ufdcimages.uflib.ufl.eduufdcimages.uflib.ufl.edu/UF/E0/04/98/56/00001/CHRZANOWSKI_S.pdf · 4...
Transcript of ufdcimages.uflib.ufl.eduufdcimages.uflib.ufl.edu/UF/E0/04/98/56/00001/CHRZANOWSKI_S.pdf · 4...
1
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
2
© 2016 Stephen Mark Chrzanowski
3
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
4
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.
5
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
6
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).
7
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
8
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
9
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
10
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
11
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
12
BIOGRAPHICAL SKETCH .......................................................................................... 258
13
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
14
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
15
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
16
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
17
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
18
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
19
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
20
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.
21
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
22
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
23
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)
60
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.
61
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
62
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
63
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°
64
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.
67
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
68
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,
70
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).
71
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.,
73
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,
74
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.
75
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.
79
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.
80
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.
81
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.
82
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
83
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
84
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
85
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.)
86
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.
87
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.
89
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
90
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
91
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
92
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
93
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.
94
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
95
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
96
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
97
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
98
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
99
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
100
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
101
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
102
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
103
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]:
104
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
105
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.
106
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
107
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
108
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;
109
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.
110
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
111
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
112
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.
113
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).
114
Figure 4-1. Radiant efficiency reaches a steady state level between 30 minutes to 12
hours following ICG an intravenous injection.
115
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
116
Figure 4-3. Schematic representation of slice selections along the length of the lower leg.
Proximal
Distal
Mid-Proximal
Middle
Mid-Distal
117
Figure 4-4. The qualitative MRI grading scale used to assess pathology within DMD
muscle.
118
Figure 4-5. Schematic study design of clinical study utilizing NIR to detect muscle
damage.
119
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.
120
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).
121
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
122
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.
123
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
124
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
125
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
126
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.
127
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,
128
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-
131
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.
132
Figure 5-1. Two-dimensional NIR optical imaging shows an increase and recovery of
fluorescent signal in muscle during reambulation following immobilization.
133
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.
134
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
136
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)
140
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
141
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.
152
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.
Control mdx gsg -/-0.0
5.0×108
1.0×109
1.5×109
2.0×109
2.5×109
To
tal R
ad
ian
t E
ffic
ien
cy (
a.u
.)
**
**
Control mdx gsg -/-20
25
30
35
MR
I-T
2 (
ms)
***
**
Control mdx gsg -/-20
25
30
35
1H
2O
-T2 (
ms)
*
***
A
B C D
F
Control mdx
young
mdx
old
gsg
young
gsg
old
20
25
30
35
40
T2 (
ms)
****
****
+++
++++
A
B
D
C
A"
B" C"
Control mdx
young
mdx
old
gsg
young
gsg
old
20
25
30
35
40
T2 (
ms)
****
****
+++
++++
A
B
D
C
A"
B" C"
Control mdx
young
mdx
old
gsg
young
gsg
old
20
25
30
35
40
T2 (
ms)
****
****
+++
++++
A
B
D
C
A"
B" C"
G H I
E
x1091.5
1.0
2.0
RadiantEfficiency
153
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
5.0×108
1.0×109
1.5×109
2.0×109
1H2O-T2 (ms)
To
tal R
ad
ian
t E
ffic
ien
cy
(a
.u.)
20 25 30 350.0
5.0×108
1.0×109
1.5×109
2.0×109
2.5×109
MRI-T2
To
tal R
ad
ian
t E
ffic
ien
cy
(a
.u.)
gsg -/-mdx
ControlA
B
154
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.
155
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.
156
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.
Baseline 6 weeks20
25
30
35
40
MR
I-T
2 (
ms
)
****
*
Baseline 6 weeks20
25
30
35
40
1H
2O
-T2 (
ms)
**
A
C D E FI J K L
G
HB
Pre-treatment Post-treatment0.0
5.0×108
1.0×109
1.5×109
To
tal R
ad
ian
t E
ffic
ien
cy (
a.u
.)
Pre-treatment Post-treatment0.0
5.0×108
1.0×109
1.5×109
2.0×109To
tal R
ad
ian
t E
ffic
ien
cy (
a.u
.)
Control
AAV
**
*
157
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.
20 25 30 350.0
5.0×108
1.0×109
1.5×109
2.0×109
1H2O-T2 (ms)
To
tal R
ad
ian
t E
ffic
ien
cy (
a.u
.)
A
B
20 25 30 350.0
5.0×108
1.0×109
1.5×109
2.0×109
MRI-T2 (ms)
To
tal R
ad
ian
t E
ffic
ien
cy (
a.u
.)AAV - Pre
Non-treatment - Pre
AAV -Post
Non-treatment - Post
158
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
159
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.
160
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).
161
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
162
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).
163
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.
164
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).
165
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.
166
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
167
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
168
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
169
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
170
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
171
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
172
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
173
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
174
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
175
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.
176
Figure 8-1. Representative size distribution (8-1A), aggregation properties (8-1B), and
fluorescence characteristics (8-1C) of ICG-PLA particles
177
Figure 8-2. Photostability at room (25°C, 8-2A) and physiologic (37°C, 8-2B)
temperature of ICG-PLA particles and ICG alone.
178
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).
179
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.
180
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).
181
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
182
(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.
183
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
184
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.
185
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
186
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.
187
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
188
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
189
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).
190
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
191
(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)
192
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
193
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.
194
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).
195
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
196
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.
197
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).
198
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.
199
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.
200
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.
201
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.
202
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.
203
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.
204
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
205
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
206
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
LIST OF REFERENCES
Aartsma-Rus, A., and van Putten, M. (2014). Assessing functional performance in the mdx mouse model. J. Vis. Exp. JoVE.
Aartsma-Rus, A., Janson, A.A.M., Kaman, W.E., Bremmer-Bout, M., van Ommen, G.-J.B., den Dunnen, J.T., and van Deutekom, J.C.T. (2004). Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am. J. Hum. Genet. 74, 83–92.
Aartsma-Rus, A., Van Deutekom, J.C.T., Fokkema, I.F., Van Ommen, G.-J.B., and Den Dunnen, J.T. (2006). Entries in the Leiden Duchenne muscular dystrophy mutation database: An overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve 34, 135–144.
Aartsma-Rus, A., Ferlini, A., Goemans, N., Pasmooij, A.M.G., Wells, D.J., Bushby, K., Vroom, E., and Balabanov, P. (2014). Translational and Regulatory Challenges for Exon Skipping Therapies. Hum. Gene Ther. 25, 885–892.
Ababneh, Z., Beloeil, H., Berde, C.B., Gambarota, G., Maier, S.E., and Mulkern, R.V. (2005). Biexponential parameterization of diffusion and T2 relaxation decay curves in a rat muscle edema model: Decay curve components and water compartments. Magn. Reson. Med. 54, 524–531.
Acharyya, S., Villalta, S.A., Bakkar, N., Bupha-Intr, T., Janssen, P.M.L., Carathers, M., Li, Z.-W., Beg, A.A., Ghosh, S., Sahenk, Z., et al. (2007). Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy. J. Clin. Invest. 117, 889–901.
Adams, G.R., and McCue, S.A. (1998). Localized infusion of IGF-I results in skeletal muscle hypertrophy in rats. J. Appl. Physiol. Bethesda Md 1985 84, 1716–1722.
Ahmad, N., Welch, I., Grange, R., Hadway, J., Dhanvantari, S., Hill, D., Lee, T.-Y., and Hoffman, L.M. (2011). Use of imaging biomarkers to assess perfusion and glucose metabolism in the skeletal muscle of dystrophic mice. BMC Musculoskelet. Disord. 12, 127.
Ahn, A.H., and Kunkel, L.M. (1993). The structural and functional diversity of dystrophin. Nat. Genet. 3, 283–291.
Akima, H., Lott, D., Senesac, C., Deol, J., Germain, S., Arpan, I., Bendixen, R., Lee Sweeney, H., Walter, G., and Vandenborne, K. (2012). Relationships of thigh muscle contractile and non-contractile tissue with function, strength, and age in boys with Duchenne muscular dystrophy. Neuromuscul. Disord. NMD 22, 16–25.
209
Alemdaroğlu, I., Karaduman, A., Yilmaz, Ö.T., and Topaloğlu, H. (2015). Different types of upper extremity exercise training in Duchenne muscular dystrophy: Effects on functional performance, strength, endurance, and ambulation. Muscle Nerve. Epub.
Alford, R., Simpson, H.M., Duberman, J., Hill, G.C., Ogawa, M., Regino, C., Kobayashi, H., and Choyke, P.L. (2009). Toxicity of organic fluorophores used in molecular imaging: literature review. Mol. Imaging 8, 341–354.
Allamand, V., and Campbell, K.P. (2000). Animal models for muscular dystrophy: valuable tools for the development of therapies. Hum. Mol. Genet. 9, 2459–2467.
Allen, D.L., Roy, R.R., and Edgerton, V.R. (1999). Myonuclear domains in muscle adaptation and disease. Muscle Nerve 22, 1350–1360.
Altinoğlu, E.I., Russin, T.J., Kaiser, J.M., Barth, B.M., Eklund, P.C., Kester, M., and Adair, J.H. (2008). Near-infrared emitting fluorophore-doped calcium phosphate nanoparticles for in vivo imaging of human breast cancer. ACS Nano 2, 2075–2084.
Amato, A.A., and Griggs, R.C. (2011). Chapter 1 - Overview of the muscular dystrophies. In Handbook of Clinical Neurology, R.C.G. and A.A. Amato, ed. (Elsevier), pp. 1–9.
Anderson, J.E., Bressler, B.H., and Ovalle, W.K. (1988). Functional regeneration in the hindlimb skeletal muscle of the mdx mouse. J. Muscle Res. Cell Motil. 9, 499–515.
Anderson, J.E., McIntosh, L.M., and Poettcker, R. (1996). Deflazacort but not prednisone improves both muscle repair and fiber growth in diaphragm and limb muscle in vivo in the mdx dystrophic mouse. Muscle Nerve 19, 1576–1585.
Angelini, C. (2007). The role of corticosteroids in muscular dystrophy: a critical appraisal. Muscle Nerve 36, 424–435.
Anthony, K., Arechavala-Gomeza, V., Taylor, L.E., Vulin, A., Kaminoh, Y., Torelli, S., Feng, L., Janghra, N., Bonne, G., Beuvin, M., et al. (2014). Dystrophin quantification: Biological and translational research implications. Neurology 83, 2062–2069.
Araki, E., Nakamura, K., Nakao, K., Kameya, S., Kobayashi, O., Nonaka, I., Kobayashi, T., and Katsuki, M. (1997). Targeted disruption of exon 52 in the mouse dystrophin gene induced muscle degeneration similar to that observed in Duchenne muscular dystrophy. Biochem. Biophys. Res. Commun. 238, 492–497.
Araujo, E.C.A., Fromes, Y., and Carlier, P.G. (2014). New Insights on Human Skeletal Muscle Tissue Compartments Revealed by In Vivo T2 NMR Relaxometry. Biophys. J. 106, 2267–2274.
210
Armstrong, R.B., Ogilvie, R.W., and Schwane, J.A. (1983). Eccentric exercise-induced injury to rat skeletal muscle. J. Appl. Physiol. 54, 80–93.
Arpan, I., Willcocks, R.J., Forbes, S.C., Finkel, R.S., Lott, D.J., Rooney, W.D., Triplett, W.T., Senesac, C.R., Daniels, M.J., Byrne, B.J., et al. (2014). Examination of effects of corticosteroids on skeletal muscles of boys with DMD using MRI and MRS. Neurology 83, 974–980.
Athanasopoulos, T., Graham, I.R., Foster, H., and Dickson, G. (2004). Recombinant adeno-associated viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy (DMD). Gene Ther. 11 Suppl 1, S109–S121.
Aziz, A., Sebastian, S., and Dilworth, F.J. (2012). The origin and fate of muscle satellite cells. Stem Cell Rev. 8, 609–622.
Babic, J., and Lenarcic, J. (2004). In vivo determination of triceps surae muscle–tendon complex viscoelastic properties. Eur. J. Appl. Physiol. 92, 477–484.
Balaban, B., Matthews, D.J., Clayton, G.H., and Carry, T. (2005). Corticosteroid treatment and functional improvement in Duchenne muscular dystrophy: long-term effect. Am. J. Phys. Med. Rehabil. Assoc. Acad. Physiatr. 84, 843–850.
Baldwin, K.M. (1996). Effects of altered loading states on muscle plasticity: what have we learned from rodents? Med. Sci. Sports Exerc. 28, S101–S106.
Baldwin, K.M., and Haddad, F. (2001). Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle. J. Appl. Physiol. Bethesda Md 1985 90, 345–357.
Ballou, B., Fisher, G.W., Waggoner, A.S., Farkas, D.L., Reiland, J.M., Jaffe, R., Mujumdar, R.B., Mujumdar, S.R., and Hakala, T.R. (1995). Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies. Cancer Immunol. Immunother. CII 41, 257–263.
Barkhof, F., Filippi, M., Miller, D.H., Scheltens, P., Campi, A., Polman, C.H., Comi, G., Adèr, H.J., Losseff, N., and Valk, J. (1997). Comparison of MRI criteria at first presentation to predict conversion to clinically definite multiple sclerosis. Brain J. Neurol. 120 ( Pt 11), 2059–2069.
Barth, J., Reha, A., Spiegel, R., Elfring, G.L., Husain, M., and Peltz, S.W. (2013). P.11.18 Design of a confirmatory phase 3, multicenter, randomized, double-blind, placebo-controlled study of ataluren in patients with nonsense mutation Duchenne muscular dystrophy. Neuromuscul. Disord. 23, 804.
Barton, E.R. (2006). Impact of sarcoglycan complex on mechanical signal transduction in murine skeletal muscle. Am. J. Physiol. - Cell Physiol. 290, C411–C419.
211
Barton, E.R. (2010). Restoration of γ-Sarcoglycan Localization and Mechanical Signal Transduction Are Independent in Murine Skeletal Muscle. J. Biol. Chem. 285, 17263–17270.
Barton, E.R., Morris, L., Musaro, A., Rosenthal, N., and Sweeney, H.L. (2002). Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice. J. Cell Biol. 157, 137–148.
Barton, E.R., Morris, L., Kawana, M., Bish, L.T., and Toursel, T. (2005). Systemic administration of L-arginine benefits mdx skeletal muscle function. Muscle Nerve 32, 751–760.
Barton-Davis, E.R., Cordier, L., Shoturma, D.I., Leland, S.E., and Sweeney, H.L. (1999). Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice. J. Clin. Invest. 104, 375–381.
Basford, J.R., Jenkyn, T.R., An, K.-N., Ehman, R.L., Heers, G., and Kaufman, K.R. (2002). Evaluation of healthy and diseased muscle with magnetic resonance elastography. Arch. Phys. Med. Rehabil. 83, 1530–1536.
Baudin, P.-Y., Marty, B., Robert, B., Schukelovitch, A., Carlier, R.Y., Azzabou, N., and Carlier, P.G. (2015). Qualitative and quantitative evaluation of skeletal muscle fatty degenerative changes using whole-body Dixon nuclear magnetic resonance imaging for an important reduction of the acquisition time. Neuromuscul. Disord. In Press, Accepted Manuscript.
Baudy, A.R., Sali, A., Jordan, S., Kesari, A., Johnston, H.K., Hoffman, E.P., and Nagaraju, K. (2011). Non-invasive Optical Imaging of Muscle Pathology in mdx Mice Using Cathepsin Caged Near-Infrared Imaging. Mol. Imaging Biol. 13, 462–470.
van Bavel, H., Drost, M.R., Wielders, J.D.L., Huyghe, J.M., Huson, A., and Janssen, J.D. (1996). Strain distribution on rat medial gastrocnemius (MG) during passive stretch. J. Biomech. 29, 1069–1074.
Beck, R.W., Chandler, D.L., Cole, S.R., Simon, J.H., Jacobs, L.D., Kinkel, R.P., Selhorst, J.B., Rose, J.W., Cooper, J.A., Rice, G., et al. (2002). Interferon beta-1a for early multiple sclerosis: CHAMPS trial subgroup analyses. Ann. Neurol. 51, 481–490.
Bendszus, M., Koltzenburg, M., Wessig, C., and Solymosi, L. (2002). Sequential MR imaging of denervated muscle: experimental study. AJNR Am. J. Neuroradiol. 23, 1427–1431.
Bengtsson, N.E., Brown, G., Scott, E.W., and Walter, G.A. (2010). lacZ as a genetic reporter for real-time MRI. Magn. Reson. Med. 63, 745–753.
Berridge, M. (1993). Inositol Trisphosphate and Calcium Signaling. Nature 361, 315–325.
212
Bertoni, C., Jarrahian, S., Wheeler, T.M., Li, Y., Olivares, E.C., Calos, M.P., and Rando, T.A. (2006). Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration. Proc. Natl. Acad. Sci. U. S. A. 103, 419–424.
Bhattacharya, S., Das, A., Dasgupta, R., and Bagchi, A. (2014). Analyses of the presence of mutations in Dystrophin protein to predict their relative influences in the onset of Duchenne Muscular Dystrophy. Cell. Signal. 26, 2857–2864.
Bishop, C., Newbould, R., Janiczek, R., and Campion, G. (2015). Magnetic Resonance Imaging Assessments of two doses of Drisapersen in the Treatment of Ambulant Boys with Duchenne Muscular Dystrophy (P7.059). Neurology 84, P7.059.
Björnsson, O.G., Murphy, R., and Chadwick, V.S. (1982). Physiochemical studies of indocyanine green (ICG): absorbance/concentration relationship, pH tolerance and assay precision in various solvents. Experientia 38, 1441–1442.
Blain, A., Greally, E., Laval, S.H., Blamire, A.M., MacGowan, G.A., and Straub, V.W. (2015). Absence of Cardiac Benefit with Early Combination ACE Inhibitor and Beta Blocker Treatment in mdx Mice. J Cardiovasc. Transl. Res. 1–10.
Blake, D.J., Weir, A., Newey, S.E., and Davies, K.E. (2002). Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol. Rev. 82, 291–329.
Blanco-Prieto, M.J., Campanero, M.A., Besseghir, K., Heimgatner, F., and Gander, B. (2004). Importance of single or blended polymer types for controlled in vitro release and plasma levels of a somatostatin analogue entrapped in PLA/PLGA microspheres. J. Controlled Release 96, 437–448.
