasion

wounds as a result of the casting procedure and animal weight was monitored. After

two weeks of immobilization, casts were removed, and animals were allowed to

undergo free cage ambulation. Data (MRI, MRS, NIR optical imaging, and tissue

assessment) were acquired at days 0, 1, 2, 3, 5, and 7 following the removal of casts (n

= 10 for each timepoint). Images were not acquired throughout the duration of casting

immobilization because the immobilized hindlimbs were unable to fit inside the MRI coil.

18 hours prior to sacrifice, 1% filter sterilized Evans blue dye (EBD; Sigma Aldrich, St.

Louis, MO) in phosphate-buffered saline (0.1 g/ml/mg) was administered to the mice

intraperitoneally as previously described (Hamer et al., 2002).

Downhill treadmill running

To induce additional damage to a dystrophic mouse phenotype, mdx mice

participated in a downhill treadmill running exercise. Because the extremities of

individuals affected with muscular dystrophy are preferentially at risk for injury (Edwards

et al., 1984), animal models that focus on these areas may be more pertinent than other

animal models. To accomplish the intentions of these experiments, mdx mice (aged 12-

32 weeks, n = 5 / group) were run on a downhill (14° decline) motorized treadmill at a

speed of 8-10 meters/minute for up to 45 minutes (Brussee et al., 1997; Lynch et al.,

1997; Whitehead et al., 2006). 5 minutes prior to the downhill running, mice were

allowed to acclimate to the treadmill environment by running horizontally at a speed of

no greater than 5 meters / minute. Mice were run in individual lanes, were supervised

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and were provided a short burst of compressed air behind them to encourage their

running. The maximum they ran was 45 minutes, but few ran as little as 5 minutes,

while most were able to run between 20-30 minutes. Following treadmill running, mice

demonstrated signs of fatigue, such as limited mobility and heavy breathing.

Recombinant adeno-associated virus administration

Recombinant human γ-sarcoglycan cDNA was loaded into AAV serotype 2/8

(rAAV) and expression was regulated by a truncated desmin promoter. A 110 μl aliquot

containing viral particles was diluted with PBS in the ratio of 1:20. 50 μl of the diluted

solution was then injected in tibialis anterior (TA) muscle and 100 μl of the diluted

solution was injected in the gastrocnemius (Gas) muscle. Hindlimbs were randomly

pre-selected to receive the AAV therapy (n = 8), or no injections (n = 4). Following

injections, mice were housed in the animal facility for six weeks, at when data (MRI,

MRS, NIR optical imaging, and histology) was collected.

Vascular perfusion experiments

Using an NIR optical imaging in house laser (780 nm, TCLDM9 TEC LD Mount,

Thorlabs, Newton NJ, USA), bandpass filter (820 nm, Hamamatsu Inc., Hamamatsu,

Japan), and camera (Pixis 1024, Princeton Instruments, Trenton, NJ, USA) setup, old

mdx mice (40-60 weeks of age) were anesthetized with isofluorane (3% knockdown,

0.75-1% maintenance), and intravenously injected with reconstituted NirawaveC in the

same manner as done in Specific Aims 1.

Image acquisition was immediately initiated, and carried on for up to 30 minutes

following injections. Through this novel in-house setup, one can visually observe, and

quantitatively differentiate the major vasculature (primarily the femoral artery) and

surrounding muscle enhancement at various times after acquisition. Additional

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experiments were conducted to visualize changes in perfusion following ischemia-

reperfusion. Specifically, the hyperemic response (Joannides et al., 1995) is exhibited

following reperfusion of major vasculature, leading to an increase of blood flow beyond

basal conditions. In this set of experiments, single hindlimbs of old mdx mice (aged 40-

60 weeks) was cuffed with a blood pressure cuff for 5 minutes. At five minutes, the cuff

was removed, and NirawaveC (1 mg / kg) was injected. Images were captured before

blood pressure cuff application, as soon as the cuffs were removed (after 5 minutes),

and for up to 1 hour in the same manner as described for the basal vasculature

assessment. Images were then analyzed based on pixel intensity using ImageJ (NIH,

Bethesda, MD) to assess the presence of dye in both muscle and the major vasculature

of the hindlimb of the mouse.

Delivery of ICG Loaded Nanoparticles to Dystrophic Muscle

An additional goal of our studies was to assess the ability of biocompatible

nanoparticles to quantitatively track the delivery of therapeutic drugs to dystrophic

muscle in an in vivo setting.

Synthesis and optimization of particles

Nanoparticles were fabricated using FDA approved poly-lactic acid (PLA) and

ICG using the water-oil-water double-emulsion method (Panyam et al., 2003; Yang et

al., 2001). To minimize ICG leaching from the particles, polyethylenimine (PEI) was

added to the compound mixture. Particle sizes were modified through use of

emulsifiers or surfactants during synthesis. Briefly, aqueous solutions of PEI and ICG

were added to the PLA polymer dissolved in dichloromethane and sonicated to obtain a

homogeneous water-oil emulsion. This emulsion was gradually added to an aqueous

solution of poly vinyl alcohol and was then sonicated, creating a water-oil-water

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emulsion. Particles were collected after stirring overnight and purified by repeated

washings with water. Residual surfactant adsorption on PLA-ICG particles was

determined by estimating the charge using zeta potential measurements.

To optimize ICG fluorescence from the ICG loaded nanoparticles, the amounts of

ICG prepared for loading into the ICG-PLA particles was varied. Fluorescence was

measured (excitation: 745 nm, emission: 820 nm) on an IVIS In Vivo Spectrum (Caliper

Life Sciences, Hopkinton, MA) in the epifluorescence mode in total radiant efficiency

units (p/sec/cm2/sr/μW/cm2) to compensate for non-uniform excitation illumination

patterns, commonly used in several in vivo systems (Shcherbakova and Verkhusha,

2013; Subach et al., 2011). Further, quantum yield (Φ), was used to optimize the ratios

of ICG, PLA, and PEI using the integrating sphere approach (Porrès et al., 2006).

To investigate stability of the particles as a variable of temperature, experiments

were conducted both at room (23°C) and physiological (37°C) temperature. For four

weeks, both PLA-ICG particle samples and ICG in reconstituted water were stored in

lightless conditions at either temperature, with daily fluorescent measurements recorded

on the IVIS daily.

In vivo capabilities of ICG loaded nanoparticles

Following optimization of particles in vitro, we wanted to translate our findings to

a preclinical model. To accomplish this, we used mdx male mice (n = 5) in this study.

First, PLA-ICG particles were administered subcutaneously. All regions of interest on

the mice were shaved prior to injections. Subcutaneous injections (n = 5 per cohort) of

either 100 µL of PLA-ICG particles (10 mg/mL), NirawaveC reconstituted according to

package instructions, or Lactated Ringer’s Buffer were injected below the loose skin

between the shoulders of mice. Images were captured for 10 days using the Xenogen

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IVIS In Vivo Spectrum (excitation: 745 nm, emission: 820 nm) and total radiant

efficiency was measured over the fixed ROI.

Following subcutaneous injections of PLA-ICG particles, PLA-ICG particles were

injected intramuscularly into the gastrocnemius of mdx mice. Reflective fluorescent

imaging was performed before and immediately following injection, and daily for 28

days. Contralateral hindlimbs were not injected, serving as each individual’s control. In

an additional cohort of mdx mice, NirawaveC (Miltenyi Biotec, Bergisch Gladbach,

Germany) was administered in the gastrocnemius, and fluorescence of the non-particle

cohort was assessed for one week.

Methods

Near Infrared Optical Imaging

For all experiments that utilized ICG enhanced NIR optical imaging to assess

muscle pathology in mice, the same protocol was followed.

One hour prior to NIR optical imaging, NirawaveC ICG (Miltenyi Biotech Inc., San

Diego, CA) was administered to the mice intravenously according to the package insert

(1 mg / kg body weight). Following an initial peak of fluorescence while ICG was in the

vascular compartments, a steady signal was maintained between 30 minutes to 12

hours post injection (Figure 4-1), thus to standardize procedures, NIR optical imaging

data was collected starting at 1 hour post-injection. Mice were anesthetized using an

oxygen and isofluorane mixture (3% induction, 0.75-1% maintenance) and NIR optical

imaging was performed using an In Vivo Fluorescence Imager (Field of view: 9 x 9 cm;

Excitation wavelength: 745 nm; Emission wavelength: 820 nm; Perkin Elmer, Waltham,

MA, USA). Acquired images were analyzed over specifically drawn regions of interest

using Living Image ® software on the same In Vivo Fluorescence Imager. Total radiant

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efficiency from all experiments was normalized to account for differences in scanning

laser power, exposure times, and scanning area selected between mice.

Magnetic Resonance Imaging and Spectroscopy

Quantitative magnetic resonance imaging (MRI) and spectroscopy (MRS) was

utilized throughout our studies, to assess and monitor muscle injury and recovery. All in

vivo MR experiments were performed on mice anesthetized with gaseous isofluorane

(3% induction, 0.75-1% maintenance) and were kept warm with a heated water tubing

system for the duration of MR procedures. Respiration rate and temperature were

monitored (Small Animal Instruments, Stony Brook, NY) throughout the scans to

ensure adequate physiologic maintenance while under anesthesia and anesthesia was

appropriately adjusted to maintain adequate vital signs. Further, all MR experiments

were performed in a 4.7 T horizontal 22.5 cm bore magnet (Agilent, Santa Clara, CA,

USA). Lower hindlimbs were inserted into a custom-built solenoid 200 MHz 1H coil (2.0

cm internal diameter).

Magnetic Resonance Imaging

MRI-T2 experiments were performed to provide quantitative feedback on the

state of health of muscles in mice. To obtain correct positioning of all subsequent

scans, localizer images in orthogonal planes were acquired using a gradient echo

sequence (TR = 30 ms, TE = 5 ms, slice thickness = 2 mm, slice number = 3 per plane,

acquisition matrix = 128 x 128, signal averages = 1). Axial proton T2 weighted multi

slice MR images were acquired along the length of all mouse lower hindlimbs (TR =

2000 ms, TE = 14 and 40 ms, FOV = 10 x 20 mm2, slice thickness = 1 mm, slice

number = 12, acquisition matrix = 128 x 256, signal averages = 2). MRI-T2 decay was

calculated assuming a single exponential curve decay and has been demonstrated to

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adequately differentiate healthy from damaged muscle (Bulfield et al., 1984; Frimel et

al., 2005a, 2005b; Mathur et al., 2011; Vohra et al., 2015). MR images were converted

from raw Varian format to digital imaging and communications in medicine (DICOM)

files for analysis. Throughout all of the experiments performed, our regions of interest

(ROIs) included the soleus (Sol), gastrocnemius (Gas) tibialis anterior (TA), anterior

compartment, and posterior compartment and were drawn using Osirix software

(Geneva, Switzerland) to calculate signal intensity (SI) and T2 relaxation times of each

of the designated muscles were calculated from the pixel intensities at TE’s of 14 and

40 ms, as described in Equation 4-1 (Frimel et al., 2005a, 2005b; Mathur et al., 2011;

Vohra et al., 2015).

𝑆𝑇2𝑚𝑎𝑝 =1

(ln(𝑆14𝑚𝑠)−ln(𝑆40𝑚𝑠)

∆𝑇𝐸) (4-1)

Magnetic Resonance Spectroscopy

1H2O spectroscopic relaxometry was assessed using a Stimulated Echo

Acquisition Mode (STEAM) sequence to assess 1H2O-T2 relaxation times of

intramuscular water (Wang et al., 2009). Voxels were placed exclusively within the

desired muscle, taking care to avoid connective tissue beyond the soleus muscle and

subcutaneous fat. For all immobilization-reambulation experiments, voxels were placed

in the soleus with the following parameters: voxel: 1 x 1 x 2 mm3, TR: 9000 ms, TE: 5-

300 ms, 16 points exponentially spaced. For treadmill exercised mdx and rAAV treated

gsg -/- mice, voxels were placed in the posterior compartment with the following

parameters: voxel: 2 x 2 x 4 mm3, TR: 9000 ms, TE: 5-300 ms, 64 points exponentially

spaced. Non-negative least squares (NNLS) analysis (number of bins: 500, bin width:

0.5 ms, minimum T2 bin: 2 ms, maximum T2 bin: 251.5 ms, regularization parameter [µ]:

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0.00085 a.u., DC offset included: yes) was performed on the spectroscopic data

acquired using in-house developed software and IDL (Elliott et al., 1999; Triplett et al.,

2014).

Tissue Analysis

At the conclusion of experiments, mice were sacrificed a day following NIR

optical imaging and MRI/MRS data capture. Morphological features of captured tissue

sections were assessed by H&E staining, and fluorescent microscopy uptake of EBD

into fibers. Furthermore, western blotting and immunofluorescence was performed to

assess the restoration of γ-sarcoglycan in rAAV treated mice. Additional tissue not used

for histology was spectrophotometrically assessed for uptake of ICG and EBD, as

described below.

Histology

EBD was dissolved in phosphate buffered saline (0.15 M NaCL, 10 mM

phosphate buffer, pH 7.4) at a concentration of 1 mg / 0.1 mL / 10 g body weight

(Hamer et al., 2002). EBD was then filtered (2 µm pore syringe filter, Nalgene,

Rochester, NY) prior to intraperitoneal injections. NirawaveC was prepared according to

the package insert. The lyophilized powder was reconstituted in 850 µL of nanopure

water, and injected intravenously into the mice (50 µL / 10 g body weight). 15-18 hours

prior to animal sacrifice, mice were administered EBD and ICG. Hindlimb muscles (Sol,

TA, Gas, extensor digitorum longus, and quadriceps), diaphragm, and heart were

carefully dissected, fixed at resting length in OCT gel (VWR, Randor, PA), frozen in

precooled isopentane, then liquid nitrogen, and stored at -80°C. Frozen muscles were

later sliced into thin sections (10 µm) at the belly of the muscle and prepared for

histology. Slides were either prepared for H&E staining, EBD fluorescent staining, or

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immunofluorescence. H&E slides were prepared by standard methods. EBD

fluorescently stained slides were prepared by mounting slides with Vectashield antifade

mounting medium with DAPI (Vector, San Mateo, CA). Slides used to assess EBD

uptake were digitized on a high sensitivity back thinned CCD (EM-CCD C9100-13,

Hamamatsu, Hamamatsu City, Japan) and EBD (Ex: 620 nm, Em: 680 nm) and DAPI

(Ex: 358 nm, Em: 462 nm) images were overlaid. H&E slides were digitized under

brightfield at 5x and 20x magnification on a DM LB microscope (Leica Microsystems,

Solms, Germany). To determine the percentage of muscle that was EBD positive,

fibers that stained positive for EBD were manually counted and divided by the total

number of fibers in each muscle’s cross sectional area.

Spectrophotometry

While muscle fiber accumulation of EBD could be easily visualized using

standard epifluorescence of cyrosectioned muscle, attempts to directly visualize ICG

were unsuccessful despite the use of an high sensitivity back thinned CCD (EM-CCD

C9100-13, Hamamatsu, Hamamatsu City, Japan); therefore, spectrophotometric

quantification ICG and EBD accumulation of individual muscles (Sol, Gas, and TA) was

performed as previously described (Yaseen et al., 2009). In brief, muscles were

pulverized in lysing matrix D tubes (MP Biomedicals, Santa Ana, CA, USA) in DMSO

(Sigma Aldrich, St. Louis, MO, USA), followed by centrifugation. EBD and ICG

absorbance was subsequently measured at 620 nm and 780 nm, respectively on a

SpectraMax 5 spectrophotometer (Molecular Devices, Sunnyvale, CA), and normalized

to tissue weights to assess passive uptake of dye into the respective muscles.

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Clinical Studies

Heterogeneous Muscle Pathology is Revealed in DMD

This study was performed to assess the ability of MRI to provide supporting

evidence that DMD does not uniformly affect muscle and that there are differences in

muscle pathology, even within the same muscle. All aspects of these study were

approved by the Institutional Review Board of the University of Florida and the

Department of Defense’s Human Research Protection Office.

Study design

In this cross sectional study, 30 boys (age, 9.9 ± 2.7; height, 1.27 ± 0.3m; weight,

34.0 ± 2.6 kg; ambulatory, 27/30; glucocorticoid positive, 30/30; Vignos median score,

25 IQR%, and 75 IQR% = 2, 1, 2.5) with DMD confirmed by molecular genetic testing

(e.g. PCR amplification) and/or immunohistochemistry from biopsy and 6 age matched

unaffected control males (age, 7.7 ± 1.9 years; height, 1.31 ± 0.11 m; weight, 26.2 ± 4.1

kg; ambulatory, 6/6; glucocorticoid positive, 0/6; Vignos median score, 25 IQR%, and 75

IQR% = 1, 1, 1) volunteered to participate in MRI and were functionally scored on the

Vignos lower extremity functional scale (Lue et al., 2009). This study was HIPAA

compliant and approved by the Institutional Review Board at the University of Florida.

Upon thorough description of the study, written consent was obtained from a parent or

legal guardian and written assent was obtained from the pediatric subjects.

Magnetic resonance acquisition and measures

Prior to scheduled testing, subjects were asked to avoid any vigorous physical

activity and to use a wheelchair or equivalent mobility device when traveling to avoid

excessive walking. Acquisitions were performed on a 3.0 Tesla whole body human

system (Achieva, Philips Medical Systems, Best, Netherlands) at the McKnight Brain

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Institute at the University of Florida. With a parent and study staff member

accompanying the subjects in the testing room, subjects were placed in a supine

position within the magnet without sedation. Each subjects’ right lower leg was placed in

an eight-channel SENSE receive-only extremity coil (Invivo, Gainesville, FL, USA) with

the proximal end of the coil aligned with the fibular head and tibial tuberosity. Padded

weights were utilized to maintain the leg in a fixed position. T1 weighted 3D gradient

echo images were acquired (field of view, 12-24 x 12-14 cm2, voxel size = 0.75 x 0.75 x

2.8 mm3, 50 slices, flip angle = 20o, TR/TE = 24/1.8, number of averages = 2).

Acquisitions were made with and without fat suppression using spectral presaturation

with inversion recovery (SPIR). During data collection, subjects were shown a movie of

their choice on an in-magnet video display system to facilitate compliance and minimize

movement artifacts during the scanning.

MRI and function data evaluation

Six muscles (tibialis anterior [TA], extensor digitorum longus [EDL], peroneus

[Per], soleus [Sol], medial gastrocnemius [MG], and lateral gastrocnemius [LG]) were

analyzed by two reviewers (Figure 4-2). For each subject, 5 fat-saturated axial images

were chosen along the length of the leg to capture using specific anatomical landmarks

in a proximal to distal direction. Slice selection was acquired based on the percentage

distance (mean ± range) down the length of the tibia (described from starting at the tibial

plateau): proximal: ~10%, mid-proximal: ~19%, middle: ~26%, mid-distal: ~35% and

distal: ~43% from the tibial plateau as shown in Figure 4-3. MRI grades (Figure 4-4) for

each muscle were based on the Mercuri grading scale, previously used for a variety of

muscular dystrophies (Fischer et al., 2008; Kinali et al., 2011; Leung et al., 2015; Liu et

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al., 1993a; Mahjneh et al., 2012; Mercuri et al., 2002; Torriani et al., 2012; Wokke et al.,

2013).

To demonstrate the distribution of pathology within all muscles, an ordinal scale

of MRI grades were assigned to images, based on pathology and disease involvement

(Figure 4-4). Note that in T1-weighted fat suppressed images shown, hypo-intense

regions may be composed of either lipid infiltrate or fibrosis, and are radiographically

indistinguishable. Therefore, MRI findings are discussed as comprehensive disease

involvement rather than specifically fatty or fibrotic infiltration of muscle. Further, MRI

scores were binned in the following categories for evaluation purposes: not affected

(MRI Score = 0), moderately affected (MRI Score = 1-2), or severely affected (MRI

Score = 3-5). To assess the overall disease involvement of the lower leg muscles, MRI

scores for all 6 muscles were summed to obtain both an overall leg MRI score

(ScoreMulti; 5 grades per slice x 5 slices per muscle x 6 muscles = total possible score of

150) and a single middle slice leg MRI score (ScoreSingle; 5 grades per slice x 1 slice x 6

muscles = total possible score of 30). Similarly, Vignos scores were binned into four

categories: 1 (able to walk and climb stairs without assistance), 2 (able to walk and

climb stairs with aid of railing), 3-4 (walks, but climbs stairs slowly [<25s for 8 standard

steps] or not at all), and 5+ (unable to rise from chair, unable to walk independently, or

unable to walk at all). Furthermore, subjects’ ages and functional ability were compared

to ScoreMulti and ScoreSingle.

Magnetic Resonance Imaging Identifies Dystrophic Muscle in the Upper Extremity

As most MR studies in DMD have studied the lower extremities to identify

disease and pathology, we sought to better establish an understanding of the disease

involvement in the upper extremities (Fischmann et al., 2014; Willcocks et al., 2014;

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Wokke et al., 2014). This study is a component of the larger ImagingDMD study (PI:

Vandenborne), and data were collected in accordance to the standard operating

procedures established by ImagingDMD.

Study design

In this study, 19 boys with DMD (age, 10.8 ± 2.4) and 5 without (age, 11.5 ± 2.5)

were enrolled. DMD was confirmed by molecular genetic testing and/or

immunohistochemistry via biopsy. The study is HIPAA compliant, and approved by the

Institutional Review Board at the University of Florida. Following a thorough description

of the study, parental consent and subject assent was obtained.

Magnetic resonance acquisition and measures

All MR acquisitions were performed on a 3.0 Tesla whole body human system

(Achieva, Philips Medical Systems, Best, Netherlands) at the McKnight Brain Institute at

the University of Florida. A parent and study staff member accompanied the subject

into the testing room. Subjects were placed in a supine position within the magnet

without sedation. A sense flex coil was positioned over the subject’s shoulder and upper

arm (Invivo, Gainesville, FL, USA), and padded weights were utilized to maintain the

subject in a comfortable stable position. T1 weighted 3D gradient echo images were

acquired to best orient and position future scans. Non-fat saturated T1, a sequence of

CLEAR T2 images with varied TE times (20, 40, 60, 80, 100 ms), and three-point Dixon

images were additionally acquired. During data collection, subjects were shown a

movie of choice to facilitate compliance and minimize movement artifacts during the

scanning.

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MRI data analysis

Three muscles of interest (deltoid [Del], biceps brachii [BB], and triceps brachii

[TB]) were analyzed. MRI-T2 was calculated using software developed within the lab

IDL (ITT Excelis, Boulder Colorado, USA) by drawing regions of interest around the

designated muscles, taking care to avoid fascia. Qualitative MRI grades were given to

each muscle based on the Mercuri grading scale (Figure 4-4), as has been done for a

number of different muscular dystrophies (Fischer et al., 2008; Kinali et al., 2011; Leung

et al., 2015; Liu et al., 1993a; Mahjneh et al., 2012; Mercuri et al., 2002; Torriani et al.,

2012; Wokke et al., 2013). MRI-T2 relaxation times and MRI qualitative grades were

compared to both subject age and PUL scores, and a linear regression analysis was

performed.

Functional evaluation

The Performance of Upper Limbs (PUL) functional assessment was utilized for

this cohort of subjects. The PUL assessment was designed specifically to assess upper

limb function in DMD (Mayhew et al., 2013; Pane et al., 2014). The PUL assessment

includes 22 items, with quantitative identifiers identifying starting function, shoulder,

upper arm, and lower arm capabilities (Mayhew et al., 2013). Higher scores indicate

greater functional ability, and the highest score possible in our study is 83.

Near Infrared Optical Imaging Detects Acute Muscle Damage

This study is being performed to assess the ability of MRI and NIR optical

imaging to detect damaged muscle, as a result of an eccentric loading exercise protocol

and the natural disease progression of DMD. Full approval has been granted to

perform this study by the Institutional Review Board of the University of Florida, the

Human Research Protection Office of the Department of Defense, and an

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Investigational New Drug Exemption was obtained to use ICG in a non-indicated

pediatric population and an Investigational Device Exemption has been granted to use

the Clinical Tomography Laser Mammography System on patients not originally

intended for by the Food and Drug Administration. This study is still in progress, but will

still be discussed in this dissertation.

Study design

This study has two cohorts, healthy male adults (n = 12) and pediatric subjects

with DMD (n = 12). A visual schematic designing study design is shown in Figure 4-5.

The first cohort of subjects are healthy male adults who undergo exercise testing and

data (MRI, MRS, contrast enhanced NIR optical imaging, blood draw, questionnaires)

collection two days later. The second subject cohort is composed of boys (ages 10-15)

with confirmed DMD. This group will undergo imaging (MRI, MRS, and optical imaging) at a

single time point to assess the status of their muscle without exercise testing. For this

second cohort, a lower age limit of 10 years was selected, since upper extremity

muscles are affected at a later age than lower extremity muscles, with hand weakness

starting at the age of 10 years (Jones H et al. 2003).

Exercise testing

The first study arm (Figure 4-5) consists of healthy subjects who will undergo

eccentric and concentric forearm contractions in opposite arms on an isokinetic

dynamometer (Biodex Corp., Shirley, NY). Eccentric exercises have been shown to cause

temporary reversible damage to muscle by T2, serum creatine kinase, and delayed onset of

soreness (DOMS), whereas concentric exercises are known to cause minimal muscle

damage (Foley et al., 1999; Sesto et al., 2008). Eccentric loading of the wrist musculature

is achieved through slowly performing 6 sets of 10 repetitions of 120% of the 1 concentric

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repetition maximum with 90 seconds between sets. This exercise protocol has previously

been shown to result in increases in various indices of acute muscle damage after 48

hours (Davies et al. 2011), including T2 (+64%), creatine kinase (CK) serum levels, and

rating of delayed onset muscle soreness (DOMS) (Cleary et al., 2002). In addition, a

parallel concentric protocol is performed by the contralateral forearm. Concentric

exercise is known to result in only minimal muscle damage (Clarkson and Hubal, 2002;

Clarkson et al., 1986). Therefore, the concentric protocol serves as a control for other

potential effects of exercise on the measurements. The order of testing and the arm that

performs each protocol is randomized.

Magnetic resonance imaging and spectroscopy

Prior to scheduled testing, subjects are asked to refrain from any unnecessary

vigorous physical activity, and DMD subjects are asked to use a wheelchair or equivalent

mobility device when travelling to avoid excessive walking. All human MRI are performed in

a 3T whole body magnet (Philips Achieva Quasar Dual 3T) in the McKnight Brain Institute

at the University of Florida. When appropriate, a parent or staff member accompany

subjects into the testing room, and subjects lay in a supine position without sedation. Fat

suppressed and non-suppressed T1 weighted images are acquired to quantify the muscle

contractile area (fat-free muscle cross sectional area [CSA]) and maximal cross-

sectional area (CSAmax) of the forearm muscles. CSA and CSAmax are then normalized

to body surface area (BSA) to account for growth and differences in body size. Additionally,

T2 weighted imaging is performed to calculate T2 relaxation times based on mono-

exponential decay curve fittings to examine the distribution of affected versus unaffected

tissue. The total affected tissue volume (percentage of pixels with T2 values > 2 standard

deviations above control values) are recorded.

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Indocyanine green enhanced near infrared optical imaging

In order to determine whether NIR optical imaging can detect damaged and

dystrophic muscle, subjects with and without DMD will undergo the same imaging

procedures as the unaffected subjects. Following the MR procedures, all subjects have

NIR optical images taken of their forearms. All optical images are be acquired on a CTLM

Model 1020 scanner (Imaging Diagnostics System Inc., Ft. Lauderdale, FL). Dynamic

image acquisition will occur both 5 minutes before and after intravenous injection of IC-

Green (0.5 mg/kg; IC-Green, Akorn Inc., Buffalo Grove, IL) to assess enhancement kinetics.

The targeted region will be the belly of the wrist flexor muscles (Hillman et al., 2001;

Miyakawa et al., 2009). Dually MR and NIR visible fidicial markers on the skin of the

forearm are used to register MRI to NIR optical images. Optical image signal

enhancement kinetics are determined on a pixel-by-pixel basis based on changes in

signal intensity in the reconstructed images. The rate of tissue enhancement as well as

the final enhancement level at 5 min are used to create a rate of enhancement, area

under the curve, and a delayed enhancement map.

Blood draws and questionnaire

Following MR and NIR optical image acquisitions, blood is intravenously drawn for

analysis of muscle damage markers, such as creatine kinase. Additionally, a

questionnaire to determine the subjective rating of pain intensity is administered. This

questionnaire includes a visual analog scale ranging from “0 = no pain” to “10 = most

pain imaginable.” The change in this reported pain level is used as a construct for

DOMS. CK activity is determined in duplicate 0.02-ml aliquots at 37°C by using

standard photometric techniques and a Sigma diagnostic test kit (CK-10, Sigma

Diagnostics, St. Louis, MO).

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Figure 4-1. Radiant efficiency reaches a steady state level between 30 minutes to 12

hours following ICG an intravenous injection.

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Figure 4-2. Fat suppressed T1 weighted image shows muscles of the lower leg in

subjects with and without DMD, with arrows pointing to the TA (solid) and Per (dashed), highlighting intramuscular differences.

DMD

Control

Proximal Middle Distal

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Figure 4-3. Schematic representation of slice selections along the length of the lower leg.

Proximal

Distal

Mid-Proximal

Middle

Mid-Distal

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Figure 4-4. The qualitative MRI grading scale used to assess pathology within DMD

muscle.

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Figure 4-5. Schematic study design of clinical study utilizing NIR to detect muscle

damage.

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CHAPTER 5 NEAR INFRARED OPTICAL IMAGING IN A PRE-CLINICAL MODEL OF ACUTE

MUSCLE DAMAGE

Introduction

Techniques to Assess Muscle Damage

Muscle damage is an important and unavoidable outcome of many pathological

states such as muscular dystrophies, inflammatory myopathies, and physical trauma.

Several pre-clinical models have been developed to induce acute muscle damage,

including eccentric loading (Armstrong et al., 1983; Clarkson and Hubal, 2002; Clarkson

et al., 1986; Proske and Morgan, 2001), immobilization-reloading (Frimel et al., 2005b),

and myotoxin injection (Gutiérrez and Ownby, 2003; Lomonte and Gutiérrez, 1989;

Lomonte et al., 1993, 2003). In particular, eccentric loading of muscle has

demonstrated ability to robustly disrupt sarcolemmal integrity in a well controlled

manner (Armstrong et al., 1983; Childers et al., 2002; Clarkson et al., 1986; Lovering

and De Deyne, 2004; Proske and Morgan, 2001). A compromised sarcolemma

releases muscle enzymes such as creatine kinase, while concurrently passively taking

up large serum proteins and markers such as Evan’s blue dye (Hamer et al., 2002) and

small inorganic dyes such as procion orange (Barton-Davis et al., 1999; Greelish et al.,

1999; Nguyen and Tidball, 2003; Palacio et al., 2002; Spencer and Mellgren, 2002;

Tidball and Wehling-Henricks, 2007; Villalta et al., 2011) into damaged muscle.

Muscle pathology has been measured by a number of techniques, all of which

possess inherent limitations. These include including muscle biopsy, serology,

functional measures, and imaging methods. Muscle biopsy, while the most direct

measure of pathology, has limited capacity to be considered a longitudinal measure of

muscle pathology in clinical trials, due to the necessity of repeated sample collections.

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Serology and functional testing, while providing a proxy to the overall state of muscle

health, fail to sensitively localize pathology, instead providing information regarding the

general health of all muscles in the body and are complicated by changes in lean body

mass typically associated with myopathy. MRI has evolved as a noninvasive method to

detect and quantify muscle pathology (Dunn and Zaim-Wadghiri, 1999; Frimel et al.,

2005a, 2005b; Kobayashi et al., 2008; Vohra et al., 2015; Walter et al., 2005), but has

several limitations, such as cost, speed of operations, contraindications for patients with

metallic implants, claustrophobia, and compliance issues (Brockmann et al., 2007; Dunn

and Zaim-Wadghiri, 1999; Lovering and De Deyne, 2004). An attractive alternative

would be the use of clinically approved fluorescent optical contrast agents to image

muscle damage in vivo similar to those currently used for traditional histological

measurements (Baudy et al., 2011; Inage et al., 2015; Kossodo et al., 2010).

Near Infrared Imaging and Indocyanine Green

Fluorescent optical imaging is a widely used technique in pre-clinical models of

disease to detect pathology by fluorescent dyes, proteins, and conjugates (Frangioni,

2003; Tan and Jiang, 2008). By utilizing optical imaging in the NIR range (700-1,000

nm), two primary advantages exist over traditional fluorophores that operate at shorter

wavelengths: deeper photon penetration within tissues and minimal tissue

autofluorescence (Frangioni, 2003; Weissleder, 2001; Weissleder and Ntziachristos,

2003). When imaging in the NIR range, penetration of signal can reach up to 30-40 cm

of tissue depth, overcoming some of the scattering limitations that other fluorescent

imaging techniques at smaller wavelengths encounter, overcoming the limitation of only

being able to image superficial surface structures (Ntziachristos et al., 2002, 2005).

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The first, and still only FDA approved NIR fluorescent contrast agent is ICG, a

775 Dalton di-sulfonated fluorescent dye with a very well characterized safety profile,

demonstrating minimal toxicity in humans (Alford et al., 2009). ICG is rapidly bound to

albumin within the circulation, thus acts as a blood pooling NIR fluorescent agent,

highlighting vasculature (Chen et al., 1999; Desmettre et al., 2000; Kobayashi et al.,

2014; Raabe et al., 2003). Additionally, ICG passively accumulates in tumors through

the enhanced permeation and retention (EPR) effect in a similar manner to gadolinium,

as used for contrast enhanced MRI (Corlu et al., 2007; Ntziachristos et al., 2000). The

EPR effect is a phenomena by which certain molecules preferentially are uptaken into

surrounding tissue. This is most frequently due to pores and fenestrations in the target

tissue or vascular endothelium supplying such tissue, as often observed during

inflammatory states and cancer (Fang et al., 2011; Maeda, 2012; Maeda et al., 2000;

Radermacher et al., 2009).