Bleicher, A.G., and Kanal, E. (2008). A Serial Dilution Study of Gadolinium-Based MR Imaging Contrast Agents. Am. J. Neuroradiol. 29, 668–673.
Bobet, J., Mooney, R.F., and Gordon, T. (1998). Force and stiffness of old dystrophic (mdx) mouse skeletal muscles. Muscle Nerve 21, 536–539.
Bogdanovich, S., Krag, T.O.B., Barton, E.R., Morris, L.D., Whittemore, L.-A., Ahima, R.S., and Khurana, T.S. (2002). Functional improvement of dystrophic muscle by myostatin blockade. Nature 420, 418–421.
Boldrin, L., Zammit, P.S., and Morgan, J.E. (2015). Satellite cells from dystrophic muscle retain regenerative capacity. Stem Cell Res. 14, 20–29.
Bonati, U., Hafner, P., Schädelin, S., Schmid, M., Naduvilekoot Devasia, A., Schroeder, J., Zuesli, S., Pohlman, U., Neuhaus, C., Klein, A., et al. Quantitative muscle MRI: A powerful surrogate outcome measure in Duchenne muscular dystrophy. Neuromuscul. Disord.
213
Bongers, H., Schick, F., Skalej, M., Jung, W.I., and Stevens, A. (1992). Localized in vivo 1H spectroscopy of human skeletal muscle: normal and pathologic findings. Magn. Reson. Imaging 10, 957–964.
Bönnemann, C.G., McNally, E.M., and Kunkel, L.M. (1996). Beyond dystrophin: current progress in the muscular dystrophies. Curr. Opin. Pediatr. 8, 569–582.
Borotto, E., Englender, J., Pourny, J.C., Naveau, S., Chaput, J.C., and Lecarpentier, Y. (1999). Detection of the fluorescence of GI vessels in rats using a CCD camera or a near-infrared video endoscope. Gastrointest. Endosc. 50, 684–688.
Bosley, K.S., Botchan, M., Bredenoord, A.L., Carroll, D., Charo, R.A., Charpentier, E., Cohen, R., Corn, J., Doudna, J., Feng, G., et al. (2015). CRISPR germline engineering--the community speaks. Nat. Biotechnol. 33, 478–486.
Botar-Jid, C., Damian, L., Dudea, S.M., Vasilescu, D., Rednic, S., and Badea, R. (2010). The contribution of ultrasonography and sonoelastography in assessment of myositis. Med. Ultrason. 12, 120–126.
Boushel, R., and Piantadosi, C. a. (2000). Near-infrared spectroscopy for monitoring muscle oxygenation. Acta Physiol. Scand. 168, 615–622.
Boushel, R., Langberg, H., Olesen, J., Nowak, M., Simonsen, L., Bülow, J., and Kjær, M. (2000). Regional blood flow during exercise in humans measured by near-infrared spectroscopy and indocyanine green. J. Appl. Physiol. 89, 1868–1878.
Brada, M., Hoang-Xuan, K., Rampling, R., Dietrich, P.Y., Dirix, L.Y., Macdonald, D., Heimans, J.J., Zonnenberg, B.A., Bravo-Marques, J.M., Henriksson, R., et al. (2001). Multicenter phase II trial of temozolomide in patients with glioblastoma multiforme at first relapse. Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. ESMO 12, 259–266.
Brancaccio, P., Maffulli, N., and Limongelli, F.M. (2007). Creatine kinase monitoring in sport medicine. Br. Med. Bull. 81-82, 209–230.
Bremer, C., Tung, C.H., and Weissleder, R. (2001). In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat. Med. 7, 743–748.
Brenman, J.E., Chao, D.S., Xia, H., Aldape, K., and Bredt, D.S. (1995). Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell 82, 743–752.
Brizidine, J., Ryan, T., Larson, R., and McCully, K. (2013). Skeletal Muscle Metabolism in Endurance Athletes with Near-Infrared Spectroscopy. Med. Sci. Sports Exerc. May 2013 45, 869–875.
214
Brockmann, M.A., Kemmling, A., and Groden, C. (2007). Current issues and perspectives in small rodent magnetic resonance imaging using clinical MRI scanners. Methods 43, 79–87.
Brussee, V., Tardif, F., and Tremblay, J.P. (1997). Muscle fibers of mdx mice are more vulnerable to exercise than those of normal mice. Neuromuscul. Disord. NMD 7, 487–492.
Bryant, N.D., Li, K., Does, M.D., Barnes, S., Gochberg, D.F., Yankeelov, T.E., Park, J.H., and Damon, B.M. (2014). Multi-parametric MRI characterization of inflammation in murine skeletal muscle. NMR Biomed. Epub.
Bulfield, G., Siller, W.G., Wight, P.A., and Moore, K.J. (1984). X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc. Natl. Acad. Sci. U. S. A. 81, 1189–1192.
Burns, D., Manning, J., O’Malley, D., and O’Halloran, K. (2015). Respiratory Function in the Mdx Mouse Model of Duchenne Muscular Dystrophy: Role of Hypoxia, Stress and Immune Factors. FASEB J. 29, 660.6.
Bushby, K., Finkel, R., Birnkrant, D.J., Case, L.E., Clemens, P.R., Cripe, L., Kaul, A., Kinnett, K., McDonald, C., Pandya, S., et al. (2010a). Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management. Lancet Neurol. 9, 77–93.
Bushby, K., Finkel, R., Birnkrant, D.J., Case, L.E., Clemens, P.R., Cripe, L., Kaul, A., Kinnett, K., McDonald, C., Pandya, S., et al. (2010b). Diagnosis and management of Duchenne muscular dystrophy, part 2: implementation of multidisciplinary care. Lancet Neurol. 9, 177–189.
Bushby, K., Finkel, R., Wong, B., Barohn, R., Campbell, C., Comi, G.P., Connolly, A.M., Day, J.W., Flanigan, K.M., Goemans, N., et al. (2014). Ataluren treatment of patients with nonsense mutation dystrophinopathy. Muscle Nerve 50, 477–487.
Campbell, K.P. (1995). Three muscular dystrophies: loss of cytoskeleton-extracellular matrix linkage. Cell 80, 675–679.
Caravan, P., Ellison, J.J., McMurry, T.J., and Lauffer, R.B. (1999). Gadolinium(III) Chelates as MRI Contrast Agents: Structure, Dynamics, and Applications. Chem. Rev. 99, 2293–2352.
Carlier, P.G., Mercuri, E., and Straub, V. (2012). Applications of MRI in muscle diseases. Neuromuscul. Disord. NMD 22 Suppl 2, S41.
Carlson, C.J., Booth, F.W., and Gordon, S.E. (1999). Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading. Am. J. Physiol. 277, R601–R606.
215
Caron, A.Z., Drouin, G., Desrosiers, J., Trensz, F., and Grenier, G. (2009). A novel hindlimb immobilization procedure for studying skeletal muscle atrophy and recovery in mouse. J. Appl. Physiol. 106, 2049–2059.
Cazzato, G., and Walton, J.N. (1968). The pathology of the muscle spindle. A study of biopsy material in various muscular and neuromuscular diseases. J. Neurol. Sci. 7, 15–70.
Cedarbaum, J.M., Stephenson, D., Rudick, R., Carrillo, M.C., Stebbins, G., Kerr, D., Heemskerk, J., Galpern, W.R., Kaufmann, P., Cella, D., et al. (2014). Commonalities and Challenges in the Development of Clinical Trial Measures in Neurology. Neurotherapeutics 1–19.
C. elegans Sequencing Consortium (1998). Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282, 2012–2018.
Cermak, N.M., Noseworthy, M.D., Bourgeois, J.M., Tarnopolsky, M.A., and Gibala, M.J. (2012). Diffusion tensor MRI to assess skeletal muscle disruption following eccentric exercise. Muscle Nerve 46, 42–50.
Cerussi, A.E., Berger, A.J., Bevilacqua, F., Shah, N., Jakubowski, D., Butler, J., Holcombe, R.F., and Tromberg, B.J. (2001). Sources of absorption and scattering contrast for near-infrared optical mammography. Acad. Radiol. 8, 211–218.
Chang, D.C., Misra, L.K., Beall, P.T., Fanguy, R.C., and Hazlewood, C.F. (1981). Nuclear magnetic resonance study of muscle water protons in muscular dystrophy of chickens. J. Cell. Physiol. 107, 139–145.
Chang, W.J., Iannaccone, S.T., Lau, K.S., Masters, B.S., McCabe, T.J., McMillan, K., Padre, R.C., Spencer, M.J., Tidball, J.G., and Stull, J.T. (1996). Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy. Proc. Natl. Acad. Sci. U. S. A. 93, 9142–9147.
Chargé, S.B.P., and Rudnicki, M.A. (2004). Cellular and molecular regulation of muscle regeneration. Physiol. Rev. 84, 209–238.
Chauveau, F., Cho, T.H., Berthezène, Y., Nighoghossian, N., and Wiart, M. (2010). Imaging inflammation in stroke using magnetic resonance imaging. Int. J. Clin. Pharmacol. Ther. 48, 718–728.
Chen, S.J., Lee, A.F., Lee, F.L., and Liu, J.H. (1999). Indocyanine green angiography of central serous chorioretinopathy. Zhonghua Yi Xue Za Zhi Chin. Med. J. Free China Ed 62, 605–613.
Cherrick, G.R., Stein, S.W., Leevy, C.M., and Davidson, C.S. (1960). Indocyanine green: observations on its physical properties, plasma decay, and hepatic extraction. J. Clin. Invest. 39, 592–600.
216
Childers, M.K., Okamura, C.S., Bogan, D.J., Bogan, J.R., Petroski, G.F., McDonald, K., and Kornegay, J.N. (2002). Eccentric contraction injury in dystrophic canine muscle. Arch. Phys. Med. Rehabil. 83, 1572–1578.
Cho, H.-S., Dong, Z., Pauletti, G.M., Zhang, J., Xu, H., Gu, H., Wang, L., Ewing, R.C., Huth, C., Wang, F., et al. (2010). Fluorescent, Superparamagnetic Nanospheres for Drug Storage, Targeting, and Imaging: A Multifunctional Nanocarrier System for Cancer Diagnosis and Treatment. Acs Nano 4, 5398–5404.
Ciciliot, S., and Schiaffino, S. (2010). Regeneration of mammalian skeletal muscle. Basic mechanisms and clinical implications. Curr. Pharm. Des. 16, 906–914.
Cirak, S., Feng, L., Anthony, K., Arechavala-Gomeza, V., Torelli, S., Sewry, C., Morgan, J.E., and Muntoni, F. (2012). Restoration of the dystrophin-associated glycoprotein complex after exon skipping therapy in Duchenne muscular dystrophy. Mol. Ther. J. Am. Soc. Gene Ther. 20, 462–467.
Clarkson, P.M., and Hubal, M.J. (2002). Exercise-induced muscle damage in humans. Am. J. Phys. Med. Rehabil. Assoc. Acad. Physiatr. 81, S52–S69.
Clarkson, P.M., Byrnes, W.C., McCormick, K.M., Turcotte, L.P., and White, J.S. (1986). Muscle soreness and serum creatine kinase activity following isometric, eccentric, and concentric exercise. Int. J. Sports Med. 7, 152–155.
Cleary, M.A., Kimura, I.F., Sitler, M.R., and Kendrick, Z.V. (2002). Temporal Pattern of the Repeated Bout Effect of Eccentric Exercise on Delayed-Onset Muscle Soreness. J. Athl. Train. 37, 32–36.
Cohn, R.D., and Campbell, K.P. (2000). Molecular basis of muscular dystrophies. Muscle Nerve 23, 1456–1471.
Cole, W.C., LeBlanc, A.D., and Jhingran, S.G. (1993). The origin of biexponential T2 relaxation in muscle water. Magn. Reson. Med. Off. J. Soc. Magn. Reson. Med. Soc. Magn. Reson. Med. 29, 19–24.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823.
Connolly, A.M., Malkus, E.C., Mendell, J.R., Flanigan, K.M., Miller, J.P., Schierbecker, J.R., Siener, C.A., Golumbek, P.T., Zaidman, C.M., McDonald, C.M., et al. (2014). Outcome reliability in non ambulatory boys/men with duchenne muscular dystrophy. Muscle Nerve . Epub.
Consolino, C.M., and Brooks, S.V. (2004). Susceptibility to sarcomere injury induced by single stretches of maximally activated muscles of mdx mice. J. Appl. Physiol. 96, 633–638.
217
Cooper, B.J., Winand, N.J., Stedman, H., Valentine, B.A., Hoffman, E.P., Kunkel, L.M., Scott, M.O., Fischbeck, K.H., Kornegay, J.N., and Avery, R.J. (1988). The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs. Nature 334, 154–156.
Cordier, L., Hack, A.A., Scott, M.O., Barton-Davis, E.R., Gao, G., Wilson, J.M., McNally, E.M., and Sweeney, H.L. (2000). Rescue of Skeletal Muscles of γ-Sarcoglycan- Deficient Mice with Adeno-Associated Virus-Mediated Gene Transfer. Mol. Ther. 1, 119–129.
Corlu, A., Choe, R., Durduran, T., Rosen, M.A., Schweiger, M., Arridge, S.R., Schnall, M.D., and Yodh, A.G. (2007). Three-dimensional in vivo fluorescence diffuse optical tomography of breast cancer in humans. Opt. Express 15, 6696–6716.
Damon, B.M., Gregory, C.D., Hall, K.L., Stark, H.J., Gulani, V., and Dawson, M.J. (2002). Intracellular acidification and volume increases explain R2 decreases in exercising muscle. Magn. Reson. Med. 47, 14–23.
Davidson, Z.E., Ryan, M.M., Kornberg, A.J., Walker, K.Z., and Truby, H. (2014). Strong Correlation Between the 6-Minute Walk Test and Accelerometry Functional Outcomes in Boys With Duchenne Muscular Dystrophy. J. Child Neurol. 0883073814530502.
De Arcangelis, V., Strimpakos, G., Gabanella, F., Corbi, N., Luvisetto, S., Magrelli, A., Onori, A., Passananti, C., Pisani, C., Rome, S., et al. (2015). Pathways Implicated in Tadalafil Amelioration of Duchenne Muscular Dystrophy. J. Cell. Physiol. Epub.
Decary, S., Mouly, V., Hamida, C.B., Sautet, A., Barbet, J.P., and Butler-Browne, G.S. (1997). Replicative potential and telomere length in human skeletal muscle: implications for satellite cell-mediated gene therapy. Hum. Gene Ther. 8, 1429–1438.
Decary, S., Hamida, C.B., Mouly, V., Barbet, J.P., Hentati, F., and Butler-Browne, G.S. (2000). Shorter telomeres in dystrophic muscle consistent with extensive regeneration in young children. Neuromuscul. Disord. NMD 10, 113–120.
Deconinck, A.E., Rafael, J.A., Skinner, J.A., Brown, S.C., Potter, A.C., Metzinger, L., Watt, D.J., Dickson, J.G., Tinsley, J.M., and Davies, K.E. (1997). Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell 90, 717–727.
Deconinck, N., Rafael, J.A., Beckers-Bleukx, G., Kahn, D., Deconinck, A.E., Davies, K.E., and Gillis, J.M. (1998). Consequences of the combined deficiency in dystrophin and utrophin on the mechanical properties and myosin composition of some limb and respiratory muscles of the mouse. Neuromuscul. Disord. NMD 8, 362–370.
218
Desguerre, I., Christov, C., Mayer, M., Zeller, R., Becane, H.-M., Bastuji-Garin, S., Leturcq, F., Chiron, C., Chelly, J., and Gherardi, R.K. (2009). Clinical Heterogeneity of Duchenne Muscular Dystrophy (DMD): Definition of Sub-Phenotypes and Predictive Criteria by Long-Term Follow-Up. PLoS ONE 4, e4347.
Desmettre, T., Devoisselle, J.M., and Mordon, S. (2000). Fluorescence Properties and Metabolic Features of Indocyanine Green (ICG) as Related to Angiography. Surv. Ophthalmol. 45, 15–27.
van Deutekom, J.C., Janson, A.A., Ginjaar, I.B., Frankhuizen, W.S., Aartsma-Rus, A., Bremmer-Bout, M., den Dunnen, J.T., Koop, K., van der Kooi, A.J., Goemans, N.M., et al. (2007). Local Dystrophin Restoration with Antisense Oligonucleotide PRO051. N. Engl. J. Med. 357, 2677–2686.
Devoisselle, J.M., Soulié-Bégu, S., Mordon, S., Desmettre, T., and Maillols, H. (1998). A preliminary study of the in vivo behaviour of an emulsion formulation of indocyanine green. Lasers Med. Sci. 13, 279–282.
DiMario, J.X., Uzman, A., and Strohman, R.C. (1991). Fiber regeneration is not persistent in dystrophic (MDX) mouse skeletal muscle. Dev. Biol. 148, 314–321.
Donahue, K., Burstein, D., Manning, W., and Gray, M. (1994). Studies of Gd-Dtpa Relaxivity and Proton-Exchange Rates in Tissue. Magn. Reson. Med. 32, 66–76.
Drakonaki, E.E., and Allen, G.M. (2010). Magnetic resonance imaging, ultrasound and real-time ultrasound elastography of the thigh muscles in congenital muscle dystrophy. Skeletal Radiol. 39, 391–396.
Drakonaki, E.E., Allen, G.M., and Wilson, D.J. (2012). Ultrasound elastography for musculoskeletal applications. Br. J. Radiol. 85, 1435–1445.
Dresner, M.A., Rose, G.H., Rossman, P.J., Muthupillai, R., Manduca, A., and Ehman, R.L. (2001). Magnetic resonance elastography of skeletal muscle. J. Magn. Reson. Imaging JMRI 13, 269–276.
Dubey, N., Shukla, J., Hazari, P.P., Varshney, R., Ganeshpurkar, A., Mishra, A.K., Trivedi, P., and Bandopadhaya, G.P. (2012). Preparation and biological evaluation of paclitaxel loaded biodegradable PCL/PEG nanoparticles for the treatment of human neuroendocrine pancreatic tumor in mice. Hell. J. Nucl. Med. 15, 9–15.
Duclos, F., Straub, V., Moore, S.A., Venzke, D.P., Hrstka, R.F., Crosbie, R.H., Durbeej, M., Lebakken, C.S., Ettinger, A.J., van der Meulen, J., et al. (1998). Progressive muscular dystrophy in alpha-sarcoglycan-deficient mice. J. Cell Biol. 142, 1461–1471.