ICG enhanced NIR optical imaging has been used for several other clinical

purposes, such as imaging of the vasculature of the retina (Chen et al., 1999;

Desmettre et al., 2000; Herbort et al., 1998; Mueller et al., 2002), breast cancer tumors

(Gurfinkel et al., 2000; Ntziachristos et al., 2000; Troyan et al., 2009; Verbeek et al.,

2014; Zelken and Tufaro, 2015), cerebral vasculature and tumors (Haglund et al., 1996;

Raabe et al., 2003), gastrointestinal vessels (Borotto et al., 1999), and cardiac

vasculature and myocardial perfusion (Nakayama et al., 2002; Taggart et al., 2003).

Despite its widespread use in other organs, there is only one recent occurrence in the

literature as method to image muscle pathology is a recent development (Inage et al.,

2015). With this in mind, we hypothesized that ICG will behave similar to EBD (Hamer

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et al., 2002), and accumulate in damaged muscle fibers, allowing for quantification of

muscle damage in a longitudinal and in vivo manner. Importantly, because there are no

FDA approved MRI blood pooling imaging contrast agents approved for use in pediatric

studies, ICG could fulfill an important role in children as a NIR optical imaging contrast

agent.

Results

Animal Procedures

Throughout the duration of experiments, all mice maintained body weight within

10% of pre-immobilization weights and three needed recasting because of abrasive

lesions developed on the skin. In these rare cases, topical antibiotics were applied to

address abrasive lesions, all with resolution. One mouse unexpectedly expired

following data collection in the MR scanner and was not used for data analysis. The

single hindlimb casting procedures were otherwise well tolerated through the duration of

experiments.

Near Infrared Imaging of Mouse Hindlimbs

When comparing pre-immobilization and day 0 reambulated hindlimbs, no

difference was observed between immobilized and non-immobilized hindlimbs.

Throughout the reambulation phase, radiant efficiency in the immobilized-reambulated

hindlimb significantly peaked by day 2 and was 3.86 fold higher than pre-casted values,

followed by a return back to baseline by day 7 (Figure 5-1). Interestingly, the

contralateral hindlimb also demonstrated an increase (2.45 fold) in total radiant

efficiency between day 2 reambulation and pre-casted values, but did not reach

significance. NIR images are also presented in the left panel of Figure 5-1, allowing for

qualitative demonstration of the immobilized (right) vs. non-immobilized (left) hindlimbs.

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The immobilized-reambulated hindlimbs at days 2 and 3 of reambulation were

significantly different than contralateral hindlimbs, pre-immobilized hindlimbs, and day 0

of the immobilized-reambulated limbs.

Magnetic Resonance Imaging and Spectroscopy Confirm Muscle Damage Following Cast Immobilization and Reambulation

Pre-immobilization hindlimbs demonstrated homogenous contrast in the all their

muscles on T2 weighted MRIs in both hindlimbs (Figure 5-2). The soleus muscle of the

immobilized-reambulated hindlimb demonstrated the greatest T2 changes, peaking at

two days following the cast removal (1.41 fold change increase in T2), and returning

values comparable to baseline by day 5 of the reambulation phase (Figure 5-2A).

Interestingly, both the gastrocnemii and tibialis anterior muscles of the immobilized-

reambulated hindlimbs demonstrated a subtle, yet significant difference (1.13 and 1.14

fold changes, respectively) from their contralateral non-immobilized limbs at the

initiation of reambulation, but this was not significant from pre-immobilization measures

(Figure 5-2B and 5-2C).

Further confirmation of the damage and recovery of the soleus muscle is

provided by 1H2O-T2 spectroscopic analysis (Figure 5-3). Representative mono-

exponential T2 curves demonstrate differences in decay rates of 1H2O-T2 signal between

the immobilized-reambulated and control hindlimbs (Figure 5-3A). Following multiple

exponential decomposition by non-negative least squares (NNLS) analysis (Bryant et

al., 2014), a representative characteristic long T2 component is shown in Figure 5-3B.

Similar to MRI-T2 measures, 1H2O-T2 values demonstrated a similar trend of damage

peaking at the second day of reambulation with a 1.28 fold change compared to pre-

casted values, followed by an eventual return to baseline (Figure 5-3C). Within the

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entire cohort of mice, long T2 components (T2 > 80ms) were present in half of mice at

the peak of muscle damage (day 2) during reambulation (Table 5-1). The occurrence of

long T2 components in both hindlimbs is summarized in Table 5-1. The only

demonstration of a long T2 component in the non-immobilized limbs occurred in 10% of

mice at the second day of reambulation.

Histology of Healthy and Damaged Muscle

Appearance of EBD accumulation at both the microscopic and macroscopic

levels within the soleus muscles confirmed the well-established time course of damage

and recovery during reloading following immobilization. Figure 5-4A qualitatively

demonstrates that EBD is minimally taken up into soleus muscle fibers immediately

following cast removal. Quantitative demonstration of dye uptake into the immobilized-

reambulated and contralateral control solei are demonstrated in Figure 5-4B. The

percentage of EBD positive fibers in the Gas and TA was not significantly elevated and

was comparable to baseline values throughout the reambulation period (data not

shown). By the second day of reambulation, fibers of the immobilized-reambulated

soleus appeared with 47.1 ± 15.6% of the fibers being EBD positive in a checkerboard

patter. EBD uptake into the contralateral soleus was less with 15.1 ± 6.3% of the being

EBD positive, but this was not significantly different than day 0 of soleus muscle the

same control limb. At the end of the reambulation week, EBD signal is again less

visible with only 5.6 ± 2% and 1.6 ± 1.8% of the fibers being EBD positive for the

immobilized-reambulated and control hindlimbs, respectively.

H&E stained sections of the immobilized and reambulated soleus at various time

points throughout the week demonstrated the well characterized histopathological

features of muscle damage and inflammation, most noticeably at the second day of

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reambulation (Frimel et al., 2005b). At the peak of damage (day 2), widened extra

cellular spaces, massive macrophage infiltration, a decreased density of muscle fibers,

and variability in fiber size were all observed. A final observation made was that

throughout the week of reambulation, there was an absence of centrally nucleated

myofibers, as centrally nucleated fibers do not typically occur until greater than ten days

following insult to muscle (Frimel et al., 2005b).

Spectrophotometric Quantification of ICG and EBD

Muscle lysates were analyzed by a spectrophotometer to quantify both EBD and

ICG accumulation within each lower leg muscle throughout the week of reambulation

(Figure 5-5). Absorbance, normalized to muscle weight, was determined at 620 nm

(EBD; Figure 5-5A) and 780 nm (ICG ; Figure 5-5B) and demonstrated significant peaks

in signal at the second day of reambulation for both dyes (EBD: 1.72 fold change and

ICG: 1.87 fold change), followed by a return back to baseline by the end of the week of

reambulation (EBD: 1.21 fold change and ICG: 1.20 fold change). Absorbance of the

gastrocnemii and tibialis anterior lysates were comparable to background noise levels

(data not shown), indicating minimal dye uptake per tissue weight into these two muscle

groups.

Correlation Between MRI and Near Infrared Optical Imaging

To determine if a correlation exists between muscle damage measures and total

radiant efficiency, 1H2O-T2, MRI-T2, and spectrophotometric absorbance values were

compared to total radiant efficiency at the peak of muscle damage. Figure 5-6 shows

the linear relationships between radiant efficiency and 1H2O-T2 (5-6A, r2 = 0.72) and

MRI-T2 (5-6B, r2 = 0.57) in the immobilized-reambulated and control muscles. Table 5-2

quantitatively shows the significance of linear regression correlations between NIR

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optical imaging radiant efficiency compared to 1H2O-T2, MRI-T2, and optical density 780

nm / mg tissue. Significant correlations (Table 5-2) are demonstrated only when

comparing the solei measures, and neither the gastrocnemii, nor tibialis anterior

muscles demonstrate any significant correlations to the total radiant efficiency.

Spectroscopy was not performed in the gastrocnemii or tibialis anterior, so correlations

could not be drawn for spectroscopy from these muscles. Finally, in order to determine

the robustness of each imaging modality, Cohen’s D effect sizes were calculated to

determine the magnitude of difference between day 0 and day 2 of the immobilized–

reambulated hindlimbs. The effect sizes for MRI-T2, 1H2O-T2, and NIR optical imaging

were 1.79, 1.39, and 1.57, suggesting comparable magnitudes of differences between

each of the imaging modalities.

Discussion

The main purpose of this study was to assess the ability of an FDA approved NIR

fluorescent contrast agent (ICG) and NIR optical imaging to noninvasively image muscle

in a well-characterized model of acute muscle damage and recovery. Muscle damage

and recovery in the soleus muscle of immobilized-reambulated mouse hindlimbs was

visualized and quantified using ICG enhanced NIR optical imaging, with further

supporting confirmation provided by MRI-T2, 1H2O-T2, histology, and spectrophotometric

assessments. The time course of muscle damage and recovery following immobilization

and free reambulation using both imaging modalities agreed with histological and

biochemical analysis of the extracted tissues. This study demonstrates the ability of

ICG enhanced NIR optical imaging to two dimensionally visualize and quantify muscle

damage in vivo.

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Near Infrared Optical Imaging as a Novel Method to Assess Muscle Damage

By utilizing a NIR optical imaging enhanced with an FDA approved contrast

agent (ICG) to assess muscle health, it is anticipated that this foundational work will be

able to quickly translate to clinical application. We chose to use ICG as a NIR

fluorescent blood-pooling agent because ICG behaves similarly to Evan’s blue dye

(Hamer et al., 2002) in that it binds to serum albumin and it was hypothesized that ICG

would accumulate in damaged muscle cells with compromised sarcolemmal

membranes. We exploited that optical imaging in the NIR range allows for deep tissue

imaging and minimal tissue autofluorescence, allowing for imaging of deep muscle

(Frangioni, 2003). Additionally, NIR optical imaging has the advantage that it can

compliment current MR techniques (Ntziachristos et al., 2000), with the additional

advantage that data acquisition can be achieved in a much more cost efficient manner

and shorter time. While NIR optical imaging of ICG has been demonstrated clinically in

other tissues of the body, the primary use of NIR optics has been to perform NIR

spectroscopic (NIRS) analyses to assess changes in perfusion status has been

performed in muscle, though the ability to image muscle damage in a rat model using

an ICG conjugate has been demonstrated (Inage et al., 2015).

In this study, we sought to quantify and visualize muscle damage through using

an established model of hindlimb immobilization followed by reambulation (Frimel et al.,

2005b). This model of hindlimb immobilization was chosen to test the ability of NIR

optical imaging to detect and quantify muscle damage for this study as the technique

demonstrates specific damage to the soleus, in the deepest of the lower hindlimb

muscles (3.54 ± 0.43 mm below the surface of the posterior skin). Furthermore, the

time course of muscle damage and recover is well characterized, and the uncasted,

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contralateral hindlimb allows for an internal control for each mouse (Frimel et al.,

2005b). NIR optical images were used to visualize muscle damage and was confirmed

by MRI-T2, mean 1H2O-T2, histology, and ICG/EBD accumulation in isolated muscles.

Importantly, a return to baseline levels of all measures of muscle damage was

observed, indicating that NIR optical imaging is can be used to image both damage and

recovery of muscle. By comparing the effect sizes of MRI-T2 (d = 1.79), 1H2O-T2 (d =

1.39) and NIR optical imaging (d = 1.57), we determined that NIR imaging was similar in

its ability to detecting muscle damage as MRI. Additionally, we found a significant linear

relationship between NIR optical imaging and 1H2O-T2 and MRI-T2, further solidifying

confirming NIR optical imaging as a capable non-invasive modality to detect muscle

damage. Even though MRI is frequently used in pediatric populations, no MRI blood

pooling contrast agents are currently clinically indicated for imaging muscle damage.

ICG, with long standing FDA approval and a safety record, could adequately fulfill this

void in the clinic arena in conjunction with NIR optical imaging (Frangioni, 2003).

From of the breadth of information that can be revealed through MR

spectroscopic analysis of muscle, we utilized this to attempt to better understand the

generation of fluorescent signal from ICG within the damaged soleus muscle (Araujo et

al., 2014; Bryant et al., 2014; Fan and Does, 2008; Frimel et al., 2005b; Hollingsworth,

2014; Walter et al., 2005). Because of significant correlations between NIR optical

imaging measures to MRI-T2, 1H2O-T2, and tissue accumulation of EBD/ICG in the

soleus, it can be hypothesized that the NIR optical imaging is pre-dominantly due to

induced pathology within the soleus rather than either the gastrocnemius or tibialis

anterior. We chose to investigate the multi-component decay of 1H2O-T2 signals,

129

allowing for differentiation between intracellular (20-40 ms), extracellular (80-120 ms),

and protein associated (<10ms) 1H2O-T2 contributions, in an attempt to elucidate what

fluid compartment ICG may end up associated with (Ababneh et al., 2005; Araujo et al.,

2014; Bryant et al., 2014; Gambarota et al., 2001). Demonstration of a long 1H2O-T2

component has been observed during edematous and inflammatory states within

muscle, indicating a large contribution of extracellular fluid within the muscle (Bryant et

al., 2014; Fan and Does, 2008). Interestingly, at the second day of reambulation

following immobilization, the long 1H2O-T2 component was present in half of the

immobilized hindlimbs. This concurrently occurred with an increased NIR optical

imaging signal, suggesting that immobilization followed by reambulation induces muscle

edema, allowing for pooling of ICG in the damaged muscles. Histological results were

consistent with previously reported data, as we observed expanded interstitial space

and infiltrating cells in only the immobilized-reambulated soleus (Bryant et al., 2014;

Frimel et al., 2005b). Lastly, biochemical analysis of tissue EBD and ICG accumulation

at the peak of muscle damage confirmed that soleus ICG content was 12.8 and 5.2 fold

higher than the gastrocnemius and tibialis anterior, respectively. The disproportionate

uptake of ICG into the soleus indicates that the immobilized-reambulated soleus

(Figures 5-5B and 5-6B) most likely is the primary contributor to fluorescent signal as

seen in Figure 5-1.

It is important to consider that MR and NIR optical imaging assess different

properties of tissue, with MR assessing inherent magnetic properties of tissue and NIR

optical imaging assessing vascular perfusion and membrane stability. NIR optical

images taken within minutes after injection (Figure 4-1) show changes in major

130

vasculature and may significantly alter the NIR signal, as previously shown in muscle

(Mancini et al., 1994; Možina, 2011). By waiting an hour after injections, we ensured

that the fluorescent signal observed was predominately from muscle uptake rather than

vascular contributions. As previously described, contralateral non-immobilized limbs

experience an overload of stimuli, which may explain the insignificant, but observable

increase in total radiant efficiency observed at day two of the non-immobilized hindlimb

in Figure 5-1 (Caron et al., 2009). For these reasons, it is suggested that NIR optical

imaging complement, rather supplement MR technology, providing additional

information in a cost and time efficient manner.

Limitations to Experiments

ICG, as a non-targeted contrast agent demonstrates both advantages and

disadvantages in this study. An advantage is that it can be used to quantitatively

assess muscle damage and recovery. Because it is not specific to the pathology

induced by the immobilization-reambulation technique used in this manuscript, it can

theoretically be applied to other pathologies and diseases affecting muscle. Though

ICG-albumin uptake is nonspecific, with future modifications, it may provide a platform

for targeting specific cell moieties to add further diagnostic and therapeutic value (Kraft

and Ho, 2014; Sheng et al., 2014). Another limitation is the lack of spatial sensitivity

while using contrast enhanced NIR optical imaging. Though NIR optical imaging was

deemed comparably sensitive to MR techniques to detect muscle damage by

magnitude of effect size assessment, additional technology development should be

pursued to increase spatial sensitivity of the technology. Due to the limit of TE sampling

in the STEAM MRS acquisitions, it is quite possible that we were only sensitive to large

differences in 1H2O-T2 fractions and potentially, with greater TE sampling and signal-to-

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noise, a long component may have been easier to resolve in damaged muscle (Bryant

et al., 2014).

Summary

I demonstrated the feasibility of using a novel technology (NIR optical imaging)

with an FDA approved fluorescent contrast dye (ICG) to tomographically assess and

image acute muscle damage and recovery in a well-characterized model of muscle

damage in mice. I have also optimized each of the imaging modalities (MRI, MRS, and

NIR Imaging) to quantify and visualize the muscle damage. Because of the cost

effectiveness, lack of ionizing radiation or radioactive substrates and longitudinal

capabilities, NIR optical imaging can be used for a diverse range of purposes (Baudy et

al., 2011; Inage et al., 2015; Možina, 2011; van de Ven et al., 2010; Verbeek et al.,

2014). By using a clinically approved contrast dye with NIR optical imaging, a

multipurpose, non-invasive, and safe imaging technology, it is anticipated that this

technology can be expeditiously applied to other diseases of the muscle, both in

animals and humans.