Dumont, N.A., Wang, Y.X., von Maltzahn, J., Pasut, A., Bentzinger, C.F., Brun, C.E., and Rudnicki, M.A. (2015). Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division. Nat. Med. advance online publication.
219
Dunn, J.F., and Zaim-Wadghiri, Y. (1999). Quantitative magnetic resonance imaging of the mdx mouse model of Duchenne muscular dystrophy. Muscle Nerve 22, 1367–1371.
Dunn, S.E., Burns, J.L., and Michel, R.N. (1999). Calcineurin is required for skeletal muscle hypertrophy. J. Biol. Chem. 274, 21908–21912.
Durham, W.J., Arbogast, S., Gerken, E., Li, Y.-P., and Reid, M.B. (2006). Progressive nuclear factor-κB activation resistant to inhibition by contraction and curcumin in mdx mice. Muscle Nerve 34, 298–303.
Eagle, M., Baudouin, S.V., Chandler, C., Giddings, D.R., Bullock, R., and Bushby, K. (2002). Survival in Duchenne muscular dystrophy: improvements in life expectancy since 1967 and the impact of home nocturnal ventilation. Neuromuscul. Disord. NMD 12, 926–929.
Ebert, B., Riefke, B., Sukowski, U., and Licha, K. (2011). Cyanine dyes as contrast agents for near-infrared imaging in vivo: acute tolerance, pharmacokinetics, and fluorescence imaging. J. Biomed. Opt. 16, 066003.
Echigoya, Y., Aoki, Y., Miskew, B., Panesar, D., Touznik, A., Nagata, T., Tanihata, J., Nakamura, A., Nagaraju, K., and Yokota, T. (2015). Long-Term Efficacy of Systemic Multiexon Skipping Targeting Dystrophin Exons 45–55 With a Cocktail of Vivo-Morpholinos in Mdx52 Mice. Mol. Ther. — Nucleic Acids 4, e225.
Edwards, R.H., Newham, D.J., Jones, D.A., and Chapman, S.J. (1984). Role of mechanical damage in pathogenesis of proximal myopathy in man. Lancet Lond. Engl. 1, 548–552.
Eghtesad, S., Jhunjhunwala, S., Little, S.R., and Clemens, P.R. (2012). Effect of rapamycin on immunity induced by vector-mediated dystrophin expression in mdx skeletal muscle. Sci. Rep. 2, 399.
Elder, C.P., Apple, D.F., Bickel, C.S., Meyer, R.A., and Dudley, G.A. (2004). Intramuscular fat and glucose tolerance after spinal cord injury – a cross-sectional study. Spinal Cord 42, 711–716.
El-Desoky, A., Seifalian, A.M., Cope, M., Delpy, D.T., and Davidson, B.R. (1999). Experimental study of liver dysfunction evaluated by direct indocyanine green clearance using near infrared spectroscopy. Br. J. Surg. 86, 1005–1011.
El Kerch, F., Ratbi, I., Sbiti, A., Laarabi, F.-Z., Barkat, A., and Sefiani, A. (2014). Carrier frequency of the c.525delT mutation in the SGCG gene and estimated prevalence of limb girdle muscular dystrophy type 2C among the Moroccan population. Genet. Test. Mol. Biomark. 18, 253–256.
220
Elliott, M.A., Walter, G.A., Swift, A., Vandenborne, K., Schotland, J.C., and Leigh, J.S. (1999). Spectral quantitation by principal component analysis using complex singular value decomposition. Magn. Reson. Med. Off. J. Soc. Magn. Reson. Med. Soc. Magn. Reson. Med. 41, 450–455.
Emery, A.E.H. (2002). The muscular dystrophies. Lancet Lond. Engl. 359, 687–695.
Engel, W.K. (1967). Muscle biopsy uses and limitations. Postgrad. Med. 41, 155–160.
Ennen, J.P., Verma, M., and Asakura, A. (2013). Vascular-targeted therapies for Duchenne muscular dystrophy. Skelet. Muscle 3, 9.
Ervasti, J.M., and Campbell, K.P. (1991). Membrane organization of the dystrophin-glycoprotein complex. Cell 66, 1121–1131.
Ervasti, J.M., and Campbell, K.P. (1993). A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J. Cell Biol. 122, 809–823.
Ervasti, J.M., Ohlendieck, K., Kahl, S.D., Gaver, M.G., and Campbell, K.P. (1990). Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle. Nature 345, 315–319.
Esper, G.J., Shiffman, C.A., Aaron, R., Lee, K.S., and Rutkove, S.B. (2006). Assessing neuromuscular disease with multifrequency electrical impedance myography. Muscle Nerve 34, 595–602.
Evans, G.F., Haller, R.G., Wyrick, P.S., Parkey, R.W., and Fleckenstein, J.L. (1998). Submaximal delayed-onset muscle soreness: correlations between MR imaging findings and clinical measures. Radiology 208, 815–820.
Fabb, S.A., Wells, D.J., Serpente, P., and Dickson, G. (2002). Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibits myofibre degeneration in nude/mdx mice. Hum. Mol. Genet. 11, 733–741.
Falzarano, M.S., Passarelli, C., Bassi, E., Fabris, M., Perrone, D., Sabatelli, P., Maraldi, N.M., Donà, S., Selvatici, R., Bonaldo, P., et al. (2013). Biodistribution and molecular studies on orally administered nanoparticle-AON complexes encapsulated with alginate aiming at inducing dystrophin rescue in mdx mice. BioMed Res. Int. 2013, 527418.
Falzarano, M.S., Bassi, E., Passarelli, C., Braghetta, P., and Ferlini, A. (2014a). Biodistribution Studies of Polymeric Nanoparticles for Drug Delivery in Mice. Hum. Gene Ther.
221
Falzarano, M.S., Passarelli, C., and Ferlini, A. (2014b). Nanoparticle delivery of antisense oligonucleotides and their application in the exon skipping strategy for Duchenne muscular dystrophy. Nucleic Acid Ther. 24, 87–100.
Fan, R.H., and Does, M.D. (2008). Compartmental relaxation and diffusion tensor imaging measurements in vivo in λ-carrageenan-induced edema in rat skeletal muscle. NMR Biomed. 21, 566–573.
Fang, J., Nakamura, H., and Maeda, H. (2011). The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv. Drug Deliv. Rev. 63, 136–151.
Farokhzad, O.C., Jon, S.Y., Khademhosseini, A., Tran, T.N.T., LaVan, D.A., and Langer, R. (2004). Nanopartide-aptamer bioconjugates: A new approach for targeting prostate cancer cells. Cancer Res. 64, 7668–7672.
Fayad, L., Deshmukh, S., Subhawong, T., and Carrino, J. (2014). Role of MR spectroscopy in musculoskeletal imaging. Indian J. Radiol. Imaging 24, 210.
Feener, C.A., Koenig, M., and Kunkel, L.M. (1989). Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus. Nature 338, 509–511.
Fenichel, G.M., Florence, J.M., Pestronk, A., Mendell, J.R., Moxley, R.T., Griggs, R.C., Brooke, M.H., Miller, J.P., Robison, J., and King, W. (1991). Long-term benefit from prednisone therapy in Duchenne muscular dystrophy. Neurology 41, 1874–1877.
Ferrante, S., Contini, D., Spinelli, L., Pedrocchi, A., Torricelli, A., Molteni, F., Ferrigno, G., and Cubeddu, R. (2009). Monitoring muscle metabolic indexes by time-domain near-infrared spectroscopy during knee flex-extension induced by functional electrical stimulation. J. Biomed. Opt. 14, 044011–044011 – 11.
Ferrari, G., Cusella-De Angelis, G., Coletta, M., Paolucci, E., Stornaiuolo, A., Cossu, G., and Mavilio, F. (1998). Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279, 1528–1530.
Ferrari, M., Mottola, L., and Quaresima, V. (2004). Principles, techniques, and limitations of near infrared spectroscopy. Can. J. Appl. Physiol. Rev. Can. Physiol. Appliquée 29, 463–487.
Ferrer, A., Foster, H., Wells, K.E., Dickson, G., and Wells, D.J. (2004). Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins. Gene Ther. 11, 884–893.
222
Finanger, E.L., Russman, B., Forbes, S.C., Rooney, W.D., Walter, G.A., and Vandenborne, K. (2012). Use of skeletal muscle MRI in diagnosis and monitoring disease progression in Duchenne muscular dystrophy. Phys. Med. Rehabil. Clin. N. Am. 23, 1–10, ix.
Finch, C.E. (1994). Longevity, Senescence, and the Genome (University of Chicago Press).
Finkel, R.S. (2010). Read-through strategies for suppression of nonsense mutations in Duchenne/ Becker muscular dystrophy: aminoglycosides and ataluren (PTC124). J. Child Neurol. 25, 1158–1164.
Fischer, D., Kley, R.A., Strach, K., Meyer, C., Sommer, T., Eger, K., Rolfs, A., Meyer, W., Pou, A., Pradas, J., et al. (2008). Distinct muscle imaging patterns in myofibrillar myopathies. Neurology 71, 758–765.
Fischmann, A., Hafner, P., Fasler, S., Gloor, M., Bieri, O., Studler, U., and Fischer, D. (2012). Quantitative MRI can detect subclinical disease progression in muscular dystrophy. J. Neurol. 259, 1648–1654.
Fischmann, A., Morrow, J.M., Sinclair, C.D.J., Reilly, M.M., Hanna, M.G., Yousry, T., and Thornton, J.S. (2014). Improved anatomical reproducibility in quantitative lower-limb muscle MRI. J. Magn. Reson. Imaging 39, 1033–1038.
Fisher, I., Abraham, D., Bouri, K., Hoffmann, E.P., Hoffman, E.P., Muntoni, F., and Morgan, J. (2005). Prednisolone-induced changes in dystrophic skeletal muscle. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 19, 834–836.
Flacke, S., Fischer, S., Scott, M.J., Fuhrhop, R.J., Allen, J.S., McLean, M., Winter, P., Sicard, G.A., Gaffney, P.J., Wickline, S.A., et al. (2001). Novel MRI contrast agent for molecular imaging of fibrin implications for detecting vulnerable plaques. Circulation 104, 1280–1285.
Flanigan, K. (2012). The Muscular Dystrophies. Semin. Neurol. 32, 255–263.
Flanigan, K.M. (2014). Duchenne and Becker Muscular Dystrophies. Neurol. Clin. 32, 671–688.
Flanigan, K.M., Voit, T., Rosales, X.Q., Servais, L., Kraus, J.E., Wardell, C., Morgan, A., Dorricott, S., Nakielny, J., Quarcoo, N., et al. (2014). Pharmacokinetics and safety of single doses of drisapersen in non-ambulant subjects with Duchenne muscular dystrophy: Results of a double-blind randomized clinical trial. Neuromuscul. Disord. 24, 16–24.
Foley, J.M., Jayaraman, R.C., Prior, B.M., Pivarnik, J.M., and Meyer, R.A. (1999). MR measurements of muscle damage and adaptation after eccentric exercise. J. Appl. Physiol. 87, 2311–2318.
223
Folli, S., Wagnières, G., Pèlegrin, A., Calmes, J.M., Braichotte, D., Buchegger, F., Chalandon, Y., Hardman, N., Heusser, C., and Givel, J.C. (1992). Immunophotodiagnosis of colon carcinomas in patients injected with fluoresceinated chimeric antibodies against carcinoembryonic antigen. Proc. Natl. Acad. Sci. U. S. A. 89, 7973–7977.
Folli, S., Westermann, P., Braichotte, D., Pèlegrin, A., Wagnières, G., van den Bergh, H., and Mach, J.P. (1994). Antibody-indocyanin conjugates for immunophotodetection of human squamous cell carcinoma in nude mice. Cancer Res. 54, 2643–2649.
Forbes, S.C., Bish, L.T., Ye, F., Spinazzola, J., Baligand, C., Plant, D., Vandenborne, K., Barton, E.R., Sweeney, H.L., and Walter, G.A. (2014a). Gene transfer of arginine kinase to skeletal muscle using adeno-associated virus. Gene Ther. 21, 387–392.
Forbes, S.C., Willcocks, R.J., Triplett, W.T., Rooney, W.D., Lott, D.J., Wang, D.-J., Pollaro, J., Senesac, C.R., Daniels, M.J., Finkel, R.S., et al. (2014b). Magnetic Resonance Imaging and Spectroscopy Assessment of Lower Extremity Skeletal Muscles in Boys with Duchenne Muscular Dystrophy: A Multicenter Cross Sectional Study. PLoS ONE 9, e106435.
Frangioni, J.V. (2003). In vivo near-infrared fluorescence imaging. Curr. Opin. Chem. Biol. 7, 626–634.
Frimel, T.N., Kapadia, F., Gaidosh, G.S., Li, Y., Walter, G.A., and Vandenborne, K. (2005a). A model of muscle atrophy using cast immobilization in mice. Muscle Nerve 32, 672–674.
Frimel, T.N., Walter, G.A., Gibbs, J.D., Gaidosh, G.S., and Vandenborne, K. (2005b). Noninvasive monitoring of muscle damage during reloading following limb disuse. Muscle Nerve 32, 605–612.
Fry, C.S., Lee, J.D., Mula, J., Kirby, T.J., Jackson, J.R., Liu, F., Yang, L., Mendias, C.L., Dupont-Versteegden, E.E., McCarthy, J.J., et al. (2015). Inducible depletion of satellite cells in adult, sedentary mice impairs muscle regenerative capacity without affecting sarcopenia. Nat. Med. 21, 76–80.
Fukada, S., Morikawa, D., Yamamoto, Y., Yoshida, T., Sumie, N., Yamaguchi, M., Ito, T., Miyagoe-Suzuki, Y., Takeda, S. ’ichi, Tsujikawa, K., et al. (2010). Genetic background affects properties of satellite cells and mdx phenotypes. Am. J. Pathol. 176, 2414–2424.
Fulford, J., Eston, R.G., Rowlands, A.V., and Davies, R.C. (2014). Assessment of magnetic resonance techniques to measure muscle damage 24 h after eccentric exercise. Scand. J. Med. Sci. Sports. Epub.
224
Galbiati, F., Engelman, J.A., Volonte, D., Zhang, X.L., Minetti, C., Li, M., Hou, H., Kneitz, B., Edelmann, W., and Lisanti, M.P. (2001). Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities. J. Biol. Chem. 276, 21425–21433.
Galvez, B.G., Sampaolesi, M., Brunelli, S., Covarello, D., Gavina, M., Rossi, B., Constantin, G., Costantin, G., Torrente, Y., and Cossu, G. (2006). Complete repair of dystrophic skeletal muscle by mesoangioblasts with enhanced migration ability. J. Cell Biol. 174, 231–243.
Gambarota, G., Cairns, B.E., Berde, C.B., and Mulkern, R.V. (2001). Osmotic effects on the T2 relaxation decay of in vivo muscle. Magn. Reson. Med. 46, 592–599.
Gieseler, K., Grisoni, K., and Ségalat, L. (2000). Genetic suppression of phenotypes arising from mutations in dystrophin-related genes in Caenorhabditis elegans. Curr. Biol. CB 10, 1092–1097.
Gillis, J.M. (1996). Membrane abnormalities and Ca homeostasis in muscles of the mdx mouse, an animal model of the Duchenne muscular dystrophy: a review. Acta Physiol. Scand. 156, 397–406.
Goemans, N.M., Tulinius, M., van den Akker, J.T., Burm, B.E., Ekhart, P.F., Heuvelmans, N., Holling, T., Janson, A.A., Platenburg, G.J., Sipkens, J.A., et al. (2011). Systemic Administration of PRO051 in Duchenne’s Muscular Dystrophy. N. Engl. J. Med. 364, 1513–1522.
Goldsmith, S.J. (1997). Receptor imaging: competitive or complementary to antibody imaging? Semin. Nucl. Med. 27, 85–93.
Gollins, H., McMahon, J., Wells, K.E., and Wells, D.J. (2003). High-efficiency plasmid gene transfer into dystrophic muscle. Gene Ther. 10, 504–512.
Gomes, A.J., Lunardi, L.O., Marchetti, J.M., Lunardi, C.N., and Tedesco, A.C. (2006). Indocyanine green nanoparticles useful for photomedicine. Photomed. Laser Surg. 24, 514–521.
Gomez-Merino, E., and Bach, J.R. (2002). Duchenne muscular dystrophy: prolongation of life by noninvasive ventilation and mechanically assisted coughing. Am. J. Phys. Med. Rehabil. Assoc. Acad. Physiatr. 81, 411–415.
Gopinath, S.P., Robertson, C.S., Grossman, R.G., and Chance, B. (1993). Near-infrared spectroscopic localization of intracranial hematomas. J. Neurosurg. 79, 43–47.
Gopinath, S.P., Robertson, C.S., Contant, C.F., Narayan, R.K., Grossman, R.G., and Chance, B. (1995). Early detection of delayed traumatic intracranial hematomas using near-infrared spectroscopy. J. Neurosurg. 83, 438–444.
225
Goyenvalle, A., Griffith, G., Babbs, A., Andaloussi, S.E., Ezzat, K., Avril, A., Dugovic, B., Chaussenot, R., Ferry, A., Voit, T., et al. (2015). Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers. Nat. Med. 21, 270–275.
Grady, R.M., Teng, H., Nichol, M.C., Cunningham, J.C., Wilkinson, R.S., and Sanes, J.R. (1997). Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy. Cell 90, 729–738.
Greelish, J.P., Su, L.T., Lankford, E.B., Burkman, J.M., Chen, H., Konig, S.K., Mercier, I.M., Desjardins, P.R., Mitchell, M.A., Zheng, X. guang, et al. (1999). Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector. Nat. Med. 5, 439–443.
Greenberg, C.R., Jacobs, H.K., Nylen, E., Rohringer, M., Averill, N., Van Ommen, G.J.B., and Wrogemann, K. (1988). Gene studies in newborn males with duchenne muscular dystrophy detected by neonatal screening. The Lancet 332, 425–427.
Greener, M.J., and Roberts, R.G. (2000). Conservation of components of the dystrophin complex in Drosophila. FEBS Lett. 482, 13–18.
Griffin, J.L., and Rosiers, C.D. (2009). Applications of metabolomics and proteomics to the mdx mouse model of Duchenne muscular dystrophy: lessons from downstream of the transcriptome. Genome Med. 1, 32.
Grisoni, K., Martin, E., Gieseler, K., Mariol, M.-C., and Ségalat, L. (2002). Genetic evidence for a dystrophin-glycoprotein complex (DGC) in Caenorhabditis elegans. Gene 294, 77–86.
Grosu, A.L., Weber, W.A., Franz, M., Stärk, S., Piert, M., Thamm, R., Gumprecht, H., Schwaiger, M., Molls, M., and Nieder, C. (2005). Reirradiation of recurrent high-grade gliomas using amino acid PET (SPECT)/CT/MRI image fusion to determine gross tumor volume for stereotactic fractionated radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 63, 511–519.
Grounds, M.D., and Torrisi, J. (2004). Anti-TNFalpha (Remicade) therapy protects dystrophic skeletal muscle from necrosis. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 18, 676–682.
Guenette, J.A., Vogiatzis, I., Zakynthinos, S., Athanasopoulos, D., Koskolou, M., Golemati, S., Vasilopoulou, M., Wagner, H.E., Roussos, C., Wagner, P.D., et al. (2008). Human respiratory muscle blood flow measured by near-infrared spectroscopy and indocyanine green. J. Appl. Physiol. 104, 1202–1210.
226
Guenette, J.A., Henderson, W.R., Dominelli, P.B., Querido, J.S., Brasher, P.M., Griesdale, D.E.G., Boushel, R., and Sheel, A.W. (2011). Blood flow index using near-infrared spectroscopy and indocyanine green as a minimally invasive tool to assess respiratory muscle blood flow in humans. Am. J. Physiol. - Regul. Integr. Comp. Physiol. 300, R984–R992.
Guglieri, M., Straub, V., Bushby, K., and Lochmüller, H. (2008). Limb-girdle muscular dystrophies. Curr. Opin. Neurol. 21, 576–584.
Guncay, A., and Yokota, T. (2015). Antisense oligonucleotide drugs for Duchenne muscular dystrophy: how far have we come and what does the future hold? Future Med. Chem. 7, 1631–1635.
Gupta, A.K., and Gupta, M. (2005). Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 26, 3995–4021.
Gurfinkel, M., Thompson, A.B., Ralston, W., Troy, T.L., Moore, A.L., Moore, T.A., Gust, J.D., Tatman, D., Reynolds, J.S., Muggenburg, B., et al. (2000). Pharmacokinetics of ICG and HPPH-car for the Detection of Normal and Tumor Tissue Using Fluorescence, Near-infrared Reflectance Imaging: A Case Study ¶. Photochem. Photobiol. 72, 94–102.
Gutiérrez, J.M., and Ownby, C.L. (2003). Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mechanisms of local and systemic myotoxicity. Toxicon Off. J. Int. Soc. Toxinology 42, 915–931.
Hack, A.A., Ly, C.T., Jiang, F., Clendenin, C.J., Sigrist, K.S., Wollmann, R.L., and McNally, E.M. (1998). γ-Sarcoglycan Deficiency Leads to Muscle Membrane Defects and Apoptosis Independent of Dystrophin. J. Cell Biol. 142, 1279–1287.
Haglund, M.M.M.D., Berger, M.S., and Hochman, D.W. (1996). Enhanced Optical Imaging of Human Gliomas and Tumor Margins. Neurosurg. Febr. 1996 38, 308–317.
Hakim, C.H., and Duan, D. (2012). A marginal level of dystrophin partially ameliorates hindlimb muscle passive mechanical properties in dystrophin-null mice. Muscle Nerve 46, 948–950.
Hakim, C.H., and Duan, D. (2013). Truncated dystrophins reduce muscle stiffness in the extensor digitorum longus muscle of mdx mice. J. Appl. Physiol. Bethesda Md 1985 114, 482–489.
Hakim, C.H., Wasala, N.B., and Duan, D. (2013). Evaluation of muscle function of the extensor digitorum longus muscle ex vivo and tibialis anterior muscle in situ in mice. J. Vis. Exp. JoVE.
227
Hallsten, K., Virtanen, K.A., Lonnqvist, F., Sipila, H., Oksanen, A., Viljanen, T., Ronnemaa, T., Viikari, J., Knuuti, J., and Nuutila, P. (2002). Rosiglitazone but not metformin enhances insulin- and exercise-stimulated skeletal muscle glucose uptake in patients with newly diagnosed type 2 diabetes. Diabetes 51, 3479–3485.
Hamaoka, T., McCully, K.K., Quaresima, V., Yamamoto, K., and Chance, B. (2007). Near-infrared spectroscopy/imaging for monitoring muscle oxygenation and oxidative metabolism in healthy and diseased humans. J. Biomed. Opt. 12, 062105–062105 – 16.
Hamer, P.W., McGeachie, J.M., Davies, M.J., and Grounds, M.D. (2002). Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability. J. Anat. 200, 69–79.
Harisinghani, M.G., Barentsz, J., Hahn, P.F., Deserno, W.M., Tabatabaei, S., van de Kaa, C.H., de la Rosette, J., and Weissleder, R. (2003). Noninvasive detection of clinically occult lymph-node metastases in prostate cancer. N. Engl. J. Med. 348, 2491–2499.
Haseler, L.J., Lin, A.P., and Richardson, R.S. (2004). Skeletal muscle oxidative metabolism in sedentary humans: 31P-MRS assessment of O2 supply and demand limitations. J. Appl. Physiol. Bethesda Md 1985 97, 1077–1081.
Hathaway, P.W., Engel, W.K., and Zellweger, H. (1970). Experimental myopathy after microarterial embolization; comparison with childhood x-linked pseudohypertrophic muscular dystrophy. Arch. Neurol. 22, 365–378.
Hayashita-Kinoh, H., Yugeta, N., Okada, H., Nitahara-Kasahara, Y., Chiyo, T., Okada, T., and Takeda, S. (2015). Intra-amniotic rAAV-mediated microdystrophin gene transfer improves canine X-linked muscular dystrophy and may induce immune tolerance. Mol. Ther.
Heckmatt, J.Z., Dubowitz, V., and Leeman, S. (1980). Detection of pathological change in dystrophic muscle with B-scan ultrasound imaging. Lancet Lond. Engl. 1, 1389–1390.
Heckmatt, J.Z., Leeman, S., and Dubowitz, V. (1982). Ultrasound imaging in the diagnosis of muscle disease. J. Pediatr. 101, 656–660.
Heemskerk, A.M., Strijkers, G.J., Drost, M.R., van Bochove, G.S., and Nicolay, K. (2007). Skeletal muscle degeneration and regeneration after femoral artery ligation in mice: monitoring with diffusion MR imaging. Radiology 243, 413–421.
Heier, C.R., Damsker, J.M., Yu, Q., Dillingham, B.C., Huynh, T., Van der Meulen, J.H., Sali, A., Miller, B.K., Phadke, A., Scheffer, L., et al. (2013). VBP15, a novel anti-inflammatory and membrane-stabilizer, improves muscular dystrophy without side effects. EMBO Mol. Med. 5, 1569–1585.
228
Henricson, E., Abresch, R., Han, J.J., Nicorici, A., Goude Keller, E., de Bie, E., and McDonald, C.M. (2013a). The 6-minute walk test and person-reported outcomes in boys with duchenne muscular dystrophy and typically developing controls: longitudinal comparisons and clinically-meaningful changes over one year. PLoS Curr. 5.
Henricson, E.K., Abresch, R.T., Cnaan, A., Hu, F., Duong, T., Arrieta, A., Han, J., Escolar, D.M., Florence, J.M., Clemens, P.R., et al. (2013b). The cooperative international neuromuscular research group Duchenne natural history study: glucocorticoid treatment preserves clinically meaningful functional milestones and reduces rate of disease progression as measured by manual muscle testing and other commonly used clinical trial outcome measures. Muscle Nerve 48, 55–67.
Herbort, C.P., LeHoang, P., and Guex-Crosier, Y. (1998). Schematic interpretation of indocyanine green angiography in posterior uveitis using a standard angiographic protocol. Ophthalmology 105, 432–440.
Heslop, L., Morgan, J.E., and Partridge, T.A. (2000). Evidence for a myogenic stem cell that is exhausted in dystrophic muscle. J. Cell Sci. 113 ( Pt 12), 2299–2308.
Heydemann, A., Huber, J.M., Demonbreun, A., Hadhazy, M., and McNally, E.M. (2005). Genetic background influences muscular dystrophy. Neuromuscul. Disord. 15, 601–609.
Hielscher, A.H., Bluestone, A.Y., Abdoulaev, G.S., Klose, A.D., Lasker, J., Stewart, M., Netz, U., and Beuthan, J. (2002). Near-Infrared Diffuse Optical Tomography. Dis. Markers 18, 313–337.
Hillman, E.M., Hebden, J.C., Schweiger, M., Dehghani, H., Schmidt, F.E., Delpy, D.T., and Arridge, S.R. (2001). Time resolved optical tomography of the human forearm. Phys. Med. Biol. 46, 1117–1130.
Hirst, R.C., McCullagh, K.J.A., and Davies, K.E. (2005). Utrophin upregulation in Duchenne muscular dystrophy. Acta Myol. Myopathies Cardiomyopathies Off. J. Mediterr. Soc. Myol. Ed. Gaetano Conte Acad. Study Striated Muscle Dis. 24, 209–216.
Hodgetts, S., Radley, H., Davies, M., and Grounds, M.D. (2006). Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNFalpha function with Etanercept in mdx mice. Neuromuscul. Disord. NMD 16, 591–602.
Hoffman, E. (2014). A rebirth for drisapersen in Duchenne muscular dystrophy? Lancet Neurol. 13, 963–965.
Hoffman, E.P., Brown Jr., R.H., and Kunkel, L.M. (1987). Dystrophin: The protein product of the duchenne muscular dystrophy locus. Cell 51, 919–928.
229
Hollinger, K., Yang, C.X., Montz, R.E., Nonneman, D., Ross, J.W., and Selsby, J.T. (2014). Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin-glycoprotein complex assembly in pig skeletal muscle. FASEB J. 28, 1600–1609.
Hollingsworth, K.G. (2014). Quantitative MRI in muscular dystrophy: An indispensable trial endpoint? Neurology 83, 956–957.
Hollingsworth, K.G., Garrood, P., Eagle, M., Bushby, K., and Straub, V. (2013). Magnetic resonance imaging in duchenne muscular dystrophy: Longitudinal assessment of natural history over 18 months. Muscle Nerve 48, 586–588.
Holzer, W., Mauerer, M., Penzkofer, A., Szeimies, R.M., Abels, C., Landthaler, M., and Bäumler, W. (1998). Photostability and thermal stability of indocyanine green. J. Photochem. Photobiol. B 47, 155–164.
Hood, D.A. (2001). Invited Review: Contractile activity-induced mitochondrial biogenesis in skeletal muscle. J. Appl. Physiol. 90, 1137–1157.
Hoogaars, W.M.H., Mouisel, E., Pasternack, A., Hulmi, J.J., Relizani, K., Schuelke, M., Schirwis, E., Garcia, L., Ritvos, O., Ferry, A., et al. (2012). Combined effect of AAV-U7-induced dystrophin exon skipping and soluble activin Type IIB receptor in mdx mice. Hum. Gene Ther. 23, 1269–1279.
Hsieh, T.-J., Wang, C.-K., Chuang, H.-Y., Jong, Y.-J., Li, C.-W., and Liu, G.-C. (2007). In Vivo Proton Magnetic Resonance Spectroscopy Assessment for Muscle Metabolism in Neuromuscular Diseases. J. Pediatr. 151, 319–321.
Hsieh, T.-J., Jaw, T.-S., Chuang, H.-Y., Jong, Y.-J., Liu, G.-C., and Li, C.-W. (2009). Muscle Metabolism in Duchenne Muscular Dystrophy Assessed by In Vivo Proton Magnetic Resonance Spectroscopy: J. Comput. Assist. Tomogr. 33, 150–154.
Hu, X., and Blemker, S.S. (2015). Musculoskeletal simulation can help explain selective muscle degeneration in duchenne muscular dystrophy. Muscle Nerve. Epub.
Huang, D., Swanson, E.A., Lin, C.P., Schuman, J.S., Stinson, W.G., Chang, W., Hee, M.R., Flotte, T., Gregory, K., Puliafito, C.A., et al. (1991). Optical coherence tomography. Science 254, 1178–1181.
Hudak, P.L., Amadio, P.C., and Bombardier, C. (1996). Development of an upper extremity outcome measure: the DASH (disabilities of the arm, shoulder and hand) [corrected]. The Upper Extremity Collaborative Group (UECG). Am. J. Ind. Med. 29, 602–608.
230
Huynh, T., Uaesoontrachoon, K., Quinn, J.L., Tatem, K.S., Heier, C.R., Van Der Meulen, J.H., Yu, Q., Harris, M., Nolan, C.J., Haegeman, G., et al. (2013). Selective modulation through the glucocorticoid receptor ameliorates muscle pathology in mdx mice. J. Pathol. 231, 223–235.
Ibraghimov-Beskrovnaya, O., Ervasti, J.M., Leveille, C.J., Slaughter, C.A., Sernett, S.W., and Campbell, K.P. (1992). Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature 355, 696–702.
Inage, K., Sakuma, Y., Yamauchi, K., Suganami, A., Orita, S., Kubota, G., Oikawa, Y., Sainoh, T., Sato, J., Fujimoto, K., et al. (2015). Longitudinal evaluation of local muscle conditions in a rat model of gastrocnemius muscle injury using an in vivo imaging system. J. Orthop. Res. Epub.
Jansen, M., van Alfen, N., Nijhuis van der Sanden, M.W.G., van Dijk, J.P., Pillen, S., and de Groot, I.J.M. (2012). Quantitative muscle ultrasound is a promising longitudinal follow-up tool in Duchenne muscular dystrophy. Neuromuscul. Disord. NMD 22, 306–317.
Janssen, B.H., Voet, N.B.M., Nabuurs, C.I., Kan, H.E., de Rooy, J.W.J., Geurts, A.C., Padberg, G.W., van Engelen, B.G.M., and Heerschap, A. (2014a). Distinct Disease Phases in Muscles of Facioscapulohumeral Dystrophy Patients Identified by MR Detected Fat Infiltration. PLoS ONE 9.
Janssen, I., Heymsfield, S.B., and Ross, R. (2002). Low relative skeletal muscle mass (sarcopenia) in older persons is associated with functional impairment and physical disability. J. Am. Geriatr. Soc. 50, 889–896.
Janssen, M.M.H.P., Bergsma, A., Geurts, A.C.H., and Groot, I.J.M. de (2014b). Patterns of decline in upper limb function of boys and men with DMD: an international survey. J. Neurol. 261, 1269–1288.
Järver, P., Coursindel, T., Andaloussi, S.E., Godfrey, C., Wood, M.J., and Gait, M.J. (2012). Peptide-mediated Cell and In Vivo Delivery of Antisense Oligonucleotides and siRNA. Mol. Ther. Nucleic Acids 1, e27.
Jerusalem, F., Engel, A.G., and Gomez, M.R. (1974). Duchenne dystrophy. I. Morphometric study of the muscle microvasculature. Brain J. Neurol. 97, 115–122.
Jiang, H., Ramesh, S., and Bartlett, M. (2000). Combined optical and fluorescence imaging for breast cancer detection and diagnosis. Crit. Rev. Biomed. Eng. 28, 371–375.
Joannides, R., Haefeli, W.E., Linder, L., Richard, V., Bakkali, E.H., Thuillez, C., and Lüscher, T.F. (1995). Nitric oxide is responsible for flow-dependent dilatation of human peripheral conduit arteries in vivo. Circulation 91, 1314–1319.
231
Jones, D.A., and Rutherford, O.M. (1987). Human muscle strength training: the effects of three different regimens and the nature of the resultant changes. J. Physiol. 391, 1–11.
Jones, D.A., Round, J.M., Edwards, R.H., Grindwood, S.R., and Tofts, P.S. (1983). Size and composition of the calf and quadriceps muscles in Duchenne muscular dystrophy. A tomographic and histochemical study. J. Neurol. Sci. 60, 307–322.
Kandarian, S.C., and Jackman, R.W. (2006). Intracellular signaling during skeletal muscle atrophy. Muscle Nerve 33, 155–165.
Kang PB, and Griggs RC (2015). Advances in muscular dystrophies. JAMA Neurol. 72, 741–742.
Kappos, L., Radue, E.-W., O’Connor, P., Polman, C., Hohlfeld, R., Calabresi, P., Selmaj, K., Agoropoulou, C., Leyk, M., Zhang-Auberson, L., et al. (2010). A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N. Engl. J. Med. 362, 387–401.
Kefi, M., Amouri, R., Driss, A., Ben Hamida, C., Ben Hamida, M., Kunkel, L.M., and Hentati, F. (2003). Phenotype and sarcoglycan expression in Tunisian LGMD 2C patients sharing the same del521-T mutation. Neuromuscul. Disord. NMD 13, 779–787.
Keiding, S., Engsted, E., and Ott, P. (1998). Sorbitol as a test substance for measurement of liver plasma flow in humans. Hepatol. Baltim. Md 28, 50–56.
Kharraz, Y., Guerra, J., Pessina, P., Serrano, A.L., and Munoz-Canoves, P. (2014). Understanding the Process of Fibrosis in Duchenne Muscular Dystrophy. BioMed Res. Int. 2014, e965631.
Kim, T.H., Chen, Y., Mount, C.W., Gombotz, W.R., Li, X., and Pun, S.H. (2010). Evaluation of temperature-sensitive, indocyanine green-encapsulating micelles for noninvasive near-infrared tumor imaging. Pharm. Res. 27, 1900–1913.
Kim, Y., Tewari, M., Pajerowski, J.D., Cai, S., Sen, S., Williams, J.H., Williams, J., Sirsi, S.R., Sirsi, S., Lutz, G.J., et al. (2009). Polymersome delivery of siRNA and antisense oligonucleotides. J. Control. Release Off. J. Control. Release Soc. 134, 132–140.
Kinali, M., Arechavala-Gomeza, V., Feng, L., Cirak, S., Hunt, D., Adkin, C., Guglieri, M., Ashton, E., Abbs, S., Nihoyannopoulos, P., et al. (2009). Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol. 8, 918–928.
232
Kinali, M., Arechavala-Gomeza, V., Cirak, S., Glover, A., Guglieri, M., Feng, L., Hollingsworth, K.G., Hunt, D., Jungbluth, H., Roper, H.P., et al. (2011). Muscle histology vs MRI in Duchenne muscular dystrophy. Neurology 76, 346–353.