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Figure 5-1. Two-dimensional NIR optical imaging shows an increase and recovery of

fluorescent signal in muscle during reambulation following immobilization.

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Figure 5-2. MRI-T2 shows damage and recovery of soleus, but not gastrocnemius nor

tibialis anterior muscles during reambulation. Representative MR images are shown along the left panel for each of the days of reambulation. Soleus (5-2A), gastrocnemius (5-2B), and tibialis anterior (5-2C) MRI-T2 relaxation times are shown before immobilization and throughout the week of reambulation.

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Figure 5-3. Spectroscopic findings confirm increase in 1H2O-T2 and reveal long T2

components in the soleus of immobilized-reambulated hindlimbs. Characteristic mono-exponential T2 decay curves are shown (5-3A), as well as a representative characteristic long T2 component (5-3B). 1H2O-T2 relaxation times before immobilization and the week of reambulation are shown (5-3C).

135

Table 5-1. Frequency of long 1H2O-T2 component in damaged hindlimbs of immobilized-reambulated mice.

Reambulation Day IMM (% with long component)

Non-IMM (% with long component)

Pre-immobilized 0 0

0 0 20

1 0 20

2 10 50

3 0 30

5 0 20

7 0 0

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Figure 5-4. Histological assessment confirms damage and recovery in the reambulated

soleus muscle of the immobilized-reambulated hindlimb at the second day of reambulation. Representative immunofluorescence and H&E images are shown (5-4A) as well as quantification of EBD positive fibers throughout the week of reambulation (5-4B).

137

Figure 5-5. Spectrophotometric assessment confirms dye uptake into the soleus muscle

at the peak of muscle damage. Absorbance, measuring EBD (5-5A) and ICG (5-5B) throughout the week of reambulation are quantified.

138

Figure 5-6. Increased radiant efficiency correlates to increased markers of damage in

the soleus muscle. Correlations between radiant efficiency and 1H2O-T2 (5-6A, r2 = 0.72) and MRI-T2 (5-6B, r2 = 0.57) are shown.

139

Table 5-2. NIR optical imaging radiant efficiency measures were correlated to 1H2O-T2, MRI-T2, and Optical Density 780 nm / mg tissue, and r2 values (with associated p values in parentheses).

1H2O-T2 MRI-T2 Absorbance 780 nm / mg

tissue

So

l

0.72 (<0.001) 0.57 (<0.001) 0.46 (0.002)

Ga

s

N/A 0.21 (0.051) 0.001 (0.156)

TA

N/A 0.12 (0.167) <0.001 (0.999)

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CHAPTER 6 QUANTIFICATION OF MUSCLE PATHOLOGY IN MDX AND GSG -/- MICE

Introduction

Muscular Dystrophies Render Muscle More Susceptible to Damage

Phenotypically, the muscular dystrophies are defined a common clinical

presentation of progressive, degenerative, and irreversible muscle weakness (Amato

and Griggs, 2011; Flanigan, 2012). With the advent of modern sequencing

technologies, over 50 genetically identifiable forms of muscular dystrophy have been

identified, based on the genetic mutation causing pathology to the muscle (Amato and

Griggs, 2011; Kang PB and Griggs RC, 2015). The most common of the muscular

dystrophies is DMD, with an incidence of 1 in 5,000 live male births (Greenberg et al.,

1988; Mah et al., 2014; Mendell et al., 2012). DMD is caused by a mutation in the

dystrophin gene, which encodes for the dystrophin protein. Another form of muscular

dystrophy is LGMD-2C, which results from mutations in the SGCG gene, which encodes

for production of the γ-sarcoglycan protein. Dystrophin connects the intracellular

contractile actin to the DAG complex, stabilizing the sarcolemmal membrane during

muscle contractions (Ervasti and Campbell, 1991; Hoffman et al., 1987). γ-sarcoglycan

is one of several sarcolemmal transmembrane glycoproteins that forms the DAG

complex, and when absent, leads to increased susceptibility to injury in the

sarcolemma, as seen in LGMD-2C (Amato and Griggs, 2011; Barton, 2006).

Mdx and Gsg -/- Mouse Models

Currently, no cures for the muscular dystrophies exist, though many promising

therapies have shown promise in preclinical and early clinical trials. Many therapies

have been developed through extensive use of protein knockout mice, which, similar to

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their human counterparts, lack proteins specific to their disease. Two commonly

studied dystrophic mouse models are the DMD (mdx) and LGMD-2C (gsg -/-) null mouse

strains (Hack et al., 1998; Hoffman et al., 1987; McNally et al., 1996a, 1996b; Sicinski et

al., 1989). These models have been effectively used to study the natural progression of

both diseases, as well as develop a variety of therapies to mitigate pathologic insult

from the diseases, such as pharmacological interventions (Anderson et al., 1996;

Barton et al., 2005; Durham et al., 2006), exon skipping (Echigoya et al., 2015;

Goyenvalle et al., 2015; Matsuo et al., 1991), viral delivery (Barton, 2010; Hayashita-

Kinoh et al., 2015), and RNA restoring therapies (Barton-Davis et al., 1999; Welch et al.,

2007). Interestingly, mdx exhibit a characteristic progression of disease, initially

experiences a large assault of inflammatory cascades, followed by a plateau of recovery

due to their ability to upregulate utrophin, a homolog of dystrophin (McDonald et al.,

2015; Vohra et al., 2015). The gsg -/- mice demonstrate a more severe phenotype,

demonstrating decreased growth, premature death, and severely dystrophic muscle as

the mice age (Hack et al., 1998). The ability to sensitively demonstrate mitigation of

disease, or worsening of pathology is a critical role of biomarkers. Therefore, a pressing

need exists for sensitive biomarkers to detect changes in disease progression,

additional pathologic insult in dystrophic muscle, and response to potential therapies in

the muscular dystrophies, both in preclinical and clinical models.

Techniques to Assess Muscle Damage Due to Dystrophies

Assessment of the ability to quantify inducible damage and therapeutic

intervention in dystrophic muscle is limited to several modalities, including histological

markers of muscle damage such as Evan’s Blue Dye (Frimel et al., 2005b; Hamer et al.,

2002) and Procion orange dye (Consolino and Brooks, 2004) and ex vivo muscle

142

contraction (Hakim et al., 2013) assessment. Though effective in animals, these

preclinical measures of muscle health are not translatable to clinical studies for ethical

and safety reasons, and translational work would be expedited by modalities that are

functional in both preclinical and clinical experiments. An optimal means of measuring

muscle function would be applicable both to animals and humans, allowing for

acceleration of preclinical findings to humans. Recently, magnetic resonance imaging

(MRI) and spectroscopy (MRS) have provided the ability to study disease in a

longitudinal and non-invasive fashion both in humans and animals with DMD (Carlier et

al., 2012; Dunn and Zaim-Wadghiri, 1999; Finanger et al., 2012; Forbes et al., 2014b;

Hollingsworth et al., 2013; Kobayashi et al., 2008; Walter et al., 2005). Changes in MRI-

T2 relaxation times reflect a number of different pathological processes that may occur

in muscle, such as generalized damage (Mathur et al., 2011), edema (Bryant et al.,

2014; Fan and Does, 2008), fatty tissue infiltration (Elder et al., 2004), and fibrosis (Li et

al., 2012). Though a plethora of helpful information can be gathered from MR

techniques, several limitations do exist, including adequate compliance of alert children,

cost, and speed of operation, and thus, NIR optical imaging may be a complimentary

and alternative technology to collect non-invasive, quantitative, and repeatable

information (Baudy et al., 2011; Brockmann et al., 2007; Kossodo et al., 2010; Lovering

et al., 2009).

Near Infrared Optical Imaging and Indocyanine Green & Current Uses

As described in the previous section, ICG enhanced NIR optical imaging has

recently developed as an effective application for several clinical applications. It has

been utilized to assess perfusion (Desmettre et al., 2000; Kobayashi et al., 2014; Raabe

et al., 2003) and identify tumor (Corlu et al., 2007; Ntziachristos et al., 2000; Zelken and

143

Tufaro, 2015). Additionally, NIR spectroscopic techniques have demonstrated the

ability to monitor blood volume, oxygenation, and flow dynamics using fluorescent dyes

(Guenette et al., 2011; Koga et al., 2012; Towse et al., 2011). To date, only two studies

have utilized contrast enhanced NIR imaging techniques to assess muscle pathology.

Previous studies have assessed muscle damage and correction of disease in mdx mice

using a caged NIR cathepsin B substrate (Baudy et al., 2011). Inage et al utilized an

acute model of muscle damage, and through ICG enhance NIR optical imaging,

detected inducible muscle damage (Inage et al., 2015). Building off of our own findings

that ICG enhanced NIR optical imaging can detect well characterized damage to muscle

(Figure 5-1), we were interested in seeing if the same principles are able to assess and

quantify muscle damage, resulting from the natural progression of two different

muscular dystrophies, as well as exacerbation and mitigation of muscle pathology

through additional interventions.

Objectives

Here, we intend to demonstrate ICG contrast enhanced NIR optical imaging as a

safe and sensitive modality to detect and quantify damage to muscle, resulting from the

natural progression of two different muscular dystrophies in vivo. Additionally, we

intend to observe increases in dye uptake of additionally damaged older mdx mouse

muscle by subjecting mice to an eccentric exercise treadmill protocol. Finally, we

attempt to quantify and visualize mitigation of disease burden on gsg -/- mice following

restoration of the missing γ-sarcoglycan protein by rAAV therapy.

144

Results

Near Infrared Optical Imaging and Magnetic Resonance Imaging and Spectroscopy of Dystrophic Mice Allows for Identification of Muscle Pathology

An increase of fluorescence was observed in mdx and gsg -/- mice as compared

to their unaffected counterparts (Figure 6-1A). Though elevated compared to control

counterparts (6-1B), the mdx (6-1C) and gsg -/- (6-1D) strains of mice were

indistinguishable from each other. MRI-T2 times of the posterior compartment in the

lower leg were significantly elevated in both dystrophic mouse models (Figure 6-1E).

Similarly, 1H2O-T2 measurements also indicated elevated relaxation times of dystrophic

muscle, as compared to healthy unaffected tissue (Figure 6-1F). Note uniformity of

control mice (6-1G) and the hyperintense patches, indicating muscle damage, within

mdx (6-1H) and gsg -/- (6-1I) hindlimbs highlighted by arrows in representative MRI

images. Upon comparison of NIR optical imaging values to MRI-T2 and 1H2O-T2

relaxation times, separation was observed for both comparisons (Figures 6-2B and 6-

2B, respectively).

Eccentric Loading by Downhill Treadmill Running Induces Quantifiable Muscle Damage to Older Mdx Mice

When compared to baseline values acquired before downhill treadmill running,

older mdx mice demonstrated significant increases of measurable fluorescence in both

the forelimbs and hindlimbs following the treadmill exercising (6-3A). Interestingly, MRI-

T2 relaxation times only showed significant increases in the forelimbs, and not in the

hindlimbs (6-3B). When measuring 1H2O-T2 before and after treadmill running, only the

hindlimbs demonstrated a significant difference, while the forelimbs did not (6-3C).

Representative NIR optical imaging (6-3D) and MRI (6-3E) images are shown.

145

Additionally, correlation plots are shown comparing NIR optical imaging to MRI-T2 (6-

4A) and 1H2O-T2 (6-4B), suggesting that a significant and linear correlation exists

between all measures.

Restoration of γ-Sarcoglycan in Muscle is Observed by Near Infrared Optical Imaging

Before, and six weeks after administration of the missing γ-sarcoglycan gene by

intramuscular AAV injections, non-invasive data (NIR optical imaging, MRI, MRS) was

collected from the gsg -/- mice. Fluorescence from ICG was decreased significantly in

the treated hindlimbs compared to both pre-injection values of the same limbs, as well

as non-injected hindlimbs (6-5A). Contrasting the findings in the hindlimbs, the

forelimbs demonstrated no significant changes in either the control or AAV groups

following intramuscular injections into the hindlimbs (6-5B). Representative NIR optical

imaging images for the baseline gsg -/- (6-5C) and to-be-treated gsg -/- (6-5D) mice, as

well as post-treatment images of non-treated gsg -/- (6-5E) and treated gsg -/- (6-5F)

mice are shown. Building upon the NIR optical imaging findings, both MRI-T2 (6-5G)

and 1H2O-T2 (6-5H) demonstrated similar trends of decreased markers of muscle

damage following the AAV treatment. Similarly, representative MRI images highlighting

hyperintense regions of pathology are shown of baseline gsg -/- (6-5I) and to-be-treated

gsg -/- (6-5J) mice, as well as post-treatment images of non-treated gsg -/- (6-5K) and

treated gsg -/- (6-5L) mice. Note that the dashed gray box indicated in figures 6-5A, 6-

5B, 6-5G, and 6-5H indicate the 95% of control values for each respective graph.

Similar to the previous experiments, correlation plots are shown comparing NIR optical

imaging to MRI-T2 (6-6A) and 1H2O-T2 (6-6B), demonstrating separation of the AAV

treated hindlimbs compared to the rest of the data.

146

Magnitude of Effect Size is Comparable Between NIR Optical Imaging, MRI, and MRS

Cohen’s D values were calculated to determine the magnitude of effect size of

NIR optical imaging versus both MRI-T2 and 1H2O-T2. NIR optical imaging demonstrated

strong capabilities to differentiate control from mdx mice, control from gsg -/- mice, the

ability of eccentric downhill treadmill running to induce damage to older mdx mice, and

the restorative capabilities of AAV therapy in gsg -/- mice (Table 6-1). Importantly, MRI-

T2 and 1H2O-T2 measures all demonstrated strong magnitudes of difference to detect

changes in muscle health as well.

Histological Assessment of Tissue Confirms Restoration of γ-Sarcoglycan

Immunofluorescence confirmed the restoration of γ-sarcoglycan in the gsg -/-

muscles that received the therapeutic AAV treatments (Figure 6-7). Tissues were co-

stained for γ-sarcoglycan, and wheat germ agluttinin to visualize the sarcolemmal

boundaries and DAPI to visualize nuclei. In the hindlimbs that received AAV treatment,

WGA and γ-sarcoglycan co-localized, but in the non-treatment group, no γ-sarcoglycan

was present.

Discussion

Major Findings

The goal of this investigation was to demonstrate efficacy of contrast enhanced

NIR optical imaging using an FDA approved contrast agent to detect and quantify

muscle pathology in two different dystrophic mouse models in a safe, repeatable and

non-invasive fashion. Mdx and gsg -/- mice demonstrated higher radiant efficiency

values than healthy counterparts, indicating uptake of the NIR fluorescent dye ICG into

damaged muscles, with further confirmation provided by MRI-T2, and 1H2O-T2 data.

147

Additional insult to muscle was implemented through an eccentric loading downhill

treadmill running protocol, with visualization and quantitative detection and spatial

visualization of pathology provided by NIR optical imaging. Finally, a restorative AAV

therapy was used to correct the γ-sarcoglycan protein deficiency in gsg -/- mice, with

confirmation of successful treatment provided by the imaging modalities and histological

assessment. To our knowledge, this is one of the first studies to use ICG enhanced NIR

optical imaging to visualize, assess, and quantify disease in muscle as well as

modification of disease through a corrective therapy.

Importance of Non-Invasive Biomarkers of Disease Progression and Regression

As clinical trials for the muscular dystrophies continue to move forward, we are

constantly reminded of the importance of sufficient outcome measures to detect natural

progression of disease and therapeutic efficacy in safe, non-invasive, repeatable, and

quantifiable manners (Bonati et al.; Connolly et al., 2014; Henricson et al., 2013b; Kinali

et al., 2011; McDonald et al., 2013; Shaibani et al., 2014; Taylor et al., 2012). A recent

shift towards quantitative MRI has drawn excitement, as a great deal of information

regarding natural progression of the muscular dystrophies (Bonati et al.; Hollingsworth,

2014) and response to treatment (Arpan et al., 2014; Bishop et al., 2015) have been

able to be provided. Additionally, data can continue to be collected following the

inevitable loss of ambulation in muscular dystrophy populations through these non-

invasive imaging techniques. Building upon another non-invasive imaging modality,

contrast enhanced NIR optical imaging, we demonstrate that muscle pathology can

similarly be detected and quantified in a safe, repeatable, and non-invasive fashion,

complimenting the findings of more expensive and timely MR procedures.