King, M.K., Lee, R.R., and Davis, L.E. (2005). Magnetic Resonance Imaging and Computed Tomography of Skeletal Muscles in Oculopharyngeal Muscular Dystrophy. J. Clin. Neuromuscul. Dis. March 2005 6, 103–108.
Kirchherr, A.-K., Briel, A., and Mäder, K. (2009). Stabilization of indocyanine green by encapsulation within micellar systems. Mol. Pharm. 6, 480–491.
Kirkpatrick, P.J., Smielewski, P., Czosnyka, M., Menon, D.K., and Pickard, J.D. (1995). Near-infrared spectroscopy use in patients with head injury. J. Neurosurg. 83, 963–970.
Knopp, M.V., Weiss, E., Sinn, H.P., Mattern, J., Junkermann, H., Radeleff, J., Magener, A., Brix, G., Delorme, S., Zuna, I., et al. (1999). Pathophysiologic basis of contrast enhancement in breast tumors. J. Magn. Reson. Imaging JMRI 10, 260–266.
Kobayashi, S., Ishikawa, T., Tanabe, J., Moroi, J., and Suzuki, A. (2014). Quantitative cerebral perfusion assessment using microscope-integrated analysis of intraoperative indocyanine green fluorescence angiography versus positron emission tomography in superficial temporal artery to middle cerebral artery anastomosis. Surg. Neurol. Int. 5, 135.
Kobayashi, Y.M., Rader, E.P., Crawford, R.W., Iyengar, N.K., Thedens, D.R., Faulkner, J.A., Parikh, S.V., Weiss, R.M., Chamberlain, J.S., Moore, S.A., et al. (2008). Sarcolemma-localized nNOS is required to maintain activity after mild exercise. Nature 456, 511–515.
Koenig, M., Monaco, A.P., and Kunkel, L.M. (1988). The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 53, 219–228.
Koenig, M., Beggs, A.H., Moyer, M., Scherpf, S., Heindrich, K., Bettecken, T., Meng, G., Müller, C.R., Lindlöf, M., and Kaariainen, H. (1989). The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am. J. Hum. Genet. 45, 498–506.
Koga, S., Kano, Y., Barstow, T.J., Ferreira, L.F., Ohmae, E., Sudo, M., and Poole, D.C. (2012). Kinetics of muscle deoxygenation and microvascular Po2 during contractions in rat: comparison of optical spectroscopy and phosphorescence-quenching techniques. J. Appl. Physiol. 112, 26–32.
Kole, R., and Krieg, A.M. (2015). Exon skipping therapy for Duchenne muscular dystrophy. Adv. Drug Deliv. Rev. 87, 104–107.
233
Koo, T., and Wood, M.J. (2013). Clinical trials using antisense oligonucleotides in duchenne muscular dystrophy. Hum. Gene Ther. 24, 479–488.
Kossodo, S., Pickarski, M., Lin, S.-A., Gleason, A., Gaspar, R., Buono, C., Ho, G., Blusztajn, A., Cuneo, G., Zhang, J., et al. (2010). Dual In Vivo Quantification of Integrin-targeted and Protease-activated Agents in Cancer Using Fluorescence Molecular Tomography (FMT). Mol. Imaging Biol. 12, 488–499.
Kraemer, W.J., Staron, R.S., Gordon, S.E., Volek, J.S., Koziris, L.P., Duncan, N.D., Nindl, B.C., Gómez, A.L., Marx, J.O., Fry, A.C., et al. (2000). The effects of 10 days of spaceflight on the shuttle Endeavor on predominantly fast-twitch muscles in the rat. Histochem. Cell Biol. 114, 349–355.
Kraft, J.C., and Ho, R.J.Y. (2014). Interactions of Indocyanine Green and Lipid in Enhancing Near-Infrared Fluorescence Properties: The Basis for Near-Infrared Imaging in Vivo. Biochemistry (Mosc.) 53, 1275–1283.
Labeit, S., and Kolmerer, B. (1995). Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science 270, 293–296.
Lai, Y., Yue, Y., Liu, M., Ghosh, A., Engelhardt, J.F., Chamberlain, J.S., and Duan, D. (2005). Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors. Nat. Biotechnol. 23, 1435–1439.
Lai, Y., Thomas, G.D., Yue, Y., Yang, H.T., Li, D., Long, C., Judge, L., Bostick, B., Chamberlain, J.S., Terjung, R.L., et al. (2009). Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy. J. Clin. Invest. 119, 624–635.
Lamminen, A.E. (1990). Magnetic resonance imaging of primary skeletal muscle diseases: patterns of distribution and severity of involvement. Br. J. Radiol. 63, 946–950.
Landsman, M.L., Kwant, G., Mook, G.A., and Zijlstra, W.G. (1976). Light-absorbing properties, stability, and spectral stabilization of indocyanine green. J. Appl. Physiol. 40, 575–583.
Lanza, I.R., Wigmore, D.M., Befroy, D.E., and Kent-Braun, J.A. (2006). In vivo ATP production during free-flow and ischaemic muscle contractions in humans. J. Physiol. 577, 353–367.
Larcher, T., Lafoux, A., Tesson, L., Remy, S., Thepenier, V., François, V., Le Guiner, C., Goubin, H., Dutilleul, M., Guigand, L., et al. (2014). Characterization of Dystrophin Deficient Rats: A New Model for Duchenne Muscular Dystrophy. PLoS ONE 9, e110371.
Larush, L., and Magdassi, S. (2011). Formation of near-infrared fluorescent nanoparticles for medical imaging. Nanomed. 6, 233–240.
234
Laurent, S., Forge, D., Port, M., Roch, A., Robic, C., Vander Elst, L., and Muller, R.N. (2008). Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chem. Rev. 108, 2064–2110.
Laval, S.H., and Bushby, K.M.D. (2004). Limb-girdle muscular dystrophies--from genetics to molecular pathology. Neuropathol. Appl. Neurobiol. 30, 91–105.
Lee, C.-H., Cheng, S.-H., Wang, Y., Chen, Y.-C., Chen, N.-T., Souris, J., Chen, C.-T., Mou, C.-Y., Yang, C.-S., and Lo, L.-W. (2009). Near-Infrared Mesoporous Silica Nanoparticles for Optical Imaging: Characterization and In Vivo Biodistribution. Adv. Funct. Mater. 19, 215–222.
Le Guiner, C., Larcher, T., Lafoux, A., Tesson, L., Remy, S., Thepenier, V., Francois, V., Goubin, H., Dutilleul, M., Guigand, L., et al. (2014). DYSTROPHIN deficient rats: generation and characterization of a new model for Duchenne muscular dystrophy. Hum. Gene Ther. 25, A28–A28.
Leinonen, H., Juntunen, J., Somer, H., and Rapola, J. (1979). Capillary circulation and morphology in Duchenne muscular dystrophy. Eur. Neurol. 18, 249–255.
Lepper, C., Partridge, T.A., and Fan, C.-M. (2011). An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration. Dev. Camb. Engl. 138, 3639–3646.
Lerner, R.M., Huang, S.R., and Parker, K.J. (1990). “Sonoelasticity” images derived from ultrasound signals in mechanically vibrated tissues. Ultrasound Med. Biol. 16, 231–239.
Leung, D.G., Carrino, J.A., Wagner, K.R., and Jacobs, M.A. (2015). Whole-body magnetic resonance imaging evaluation of facioscapulohumeral muscular dystrophy. Muscle Nerve. Epub.
Lexell, J., and Taylor, C.C. (1989). Variability in muscle fibre areas in whole human quadriceps muscle. How much and why? Acta Physiol. Scand. 136, 561–568.
Li, J., Geisbush, T.R., Rosen, G.D., Lachey, J., Mulivor, A., and Rutkove, S.B. (2014). Electrical impedance myography for the in vivo and ex vivo assessment of muscular dystrophy (mdx) mouse muscle. Muscle Nerve 49, 829–835.
Li, Z.B., Zhang, J., and Wagner, K.R. (2012). Inhibition of myostatin reverses muscle fibrosis through apoptosis. J. Cell Sci. 125, 3957–3965.
Lieber, R.L., and Fridén, J. (2000). Functional and clinical significance of skeletal muscle architecture. Muscle Nerve 23, 1647–1666.
Liu, C.H., D’Arceuil, H.E., and de Crespigny, A.J. (2004). Direct CSF injection of MnCl(2) for dynamic manganese-enhanced MRI. Magn. Reson. Med. 51, 978–987.
235
Liu, G.C., Jong, Y.J., Chiang, C.H., and Jaw, T.S. (1993a). Duchenne muscular dystrophy: MR grading system with functional correlation. Radiology 186, 475–480.
Liu, M., Chino, N., and Ishihara, T. (1993b). Muscle damage progression in Duchenne muscular dystrophy evaluated by a new quantitative computed tomography method. Arch. Phys. Med. Rehabil. 74, 507–514.
Lomonte, B., and Gutiérrez, J.M. (1989). A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo). Toxicon Off. J. Int. Soc. Toxinology 27, 725–733.
Lomonte, B., Tarkowski, A., and Hanson, L.A. (1993). Host response to Bothrops asper snake venom. Analysis of edema formation, inflammatory cells, and cytokine release in a mouse model. Inflammation 17, 93–105.
Lomonte, B., Angulo, Y., and Calderón, L. (2003). An overview of lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action. Toxicon Off. J. Int. Soc. Toxinology 42, 885–901.
Lovering, R.M., and De Deyne, P.G. (2004). Contractile function, sarcolemma integrity, and the loss of dystrophin after skeletal muscle eccentric contraction-induced injury. Am. J. Physiol. Cell Physiol. 286, C230–C238.
Lovering, R.M., McMillan, A.B., and Gullapalli, R.P. (2009). Location of myofiber damage in skeletal muscle after lengthening contractions. Muscle Nerve 40, 589–594.
Lue, Y.-J., Lin, R.-F., Chen, S.-S., and Lu, Y.-M. (2009). Measurement of the Functional Status of Patients with Different Types of Muscular Dystrophy. Kaohsiung J. Med. Sci. 25, 325–333.
Lund, E., Kendall, S.A., Janerot-Sjøberg, B., and Bengtsson, A. (2003). Muscle metabolism in fibromyalgia studied by P-31 magnetic resonance spectroscopy during aerobic and anaerobic exercise. Scand. J. Rheumatol. 32, 138–145.
Lutty, G. (1978). Acute Intravenous Toxicity of Biological Stains, Dyes, and Other Fluorescent Substances. Toxicol. Appl. Pharmacol. 44, 225–249.
Lynch, G.S., Fary, C.J., and Williams, D.A. (1997). Quantitative measurement of resting skeletal muscle [Ca2+]i following acute and long-term downhill running exercise in mice. Cell Calcium 22, 373–383.
Lynch, G.S., Hinkle, R.T., Chamberlain, J.S., Brooks, S.V., and Faulkner, J.A. (2001). Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old. J. Physiol. 535, 591–600.
236
Ma, Y., Sadoqi, M., and Shao, J. (2012). Biodistribution of indocyanine green-loaded nanoparticles with surface modifications of PEG and folic acid. Int. J. Pharm. 436, 25–31.
Maeda, H. (2012). Vascular permeability in cancer and infection as related to macromolecular drug delivery, with emphasis on the EPR effect for tumor-selective drug targeting. Proc. Jpn. Acad. Ser. B-Phys. Biol. Sci. 88, 53–71.
Maeda, H., Wu, J., Sawa, T., Matsumura, Y., and Hori, K. (2000). Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J. Controlled Release 65, 271–284.
Mah, J.K., Korngut, L., Dykeman, J., Day, L., Pringsheim, T., and Jette, N. (2014). A systematic review and meta-analysis on the epidemiology of Duchenne and Becker muscular dystrophy. Neuromuscul. Disord. 24, 482–491.
Mahjneh, I., Bashir, R., Kiuru-Enari, S., Linssen, W., Lamminen, A., and Visser, M. de (2012). Selective pattern of muscle involvement seen in distal muscular dystrophy associated with anoctamin 5 mutations: a follow-up muscle MRI study. Neuromuscul. Disord. NMD 22 Suppl 2, S130–S136.
Mahmood, U., Tung, C.H., Bogdanov, A., and Weissleder, R. (1999). Near-infrared optical imaging of protease activity for tumor detection. Radiology 213, 866–870.
Manchanda, R., Fernandez-Fernandez, A., Nagesetti, A., and McGoron, A.J. (2010). Preparation and characterization of a polymeric (PLGA) nanoparticulate drug delivery system with simultaneous incorporation of chemotherapeutic and thermo-optical agents. Colloids Surf. B Biointerfaces 75, 260–267.
Mancini, D.M., Bolinger, L., Li, H., Kendrick, K., Chance, B., and Wilson, J.R. (1994). Validation of near-infrared spectroscopy in humans. J. Appl. Physiol. 77, 2740–2747.
Manduca, A., Oliphant, T.E., Dresner, M.A., Mahowald, J.L., Kruse, S.A., Amromin, E., Felmlee, J.P., Greenleaf, J.F., and Ehman, R.L. (2001). Magnetic resonance elastography: Non-invasive mapping of tissue elasticity. Med. Image Anal. 5, 237–254.
Mariappan, Y.K., Glaser, K.J., and Ehman, R.L. (2010). Magnetic Resonance Elastography: A Review. Clin. Anat. N. Y. N 23, 497–511.
Martin, E.A., Barresi, R., Byrne, B.J., Tsimerinov, E.I., Scott, B.L., Walker, A.E., Gurudevan, S.V., Anene, F., Elashoff, R.M., Thomas, G.D., et al. (2012). Tadalafil alleviates muscle ischemia in patients with Becker muscular dystrophy. Sci. Transl. Med. 4, 162ra155.
Martins-Bach, A.B., Bloise, A.C., Vainzof, M., and Rahnamaye Rabbani, S. (2012). Metabolic profile of dystrophic mdx mouse muscles analyzed with in vitro magnetic resonance spectroscopy (MRS). Magn. Reson. Imaging 30, 1167–1176.
237
Mathur, S., Vohra, R.S., Germain, S.A., Forbes, S., Bryant, N.D., Vandenborne, K., and Walter, G.A. (2011). Changes in muscle T2 and tissue damage after downhill running in mdx Mice. Muscle Nerve 43, 878–886.
Matsuo, M., Masumura, T., Nishio, H., Nakajima, T., Kitoh, Y., Takumi, T., Koga, J., and Nakamura, H. (1991). Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe. J. Clin. Invest. 87, 2127–2131.
Maurits, N.M., Beenakker, E.A.C., van Schaik, D.E.C., Fock, J.M., and van der Hoeven, J.H. (2004). Muscle ultrasound in children: normal values and application to neuromuscular disorders. Ultrasound Med. Biol. 30, 1017–1027.
Mayhew, A., Mazzone, E.S., Eagle, M., Duong, T., Ash, M., Decostre, V., Vandenhauwe, M., Klingels, K., Florence, J., Main, M., et al. (2013). Development of the Performance of the Upper Limb module for Duchenne muscular dystrophy. Dev. Med. Child Neurol. 55, 1038–1045.
Mázala, D.A.G., Grange, R.W., and Chin, E.R. (2015). The role of proteases in excitation-contraction coupling failure in muscular dystrophy. Am. J. Physiol. Cell Physiol. 308, C33–C40.
McCully, K.K., and Hamaoka, T. (2000). Near-infrared spectroscopy: what can it tell us about oxygen saturation in skeletal muscle? Exerc. Sport Sci. Rev. 28, 123–127.
McDonald, A.A., Hebert, S.L., Kunz, M.D., Ralles, S.J., and McLoon, L.K. (2015). Disease course in mdx:utrophin+/− mice: comparison of three mouse models of Duchenne muscular dystrophy. Physiol. Rep. Epub.
McDonald, C.M., Henricson, E.K., Abresch, R.T., Florence, J., Eagle, M., Gappmaier, E., Glanzman, A.M., PTC124-GD-007-DMD Study Group, Spiegel, R., Barth, J., et al. (2013). The 6-minute walk test and other clinical endpoints in duchenne muscular dystrophy: reliability, concurrent validity, and minimal clinically important differences from a multicenter study. Muscle Nerve 48, 357–368.
McDouall, R.M., Dunn, M.J., and Dubowitz, V. (1990). Nature of the mononuclear infiltrate and the mechanism of muscle damage in juvenile dermatomyositis and Duchenne muscular dystrophy. J. Neurol. Sci. 99, 199–217.
McIntosh, L.M., Baker, R.E., and Anderson, J.E. (1998). Magnetic resonance imaging of regenerating and dystrophic mouse muscle. Biochem. Cell Biol. Biochim. Biol. Cell. 76, 532–541.
McNally, E.M., Passos-Bueno, M.R., Bönnemann, C.G., Vainzof, M., de Sá Moreira, E., Lidov, H.G., Othmane, K.B., Denton, P.H., Vance, J.M., Zatz, M., et al. (1996a). Mild and severe muscular dystrophy caused by a single gamma-sarcoglycan mutation. Am. J. Hum. Genet. 59, 1040–1047.
238
McNally, E.M., Duggan, D., Gorospe, J.R., Bönnemann, C.G., Fanin, M., Pegoraro, E., Lidov, H.G., Noguchi, S., Ozawa, E., Finkel, R.S., et al. (1996b). Mutations that disrupt the carboxyl-terminus of gamma-sarcoglycan cause muscular dystrophy. Hum. Mol. Genet. 5, 1841–1847.
McNally, E.M., Kaltman, J.R., Benson, D.W., Canter, C.E., Cripe, L.H., Duan, D., Finder, J.D., Groh, W.J., Hoffman, E.P., Judge, D.P., et al. (2015). Contemporary cardiac issues in Duchenne muscular dystrophy. Working Group of the National Heart, Lung, and Blood Institute in collaboration with Parent Project Muscular Dystrophy. Circulation 131, 1590–1598.
McPherron, A.C., and Lee, S.J. (1997). Double muscling in cattle due to mutations in the myostatin gene. Proc. Natl. Acad. Sci. U. S. A. 94, 12457–12461.
McPherron, A.C., Lawler, A.M., and Lee, S.J. (1997). Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387, 83–90.