148

Importance of NIR Optical Imaging’s Contribution as an Outcome Measure Across Animals and Humans

This is one of the first studies to demonstrate ICG enhanced NIR optical imaging

to detect perturbation to muscle health in a preclinical model (Inage et al., 2015). To

date, two groups have utilized NIR optical imaging to detect muscle damage in the mice

(Baudy et al., 2011; Inage et al., 2015). Baudy and colleagues elegantly demonstrated

that a caged NIR cathepsin B substrate could be used to sensitively visualize damage,

inflammation, regeneration, and response to therapy within dystrophic skeletal muscle

(Baudy et al., 2011). Using ICG enhanced NIR optical imaging, Inage et al identified

induced muscle damage in rats (Inage et al., 2015). As ICG enhanced NIR optical

imaging has not been previously utilized to quantify and assess muscle pathology in the

muscular dystrophies, we demonstrated differentiation in fluorescent signal between

unaffected control mice and two models of dystrophic mice, the mdx and gsg -/-,

indicating uptake of ICG into damaged sarcolemmal membranes (Figure 5-7A). Further

confirmation of muscle pathology was demonstrated through several MR measures,

such as MRI-T2 (Figure 5-7E), and 1H2O-T2 (Figure 5-7F), indicating muscle pathology

in muscle in both mdx and gsg -/- mice. Elevated MRI-T2 and 1H2O-T2 values are

presumed to indicate active degeneration and regeneration that occurs as a result of the

disease, as previously demonstrated (Mathur et al., 2011; McIntosh et al., 1998; Pacak

et al., 2007; Vohra et al., 2015; Walter et al., 2005). To ensure that both non-invasive

modalities agreed, data were plotted against each other, and significant correlations

were drawn comparing both NIR optical imaging to MRI-T2 (Figure 5-8A) as well as NIR

optical imaging to 1H2O-T2 (Figure 5-8B).

149

Additional to being able to detect baseline differences in the state of health of

muscle, it is critical to be able to differentiate worsening or amelioration of disease

states, as the muscular dystrophies are not static diseases. Worsening of pathology

was able to be studied through a downhill treadmill running protocol, which is known to

cause eccentric loading damage to muscles (Mathur et al., 2011). When comparing

data before and after treadmill running, the mice demonstrated a significant increase in

fluorescent dye uptake into the muscles (Figure 5-9A), with further confirmation of

damage provided by MRI-T2 (Figure 5-9B) and 1H2O-T2 (Figure 5-9C). Interestingly, the

MRI-T2 and 1H2O-T2 did not show significant differences in the hindlimbs and forelimbs,

respectively. This may be due to the already heavy disease burden that dystrophic

muscles face and heterogeneous distribution of disease in dystrophic muscle. On the

contrary, NIR optical imaging demonstrated significant differences for both the forelimbs

and hindlimbs before and after treadmill running.

Perhaps the most critical task of an outcome measure is to be able to detect

changes in the state of health following therapeutic intervention. Many unanswered

questions have resulted from recent clinical trials (Bushby et al., 2014; Mendell et al.,

2013; Voit et al., 2014), and what outcome measures are optimal, whether functional

and strength measures, or MRI measures (Arpan et al., 2014; Hollingsworth, 2014). In

this study, we demonstrate the mitigation of LGMD-2C disease burden through

restoration of the missing protein through intramuscular injections of human γ-

sarcoglycan. Non-invasive amelioration of the disease is observed by NIR optical

imaging (Figure 5-11A), MRI-T2 (Figure 5-11F), 1H2O-T2 (Figure 5-11G), and

immunofluorescence (Figure 5-13).

150

Comparison Between NIR Optical Imaging and MR

Both non-invasive technologies, NIR optical imaging and MR, demonstrate

competency to detect muscle pathology cross sectionally, further insult to muscle, and

correction of disease in muscle. Compromises are made when using each technology –

MRI provides great spatial resolution, but limited spectral information, vice versa using

MRS, and NIR optical imaging demonstrates high sensitivity and effect size with limited

information regarding the composition and spatial resolution of regions of interest. A

more common use of ICG enhanced NIR optical imaging is to study perfusion and

vascular phenomena in vivo (Mancini et al., 1994; Možina, 2011), by acquiring data

immediately after injection. However, in this study, we collected NIR optical imaging

data an hour after ICG administration, which allowing ensured that the fluorescent

signal observed was predominantly due to dye uptake in muscle. Interestingly,

following AAV restoration of γ-sarcoglycan in gsg -/- mice (Figure 5-11), both MR

parameters return to control levels (indicated by the dashed gray boxes in Figure 5-11),

but NIR optical imaging data does not return to baseline levels. This may be due to a

number of reasons, including incomplete disease correction by the AAV or that the

disease had deposited too much fibrotic tissue prior to AAV therapy to limit delivery of

the AAV throughout the diseased muscle. These conflicting findings suggest that NIR

optical imaging and MR may be complimentary, rather than supplementary

technologies, each providing different valuable information that the other technology is

unable to provide.

Limitations

In these experiments, we demonstrate a novel use of NIR optical imaging to

assess and quantify diseased and damaged muscle, as well as amelioration of disease

151

through rAAV therapy, but this study is not without limitations. First, ICG is a non-

specific contrast agent, which has both advantages and disadvantages. It may be

advantageous to use because it can ubiquitously be applied for several different

applications within pathologies that affect muscle. For the same reasons, this may be

viewed as a disadvantage, as it is taken up non-specifically anywhere where there may

be a compromised membrane. Another advantage of ICG is that it is an FDA approved

contrast agent, allowed to be used in pediatric populations, to which there are none

currently available for use in MR studies. Although NIR optical imaging was deemed to

be comparable to MRI and MRS by effect size measurements, the development of

technology to provide better spatial resolution (i.e., assessment in different planes)

would provide much benefit to NIR imaging. Future studies warrant longitudinal

investigations of muscular dystrophies and using other disease modifying agents to

determine if NIR optical imaging can similarly detect amelioration of disease both in pre-

clinical and clinical models.

Summary

In summary, we demonstrate the utilization of NIR optical imaging with an FDA

approved NIR fluorophore, ICG, to spatially assess and quantify pathology resulting

from two different muscular dystrophies in mice, worsening of muscle pathology through

eccentric loading, as well as correction of disease through an AAV restorative therapy.

By its comparable demonstration of disease detection to MRI and MRS, NIR optical

imaging serve as a multipurpose, non-invasive, safe imaging technology that can be

applied to other disorders of muscle in both animals and humans, available for rapid

translation in clinical trials.

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Figure 6-1. Dystrophy induced muscle pathology can be detected by NIR optical

imaging, MRI, and MRS. NIR optical imaging quantification (6-1A) is shown between healthy control (6-1B), mdx (6-1C), and gsg -/- (6-1D) mice. Additionally, differences by MRI-T2 (6-1E) and 1H2O- T2 (6-1F) are shown with representative healthy control (6-1G), mdx (6-1H), and gsg -/- (6-1I) mice.

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Figure 6-2. Increased radiant efficiency correlates with increased magnetic resonance

measures in healthy and dystrophic mice. Correlations comparing radiant efficiency to MRI-T2 (6-2A) and 1H2O- T2 (6-2B) are shown.

20 25 30 350.0

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Figure 6-3. NIR optical imaging, MRI, and MRS confirm increased damage to muscle

following treadmill exercising in mdx mice. Quantitative differences for mouse forelimbs and hindlimbs before and after treadmill running are shown by way of NIR optical imaging (6-3A), MRI-T2 (6-3B) and 1H2O- T2 (6-3C). Representative NIR optical images (6-3D) and MR images (6-3E) are also shown.

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Figure 6-4. Increased total radiant efficiency correlates with increased magnetic

resonance measures before and after damage induced by treadmill running. Correlations comparing radiant efficiency to MRI-T2 (6-4A) and 1H2O-T2 (6-4B) are shown.

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Figure 6-5. gsg -/- mice treated with AAV demonstrate decreased near infrared

fluorescence and lower MRI-T2 and 1H2O-T2 relaxation times following treatment. NIR optical imaging was quantitatively assessed for the hindlimbs (6-5A) and forelimbs (6-5B). Representative NIR optical images are shown for baseline control gsg -/- (6-5C), to-be-treated gsg -/- mice (6-5D) as well as control gsg -/- (6-5E) and AAV treated gsg -/- (6-5F) mice. MRI-T2 and 1H2O-T2 data were quantified for both cohorts before and 6 weeks after intervention. In a parallel fashion to 6-5C-F, MR images of baseline control gsg -/- (6-5I), to-be-treated gsg -/- mice (6-5J) as well as control gsg -/- (6-5K) and AAV treated gsg -/- (6-5L) mice. Note that the dashed gray box indicated in figures 6-5A, 6-5B, 6-5G, and 6-5H indicate the 95th percentile for control values of each of the measures.

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Figure 6-6. Increased total radiant efficiency correlates with increased magnetic

resonance measures in gsg -/- mice with and without restorative AAV therapy. Correlations comparing radiant efficiency to MRI-T2 (6-6A) and 1H2O-T2 (6-6B) are shown.

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Table 6-1. Effect size magnitude demonstrates comparable differences between NIR optical imaging and MR measures.

NIR optical imaging MRI-T2 1H2O-T2

Control vs. mdx 2.57 3.63 2.93

Control vs. gsg -/- 4.14 3.15 1.89

Treadmill induced damage 1.88 0.73 1.96

AAV delivery of γ-sarcoglycan 3.30 1.51 1.59

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Figure 6-7. Representative immunofluorescence images with and without AAV delivery

of γ-sarcoglycan. Shown are stains for γ-sarcoglycan, wheat germ agglutinin, DAPI, and combined images.

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CHAPTER 7 NIR OPTICAL IMAGING CAN DETECT CHANGES IN MAJOR VASCULATURE

Introduction

One area that has received increased attention in the muscular dystrophy world

are the vascular defects of dystrophic muscle (Thomas, 2013). Prior to the discovery of

dystrophin, it was noted that small random groups of muscle fibers appeared to be

necrotic, and it was proposed that this focal insult was due to pathologies in local

microvasculature (Cazzato and Walton, 1968; Engel, 1967). This theory was supported

through early experiments, that caused localized ischemia in muscle (Hathaway et al.,

1970; Mendell et al., 1971). Support for this theory diminished as dystrophic muscle

microvasculature was found to be comparable to control muscle (Jerusalem et al., 1974;

Leinonen et al., 1979; Musch et al., 1975). Upon discovery of the co-localization of

neuronal nitric oxide synthase µ (nNOSµ) and dystrophin to the subsarcolemmal

surface, studies investigating the importance of vascular defects in dystrophic muscle

were reinvigorated (Brenman et al., 1995; Chang et al., 1996; Lai et al., 2009). nNOSµ

produces nitric oxide (NO), which acts as a local paracrine vasodilatory signal (Nathan,

1992). When dystrophin is deficient in dystrophic muscle, nNOSµ concurrently is

mislocalized, leading to a state of functional ischemia in dystrophic muscle. Through

restoration of nNOSµ through a dystrophin mini-gene that contains the nNOSµ binding

sites, exercise tolerance was improved (Lai et al., 2009; Zhang et al., 2013). Similarly,

when treated with phosphodiesterase-5 inhibitors, which mitigate the degradation of

NO, and allow vasodilation to occur, damage to dystrophic muscle is lessened

(Kobayashi et al., 2008).

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These discoveries support the importance of appropriate vasculature

maintenance in dystrophic muscle. Several mechanisms exist to measure changes in

blood flow, such as ultrasound and near infrared spectrometry (Ahmad et al., 2011). For

our interests, we study ICG enhanced NIR optical imaging of vasculature. Because ICG

rapidly binds to albumin, it provides contrast of vascular compartments. It is rapidly

cleared out of circulation via the liver, and passively taken up into cells with

compromised membranes, as we demonstrated occurs in muscle in previous sections.

Previously, contrast enhanced NIR optical imaging has been used to asses

vasculature in a variety of settings, including imaging of the vasculature of the retina

(Chen et al., 1999; Desmettre et al., 2000; Herbort et al., 1998; Mueller et al., 2002),

breast cancer tumors (Gurfinkel et al., 2000; Ntziachristos et al., 2000; Troyan et al.,

2009; Verbeek et al., 2014; Zelken and Tufaro, 2015), cerebral vasculature and tumors

(Haglund et al., 1996; Raabe et al., 2003), gastrointestinal vessels (Borotto et al., 1999),

and cardiac vasculature and myocardial perfusion (Nakayama et al., 2002; Taggart et

al., 2003). As it has been used to image vasculature in a number of different tissues,

we hoped to expand on this knowledge by imaging vasculature in the leg. It was our

intention to demonstrate contrast enhanced NIR optical imaging to visualize, and

quantitate changes in blood flow in muscle. To first demonstrate proof of principle, we

chose a robust model of perturbations of blood flow, assessing if we can detect and

quantify the inducible hyperemic response in hindlimb of mice (Joannides et al., 1995).

In experiments assessing damage to muscle, all NIR optical imaging data was collected

greater than 1 hour following ICG injection, but we hypothesize that by imaging ICG

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enhancement immediately following delivery, we will be able to measure vascular flow

and muscle perfusion.

Results

Proof of principle experiments demonstrated the capability to image the

vasculature of the lower hindlimb of mice. The major lower hindlimb’s major vasculature

was able to be quantitatively visualized in a time dependent manner, and distinguished

from the surrounding muscle (Figure 7-1A). Differences between the femoral artery and

surrounding muscle are able to be quantified (Figure 7-1A) and visualized at several

representative timepoints 1 second, 15 seconds, and 180 seconds after ICG injection

(Figures 7-1B, 7-1C, and 7-1D). Immediately following injections, fluorescence from ICG

rapidly peaked as ICG disseminated throughout the vasculature of the body.

Fluorescence precipitously declined at several timepoints (Figures 7-1B to 7-1D)

following injection, and quickly fluorescence of muscle and vasculature became similar.

Following removal of a blood pressure cuff, a characteristic hyperemic response

was observed by ICG enhanced NIR optical imaging (Figure 7-2). Following removal of

a blood pressure cuff, a hyperemic response can be observed in the cuff-and-released

limb as compared to the contralateral control hindlimb (Figure 7-2A). Representative

images 300s (Figure 7-2B) and 2000s after injection (Figure 7-2C) are shown. When

comparing the control limb that did not undergo blood pressure cuff occlusion to the

variable experimental limb, similar initial fluorescence values were observed in both

hindlimbs, but the cuff-and-release limb maintained higher fluorescence longer than the

contralateral control limb (Figure 7-2).

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Discussion and Summary

Those not yet fully completed, these preliminary experiments present proof of

principle findings that ICG enhanced NIR optical imaging can quantitatively image

normal and modified vascular flow within muscles in a preclinical model. Differentiation

between surrounding muscle, and the primary vasculature of the lower leg are

distinguished (Figure 7-1). Further, a characteristic hyperemic response is observed,

with greater blood flow in the cuff-and-release hindlimb as compared to contralateral

control limbs (Figure 7-2). Recently, contrast enhanced NIR optical imaging recently has

developed as an effective way for several clinical applications, such as perfusion

studies (Desmettre et al., 2000; Kobayashi et al., 2014; Raabe et al., 2003) and tumor

identification (Corlu et al., 2007; Ntziachristos et al., 2000; Zelken and Tufaro, 2015).

These experiments lay the foundational work necessary to develop further experiments

in studying the vasculature, and changes to vasculature in both healthy and dystrophic

muscle.

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Figure 7-1. Differences between major vasculature and surrounding muscle are able to

be spatially and temporally identified. Quantitation of muscle and vessel fluorescent intensity is quantified (7-1A). Further, representative images from 1 second, 15 seconds, and 180 seconds after ICG injection are shown (7-1B, 7-1C, and 7-1D, representatively).

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Figure 7-2. A hyperemic response is able to be quantified through NIR optical imaging.

Quantitation of the hyperemic response observed is presented (7-2A), as are representative NIR images 300 and 2000 seconds after cuff release (7-2B and 7-2C, representatively.

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CHAPTER 8 POTENTIAL OF NEAR INFRARED RESPONSIVE PARTICLES AND

QUANTIFICATION OF DRUG DELIVERY

Introduction

The utilization of contrast sensitive biocompatible particles has great potential to

aid the understanding of the delivery of therapeutic agents to targets. In muscle,

contrast enhanced NIR imaging without particles has revealed great insight into

pathology within muscle, in a rat model of muscle damage (Inage et al., 2015) and a

mouse model of muscular dystrophy (Baudy et al., 2011; Huynh et al., 2013).

However, tracking of the delivery of therapeutic treatments to disease models is only

possible through later analysis of tissue, and real time assessment is not available

through current methods. Delivery and biodistribution of particles carrying antisense

oligonucleotides (AONs) that were concurrently NIR fluorescently responsive has been

performed in the mdx mouse model (Falzarano et al., 2014a, 2014b). As more

efficacious strategies to treat the muscular dystrophies continue to progress, we intend

to boost the efficacy of therapeutics through theranostic vehicles, capable of delivering

therapeutic drugs and providing real time in vivo tracking of the delivery of these

particles.

As with all systemically delivery therapeutic agents, the ability to track local

delivery of agents in a highly efficacious manner is critical to confirm positive delivery of

therapy to target tissue. In dystrophic muscle, this is a particular challenge due to

muscle’s perfusion defects and fibrotic deposition, as well as the required systemic

delivery of therapy. As much excitement surrounds different genetic therapies,

efficacious manners to deliver drugs are continually investigated. In DMD, AONs have

emerged as a promising therapeutic option to induce functional dystrophin and are

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currently undergoing clinical trials (Bishop et al., 2015; Flanigan et al., 2014; Hoffman,

2014; Koo and Wood, 2013; Mendell et al., 2013, 2016; Sazani et al., 2014). While

naked AONs are reasonably stable in circulation, their bioavailability is limited by poor

cell trafficking and endosomal entrapment, requiring repeated and high doses to render

clinical efficacy (Järver et al., 2012). With a pressing need to discover a capable vehicle

to transport and protect AONs, it is our intention to develop an optimal drug delivery

system, capable of being tracked in vivo in real time, as well as delivering therapeutic

agents. Particle mediated delivery of AONs may resolve these issues, resulting in

improved therapeutic outcomes.

Particle based drug delivery systems have been investigated, balancing positive

benefits with negative consequences of different modalities. Previously, liposomes,

polymers, and cell-penetrating peptides have all utilized as vehicles to deliver drugs,

with each having their own benefits and shortcomings. Such shortcomings include a

lack of tropism to hone in effective specific targeting and difficulty controlling release of

the packaged contents. Several issues to consider in the synthesis and delivery of drug

conjugated particles include: (1) ability to be systemically distributed, (2) consistent

tissue targeting, (3) adequate in vivo stability of the drug-particle complex, (4) optimizing

the intracellular uptake, (5) ability to track and determine the biodistribution in vivo. The

lack of noninvasive tools to monitor and ensure the drug delivery to the target tissue

under in vivo conditions increases the uncertainty of clinical effectiveness. An approach

that allows for non-invasive tracking and monitoring of drug delivery to dystrophic

muscle sites in vivo using biocompatible biodegradable particles would help to relate

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drug delivery with therapeutic outcome and help in developing effective treatment

options.

In the effort to design optimal particles, poly-lactic acid (PLA), a biocompatible

polymer, was chosen to compose the particles. Previously, polymeric particles such as

those made of poly(methylmethacrylate) (PMMA) have been successfully employed as

carrier for delivering AONs in mdx mice (Falzarano et al., 2013; Rimessi et al., 2009).

As we additionally sought to track particles in real time, we conjugated ICG, an FDA

approved NIR fluorescent dye, to our PLA particles. Silica particles have been

developed with adsorbed ICG to provide an in vivo means to optically image the

distribution of such particles (Lee et al., 2009). ICG is an ideal NIR fluorescent contrast

agent to conjugate to biocompatible nanoparticles because of its negligible side effects

and low toxicity (Lutty, 1978). ICG is a blood-pool NIR contrast agent, clinically

approved for use in retinal angiography (Yannuzzi, 2011), blood flow measurements (El-

Desoky et al., 1999; Keiding et al., 1998), guiding biopsies (Motomura et al., 1999),

perfusion studies (Guenette et al., 2011), and lymphatic mapping (Rasmussen et al.,

2009). Despite multiple applications, there are inherent challenges regarding ICG

stability in solution and biological systems, including dependence of physical and optical

properties of ICG on pH, temperature, and exposure to light (Björnsson et al., 1982;

Holzer et al., 1998). Additionally, ICG has demonstrated optical instability in

physiologically relevant solutions (Desmettre et al., 2000; El-Desoky et al., 1999;

Landsman et al., 1976; Muckle, 1976). While beneficial when interested in vascular

properties, a rapid clearance from circulation (2-4 minute half life), is disadvantageous

when trying to assess uptake into damaged tissue (Wolfe and Csaky, 2004). The

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encapsulation of ICG into biocompatible particles offers great potential to maintain the

optical properties of ICG in an in vivo setting.

With the progress of new therapeutic strategies and investigations to mitigate the

pathologies of the muscular dystrophies, ICG enhanced NIR imaging provides a

minimally invasive longitudinal modality to monitor drug delivery and therapeutic

response in vivo. Development, characterization, and application of biocompatible poly-

lactic acid particles with encapsulated ICG are necessary to better understand the

capabilities of these particles. Through development of biocompatible particles that

encapsulated ICG that were tested in vitro and in vivo, we have laid the groundwork

necessary to accomplish this.

Results

Synthesis and Characterization of ICG-PLA Particles

First, physical and optical properties of ICG-PLA particles were characterized.

Size distribution was determined by digital light scattering, and found to range from 40-

100 nm (Figure 8-1A). Quantum yield (Φ) is the ratio of emitted to absorbed photons in

fluorophores and was determined to be 0.032, which is better than the reported range

for ICG dye in aqueous solutions (0.027 to 0.01) (Larush and Magdassi, 2011; Russin et

al., 2010). Corresponding scanning electron microscope (SEM) images demonstrated

size and morphologic homogeneity amongst the ICG loaded particles (Figure 8-1B).

Fluorescence of the ICG-PLA particles was also determined, and the peak of

fluorescence in ICG-PLA particles was found to be comparable to ICG in solution at 815

nm, with a small (~10 nm) red shift compared to the ICG monomers (Figure 8-1C),

comparable to previous studies (Gomes et al., 2006; Ranjan et al., 2011; Saxena et al.,

2004). Furthermore, the encapsulation efficiency of ICG in the PLA particles was

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determined to be ~ 70%, which is comparable to the ranges reported for electrostatically

assembled mesocapsules and micelles (Kim et al., 2010, 2010). The zeta potential of

the ICG - PLA particles ranges from -30 to -38 mV (SD) and +26 to +37mv range (SD)

for particles prepared in polyvinyl alcohol (PVA) and didodecyldimethylammonium

(DDAB) surfactants, respectively.

Photostability at Room and Physiological Temperatures

To assess the effect of temperature and time on the photostability of ICG and

ICG-PLA particles, samples were left at either 23°C (Figure 8-2A) or 37°C (Figure 8-2B)

for up to four weeks. Total radiant efficiency was recorded throughout the month, and it

was demonstrated that the ICG-PLA particles retained much of initial fluorescence at

room (90%) and physiological (83%) temperatures, respectively. Comparatively, the

fluorescence from ICG dye alone rapidly decayed at both temperatures.

In Vivo Contrast Enhanced NIR Optical Imaging of PLA-ICG via Subcutaneous Injections:

Intrascapular subcutaneous injections of ICG-PLA particles, ICG dye alone, or

lactated ringer’s buffer (LRB) were given to mice (Figure 8-3). As expected, the ICG

dye alone caused an initial increase in signal, but quickly returned back to baseline

values. Furthermore, fluorescent signal was maintained only the ICG-PLA particle

injected mice over the course of 10 days. The injection of lactated ringer’s solution

demonstrated no change in signal from baseline, serving as the negative control.

Representative images immediately following injections, as well as two days after

demonstrate the lack of fluorescent signal in mice that received LRB, the quick

deterioration of fluorescent in the ICG cohort, and maintenance of signal in mice that

received ICG-PLA particles. Besides day 0 measurements, all ICG-PLA particles are

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significantly elevated as compared to ICG alone measures. Additionally, ICG-PLA

particles become significantly lower than day 0 values after 4 days.

In Vivo Contrast Enhanced NIR Optical Imaging of PLA-ICG via Intramuscular Injections

Intramuscular injections of ICG-PLA particles into gastrocnemii demonstrate

maintenance of fluorescent signal for up to 2 weeks (Figure 8-4). Similar to the

subcutaneous injection experiments, PLA-ICG particles demonstrate prolonged stability

of signal within target tissue. Quantitatively, the injected hindlimbs had significantly

elevated fluorescence for approximately 10 days, as compared to contralateral non-

injected hindlimbs (Figure 8-4A). Representative images at 1 (Figure 8-4B) and 28

(Figure 8-4C) days following injections demonstrated a preservation of fluorescent

signal near the injection site. Ex vivo assessments were performed on three cohorts of

muscle from additional experiments: control (non-injected), ICG injected at 1 week, ICG-

PLA particles at 1 week, and ICG-PLA particles at 1 month (Figure 8-5). Analyzed

tissues demonstrated elevated fluorescence in the gastrocnemius, but none of the other

hindlimbs muscles, as shown visually (Figure 8-5A) and quantitatively (Figure 8-5B).

Discussion

The experiments presented in this section lay the necessary foundational work to

utilize contrast sensitive biocompatible particles to help understand the delivery of

therapeutic agents to muscle.

Particle Synthesis and Characterization

Particles were optimized by characterizing their size, morphology, conjugation

with ICG, and temporal and thermal stability. Though ICG has previously been

encapsulated in other mediums, such as liposomes (Proulx et al., 2010), inorganic

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materials (Altinoğlu et al., 2008), micelles (Kim et al., 2010; Kirchherr et al., 2009), and

silica (Sharma et al., 2010), PLA polymers are attractive for our interests because of

their biocompatible and biodegradable properties, and because they are currently used

in clinical applications (Mikos et al., 1994; Rosler et al., 2001). Currently, several drug

delivery formulations exist for treating prostate cancer (Cho et al., 2010; Farokhzad et

al., 2004) and neuroendocrine tumors (Blanco-Prieto et al., 2004; Dubey et al., 2012).

Additionally, particles have also been used preclinically to deliver AONs intramuscularly

(Sirsi et al., 2009) and immunosuppressant drugs intravenously (Eghtesad et al., 2012)

in dystrophic mdx mice.

Upon demonstration of sufficient particle sizing (Figure 8-1B) and morphology

(Figure 8-1B), it was necessary to optimize fluorescence of the ICG associated

particles. Optical properties of ICG are influenced by concentration, exposure to light,

solvent used during preparation, and encapsulation into polymeric particles (Björnsson

et al., 1982; Desmettre et al., 2000; Devoisselle et al., 1998; Gomes et al., 2006;

Landsman et al., 1976; Mordon et al., 1998; Saxena et al., 2004). Furthermore, the

amphiiphilic nature of ICG and its poor chemical and photostability in highly alkaline

conditions, such as those present in the PLA particle synthesis, (Devoisselle et al.,

1998; Mordon et al., 1998) provide challenges when encapsulating ICG into particles.

Further, an additional concern when encapsulating ICG into PLA particles is that the

optical properties of ICG may be affected during the synthesis process. Figure 8-1C

demonstrates that peak of fluorescence experiences a subtle red shift as shown by

other groups (Gomes et al., 2006; Ranjan et al., 2011; Saxena et al., 2004; Yu et al.,

2010; Zheng et al., 2011), but the magnitude of fluorescence is not altered. This red

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shift may be due to aggregation of ICG within the particle matrices and changes in the

microenvironment of ICG. Additionally, the quantum yield (Φ) of our ICG-PLA particles

demonstrates that the encapsulation of ICG within the polymer and its interaction with

PEI does not adversely impact its fluorescence efficiency. It is noted that in comparison

to other ICG containing vehicles, such as liposomes (Portnoy et al., 2011), calcium

phosphate (Altinoğlu et al., 2008), and micellar systems (Kirchherr et al., 2009), the Φ

values for our PLA-ICG particles are comparable.

Another important element to consider when developing contrast enhance

particles for in vivo settings is their optical stability. Though encapsulation of ICG into

lipids (Kraft and Ho, 2014), inorganic materials (Altinoğlu et al., 2008; Sharma et al.,

2010), and micelles (Kirchherr et al., 2009) have all improved the photostability profile of

ICG, we wished to further increase stabilization in vivo. Previous studies have

demonstrated sufficient, but higher rates of release of ICG from particles in vivo than

what we desire (Ma et al., 2012; Saxena et al., 2006). Particles co-conjugating ICG and

doxorubicin have similarly demonstrated release of up to 70% of ICG in a day from the

co-encapsulated particles (Manchanda et al., 2010). A main purpose of these studies

are to retain optical properties in our particles, and one method to accomplish this may

be through utilizing electrostatic interactions between ICG and the substrate

polyethylenimine (PEI). Incorporation of PEI (10,000 molecular weight) into the PLA

particles demonstrated enhanced photostability between conjugated ICG and free ICG

in an aqueous medium (Figure 8-2).

Application of Particles to Animal Models

Following optimization of the particle sizing, surface charge ratio, and

encapsulation of ICG, the next set of experiments focused on translating our in vitro

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findings to an in vivo environment. In vivo, particles demonstrated significant

maintenance of signal following subcutaneous and intramuscular injections compared to

injections of ICG in an aqueous solution. Providing the ability to track particles in vivo

and monitor the localization of the particles for weeks demonstrate the potential to

deliver and prolong localized release of therapeutic agents, while concurrently

performing longitudinal imaging to assess the location of delivery of drugs.

First, ICG-PLA particles, ICG, and LRB were subcutaneously administered to

mice. Positive fluorescent signal was initially observed in the mice that received ICG-

PLA particles and ICG, but the ICG-PLA particles additionally retained signal for up to

four weeks following injections, while the ICG cohort’s fluorescence quickly extinguished

(Figure 8-3). Upon confirmation that fluorescent signal could be identified by

subcutaneous injections, intramuscular injections were performed to similarly see if

fluorescent signal could be identified. As expected, injections into the gastrocnemii of

ICG-PLA particles were able to be measured for up to 4 weeks post-injection (Figure 8-

3). Importantly, these experiments demonstrate that in vivo imaging with ICG-PLA

particles are able to maintain prolonged signal in vivo and avoid autofluorescence

issues that often arise with fluorophores of shorter wavelengths (Frangioni, 2003), and

image within internal anatomical compartments rather than only the surface of the skin.

This set of experiments demonstrated the feasibility to detect a signal from the ICG in

vivo, as well as to demonstrate the efficacy of signal stability through administration of

ICG loaded PLA particles.

The gradual decline of fluorescent signal from the injected ICG-PLA particles

may be caused by several phenomena. Degradation of either ICG itself or the particles

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containing ICG may cause a decline in observed fluorescent signal. As the

subcutaneous experiment demonstrated, fluorescence of the unencapsulated ICG alone

quickly diminishes compared to that of the ICG encapsulated within the PLA particles

(Figure 8-3). The presence of fluorescent signal beyond two weeks after injections

indicate that residual particles may remain entrapped at the site of injection, potentially

allowing for longitudinal tracking in vivo. These results demonstrate high photostability

of ICG-PLA particles and the ability to perform in vivo imaging much longer than using

ICG alone would permit.

Summary of Delivery of Nanoparticles

Building off of these findings, the next steps of our project include performing

several experiments to further characterize the delivery of drugs to dystrophic muscle.

Unique to this study is that we have designed NIR probes comprising FDA approved

PLA and ICG allowing for eventual clinical transition. Even though ICG was the first NIR

contrast agent approved the FDA, its clinical implementation has been limited by low

photostability and rapid in vivo elimination. In this investigation, we optimized the

composition of a biocompatible PLA based particle capable of efficiently encapsulating

ICG as a longitudinal NIR imaging agent for both in vitro and in vivo purposes.

Encapsulation of the ICG within PLA particles allowed for greater preservation of

fluorescent signal, both in vitro and in vivo, demonstrating the beneficial thermostability

and photostability effects of the PLA based particles. Moving forward, we will optimize

AON conjugation to the ICG loaded particles, and to both myoblasts and mice, deliver

unencapsulated AONs + ICG-PLA particles or AON-ICG-PLA particles, assessing the

distribution and therapeutic efficacy of each intervention.

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Figure 8-1. Representative size distribution (8-1A), aggregation properties (8-1B), and

fluorescence characteristics (8-1C) of ICG-PLA particles

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Figure 8-2. Photostability at room (25°C, 8-2A) and physiologic (37°C, 8-2B)

temperature of ICG-PLA particles and ICG alone.

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Figure 8-3. Subcutaneous injections of PLA-ICG show prolonged maintained signal

compared to Lactated Ringer’s Solution and ICG alone visually (8-3A) and quantitatively (8-3B).

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Figure 8-4. Intramuscularly injected PLA-ICG particles maintain prolonged fluorescent

signal (8-4A) at 1 (8-4B) and 28 (8-4C) days following injections.

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Figure 8-5. Ex vivo NIR optical images of excised muscles following intramuscular

injections into the gastrocnemius demonstrate in vivo stability of PLA-ICG particles visually (8-5A) and quantitatively (8-5B).

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CHAPTER 9 DAMAGED AND DYSTROPHIC MUSCLE IN HUMANS

Building off of the findings that have been established in the prior chapters at the

pre-clinical level, we attempted to translate our NIR optical imaging methodologies to

study pathology in human muscle. My human studies focus on two primary projects:

assessing the spatial geographic (WC) differences in pathology in boys with DMD by

MRI, and to develop NIR optical imaging as an complimentary non-invasive modality to

image and quantify muscle damage in a model of inducible muscle damage in healthy

individuals.