Melacini, P., Vianello, A., Villanova, C., Fanin, M., Miorin, M., Angelini, C., and Dalla Volta, S. (1996). Cardiac and respiratory involvement in advanced stage Duchenne muscular dystrophy. Neuromuscul. Disord. NMD 6, 367–376.
Mendell, J.R., Engel, W.K., and Derrer, E.C. (1971). Duchenne muscular dystrophy: functional ischemia reproduces its characteristic lesions. Science 172, 1143–1145.
Mendell, J.R., Rodino-Klapac, L.R., Rosales-Quintero, X., Kota, J., Coley, B.D., Galloway, G., Craenen, J.M., Lewis, S., Malik, V., Shilling, C., et al. (2009). Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins. Ann. Neurol. 66, 290–297.
Mendell, J.R., Shilling, C., Leslie, N.D., Flanigan, K.M., al-Dahhak, R., Gastier-Foster, J., Kneile, K., Dunn, D.M., Duval, B., Aoyagi, A., et al. (2012). Evidence-based path to newborn screening for duchenne muscular dystrophy. Ann. Neurol. 71, 304–313.
Mendell, J.R., Rodino-Klapac, L.R., Sahenk, Z., Roush, K., Bird, L., Lowes, L.P., Alfano, L., Gomez, A.M., Lewis, S., Kota, J., et al. (2013). Eteplirsen for the treatment of Duchenne muscular dystrophy. Ann. Neurol. 74, 637–647.
Mendell, J.R., Goemans, N., Lowes, L.P., Alfano, L.N., Berry, K., Shao, J., Kaye, E.M., Mercuri, E., and for the Eteplirsen Study Group and Telethon Foundation DMD Italian Network (2016). Longitudinal effect of eteplirsen versus historical control on ambulation in Duchenne muscular dystrophy. Ann. Neurol. Epub.
Mercuri, E., and Muntoni, F. (2013). Muscular dystrophy: new challenges and review of the current clinical trials. Curr. Opin. Pediatr. 25, 701–707.
239
Mercuri, E., Talim, B., Moghadaszadeh, B., Petit, N., Brockington, M., Counsell, S., Guicheney, P., Muntoni, F., and Merlini, L. (2002). Clinical and imaging findings in six cases of congenital muscular dystrophy with rigid spine syndrome linked to chromosome 1p (RSMD1). Neuromuscul. Disord. 12, 631–638.
Mercuri, E., Pichiecchio, A., Allsop, J., Messina, S., Pane, M., and Muntoni, F. (2007). Muscle MRI in inherited neuromuscular disorders: past, present, and future. J. Magn. Reson. Imaging JMRI 25, 433–440.
Merlini, L., and Sabatelli, P. (2015). Improving clinical trial design for Duchenne muscular dystrophy. BMC Neurol. 15, 153.
Messere, A., and Roatta, S. (2015). Local and remote thermoregulatory changes affect NIRS measurement in forearm muscles. Eur. J. Appl. Physiol. 1–11.
Mikos, A., Thorsen, A., Czerwonka, L., Bao, Y., Langer, R., Winslow, D., and Vacanti, J. (1994). Preparation and Characterization of Poly(l-Lactic Acid) Foams. Polymer 35, 1068–1077.
Misra, L.K., Smith, N.K., Chang, D.C., Sparks, R.L., Cameron, I.L., Beall, P.T., Harrist, R., Nichols, B.L., Fanguy, R.C., and Hazlewood, C.F. (1980). Intracellular concentration of elements in normal and dystrophic skeletal muscles of the chicken. J. Cell. Physiol. 103, 193–200.
Misselwitz, B., Platzek, J., Raduechel, B., Oellinger, J.J., and Weinmann, H.-J. (1999). Gadofluorine 8: initial experience with a new contrast medium for interstitial MR lymphography. Magn. Reson. Mater. Phys. Biol. Med. 8, 190–195.
Miyakawa, M., Nagai, D., and Tanikawa, Y. (2009). Imaging of forearm-muscle activities by CP-MCT and TR-DOT. In Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2009. EMBC 2009, pp. 5833–5837.
Moizard, M.P., Toutain, A., Fournier, D., Berret, F., Raynaud, M., Billard, C., Andres, C., and Moraine, C. (2000). Severe cognitive impairment in DMD: obvious clinical indication for Dp71 isoform point mutation screening. Eur. J. Hum. Genet. EJHG 8, 552–556.
Monaco, A.P., Bertelson, C.J., Liechti-Gallati, S., Moser, H., and Kunkel, L.M. (1988). An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 2, 90–95.
Mordon, S., Devoisselle, J.M., Soulie-Begu, S., and Desmettre, T. (1998). Indocyanine green: physicochemical factors affecting its fluorescence in vivo. Microvasc. Res. 55, 146–152.
Mornet, S., Vasseur, S., Grasset, F., and Duguet, E. (2004). Magnetic nanoparticle design for medical diagnosis and therapy. J. Mater. Chem. 14, 2161–2175.
240
Motomura, K., Inaji, H., Komoike, Y., Kasugai, T., Noguchi, S., and Koyama, H. (1999). Sentinel node biopsy guided by indocyanine green dye in breast cancer patients. Jpn. J. Clin. Oncol. 29, 604–607.
Mouly, V., Aamiri, A., Bigot, A., Cooper, R.N., Di Donna, S., Furling, D., Gidaro, T., Jacquemin, V., Mamchaoui, K., Negroni, E., et al. (2005). The mitotic clock in skeletal muscle regeneration, disease and cell mediated gene therapy. Acta Physiol. Scand. 184, 3–15.
Možina, H. (2011). Near-infrared spectroscopy for evaluation of global and skeletal muscle tissue oxygenation. World J. Cardiol. 3, 377.
Muckle, T.J. (1976). Plasma proteins binding of indocyanine green. Biochem. Med. 15, 17–21.
Mueller, A.J., Freeman, W.R., Schaller, U.C., Kampik, A., and Folberg, R. (2002). Complex microcirculation patterns detected by confocal indocyanine green angiography predict time to growth of small choroidal melanocytic tumors: MuSIC Report II. Ophthalmology 109, 2207–2214.
Muntoni, F., Torelli, S., and Ferlini, A. (2003). Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol. 2, 731–740.
Murry, C.E., Wiseman, R.W., Schwartz, S.M., and Hauschka, S.D. (1996). Skeletal myoblast transplantation for repair of myocardial necrosis. J. Clin. Invest. 98, 2512–2523.
Musch, B.C., Papapetropoulos, T.A., McQueen, D.A., Hudgson, P., and Weightman, D. (1975). A comparison of the structure of small blood vessels in normal, denervated and dystrophic human muscle. J. Neurol. Sci. 26, 221–234.
Muthupillai, R., and Ehman, R.L. (1996). Magnetic resonance elastography. Nat. Med. 2, 601–603.
Muthupillai, R., Lomas, D.J., Rossman, P.J., Greenleaf, J.F., Manduca, A., and Ehman, R.L. (1995). Magnetic resonance elastography by direct visualization of propagating acoustic strain waves. Science 269, 1854–1857.
Muthupillai, R., Rossman, P.J., Lomas, D.J., Greenleaf, J.F., Riederer, S.J., and Ehman, R.L. (1996). Magnetic resonance imaging of transverse acoustic strain waves. Magn. Reson. Med. 36, 266–274.
Nakatani, M., Takehara, Y., Sugino, H., Matsumoto, M., Hashimoto, O., Hasegawa, Y., Murakami, T., Uezumi, A., Takeda, S. ’ichi, Noji, S., et al. (2008). Transgenic expression of a myostatin inhibitor derived from follistatin increases skeletal muscle mass and ameliorates dystrophic pathology in mdx mice. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 22, 477–487.
241
Nakayama, A., del Monte, F., Hajjar, R.J., and Frangioni, J.V. (2002). Functional near-infrared fluorescence imaging for cardiac surgery and targeted gene therapy. Mol. Imaging 1, 365–377.
Nathan, C. (1992). Nitric oxide as a secretory product of mammalian cells. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 6, 3051–3064.
Nelson, M.D., Rader, F., Tang, X., Tavyev, J., Nelson, S.F., Miceli, M.C., Elashoff, R.M., Sweeney, H.L., and Victor, R.G. (2014). PDE5 inhibition alleviates functional muscle ischemia in boys with Duchenne muscular dystrophy. Neurology 82, 2085–2091.
Nelson, S.F., Crosbie, R.H., Miceli, M.C., and Spencer, M.J. (2009). Emerging genetic therapies to treat Duchenne muscular dystrophy. Curr. Opin. Neurol. 22, 532–538.
Nelson, S.J., Vigneron, D.B., and Dillon, W.P. (1999). Serial evaluation of patients with brain tumors using volume MRI and 3D 1H MRSI. NMR Biomed. 12, 123–138.
Nguyen, H.X., and Tidball, J.G. (2003). Expression of a muscle-specific, nitric oxide synthase transgene prevents muscle membrane injury and reduces muscle inflammation during modified muscle use in mice. J. Physiol. 550, 347–356.
Nigro, V., and Piluso, G. Spectrum of muscular dystrophies associated with sarcolemmal-protein genetic defects. Biochim. Biophys. Acta BBA - Mol. Basis Dis.
Noguchi, S., McNally, E.M., Ben Othmane, K., Hagiwara, Y., Mizuno, Y., Yoshida, M., Yamamoto, H., Bönnemann, C.G., Gussoni, E., Denton, P.H., et al. (1995). Mutations in the dystrophin-associated protein gamma-sarcoglycan in chromosome 13 muscular dystrophy. Science 270, 819–822.
Ntziachristos, V., Yodh, A.G., Schnall, M., and Chance, B. (2000). Concurrent MRI and diffuse optical tomography of breast after indocyanine green enhancement. Proc. Natl. Acad. Sci. 97, 2767–2772.
Ntziachristos, V., Tung, C.-H., Bremer, C., and Weissleder, R. (2002). Fluorescence molecular tomography resolves protease activity in vivo. Nat. Med. 8, 757–761.
Ntziachristos, V., Bremer, C., and Weissleder, R. (2003). Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur. Radiol. 13, 195–208.
Ntziachristos, V., Ripoll, J., Wang, L.V., and Weissleder, R. (2005). Looking and listening to light: the evolution of whole-body photonic imaging. Nat. Biotechnol. 23, 313–320.
242
Ogan, M., Schmiedl, U., Moseley, M., Grodd, W., Paajanen, H., and Brasch, R. (1987). Albumin Labeled with Gd-Dtpa - an Intravascular Contrast-Enhancing Agent for Magnetic-Resonance Blood Pool Imaging - Preparation and Characterization. Invest. Radiol. 22, 665–671.
Ohlendieck, K., and Campbell, K.P. (1991). Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice. J. Cell Biol. 115, 1685–1694.
Olivier, N., Boissière, J., Allart, E., Mucci, P., Thevenon, A., Daussin, F., and Tiffreau, V. Evaluation of muscle oxygenation by near infrared spectroscopy in patients with facioscapulohumeral muscular dystrophy. Neuromuscul. Disord.
van Oosten, B.W., Barkhof, F., Truyen, L., Boringa, J.B., Bertelsmann, F.W., von Blomberg, B.M., Woody, J.N., Hartung, H.P., and Polman, C.H. (1996). Increased MRI activity and immune activation in two multiple sclerosis patients treated with the monoclonal anti-tumor necrosis factor antibody cA2. Neurology 47, 1531–1534.
Ousterout, D.G., Kabadi, A.M., Thakore, P.I., Majoros, W.H., Reddy, T.E., and Gersbach, C.A. (2015). Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat. Commun. 6.
Pacak, C.A., Walter, G.A., Gaidosh, G., Bryant, N., Lewis, M.A., Germain, S., Mah, C.S., Campbell, K.P., and Byrne, B.J. (2007). Long-term Skeletal Muscle Protection After Gene Transfer in a Mouse Model of LGMD-2D. Mol. Ther. 15, 1775–1781.
Padhani, A.R., Gapinski, C.J., Macvicar, D.A., Parker, G.J., Suckling, J., Revell, P.B., Leach, M.O., Dearnaley, D.P., and Husband, J.E. (2000). Dynamic contrast enhanced MRI of prostate cancer: correlation with morphology and tumour stage, histological grade and PSA. Clin. Radiol. 55, 99–109.
Palacio, J., Gāldiz, J.B., Alvarez, F.J., Orozco-Levi, M., Lloreta, J., and Gea, J. (2002). Procion orange tracer dye technique vs. identification of intrafibrillar fibronectin in the assessment of sarcolemmal damage. Eur. J. Clin. Invest. 32, 443–447.
Pane, M., Mazzone, E.S., Fanelli, L., De Sanctis, R., Bianco, F., Sivo, S., D’Amico, A., Messina, S., Battini, R., Scutifero, M., et al. (2014). Reliability of the Performance of Upper Limb assessment in Duchenne muscular dystrophy. Neuromuscul. Disord. NMD 24, 201–206.
Pankhurst, Q.A., Connolly, J., Jones, S.K., and Dobson, J. (2003). Applications of magnetic nanoparticles in biomedicine. J. Phys. -Appl. Phys. 36, R167–R181.
Panyam, J., Dali, M.M., Sahoo, S.K., Ma, W., Chakravarthi, S.S., Amidon, G.L., Levy, R.J., and Labhasetwar, V. (2003). Polymer degradation and in vitro release of a model protein from poly(D,L-lactide-co-glycolide) nano- and microparticles. J. Control. Release Off. J. Control. Release Soc. 92, 173–187.
243
Park, J.H., Olsen, N.J., King, L., Jr, Vital, T., Buse, R., Kari, S., Hernanz-Schulman, M., and Price, R.R. (1995). Use of magnetic resonance imaging and P-31 magnetic resonance spectroscopy to detect and quantify muscle dysfunction in the amyopathic and myopathic variants of dermatomyositis. Arthritis Rheum. 38, 68–77.
Parker, K.J., Huang, S.R., Musulin, R.A., and Lerner, R.M. (1990). Tissue response to mechanical vibrations for “sonoelasticity imaging.” Ultrasound Med. Biol. 16, 241–246.
Pasternak, C., Wong, S., and Elson, E.L. (1995). Mechanical function of dystrophin in muscle cells. J. Cell Biol. 128, 355–361.
Pastoret, C., and Sebille, A. (1995). mdx mice show progressive weakness and muscle deterioration with age. J. Neurol. Sci. 129, 97–105.
Péault, B., Rudnicki, M., Torrente, Y., Cossu, G., Tremblay, J.P., Partridge, T., Gussoni, E., Kunkel, L.M., and Huard, J. (2007). Stem and Progenitor Cells in Skeletal Muscle Development, Maintenance, and Therapy. Mol. Ther. 15, 867–877.
Pegoraro, E., and Hoffman, E.P. (1993). Limb-Girdle Muscular Dystrophy Overview. In GeneReviews(®), R.A. Pagon, M.P. Adam, H.H. Ardinger, S.E. Wallace, A. Amemiya, L.J. Bean, T.D. Bird, C.-T. Fong, H.C. Mefford, R.J. Smith, et al., eds. (Seattle (WA): University of Washington, Seattle),.
Pèlegrin, A., Folli, S., Buchegger, F., Mach, J.P., Wagnières, G., and van den Bergh, H. (1991). Antibody-fluorescein conjugates for photoimmunodiagnosis of human colon carcinoma in nude mice. Cancer 67, 2529–2537.
Perkins, K.J., and Davies, K.E. (2002). The role of utrophin in the potential therapy of Duchenne muscular dystrophy. Neuromuscul. Disord. NMD 12 Suppl 1, S78–S89.
Petrof, B. (2002). Molecular Pathophysiology of Myofiber Injury in Deficiencies of the Dystrophin-Glycoprotein Complex. Am J Phy Med Rehabil 81, S162–S174.
Petrof, B.J., Shrager, J.B., Stedman, H.H., Kelly, A.M., and Sweeney, H.L. (1993). Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc. Natl. Acad. Sci. U. S. A. 90, 3710–3714.
Pillen, S., van Keimpema, M., Nievelstein, R.A.J., Verrips, A., van Kruijsbergen-Raijmann, W., and Zwarts, M.J. (2006). Skeletal muscle ultrasonography: Visual versus quantitative evaluation. Ultrasound Med. Biol. 32, 1315–1321.
Pillen, S., Arts, I.M.P., and Zwarts, M.J. (2008). Muscle ultrasound in neuromuscular disorders. Muscle Nerve 37, 679–693.
244
Pillen, S., Tak, R.O., Zwarts, M.J., Lammens, M.M.Y., Verrijp, K.N., Arts, I.M.P., van der Laak, J.A., Hoogerbrugge, P.M., van Engelen, B.G.M., and Verrips, A. (2009). Skeletal muscle ultrasound: correlation between fibrous tissue and echo intensity. Ultrasound Med. Biol. 35, 443–446.
Pimorady-Esfahani, A., Grounds, M.D., and McMenamin, P.G. (1997). Macrophages and dendritic cells in normal and regenerating murine skeletal muscle. Muscle Nerve 20, 158–166.
Pistilli, E.E., Bogdanovich, S., Goncalves, M.D., Ahima, R.S., Lachey, J., Seehra, J., and Khurana, T. (2011). Targeting the Activin Type IIB Receptor to Improve Muscle Mass and Function in the mdx Mouse Model of Duchenne Muscular Dystrophy. Am. J. Pathol. 178, 1287–1297.
Poellinger, A., Persigehl, T., Mahler, M., Bahner, M., Ponder, S.L., Diekmann, F., Bremer, C., and Moesta, T. (2011). Near-infrared imaging of the breast using omocianine as a fluorescent dye: results of a placebo-controlled, clinical, multicenter trial. Invest. Radiol. 46, 697–704.
Polman, C.H., O’Connor, P.W., Havrdova, E., Hutchinson, M., Kappos, L., Miller, D.H., Phillips, J.T., Lublin, F.D., Giovannoni, G., Wajgt, A., et al. (2006). A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N. Engl. J. Med. 354, 899–910.
Porrès, L., Holland, A., Pålsson, L.-O., Monkman, A.P., Kemp, C., and Beeby, A. (2006). Absolute measurements of photoluminescence quantum yields of solutions using an integrating sphere. J. Fluoresc. 16, 267–272.
Porter, J.D., Khanna, S., Kaminski, H.J., Rao, J.S., Merriam, A.P., Richmonds, C.R., Leahy, P., Li, J., Guo, W., and Andrade, F.H. (2002). A chronic inflammatory response dominates the skeletal muscle molecular signature in dystrophin-deficient mdx mice. Hum. Mol. Genet. 11, 263–272.