A Multislice Analysis Reveals Heterogeneity within Lower Limbs of Boys with DMD

Introduction

Duchenne muscular dystrophy (DMD), an X-linked recessive genetic disorder

with an incidence of 10.7 to 27.7 per 100,000, is caused by a mutation in the dystrophin

gene, resulting in an absence or dysfunction of the protein dystrophin (Hoffman et al.,

1987; Mah et al., 2014). Structurally, progressive pathological changes in skeletal

muscle resulting from DMD are well described and include inflammation (McDouall et

al., 1990), lipid infiltration (Bongers et al., 1992), and fibrotic deposition (Kharraz et al.,

2014). Clinical manifestations and natural history of DMD are understood, and include

progressive muscle weakness, a loss of functional abilities, and premature mortality

(Bushby et al., 2010a, 2010b; Flanigan, 2014). Though much is known about DMD, a

paucity of information exists regarding the pathology along the length differences of

disease pathology along the length of muscles in individuals with the disease.

Magnetic resonance imaging (MRI) has developed as a safe and effective

modality to qualitatively investigate patterns of disease pathology within muscle in DMD

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(Baudin et al., 2015; Kinali et al., 2011; Liu et al., 1993a; Mercuri et al., 2007; Schreiber

et al., 1987; Torriani et al., 2012; Wokke et al., 2013). Importantly, MRI allows for

excellent three-dimensional spatial resolution, which allows for differentiation of muscle

architecture and visualization of deeper muscle groups in an objective, non-invasive,

sensitive, and specific manner, independent of patient effort. Traditionally, most MRI

study protocols analyze muscle data from either single or several consecutive slices,

selecting slices based on anatomical landmarks such as the maximum cross sectional

area of a muscle (Torriani et al., 2012; Wokke et al., 2013). A ordinal MRI grading scale

was developed to qualitatively assess the muscle health in congenital muscular

dystrophy, providing information on both severity of disease presentation, as well as

characteristics of disease involvement within muscle (Mercuri et al., 2002). This scale

has been utilized, and modified for a number of studies, providing information on the

distribution and characterization of disease in muscle in limb-girdle (Sarkozy et al.,

2012; Stramare et al., 2010; Willis et al., 2014), fascioscapulohumeral (Janssen et al.,

2014a; Leung et al., 2015), oculopharyngeal (Fischmann et al., 2012), and distal

(Mahjneh et al., 2012) muscular dystrophies, as well as a spectrum of other myopathies

(Baudin et al., 2015; Fischer et al., 2008). In DMD, where muscle injury leads to variable

fatty deposition across muscles, a three dimensional evaluation may provide greater

insight into the understanding of the disease process (Kinali et al., 2011; Torriani et al.,

2012; Wokke et al., 2013). Based on the aforementioned work in other muscular

dystrophies, we are interested in performing a multi-slice qualitative evaluation between

and within individual muscles in DMD to provide more comprehensive insight and

understanding of DMD.

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To optimally manage and treat those affected by DMD, a foundational

understanding of how DMD affects muscle is required. While the molecular basis and

gross clinical manifestations of the disease are well characterized, investigation into the

intramuscular characterization of disease has been less explored. Through pre-clinical

studies, a greater understanding of the geographic vulnerabilities to disease pathology

can be better observed, specifically an increased susceptibility to disease at the muscle-

tendon junction. Though muscles are initially rendered susceptible to damage as a

result of dystrophin lacking, further properties may influence the ability of tissue to

succumb to the progression of disease, including the biological composition of tissue

(Babic and Lenarcic, 2004; Suydam et al., 2015), the distribution of strain (van Bavel et

al., 1996), the cross sectional area (Sun et al., 1994), speed and type of contractions

(Sharafi et al., 2011), and other passive viscoelastic properties (Pasternak et al., 1995).

Through MRI, we are able to acquire data along the length of muscle, allowing for

observation of how DMD appears to affect different portions of the muscle uniquely.

This study expands upon the current knowledge base of DMD pathophysiology

by assessing individual lower leg muscles using a multi-slice evaluation in boys with

DMD to better understand the heterogeneous nature of disease involvement using a T1

weighted multi-slice qualitative MRI assessment. Through using an ordinal MRI grading

scale of muscle pathology, it was hypothesized that differences of involvement would be

observed within individual muscles, with greater involvement observed at the proximal

and distal ends when compared to the mid-bellies of muscle. The objectives of the

present study were (a) to utilize multi-slice MRI in lower legs of boys with DMD to study

tendon to muscle differences of disease involvement within and between individual

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lower leg muscles and (b) to determine if evaluation in a multi-planar fashion reveals

stronger correlations to age and function than single-slice analyses in boys with DMD.

Results

Involvement of DMD in muscle presents in non-uniform manner

In order to assess spatial heterogeneity and pathology, MR images were

acquired along the lengths of lower legs of boys with DMD. Representative cross

sectional images showed variability of disease involvement between muscles, with the

worst pathology in the peroneal and tibialis anterior muscles. Representative images

that visually highlight disease involvement in these particular muscle groups were

previously identified in the methodology chapter (Figure 4-4). When investigating

pathology along the length of muscles, variability was observed, with greater amounts of

disease at the most proximal and distal ends of muscle as compared to the midbelly.

Visually, differences of spatial pathology between proximal, midbelly, and distal portions

of the muscle can be appreciated in the peroneals (Figure 4-2, dashed arrow) and

tibialis anterior (Figure 4-2, solid arrow). The spectrum of MRI grades and how different

pathology can manifest along different geographic regions of the same muscle are also

shown (Figure 9-1). Furthermore, while boys with DMD typically demonstrated worse

pathology at the ends of muscle, not all subjects demonstrated identical patterns of

disease progression. Through presentation of each subjects’ muscle MRI grades

(Figure 9-2), one can appreciate the broad distribution of disease involvement amongst

subjects.

Relationship between MRI scores, function and age

Additionally, a goal of this study was to see if a comprehensive multi-slice

assessment could better correlate with clinical measures than single slice analyses.

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Figure 7-3b and 7-3d show the relationship between subjects binned by their Vignos

score and the summative multi-slice and middle slice MRI scores, respectfully, by

median and 25th and 75th percentiles. In figures 9-3b and 9-3d, comparison to Vignos

grade 5+ is indicated by asterisks where * indicates p < 0.5, ** indicates p < 0.01, ****

indicates p < 0.0001, and comparison to Vignos grade 3-4 is indicated by daggers,

where † indicates p < 0.05. With increasing ages of subjects, ScoreMulti MRI score

concurrently increased (rho = 0.63, p = 0.006), confirming that disease involvement

within muscle increases as children age (Figure 9-3A), whereas ScoreSingle (Figure 9-

3C) did not correlate with increasing age of subjects (rho = -0.21, p = 0.32).

Additionally, functional status was measured by Vignos scoring (Lue et al., 2009) to

assess if MRI grading may be related to functional capabilities. Increases in both

ScoreMulti (p = 0.0035, Figure 9-3B) and ScoreSingle (p = 0.0047, Figure 9-3D) were

observed with decreased functional status.

Discussion

The primary purpose of this paper was to investigate intramuscular heterogeneity

of disease involvement in the lower legs of 5 – 15 year old boys with DMD through a

simple multislice MRI acquisition that can be performed on most clinical MRI scanners.

Building upon developments of MRI as a feasible modality to investigate muscle

pathology in DMD, continuing to understand the heterogeneous nature of DMD is critical

to further advance our knowledge of this irreversibly lethal disease (Baudin et al., 2015;

Kinali et al., 2011; Liu et al., 1993a; Schreiber et al., 1987; Torriani et al., 2012; Wokke

et al., 2013). Through this investigation, we demonstrate that by utilization of a

multislice MRI acquisition of subjects with DMD, muscles of the lower leg revealed

differential disease involvement within muscles, most prominently at the myotendinous

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junction, that heterogeneity of pathology exists between subjects, and that a multislice

grading scheme reflected functional disease progression.

Previously, a qualitative MRI grading scheme was developed for neuromuscular

disorders (Mercuri et al., 2002), and adopted by several studies investigating disease

progression in DMD (Baudin et al., 2015; Kinali et al., 2011; Torriani et al., 2012; Wokke

et al., 2013). Unique to this study was the use of a mutlislice acquisition along the lower

leg, allowing for investigation of intramuscular differences in disease pathology,

especially at the myotendinous junction, which has previously not be investigated.

Through such an investigation, we were visually able to appreciate differences between

the myotendinous region versus midbelly of several muscles, highlighted in the tibialis

anterior and peroneus, as observed in Figure 4-2. The concept that disease

involvement in muscle is not homogenous is highlighted further in Figure 4-3, seeing

how two individuals’ muscles are non-uniformly affected between subjects and within

individual muscles. This may suggest that the results of data collection, such as biopsy

or MR slice selections, may be largely dependent on the location of muscle

investigated. If one were to study only the ‘middle’ portion of Subject A’s tibialis

anterior, the conclusion that the disease has not progressed much could be easily

made, whereas the contrary conclusion could be drawn if either of the myotendinous

junctions of the same muscle were investigated as the ends of the TA demonstrate

much greater damage than the middle. Taking into account the remainder of the

subject population, a broad distribution of disease involvement is observed throughout

the length of lower leg muscle (Figure 9-1). A general trend was observed in that an

increase of disease pathology can be observed towards the myotendinous junctions.

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These results highlight the differences of muscle pathology that individuals with DMD

can present with.

The final analyses performed in this study were to see if the MRI scores

generated could be related to other measures of disease progression. As individuals

with DMD age, muscles continually accumulate insults of the disease in their muscles,

evident by the clinical progression of DMD (Bushby et al., 2010a; Kinali et al., 2011;

Torriani et al., 2012; Willcocks et al., 2014). While our ScoreMulti positively correlated

with the age of subjects, ScoreSingle did not, suggesting that a multi-slice assessment.

Conflicting with the age correlations, increases in the Vignos lower extremity scale

demonstrating progressive decline in functional ability paralleled increases in both

ScoreMulti and ScoreSingle MRI grades (Lue et al., 2009). Effectively, this demonstrates

that with increasing age and decreasing function, lower leg muscles of boys with DMD

have increasingly progressive disease involvement, and that more accurate

representation of disease may be able to be demonstrated through investigating greater

geography of muscle rather than a single slice.

Together, with observation of differential disease involvement in our clinical study

and pre-clinical results of the effects of DMD, one can speculate why it similarly does

not affect individuals identically. The exact reasons for why DMD does not affect muscle

homogenously eludes researchers, but several studies may help elucidate mechanisms

for its heterogeneous pathology. Differing amounts of eccentric contractions that

muscles experience during gait have been shown to strongly correlate with lower limb

fat fraction, a marker of disease progression (Baudin et al., 2015; Hu and Blemker,

2015). In the mdx mouse, the stress relaxation rate of the extensor digitorum longus

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was found to be increased in mdx mice compared to healthy counterparts, and

recoverable upon micro and mini dystrophin treatment, suggesting that dystrophic

muscle itself has different passive properties than healthy muscle (Hakim and Duan,

2012, 2013). In other studies, strain measures of the gastrocnemii belly (20-30%) were

found to be greater than those of the aponeurosis (1-5%) (van Bavel et al., 1996) and

that the tapered myotendinous junction experiences greater stress than the muscle belly

(Sharafi et al., 2011). This suggests that different passive mechanical properties exist

between different geographical segments of muscles, rendering the myotendinous

junctions more vulnerable to the pathologic insult observed in DMD. Sun et al elegantly

demonstrated microfailure of the muscle-tendon unit using peroneus longus muscles of

rabbits, suggesting that the weakest region of muscle is at the myotendinous junction,

which further supports MRI data shown from our patient cohort (Sun et al., 1994). For

obvious reasons, many of these pre-clinical experiments are not feasible to perform in

clinical subjects because of their invasive nature, so extrapolation of pre-clinical findings

to humans is appropriate to compare to our findings.

Limitations

This study is not without limitations, as addressed below. A primary limitation of

this study is the lack of full geographic capture of muscle from tendon to tendon. This

study is a subset of a larger study (RO1, AR0569373, PI: Vandenborne), and images

were captured to meet the needs to the larger study. Expectedly, the gastrocnemii

muscles tend to show substantial involvement towards the most distal portions of the

muscle; however, less involvement was seen at the proximal slices, as the MRI protocol

employed did not capture the proximal half of these muscles. Additionally, the soleus

seems to counter the general trend of increased involvement towards the most proximal

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slice, but is explained because the soleus muscle is physically longer than the other

muscles and the pre-selected slices do not capture the anatomical ends of these

muscles where greater disease involvement may be more likely to be observed. An

additional limitation of our study may argued to be the subjective grading of muscular

involvement by MRI. While more quantitative measure of lipid deposition exists such

as, 3-Point Dixon (Wokke et al., 2013), spectroscopy (Torriani et al., 2012), and T2

weighting (Willcocks et al., 2014), these techniques are not all readily available on

clinical MR scanners and may require more advanced MR software or data evaluation

techniques that are not readily available to use and interpret on traditional clinical

systems. In a population of several muscular dystrophies, Baudin et al demonstrated

statistical equivalency between T1 weighted imaging to longer Dixon scan methods

(Baudin et al., 2015). Because disease involvement of DMD muscle includes both T1

shortening fibrotic effects and T1 lengthening lipid effects on muscle, we employed T1

weighting with fat suppression techniques in our study in a similar manner to a study

performed by Leung et al in fascioscapulohumeral muscular dystrophy (Leung et al.,

2015) to comprehensively address all DMD pathologies. Future studies looking at intra-

muscular heterogeneity would benefit greatly from larger spatial coverage of slice

selection and investigating differences in composition throughout muscle. In a broader

scope, further studies are warranted in other neuromuscular disorders to see if other

diseases demonstrate unique intra-muscular disease heterogeneity, as is observed in

DMD in this study.

Summary of a Multislice Assessment of the Lower Leg in DMD

In this investigation, we performed a qualitative multi-slice evaluation of the

pathology of lower leg muscles of boys with Duchenne muscular dystrophy (DMD).

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There were four major conclusions from this study: (i) the six individual muscles of the

lower leg were not affected equally by the disease; (ii) there was more muscle

involvement at the myotendinous junctions rather than the midbelly of muscle; (iii)

differential disease involvement was found between subjects; and (iv) MRI mutlislice

grades are related to age and functional ability. In summary, our results show a unique

distribution of involvement both inter- and intra- muscularly in the lower legs of boys with

DMD. This study merits further investigation of the local geographic pathologic

differences within dystrophic muscle, which may potentially be utilized in assessing the

natural progression and therapeutic intervention. Caution may be warranted when using

single slice acquisitions, as they may represent localized geographic (WC) disease

pathology in muscle, and that capture of entire muscles, including the myotendinous

junction may more appropriately represent overall disease status in individuals with

DMD.

Preliminary Assessment of the Upper Extremity in DMD by MRI

Introduction

Previously, most MR imaging studies for the muscular dystrophies have focused

on the lower extremities (Arpan et al., 2014; Fischmann et al., 2014; Forbes et al.,

2014b; Wary et al., 2015; Willcocks et al., 2014; Wokke et al., 2014). Recently, our lab

has begun to investigate the upper extremity, in regards to function and imaging, to

better establish an understanding of the progression of DMD in the arms.

Understanding the progression of disease in the arms is important for two primary

reasons. First, inclusion and exclusion criteria in many clinical studies require sufficient

ambulation to participate in trials, which means that individuals who have become non-

ambulatory are not allowed to potentially benefit from participation in clinical trials

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(Cedarbaum et al., 2014; Merlini and Sabatelli, 2015; Ricotti et al., 2015; Scully et al.,

2012). Furthermore, upon loss of ambulation, the use and function of the upper

extremity becomes critically important to maintain independence in this patient

population (Alemdaroğlu et al., 2015; Janssen et al., 2014b; Pane et al., 2014).

Because of the paucity of research that exists in this area, it was our interest to

foundationally investigate the upper extremities, and the relationship between

progression of disease, age, and function.

Results

In this preliminary investigation, we sought to identify pathology in the upper

extremities of boys with DMD. First, we performed a cross sectional analysis,

comparing individual muscles of boys with DMD to age matched controls (Figure 9-4).

MRI-T2 relaxation times in dystrophic muscle was elevated in the deltoid, biceps brachii,

and triceps brachii muscle groups when compared to control muscle (Figure 9-4A).

Forearm muscles were elevated, but insignificantly different compared to control MRI-T2

relaxation times (Figure 9-4A). The previously described qualitative MRI grading

scheme (Figure 4-2) was utilized to again qualitatively assess muscle pathology (Figure

4-5B). Expectedly, all MRI grades of control muscle were graded ‘0’, indicating no

pathology within the muscle, and again, the deltoids, biceps brachii, and triceps brachii

were significantly elevated compared to the healthy muscle. Interestingly, the proximal

deltoids were also elevated when compared to the distal anterior forearm muscles.

The final assessment performed in this sub-study was to compare age and

function to MR imaging findings. Age was correlated to MRI-T2 (Figure 9-5A) and

qualitative MRI Scores (Figure 9-5B), showing significant positive correlation (r2 = 0.25

and r2 = 0.37, respectively) to both measures. Furthermore, the MRI-T2 (Figure 9-5C)

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and qualitative MRI Scores (Figure 9-5D), were additionally correlate to PUL functional

assessment findings, demonstrating significant correlation (r2 = 0.49 and r2 = 0.42,

respectively) for both measures.

Discussion

These investigations of the upper extremities provide foundational data to further

understand the progression of DMD beyond the traditionally studied skeletal muscles of

the lower extremity. While loss of function in the lower extremity is more visibly

apparent as boys transition to wheelchairs, maintenance of function the arms is

arguably more critical for males to retain independence throughout their lives

(Alemdaroğlu et al., 2015; Janssen et al., 2014b; Pane et al., 2014). Preservation of arm

function, even after loss of ambulation, provides males the ability to live their lives in as

independent of a state as possible, allowing them to achieve activities of daily living.

To the muscular dystrophy community, clinical trials remain a beacon of hope for

the potential of a cure. Participation in such trials remains a major hurdle for many

individuals because of study design requirements, and rigid inclusion and exclusion

criteria (Mercuri and Muntoni, 2013; Ricotti et al., 2015). In many studies, adequate

ambulatory function is a requirement to allow participation in trials, and because of this,

many males with DMD are simply not eligible to participate and potentially benefit from

such trials. Therefore, developing a better understanding of how DMD affects the upper

extremities was a major goal of this study. Proximal muscles, such as the deltoids,

were shown to be affected greater from the disease than the distal forearm muscles, in

agreement with the described proximal to distal time course of DMD (Figure 9-4)

(Bushby et al., 2010a; Fischmann et al., 2012; Willcocks et al., 2014). Further, as

males aged, MR measures showed more significant markers of damage (Figures 9-5A

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and 9-5B). When comparing functional measures of the upper extremity to MR

measures, they again significantly correlated with each other (Figures 9-5C and 9-5D),

suggesting that MR may be an appropriate proxy to assess the state of muscle health in

DMD.

This study is our first investigation of utilizing MRI to assess muscle in the upper

extremity in DMD and is not without limitations. As the study is still being optimized, not

all subjects underwent PUL functional testing, and therefore some MR data does not

have a corresponding functional measure. Furthermore, the sequences and scans

being utilized have undergone optimization, and we hope that with further data

collection, standard operating procedures will be able to be established.

Summary of Upper Extremity Findings

In summary, this preliminary study assesses the state of muscle health in the

upper extremity, demonstrating proximal versus distal differences in the amount of

pathology caused by DMD. Furthermore, both age and function demonstrate significant

correlations to the MR measures performed in this study, suggesting that MRI may be

an adequate proxy measure of disease progression in the upper extremities of males

with DMD.

Differences Between Concentric and Eccentric Lower Arm Exercises

Introduction

The ultimate goal of our studies is to translate NIR optical imaging from

conceptual preclinical studies to practical human studies, demonstrating the ability of

NIR optical imaging in humans to detect damaged and diseased muscle. The clinical

study discussed in the former half of this chapter served to demonstrate the ability of

MRI to detect muscle that has been damaged as a result of natural disease in humans.

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In a parallel manner, it is the intention of the experiments described below to

demonstrate that NIR optical imaging can longitudinally, quantitatively, and repeatedly

assess the state of muscle health.

Optical imaging in the NIR range in humans is relatively unexplored, and to date,

has been limited primarily to research of the breast (Altinoğlu et al., 2008; Jiang et al.,

2000; Poellinger et al., 2011; Schneider et al., 2011), exercising muscle (Boushel and

Piantadosi, 2000; Brizidine et al., 2013; Guenette et al., 2008; Hamaoka et al., 2007),

brain (Wolf et al., 2007), and joints (Yuan et al., 2007). A redistribution of tissue water

and blood after exercising results in optical signal changes, which can be detected with

clinical NIR optical imaging. It is our intention to utilize these intrinsic properties of the

body to investigate changes in muscle permeability caused by an exercise routine in a

healthy population and in the natural progression of DMD affected children. Although this

study is still in progress, preliminary results are reported in this dissertation.

Results

To assess temporary and reversible exercise-induced muscle damage resulting

from forearm exercise in healthy subjects, a detailed MR characterization of both

forearms in all subjects is performed. Each subject serves as his own control as

concentric exercise causes minimal muscle damage compared to eccentric exercises.

Fat suppressed T2 weighted images of concentrically (Figure 9-6A) and eccentrically

(Figure 9-6B) exercised forearms are shown. Muscles targeted to be damaged through

our exercise protocol are outlined with a dashed red line in Figure 9-6B. Quantitative

MRI analysis revealed that eccentric contractions increase muscle T2 by ~5% (p<0.05)

when compared to the concentrically exercised forearm (Figure 9-6C).

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Similar to the developmental stage of the MR data, the NIR optical imaging

demonstrate that the concentrically exercised forearm (Figure 9-7A) demonstrate less

signal than the eccentric exercised forearm (Figure 9-7B). The optical images show

increased blood content of the forearm due to elevated blood flow and fluid retention

within the eccentrically exercised forearm when compared to the concentrically loaded

forearm.

Discussion

Early studies have shown MR and optical measurements are sensitive to

eccentrically induced, acute muscle damage in unaffected control subjects (Cermak et

al., 2012; Fulford et al., 2014; Sesto et al., 2008). In addition, pilot studies in the arms

of DMD boys indicate that unlike the upper arm and the shoulder the forearm is

relatively preserved when considering the degree of fatty tissue deposition determined

by MRI (Alemdaroğlu et al., 2015; Bushby et al., 2010a; Hudak et al., 1996). This is

advantageous for optical imaging, due to the presence of chronic muscle damage not

being obscured by the highly scattering lipids (Cerussi et al., 2001). The DMD forearm

is ideal for our imaging applications for the following reasons: 1) early disease

involvement based on quantitative T2 measures, 2) low levels of fatty tissue deposition

to minimize confounding light scattering results resulting from light scatter by lipid and

atrophic muscle, 3) its anatomical location allows it to be easily inserted to the CTLM

optical imaging device for imaging even while seated in a wheelchair.

Elevated T2 values have been commonly observed following eccentric

contractions grossly in larger muscles (Fulford et al., 2014; Sesto et al., 2008), these

results illustrate that MRI possesses adequate sensitivity and spatial resolution to image

acute T2 changes in the much smaller wrist flexor muscles of the forearm after eccentric

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exercise, as we observe in this study (Figure 9-6). Quantitative changes in muscle T2

and volume of affected tissue (calculated from T2 imaging maps) are expected to

correlate to changes observed with NIR-OI of the forearm. Building off of the MR

findings, NIR optical imaging also demonstrates the capability to distinguish

eccentrically from concentrically exercised forearms (Figure 9-7). During the

sarcolemmal damaging contractions that the eccentric exercises induce onto muscle,

edematous inflammation occurs, leading to a detectable signal by NIR optical imaging.

This is visible by the elevation of hyperintensities in the eccentrically loaded forearms as

compared to the contralateral concentrically exercised forearms. Though this study is

incomplete, the data collected thus far is encouraging. Moving forward, the study simply

needs to be performed. Because of several hardware issues with the CTLM System,

recruitment and enrollment into the study has been delayed, but is again underway.

Summary Concentric and Eccentric Lower Arm Exercises

This study provides preliminary data that supports the ability of NIR optical imaging as a

feasible technology to assess the state of muscle health in muscle that has been

damaged by eccentric exercising and from the natural progression of DMD. Differences

between eccentric and concentrically exercised forearm muscles suggest that the

protocols implemented in these studies are appropriately designed to test the

capabilities of NIR optical imaging to assess muscle health.

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Figure 9-1. Qualitative MRI Scores from two representative DMD patients

demonstrating differences in involvement along the length of six lower leg muscle groups. X axes are labeled with P (proximal), MP (mid-proximal), M (middle), MD (mid-distal), and D (distal).

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Figure 9-2. Comprehensive degree of involvement in all slices of all subjects’ muscles

(A: Peroneus, B: Extensor Digitorum Longus, C: Tibialis Anterior, D: Soleus, E: Medial Gastrocnemius, F: Lateral Gastrocnemius), ranging from 0 (white) to 5 (black). A diagonal line over a point indicates that the data was deemed unreliable due to a low SNR or the muscle of interest was not present at the selected slice.

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Figure 9-3. Age and function are related to MRIsingle and MRImulti scores. Correlations

between age and ScoreMulti and ScoreSingle are shown in 9-3A, and 9-3B, respectively. Comparison to Vignos functional scores and ScoreMulti and ScoreSingle are shown in 9-3C and 9-3D, respectively.

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Figure 9-4. Cross sectional analysis of upper extremity muscles in boys with DMD.

Quantitative MRI-T2 measures demonstrate significant differences between control and DMD subjects in the deltoid, biceps, and triceps, but not the forearm muscles (9-4A). Qualitative MRI scores are shown for DMD subjects, and the only significant difference was between the anterior forearm and deltoid muscles (9-4B). Note that all control subjects scored ‘0’ for their qualitative MRI scores, and are not shown.

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Figure 9-5. Age and PUL function as related to MRI-T2 and MRI qualitative scores.

Correlations between age and MRI-T2 and MRI qualitative scores are shown in 9-5A, and 9-5B, respectively. Comparison to PUL functional scores and MRI-T2 and MRI qualitative scores are shown in 9-5C and 9-5D, respectively.

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Figure 9-6. Fat suppressed axial MR images of concentrically (9-6A) and eccentrically

(9-6B) exercised human forearms with quantification (9-6C) of T2 relaxation times taken from the deep flexor muscles of the forearms.

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Figure 9-7. Three dimensional absorbance reconstructions of human forearms were

taken two days following eccentric (9-7A) and concentric (9-7B) exercise. Hyperintensities in the eccentric (9-7A) arms indicate elevated fluid retention as compared to the contralateral concentrically exercised (9-7B) forearms.

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CHAPTER 10 CONCLUSION

Overview

The muscular dystrophies are a collection of progressive and irreversible muscle

wasting disorders with no current curative therapy. Modern treatments include non-

curative interventions, such as mechanisms to increase muscle mass, correct blood

flow perturbations, minimize inflammation and fibrosis, and correct calcium handling.

Investigations that may ultimately provide a cure to the different muscular dystrophies

include protein, transcriptome, and genome restoring remedies. These therapies and

developments to treat and mitigate the pathologies have been developed from basal

understandings of muscle physiology and growth and repair mechanisms. Though

clinical trials offer great hope to the muscular dystrophy family, all have experienced

setbacks and failures thus far. Inadequate measures of muscle health, namely biopsies

and functional tests, effectively null any positive benefits that drugs in clinical trials may

possess, necessitating the development of other outcome measures. Such outcome

measures should be non-invasive, objective requiring minimal subject involvement,

safe, repeatable, and quantifiable.

NIR optical imaging and MRI offer the ability to non-invasively, longitudinally, and

objectively quantify the state of muscle health in complimentary manners. In this

dissertation, I have presented a collection of non-invasive techniques that assess and

monitor basic processes in healthy, exercised damaged, disease damaged, and

disease treated muscle. Both technologies possess their own advantages and

limitations, but in combination, reveal complimentary information regarding the state of

muscle health. This allowed me to quantitatively track progression of disease, or

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conversely, regression of pathology resulting from therapeutic intervention. Importantly,

these measures are longitudinal, non-invasive, objective, and quantifiable. Confirmatory

support of these measures is provided by histology and tissue spectrophotometry. All

together, these non-invasive imaging techniques hold great promise to fulfill the need

for a non-invasive imaging method to monitor and quantify cellular damage, muscle

perfusion, and drug delivery to accelerate testing of drug efficacy in clinical trials for

muscular dystrophy and potentially other muscle disorders.

Summary of Experiments

Capabilities of ICG Enhanced NIR Optical Imaging in Preclinical Models

ICG enhanced NIR optical imaging detected damaged and dystrophic muscle in

several preclinical models. In an acute model of muscle damage, caused by

immobilization followed by reambulation to murine hindlimbs, a characteristic

timecourse of muscle damage and recovery was observed by ICG enhanced NIR

optical imaging. Further confirmatory measures were performed using MRI, MRS,

histology, and tissue spectroscopy. A second round of experiments demonstrating the

ability of ICG enhanced NIR optical imaging to cross sectionally detect muscle that has

been damaged due to two different muscular dystrophies was performed. Additional

insult to muscle, by way of downhill treadmill running demonstrated that exacerbation of

damage to dystrophic muscle is able to be measured. Therapeutic treatment of

dystrophic muscle, through intramuscular administration of AAVs containing the missing

gene of interest, was able to be quantified using ICG enhanced NIR optical imaging.

Similar to the immobilization-reambulation experiments, MRI, MRS, histology, and

tissue spectrophotometry confirmed the NIR optical imaging findings. In all, these

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preclinical studies demonstrate the capabilities of ICG enhanced optical imaging as a

potent modality to assess the state of static and dynamic muscle health in relevant

mouse models.

Potential of Near Infrared Responsive Particles

First, preliminary experiments have demonstrated the capabilities of ICG

enhanced NIR optical imaging to quantify baseline blood flow of the major vasculature

of the mouse hindlimbs, as well as perturbations to blood flow. Furthermore, through

conjugation of ICG to biocompatible PLA particles, we have laid the foundational work

to track the delivery of disease modifying therapies to dystrophic muscle. Through our

findings in these sets of experiments, I demonstrated the prolongation of physical

stability and optical fluorescence of ICG in in vitro and in vivo settings. As these

experiments are still in the developmental stages, my future steps include the

incorporation of disease correcting drugs within the NIR responsive ICG loaded PLA

particles.

Clinical Application of MRI and NIR Optical Imaging

Translation of preclinical findings to the clinical arena is the ultimate goal of all scientific

endeavors. In the first clinical investigation performed, I demonstrated the ability of MRI

to differentiate disease pathology along the lengths of several lower leg muscles of boys

affected by DMD. Through use of a multi-slice evaluation of the lower legs of boys with

DMD, I showed that muscles are not affected equally, disease involvement is more

severe near the myotendinous junctions, individuals are affected uniquely, and that

qualitative MRI grades correlate to age and function. Next, I assessed progression of

disease in the upper extremities of males with DMD, identifying a proximal to distal

pattern of disease progression that correlates to age and function. Though still in

207

progress, the final clinical study suggests encouraging results in that NIR optical

imaging may be an adequate modality to detect and differentiate damaged from healthy

muscle.

208

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BIOGRAPHICAL SKETCH

The impetus for Steve’s motivation to research the muscular dystrophies began at a

young age, when he began to volunteer at summer Muscular Dystrophy Association (MDA)

camps for individuals with neuromuscular disorders. During the era that Steve began

working with the MDA, much noise, ruckus, and publicity was raised to help raise awareness

for the muscular dystrophies. Though many knew what the muscular dystrophies were

because of his efforts, something was clearly still lacking – a cure. Having seen many

friends’ lives prematurely because of the muscular dystrophies, Steve observed that clinical

management was the best that clinicians could provide to this population. A clear calling to

do research stemmed from this realization.

Unsure of the optimal route to pursue his interests, Steve serendipitously ventured to

study Biomedical Engineering at the University of Cincinnati. The years at the University of

Cincinnati have molded Steve’s personal and professional life in many ways. Through the

Co-op Program at the University of Cincinnati, Steve was introduced to Dr. Christy Holland,

who still remains a close confidante to this day. In her research lab, Dr. Holland took Steve

under her wing, wisely providing appropriate motivation and encouragement, to help Steve

co-author two papers. More importantly, Dr. Holland planted the seed of excitement in Steve

of the possibilities of research, introducing him to a network of positive influences that have

proved vital to his successes down the road. She introduced Steve to many MD-PhD’s,

including Drs. Kate Hitchcock, Chip Shaw, Patrick Kee, and Shaoling Huang, who have been

successful both personally and professionally, showing Steve opportunities that he did not

know existed. With great guidance and input from Dr. Kate Hitchcock, Steve sought

application to MD-PhD programs, and despite many curveballs, found a future home at the

University of Florida.

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At the University of Florida, Steve completed the first two years of medical school and

completed the USMLE 1, at which time, his pre-doctoral graduate training was to commence.

By joining the combined lab of Drs. Glenn Walter and Krista Vandenborne, Steve sought to

be trained in a well-rounded, translational lab studying the muscular dystrophies. With Dr.

Walter as his primary mentor, Steve joined the Department of Physiology and Functional

Genomics and soon after, received two T-32 Training Grants (Neuromuscular Plasticity and

Hypertension) to perform his research. His current work, which has formed the bulk of this

dissertation, has been focused on developing non-invasive biomarkers using near infrared

optical imaging and magnetic resonance imaging.

Steve’s long-term interest involves investigating the development and quantification

of novel therapeutic treatments of the spectrum of muscular dystrophies as a physician-

scientist. Beyond the lab life, Steve enjoys music, beer brewing, traveling, exercising, and

the company of friends and family.