Portnoy, E., Lecht, S., Lazarovici, P., Danino, D., and Magdassi, S. (2011). Cetuximab-labeled liposomes containing near-infrared probe for in vivo imaging. Nanomedicine Nanotechnol. Biol. Med. 7, 480–488.
Proske, U., and Morgan, D.L. (2001). Muscle damage from eccentric exercise: mechanism, mechanical signs, adaptation and clinical applications. J. Physiol. 537, 333–345.
Proulx, S.T., Luciani, P., Derzsi, S., Rinderknecht, M., Mumprecht, V., Leroux, J.-C., and Detmar, M. (2010). Quantitative Imaging of Lymphatic Function with Liposomal Indocyanine Green. Cancer Res. 70, 7053–7062.
Raabe, A.M.D., Beck, J., Gerlach, R., Zimmermann, M.M.D., and Seifert, V.M.D. (2003). Near-infrared Indocyanine Green Video Angiography: A New Method for Intraoperative Assessment of Vascular Flow. Neurosurg. January 2003 52, 132–139.
245
Radermacher, K.A., Beghein, N., Boutry, S., Laurent, S., Elst, L.V., Muller, R.N., Jordan, B.F., and Gallez, B. (2009). In Vivo Detection of Inflammation Using Pegylated Iron Oxide Particles Targeted at E-Selectin A Multimodal Approach Using MR Imaging and EPR Spectroscopy. Invest. Radiol. 44, 398–404.
Ranjan, A.P., Zeglam, K., Mukerjee, A., Thamake, S., and Vishwanatha, J.K. (2011). A sustained release formulation of chitosan modified PLCL:poloxamer blend nanoparticles loaded with optical agent for animal imaging. Nanotechnology 22, 295104.
Rasmussen, J.C., Tan, I.-C., Marshall, M.V., Fife, C.E., and Sevick-Muraca, E.M. (2009). Lymphatic imaging in humans with near-infrared fluorescence. Curr. Opin. Biotechnol. 20, 74–82.
Reeves, E.K.M., Hoffman, E.P., Nagaraju, K., Damsker, J.M., and McCall, J.M. (2013). VBP15: Preclinical characterization of a novel anti-inflammatory delta 9,11 steroid. Bioorg. Med. Chem. 21, 2241–2249.
Reimers, C.D., Schlotter, B., Eicke, B.M., and Witt, T.N. (1996). Calf enlargement in neuromuscular diseases: a quantitative ultrasound study in 350 patients and review of the literature. J. Neurol. Sci. 143, 46–56.
Relaix, F., Rocancourt, D., Mansouri, A., and Buckingham, M. (2005). A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature 435, 948–953.
Renault, V., Piron-Hamelin, G., Forestier, C., DiDonna, S., Decary, S., Hentati, F., Saillant, G., Butler-Browne, G.S., and Mouly, V. (2000). Skeletal muscle regeneration and the mitotic clock. Exp. Gerontol. 35, 711–719.
Ricotti, V., Muntoni, F., and Voit, T. (2015). Challenges of clinical trial design for DMD. Neuromuscul. Disord. NMD 25, 932–935.
Rimessi, P., Sabatelli, P., Fabris, M., Braghetta, P., Bassi, E., Spitali, P., Vattemi, G., Tomelleri, G., Mari, L., Perrone, D., et al. (2009). Cationic PMMA Nanoparticles Bind and Deliver Antisense Oligoribonucleotides Allowing Restoration of Dystrophin Expression in the mdx Mouse. Mol. Ther. 17, 820–827.
Ringleb, S.I., Bensamoun, S.F., Chen, Q., Manduca, A., An, K.-N., and Ehman, R.L. (2007). Applications of magnetic resonance elastography to healthy and pathologic skeletal muscle. J. Magn. Reson. Imaging JMRI 25, 301–309.
Rittweger, J., Frost, H.M., Schiessl, H., Ohshima, H., Alkner, B., Tesch, P., and Felsenberg, D. (2005). Muscle atrophy and bone loss after 90 days’ bed rest and the effects of flywheel resistive exercise and pamidronate: results from the LTBR study. Bone 36, 1019–1029.
246
Rosler, A., Vandermeulen, G.W.M., and Klok, H.A. (2001). Advanced drug delivery devices via self-assembly of amphiphilic block copolymers. Adv. Drug Deliv. Rev. 53, 95–108.
Roy, R.R., Baldwin, K.M., and Edgerton, V.R. (1991). The plasticity of skeletal muscle: effects of neuromuscular activity. Exerc. Sport Sci. Rev. 19, 269–312.
Ruehm, S.G., Schroeder, T., and Debatin, J.F. (2001). Interstitial MR lymphography with gadoterate meglumine: Initial experience in humans. Radiology 220, 816–821.
Ruotsalainen, U., Raitakari, M., Nuutila, P., Oikonen, V., Sipila, H., Teras, M., Knuuti, M.J., Bloomfield, P.M., and Iida, H. (1997). Quantitative blood flow measurement of skeletal muscle using oxygen-15-water and PET. J. Nucl. Med. 38, 314–319.
Russin, T.J., Altınoğlu, E.İ., Adair, J.H., and Eklund, P.C. (2010). Measuring the fluorescent quantum efficiency of indocyanine green encapsulated in nanocomposite particulates. J. Phys. Condens. Matter Inst. Phys. J. 22, 334217.
Rutkove, S.B. (2009). Electrical Impedance Myography: Background, Current State, and Future Directions. Muscle Nerve 40, 936–946.
Rutkove, S.B., and Darras, B.T. (2013). Electrical impedance myography for the assessment of children with muscular dystrophy: a preliminary study. J. Phys. Conf. Ser. 434.
Rutkove, S.B., Esper, G.J., Lee, K.S., Aaron, R., and Shiffman, C.A. (2005). Electrical impedance myography in the detection of radiculopathy. Muscle Nerve 32, 335–341.
Rutkove, S.B., Zhang, H., Schoenfeld, D.A., Raynor, E.M., Shefner, J.M., Cudkowicz, M.E., Chin, A.B., Aaron, R., and Shiffman, C.A. (2007). Electrical impedance myography to assess outcome in amyotrophic lateral sclerosis clinical trials. Clin. Neurophysiol. 118, 2413–2418.
Rutkove, S.B., Shefner, J.M., Gregas, M., Butler, H., Caracciolo, J., Lin, C., Fogerson, P.M., Mongiovi, P., and Darras, B.T. (2010). Characterizing Spinal Muscular Atrophy with Electrical Impedance Myography. Muscle Nerve 42, 915–921.
Rutkove, S.B., Caress, J.B., Cartwright, M.S., Burns, T.M., Warder, J., David, W.S., Goyal, N., Maragakis, N.J., Clawson, L., Benatar, M., et al. (2012). Electrical impedance myography as a biomarker to assess ALS progression. Amyotroph. Lateral Scler. 13, 439–445.
Rutkove, S.B., Geisbush, T.R., Mijailovic, A., Shklyar, I., Pasternak, A., Visyak, N., Wu, J.S., Zaidman, C., and Darras, B.T. (2014). Cross-sectional Evaluation of Electrical Impedance Myography and Quantitative Ultrasound for the Assessment of Duchenne Muscular Dystrophy in a Clinical Trial Setting. Pediatr. Neurol. 51, 88–92.
247
Ryan, N.J. (2014). Ataluren: First Global Approval. Drugs 74, 1709–1714.
Sacco, A., Mourkioti, F., Tran, R., Choi, J., Llewellyn, M., Kraft, P., Shkreli, M., Delp, S., Pomerantz, J.H., Artandi, S.E., et al. (2010). Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice. Cell 143, 1059–1071.
Sack, I., Bernarding, J., and Braun, J. (2002). Analysis of wave patterns in MR elastography of skeletal muscle using coupled harmonic oscillator simulations. Magn. Reson. Imaging 20, 95–104.
Saleh, A., Schroeter, M., Jonkmanns, C., Hartung, H.-P., Mödder, U., and Jander, S. (2004). In vivo MRI of brain inflammation in human ischaemic stroke. Brain J. Neurol. 127, 1670–1677.
Sampaolesi, M., Blot, S., D’Antona, G., Granger, N., Tonlorenzi, R., Innocenzi, A., Mognol, P., Thibaud, J.-L., Galvez, B.G., Barthélémy, I., et al. (2006). Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature 444, 574–579.
Sandri, M., El Meslemani, A.H., Sandri, C., Schjerling, P., Vissing, K., Andersen, J.L., Rossini, K., Carraro, U., and Angelini, C. (2001). Caspase 3 expression correlates with skeletal muscle apoptosis in Duchenne and facioscapulo human muscular dystrophy. A potential target for pharmacological treatment? J. Neuropathol. Exp. Neurol. 60, 302–312.
Sarkozy, A., Deschauer, M., Carlier, R.-Y., Schrank, B., Seeger, J., Walter, M.C., Schoser, B., Reilich, P., Leturq, F., Radunovic, A., et al. (2012). Muscle MRI findings in limb girdle muscular dystrophy type 2L. Neuromuscul. Disord. NMD 22 Suppl 2, S122–S129.
Saxena, V., Sadoqi, M., and Shao, J. (2004). Indocyanine green-loaded biodegradable nanoparticles: preparation, physicochemical characterization and in vitro release. Int. J. Pharm. 278, 293–301.
Saxena, V., Sadoqi, M., and Shao, J. (2006). Polymeric nanoparticulate delivery system for Indocyanine green: Biodistribution in healthy mice. Int. J. Pharm. 308, 200–204.
Sazani, P., Magee, T., Charleston, J.S., Shanks, C., Zhang, J., Carver, M., Rodino-Klapac, L., Sahenk, Z., Roush, K., Bird, L., et al. (2014). Safety and pharmacokinetic profile of eteplirsen, SRP-4045, and SRP-4053, three phosphorodiamidate morpholino oligomers (PMOs) for the treatment of patients with Duchenne muscular dystrophy (DMD). Neuromuscul. Disord. 24, 828.
248
Schneider, P., Piper, S., Schmitz, C.H., Schreiter, N.F., Volkwein, N., Lüdemann, L., Malzahn, U., and Poellinger, A. (2011). Fast 3D Near-infrared breast imaging using indocyanine green for detection and characterization of breast lesions. RöFo Fortschritte Auf Dem Geb. Röntgenstrahlen Nukl. 183, 956–963.
Schreiber, A., Smith, W.L., Ionasescu, V., Zellweger, H., Franken, E.A., Dunn, V., and Ehrhardt, J. (1987). Magnetic resonance imaging of children with Duchenne muscular dystrophy. Pediatr. Radiol. 17, 495–497.
Schultz, E. (1985). Satellite cells in normal, regenerating and dystrophic muscle. Adv. Exp. Med. Biol. 182, 73–84.
Schultz, E., Jaryszak, D.L., and Valliere, C.R. (1985). Response of satellite cells to focal skeletal muscle injury. Muscle Nerve 8, 217–222.
Schwartz, D.P., Dastgir, J., Salman, A., Lear, B., Bönnemann, C.G., and Lehky, T.J. (2016). Electrical impedance myography discriminates congenital muscular dystrophy from controls. Muscle Nerve 53, 402–406.
Scully, M.A., Pandya, S., and Moxley, R.T. (2012). Review of Phase II and Phase III clinical trials for Duchenne muscular dystrophy. Expert Opin. Orphan Drugs 1, 33–46.
Selsby, J.T., Ross, J.W., Nonneman, D., and Hollinger, K. (2015). Porcine Models of Muscular Dystrophy. Ilar J. 56, 116–126.
Sesto, M.E., Chourasia, A.O., Block, W.F., and Radwin, R.G. (2008). Mechanical and magnetic resonance imaging changes following eccentric or concentric exertions. Clin. Biomech. 23, 961–968.
Sevick-Muraca, E.M., Houston, J.P., and Gurfinkel, M. (2002). Fluorescence-enhanced, near infrared diagnostic imaging with contrast agents. Curr. Opin. Chem. Biol. 6, 642–650.
Shaibani, A., Jabari, D., Jabbour, M., Arif, C., Lee, M., and Rahbar, M.H. (2014). Diagnostic outcome of muscle biopsy. Muscle Nerve. Epub.
Sharafi, B., Ames, E.G., Holmes, J.W., and Blemker, S.S. (2011). Strains at the myotendinous junction predicted by a micromechanical model. J. Biomech. 44, 2795–2801.
Sharma, P., Singh, A., Brown, S.C., Bengtsson, N., Walter, G.A., Grobmyer, S.R., Iwakuma, N., Santra, S., Scott, E.W., and Moudgil, B.M. (2010). Multimodal nanoparticulate bioimaging contrast agents. Methods Mol. Biol. Clifton NJ 624, 67–81.
249
Sharp, N.J., Kornegay, J.N., Van Camp, S.D., Herbstreith, M.H., Secore, S.L., Kettle, S., Hung, W.Y., Constantinou, C.D., Dykstra, M.J., and Roses, A.D. (1992). An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy. Genomics 13, 115–121.
Shavlakadze, T., White, J., Hoh, J.F.Y., Rosenthal, N., and Grounds, M.D. (2004). Targeted expression of insulin-like growth factor-I reduces early myofiber necrosis in dystrophic mdx mice. Mol. Ther. J. Am. Soc. Gene Ther. 10, 829–843.
Shaye, D.D., and Greenwald, I. (2011). OrthoList: a compendium of C. elegans genes with human orthologs. PloS One 6, e20085.
Shcherbakova, D.M., and Verkhusha, V.V. (2013). Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat. Methods 10, 751–754.
Shellock, F.G., Fukunaga, T., Mink, J.H., and Edgerton, V.R. (1991). Exertional muscle injury: evaluation of concentric versus eccentric actions with serial MR imaging. Radiology 179, 659–664.
Sheng, Z., Hu, D., Zheng, M., Zhao, P., Liu, H., Gao, D., Gong, P., Gao, G., Zhang, P., Ma, Y., et al. (2014). Smart Human Serum Albumin-Indocyanine Green Nanoparticles Generated by Programmed Assembly for Dual-Modal Imaging-Guided Cancer Synergistic Phototherapy. ACS Nano.
Shimatsu, Y., Katagiri, K., Furuta, T., Nakura, M., Tanioka, Y., Yuasa, K., Tomohiro, M., Kornegay, J.N., Nonaka, I., and Takeda, S. ’ichi (2003). Canine X-linked muscular dystrophy in Japan (CXMDJ). Exp. Anim. Jpn. Assoc. Lab. Anim. Sci. 52, 93–97.
Shimatsu, Y., Yoshimura, M., Yuasa, K., Urasawa, N., Tomohiro, M., Nakura, M., Tanigawa, M., Nakamura, A., and Takeda, S. ’ichi (2005). Major clinical and histopathological characteristics of canine X-linked muscular dystrophy in Japan, CXMDJ. Acta Myol. Myopathies Cardiomyopathies Off. J. Mediterr. Soc. Myol. Ed. Gaetano Conte Acad. Study Striated Muscle Dis. 24, 145–154.
Shklyar, I., Pasternak, A., Kapur, K., Darras, B.T., and Rutkove, S.B. (2015). Composite biomarkers for assessing Duchenne muscular dystrophy: an initial assessment. Pediatr. Neurol. 52, 202–205.
Sicinski, P., Geng, Y., Ryder-Cook, A.S., Barnard, E.A., Darlison, M.G., and Barnard, P.J. (1989). The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 244, 1578–1580.
Sirsi, S.R., Schray, R.C., Wheatley, M.A., and Lutz, G.J. (2009). Formulation of polylactide-co-glycolic acid nanospheres for encapsulation and sustained release of poly(ethylene imine)-poly(ethylene glycol) copolymers complexed to oligonucleotides. J. Nanobiotechnology 7, 1.
250
Skuk, D. (2004). Myoblast transplantation for inherited myopathies: a clinical approach. Expert Opin. Biol. Ther. 4, 1871–1885.
Sorichter, S., Koller, A., Haid, C., Wicke, K., Judmaier, W., Werner, P., and Raas, E. (1995). Light concentric exercise and heavy eccentric muscle loading: effects on CK, MRI and markers of inflammation. Int. J. Sports Med. 16, 288–292.
Soukos, N.S., Hamblin, M.R., Keel, S., Fabian, R.L., Deutsch, T.F., and Hasan, T. (2001). Epidermal growth factor receptor-targeted immunophotodiagnosis and photoimmunotherapy of oral precancer in vivo. Cancer Res. 61, 4490–4496.
Spencer, M.J., and Mellgren, R.L. (2002). Overexpression of a calpastatin transgene in mdx muscle reduces dystrophic pathology. Hum. Mol. Genet. 11, 2645–2655.
Spudich, J., and Watt, S. (1971). Regulation of Rabbit Skeletal Muscle Contraction .1. Biochemical Studies. J. Biol. Chem. 246, 4866 – &.
Stern, L.M., Caudrey, D.J., Perrett, L.V., and Boldt, D.W. (1984). Progression of muscular dystrophy assessed by computed tomography. Dev. Med. Child Neurol. 26, 569–573.
Stramare, R., Beltrame, V., Borgo, R.D., Gallimberti, L., Frigo, A.C., Pegoraro, E., Angelini, C., Rubaltelli, L., and Feltrin, G.P. (2010). MRI in the assessment of muscular pathology: a comparison between limb-girdle muscular dystrophies, hyaline body myopathies and myotonic dystrophies. Radiol. Med. (Torino) 115, 585–599.
Subach, O.M., Patterson, G.H., Ting, L.-M., Wang, Y., Condeelis, J.S., and Verkhusha, V.V. (2011). A photoswitchable orange-to-far-red fluorescent protein, PSmOrange. Nat. Methods 8, 771–777.
Sun, C., Lee, J.S.H., and Zhang, M. (2008). Magnetic nanoparticles in MR imaging and drug delivery. Adv. Drug Deliv. Rev. 60, 1252–1265.
Sun, J.S., Tsaung, Y.H., Hou, S.M., Hang, Y.S., Liu, T.K., Cheng, C.K., and Tsao, K.T. (1994). Microfailure of peroneus longus muscle during passive extension. Proc. Natl. Sci. Counc. Repub. China B 18, 24–29.
Suydam, S.M., Soulas, E.M., Elliott, D.M., Gravare Silbernagel, K., Buchanan, T.S., and Cortes, D.H. (2015). Viscoelastic properties of healthy achilles tendon are independent of isometric plantar flexion strength and cross-sectional area. J. Orthop. Res. 33, 926–931.
Taggart, D.P., Choudhary, B., Anastasiadis, K., Abu-Omar, Y., Balacumaraswami, L., and Pigott, D.W. (2003). Preliminary experience with a novel intraoperative fluorescence imaging technique to evaluate the patency of bypass grafts in total arterial revascularization. Ann. Thorac. Surg. 75, 870–873.
251
Tan, Y., and Jiang, H. (2008). Diffuse optical tomography guided quantitative fluorescence molecular tomography. Appl. Opt. 47, 2011–2016.
Tarulli, A., Esper, G.J., Lee, K.S., Aaron, R., Shiffman, C.A., and Rutkove, S.B. (2005). Electrical impedance myography in the bedside assessment of inflammatory myopathy. Neurology 65, 451–452.
Taylor, L.E., Kaminoh, Y.J., Rodesch, C.K., and Flanigan, K.M. (2012). Quantification of dystrophin immunofluorescence in dystrophinopathy muscle specimens. Neuropathol. Appl. Neurobiol. 38, 591–601.
Tedesco, F.S., Dellavalle, A., Diaz-Manera, J., Messina, G., and Cossu, G. (2010). Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. J. Clin. Invest. 120, 11–19.
Tesch, P.A., Trieschmann, J.T., and Ekberg, A. (2004). Hypertrophy of chronically unloaded muscle subjected to resistance exercise. J. Appl. Physiol. Bethesda Md 1985 96, 1451–1458.
Thomas, G.D. (2013). Functional muscle ischemia in Duchenne and Becker muscular dystrophy. Front. Physiol. 4.
Thomas, G.D., Ye, J., De Nardi, C., Monopoli, A., Ongini, E., and Victor, R.G. (2012). Treatment with a nitric oxide-donating NSAID alleviates functional muscle ischemia in the mouse model of Duchenne muscular dystrophy. PloS One 7, e49350.
Thomas, M., Langley, B., Berry, C., Sharma, M., Kirk, S., Bass, J., and Kambadur, R. (2000). Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J. Biol. Chem. 275, 40235–40243.
Tidball, J.G. (1995). Inflammatory cell response to acute muscle injury. Med. Sci. Sports Exerc. 27, 1022–1032.
Tidball, J.G. (2005). Inflammatory processes in muscle injury and repair. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288, R345–R353.
Tidball, J.G. (2011). Mechanisms of muscle injury, repair, and regeneration. Compr. Physiol. 1, 2029–2062.
Tidball, J.G., and Wehling-Henricks, M. (2007). Macrophages promote muscle membrane repair and muscle fibre growth and regeneration during modified muscle loading in mice in vivo. J. Physiol. 578, 327–336.
Tinsley, J., Deconinck, N., Fisher, R., Kahn, D., Phelps, S., Gillis, J.M., and Davies, K. (1998). Expression of full-length utrophin prevents muscular dystrophy in mdx mice. Nat. Med. 4, 1441–1444.
252
Torriani, M., Townsend, E., Thomas, B.J., Bredella, M.A., Ghomi, R.H., and Tseng, B.S. (2012). Lower leg muscle involvement in Duchenne muscular dystrophy: an MR imaging and spectroscopy study. Skeletal Radiol. 41, 437–445.
Torricelli, A., Quaresima, V., Pifferi, A., Biscotti, G., Spinelli, L., Taroni, P., Ferrari, M., and Cubeddu, R. (2004). Mapping of calf muscle oxygenation and haemoglobin content during dynamic plantar flexion exercise by multi-channel time-resolved near-infrared spectroscopy. Phys. Med. Biol. 49, 685.
Towse, T.F., Slade, J.M., Ambrose, J.A., DeLano, M.C., and Meyer, R.A. (2011). Quantitative analysis of the postcontractile blood-oxygenation-level-dependent (BOLD) effect in skeletal muscle. J. Appl. Physiol. Bethesda Md 1985 111, 27–39.
Triplett, W.T., Baligand, C., Forbes, S.C., Willcocks, R.J., Lott, D.J., DeVos, S., Pollaro, J., Rooney, W.D., Sweeney, H.L., Bönnemann, C.G., et al. (2014). Chemical shift-based MRI to measure fat fractions in dystrophic skeletal muscle. Magn. Reson. Med. 72, 8–19.
Troyan, S.L., Kianzad, V., Gibbs-Strauss, S.L., Gioux, S., Matsui, A., Oketokoun, R., Ngo, L., Khamene, A., Azar, F., and Frangioni, J.V. (2009). The FLARETM Intraoperative Near-Infrared Fluorescence Imaging System: A First-in-Human Clinical Trial in Breast Cancer Sentinel Lymph Node Mapping. Ann. Surg. Oncol. 16, 2943–2952.
Tung, C.H., Bredow, S., Mahmood, U., and Weissleder, R. (1999). Preparation of a cathepsin D sensitive near-infrared fluorescence probe for imaging. Bioconjug. Chem. 10, 892–896.
Turner, N.J., and Badylak, S.F. (2012). Regeneration of skeletal muscle. Cell Tissue Res. 347, 759–774.
Turner, P.R., Westwood, T., Regen, C.M., and Steinhardt, R.A. (1988). Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice. Nature 335, 735–738.
Tyler, K.L. (2003). Origins and early descriptions of “Duchenne muscular dystrophy.” Muscle Nerve 28, 402–422.
Valentine, B.A., Cooper, B.J., de Lahunta, A., O’Quinn, R., and Blue, J.T. (1988). Canine X-linked muscular dystrophy. An animal model of Duchenne muscular dystrophy: clinical studies. J. Neurol. Sci. 88, 69–81.
Vasilescu, D., Vasilescu, D., Dudea, S., Botar-Jid, C., Sfrângeu, S., and Cosma, D. (2010). Sonoelastography contribution in cerebral palsy spasticity treatment assessment, preliminary report: a systematic review of the literature apropos of seven patients. Med. Ultrason. 12, 306–310.
253
van de Ven, S., Wiethoff, A., Nielsen, T., Brendel, B., van der Voort, M., Nachabe, R., Van der Mark, M., Van Beek, M., Bakker, L., Fels, L., et al. (2010). A Novel Fluorescent Imaging Agent for Diffuse Optical Tomography of the Breast: First Clinical Experience in Patients. Mol. Imaging Biol. 12, 343–348.
Verbeek, F.P.R., Troyan, S.L., Mieog, J.S.D., Liefers, G.-J., Moffitt, L.A., Rosenberg, M., Hirshfield-Bartek, J., Gioux, S., Velde, C.J.H. van de, Vahrmeijer, A.L., et al. (2014). Near-infrared fluorescence sentinel lymph node mapping in breast cancer: a multicenter experience. Breast Cancer Res. Treat. 143, 333–342.
Villalta, S.A., Deng, B., Rinaldi, C., Wehling-Henricks, M., and Tidball, J.G. (2011). IFNγ promotes muscle damage in the mdx mouse model of Duchenne muscular dystrophy by suppressing M2 macrophage activation and inhibiting muscle cell proliferation. J. Immunol. Baltim. Md 1950 187, 5419–5428.
Vogiatzis, I., Athanasopoulos, D., Boushel, R., Guenette, J.A., Koskolou, M., Vasilopoulou, M., Wagner, H., Roussos, C., Wagner, P.D., and Zakynthinos, S. (2008). Contribution of respiratory muscle blood flow to exercise-induced diaphragmatic fatigue in trained cyclists. J. Physiol. 586, 5575–5587.
Vohra, R.S., Mathur, S., Bryant, N.D., Forbes, S., Vandenborne, K., and Walter, G.A. (2015). Age-related T2 changes in hindlimb muscles of mdx mice. Muscle Nerve. Epub.
Voit, T., Topaloglu, H., Straub, V., Muntoni, F., Deconinck, N., Campion, G., De Kimpe, S.J., Eagle, M., Guglieri, M., Hood, S., et al. (2014). Safety and efficacy of drisapersen for the treatment of Duchenne muscular dystrophy (DEMAND II): an exploratory, randomised, placebo-controlled phase 2 study. Lancet Neurol. 13, 987–996.
Wadosky, K.M., Li, L., Rodríguez, J.E., Min, J.-N., Bogan, D., Gonzalez, J., Patterson, C., Kornegay, J.N., and Willis, M. (2011). Regulation of the calpain and ubiquitin-proteasome systems in a canine model of muscular dystrophy. Muscle Nerve 44, 553–562.
Wagner, K.R., Hamed, S., Hadley, D.W., Gropman, A.L., Burstein, A.H., Escolar, D.M., Hoffman, E.P., and Fischbeck, K.H. (2001). Gentamicin treatment of Duchenne and Becker muscular dystrophy due to nonsense mutations. Ann. Neurol. 49, 706–711.
Walter, G., Cordier, L., Bloy, D., and Lee Sweeney, H. (2005). Noninvasive monitoring of gene correction in dystrophic muscle. Magn. Reson. Med. 54, 1369–1376.
Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., Zhang, F., and Jaenisch, R. (2013). One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918.
254
Wang, L., Salibi, N., Wu, Y., Schweitzer, M.E., and Regatte, R.R. (2009). Relaxation times of skeletal muscle metabolites at 7T. J. Magn. Reson. Imaging 29, 1457–1464.
Warnke, P.C., Kreth, F.W., and Ostertag, C.B. (1995). Early postoperative magnetic resonance imaging after resection of malignant glioma: objective evaluation of residual tumor and its influence on regrowth and prognosis. Neurosurgery 36, 872–874.
Wary, C., Azzabou, N., Giraudeau, C., Le Louër, J., Montus, M., Voit, T., Servais, L., and Carlier, P. (2015). Quantitative NMRI and NMRS identify augmented disease progression after loss of ambulation in forearms of boys with Duchenne muscular dystrophy. NMR Biomed. Epub.
Weidman, E.R., Charles, H.C., Negro-Vilar, R., Sullivan, M.J., and MacFall, J.R. (1991). Muscle activity localization with 31P spectroscopy and calculated T2-weighted 1H images. Invest. Radiol. 26, 309–316.
Weinmann, H.J., Ebert, W., Misselwitz, B., and Schmitt-Willich, H. (2003). Tissue-specific MR contrast agents. Eur. J. Radiol. 46, 33–44.
Weissleder, R. (2001). A clearer vision for in vivo imaging. Nat. Biotechnol. 19, 316–317.
Weissleder, R., and Ntziachristos, V. (2003). Shedding light onto live molecular targets. Nat. Med. 9, 123–128.
Weissleder, R., Tung, C.H., Mahmood, U., and Bogdanov, A. (1999). In vivo imaging of tumors with protease-activated near-infrared fluorescent probes. Nat. Biotechnol. 17, 375–378.
Welch, E.M., Barton, E.R., Zhuo, J., Tomizawa, Y., Friesen, W.J., Trifillis, P., Paushkin, S., Patel, M., Trotta, C.R., Hwang, S., et al. (2007). PTC124 targets genetic disorders caused by nonsense mutations. Nature 447, 87–91.
Weller, B., Karpati, G., and Carpenter, S. (1990). Dystrophin-deficient mdx muscle fibers are preferentially vulnerable to necrosis induced by experimental lengthening contractions. J. Neurol. Sci. 100, 9–13.
Whitehead, N.P., Streamer, M., Lusambili, L.I., Sachs, F., and Allen, D.G. (2006). Streptomycin reduces stretch-induced membrane permeability in muscles from mdx mice. Neuromuscul. Disord. NMD 16, 845–854.
Wilhelm, C., Billotey, C., Roger, J., Pons, J.N., Bacri, J.C., and Gazeau, F. (2003). Intracellular uptake of anionic superparamagnetic nanoparticles as a function of their surface coating. Biomaterials 24, 1001–1011.
255
Willcocks, R.J., Arpan, I.A., Forbes, S.C., Lott, D.J., Senesac, C.R., Senesac, E., Deol, J., Triplett, W.T., Baligand, C., Daniels, M.J., et al. (2014). Longitudinal measurements of MRI-T2 in boys with Duchenne muscular dystrophy: Effects of age and disease progression. Neuromuscul. Disord. 24, 393–401.
Williams, M.S. (2004). Myostatin mutation associated with gross muscle hypertrophy in a child. N. Engl. J. Med. 351, 1030–1031; author reply 1030–1031.
Willis, T.A., Hollingsworth, K.G., Coombs, A., Sveen, M.-L., Andersen, S., Stojkovic, T., Eagle, M., Mayhew, A., de Sousa, P.L., Dewar, L., et al. (2014). Quantitative magnetic resonance imaging in limb-girdle muscular dystrophy 2I: a multinational cross-sectional study. PloS One 9, e90377.
Willmann, R., Possekel, S., Dubach-Powell, J., Meier, T., and Ruegg, M.A. (2009). Mammalian animal models for Duchenne muscular dystrophy. Neuromuscul. Disord. NMD 19, 241–249.
Wokke, B.H., Bos, C., Reijnierse, M., van Rijswijk, C.S., Eggers, H., Webb, A., Verschuuren, J.J., and Kan, H.E. (2013). Comparison of dixon and T1-weighted MR methods to assess the degree of fat infiltration in duchenne muscular dystrophy patients. J. Magn. Reson. Imaging 38, 619–624.
Wokke, B.H., van den Bergen, J.C., Versluis, M.J., Niks, E.H., Milles, J., Webb, A.G., van Zwet, E.W., Aartsma-Rus, A., Verschuuren, J.J., and Kan, H.E. (2014). Quantitative MRI and strength measurements in the assessment of muscle quality in Duchenne muscular dystrophy. Neuromuscul. Disord. 24, 409–416.
Wolf, M., Ferrari, M., and Quaresima, V. (2007). Progress of near-infrared spectroscopy and topography for brain and muscle clinical applications. J. Biomed. Opt. 12, 062104–062104 – 14.
Wolfe, J.D., and Csaky, K.G. (2004). Indocyanine green enhanced retinal vessel laser closure in rats: histologic and immunohistochemical observations. Exp. Eye Res. 79, 631–638.
Wolff, J.A., and Budker, V. (2005). The mechanism of naked DNA uptake and expression. Adv. Genet. 54, 3–20.
Wrogemann, K., and Pena, S.D. (1976). Mitochondrial calcium overload: A general mechanism for cell-necrosis in muscle diseases. Lancet Lond. Engl. 1, 672–674.
Yamamoto, Y., Yamaguchi, S., Sasho, T., Fukawa, T., Akatsu, Y., Nagashima, K., and Takahashi, K. (2016). Quantitative Ultrasound Elastography With an Acoustic Coupler for Achilles Tendon Elasticity: Measurement Repeatability and Normative Values. J. Ultrasound Med. Off. J. Am. Inst. Ultrasound Med. 35, 159–166.
256
Yang, Y.Y., Chung, T.S., and Ng, N.P. (2001). Morphology, drug distribution, and in vitro release profiles of biodegradable polymeric microspheres containing protein fabricated by double-emulsion solvent extraction/evaporation method. Biomaterials 22, 231–241.
Yannuzzi, L.A. (2011). Indocyanine green angiography: a perspective on use in the clinical setting. Am. J. Ophthalmol. 151, 745–751.e1.
Yaseen, M.A., Yu, J., Jung, B., Wong, M.S., and Anvari, B. (2009). Biodistribution of Encapsulated Indocyanine Green in Healthy Mice. Mol. Pharm. 6, 1321–1332.
Yu, J., Javier, D., Yaseen, M.A., Nitin, N., Richards-Kortum, R., Anvari, B., and Wong, M.S. (2010). Self-assembly synthesis, tumor cell targeting, and photothermal capabilities of antibody-coated indocyanine green nanocapsules. J. Am. Chem. Soc. 132, 1929–1938.
Yuan, Z., Zhang, Q., Sobel, E., and Jiang, H. (2007). Three-dimensional diffuse optical tomography of osteoarthritis: initial results in the finger joints. J. Biomed. Opt. 12, 034001.
Yuasa, K., Sakamoto, M., Miyagoe-Suzuki, Y., Tanouchi, A., Yamamoto, H., Li, J., Chamberlain, J.S., Xiao, X., and Takeda, S. (2002). Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product. Gene Ther. 9, 1576–1588.
Yugeta, N., Urasawa, N., Fujii, Y., Yoshimura, M., Yuasa, K., Wada, M.R., Nakura, M., Shimatsu, Y., Tomohiro, M., Takahashi, A., et al. (2006). Cardiac involvement in Beagle-based canine X-linked muscular dystrophy in Japan (CXMDJ): electrocardiographic, echocardiographic, and morphologic studies. BMC Cardiovasc. Disord. 6, 47.
Zaheer, A., Lenkinski, R.E., Mahmood, A., Jones, A.G., Cantley, L.C., and Frangioni, J.V. (2001). In vivo near-infrared fluorescence imaging of osteoblastic activity. Nat. Biotechnol. 19, 1148–1154.
Zaidman, C.M., Wu, J.S., Wilder, S., Darras, B.T., and Rutkove, S.B. (2014). Minimal training is required to reliably perform quantitative ultrasound of muscle. Muscle Nerve 50, 124–128.
Zaidman, C.M., Malkus, E.C., and Connolly, A.M. (2015). Muscle ultrasound quantifies disease progression over time in infants and young boys with duchenne muscular dystrophy. Muscle Nerve 52, 334–338.
Zelken, J.A., and Tufaro, A.P. (2015). Current Trends and Emerging Future of Indocyanine Green Usage in Surgery and Oncology: An Update. Ann. Surg. Oncol. 1–13.
257
Zhang, Y., Yue, Y., Li, L., Hakim, C.H., Zhang, K., Thomas, G.D., and Duan, D. (2013). Dual AAV therapy ameliorates exercise-induced muscle injury and functional ischemia in murine models of Duchenne muscular dystrophy. Hum. Mol. Genet. 22, 3720–3729.
Zheng, X., Xing, D., Zhou, F., Wu, B., and Chen, W.R. (2011). Indocyanine green-containing nanostructure as near infrared dual-functional targeting probes for optical imaging and photothermal therapy. Mol. Pharm. 8, 447–456.
Ziv, K., Meir, G., Harmelin, A., Shimoni, E., Klein, E., and Neeman, M. (2010). Ferritin as a reporter gene for MRI: chronic liver over expression of H-ferritin during dietary iron supplementation and aging. NMR Biomed. 23, 523–531.
258
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.
259
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.