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
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NEAR INFRARED OPTICAL IMAGING AND MAGNETIC RESONANCE CHARACTERIZATION OF DYSTROPHIC AND DAMAGED MUSCLE
By
STEPHEN MARK CHRZANOWSKI
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
My family, friends and mentors, who carried me through the troughs and lifted me to the
peaks during the PhD - I could not have done this without you
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ACKNOWLEDGMENTS
We are like dwarfs on the shoulders of giants, so that we can see more than they, and things at a greater distance, not by virtue of any sharpness of sign on our part, or any physical distinction, but because we are carried high and raised up by their giant size.
–Benand of Chartres, circa 1130
Dr. Glenn A. Walter’s substantial scientific accolades are internationally
respected, but his accomplishments as a human being far outweigh what he has
achieved in the world of science. Throughout our personal and professional relationship
as mentor-mentee, he was continually my ever-optimistic guiding force throughout my
pre-doctoral training years. I am confident to say that his mentorship will not conclude
following the completion of my training period under his protective shield.
I am a product of the village of individuals that have guided me along this route,
each enlightening our journey in their own unique ways. Dr. Krista Vandenborne
encouraged me to ‘enjoy the journey,’ despite my greatest efforts to only focus on the
results. The remainder of my advisory committee, including Drs. Barry Byrne, Peter
Sayeski, and Huabei Jiang, each contributed vital seeds of knowledge to foster me into
the evolving physician-scientist in training that I’ve become today. I would not be here if
it wasn’t for the renegade Dr. Steve Hsu, who while program director of the University of
Florida’s MD-PhD program, provided the initial opportunity to begin my journey as an
MD-PhD. I must go about thanking Dr. Robbie Regenhardt as well, who asked me one
evening stumbling through midtown to be his replacement as the MD-PhD student
advocate, allowing me a glimpse of how our College of Medicine runs. The current
Executive Committee of Drs. William “Stratford” May, Al Lewin, Lisa Spyrida, and Linda
Bloom have been constant golden examples of exemplary scientists and mentors.
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Drs. Celine Baligand, Ravneet Vohra, Fan Ye, and Rebecca Willcocks are the
“boots on the ground” field generals who have demonstrated exemplary patience,
teaching me all of the applicable skills that are used on a daily basis in the clinical and
preclinical laboratories. My colleagues, Brittany Lee, Abhinandan Batra, Wootaek Lim,
Umar Alabasi, Harneet Arora, Alison Barnard, and Ishu Arpan, have been fantastic
peers to provide humor, scientific inquiry, perspective, and always have been a helping
hand in our many projects. Much gratitude is necessary to the oft unheralded behind
the scenes team of Christa Stout, Jenny Fairfield, Hilary and Renee Cunkle, Cathy
Powers, Seth Panayiotou, and Andres Saagova, as they have consistently kept me on
track to succeed in the lab, without receiving due credit themselves.
Beyond the lab, my journey as a member of the muscular dystrophy family began
long before my time at the University of Florida. The Kapusta and Trevis families have
greatly shaped my life journey, and any contribution of scientific knowledge I may
compose pales in comparison to the contribution their families have made to my life.
Also, the Muscular Dystrophy Association camps in Cleveland, Cincinnati, and
Jacksonville have provided a lifetime of stories through the inappropriate sense of
humor that many of the campers possess.
I’m incredibly lucky to have not one best friend, but rather four in Bryan Trevis
and Drs. Nicholas Peter James Perry, Narayanasarma Singam, and Damon Fu, who
have been my security net of reassurance, humor, perspective, and enlightenment
throughout this PhD. Through our years at Cincinnati together, Sarma, Fu, and Nic
consistently provided positive encouragement in and beyond the classroom. When
Bryan and I journeyed 10,000 miles across the USA, he taught me that it is not the
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destination that matters, but rather the journey to get there. Rosha Poudyal, more than
anyone else has encouraged me through the troughs and celebrated my successes
throughout graduate school, and words do not suffice to tell my appreciation for the
support and love she has provided during this journey.
A special token of appreciation is warranted to Drs. Christy Holland and Kate
Hitchcock, who both saw potential in me, and taught me to believe in myself, more than
I thought possible. Dr. Hitchcock in particular deserves gratitude, for not only showing
me how to be a successful physician-scientist, but for also serendipitously providing
fantastic clinical care halfway across the country to my significant other, as she battled
meningitis.
And finally, I must thank my family, near and far, for instilling in me a sense of
love for each other, an interest in science, and a stubborn tenacious nature.
Funding for this research was provided by the Department of Defense
Other strategies ......................................................................................... 43 Challenges of Therapeutic Trials ...................................................................... 45
2 NON-INVASIVE ASSESSMENTS OF MUSCLE HEALTH ..................................... 50
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
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
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
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
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
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
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
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
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
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
Involvement of DMD in muscle presents in non-uniform manner ............. 184 Relationship between MRI scores, function and age ............................... 184
Summary of a Multislice Assessment of the Lower Leg in DMD .................... 189 Preliminary Assessment of the Upper Extremity in DMD by MRI .......................... 190
Summary of Upper Extremity Findings ........................................................... 193 Differences Between Concentric and Eccentric Lower Arm Exercises ................. 193
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
5-2 NIR optical imaging radiant efficiency measures were correlated to 1H2O-T2, MRI-T2, and Optical Density 780 nm / mg tissue, and r2 values (with associated p values in parentheses). ................................................................................. 139
6-1 Effect size magnitude demonstrates comparable differences between NIR optical imaging and MR measures. .................................................................. 158
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LIST OF FIGURES
Figure page 1-1 The sarcolemma and dystrophin associated glycoprotein complex. ................... 49
1-2 Binding sites and protein structure of dystrophin. Numbers refer to the spectrin-like repeats throughout the protein. NTD, N terminal domain; CR, Cysteine Rich region; CTD, C terminal domain. ................................................. 49
2-1 A spin echo sequence showing the initial 90° RF pulse, followed by the generated FID, the refocusing 180° RF pulse, and the additional 90° pulse of the next sequence. ............................................................................................. 81
2-2 Longitudinal (T1) relaxation curves showing the difference in relaxation between fat and muscle, and how different TR acquisitions (along the x-axis) alter the difference in signal generated between tissue types. ........................... 82
2-3 Transverse (T2) relaxation curves showing the difference in relaxation between muscle and edema, and how different TE acquisitions (along the x-axis) alter the difference in signal decay between tissue types. ......................... 83
2-4 Inversion recovery technique to calculate T1 demonstrating representative signal recovery profiles for edema, muscle, and lipid. ........................................ 84
2-5 Progressive saturation technique demonstrating how different acquisition times within the same recovery curve can be used to calculate T1. .................... 85
2-6 A Carr-Purcell sequence showing the initial 90° RF pulse, followed by a train of 180°RF pulses in the X plane, with each refocusing the FID in the opposite direction. ............................................................................................................. 86
2-7 A Carr-Purcell-Meiboom-Gill Pulse sequence showing the initial 90° RF pulse, followed by a train of 180°RF pulses given in the rotating frame, with each refocusing the FID in the same direction. .................................................. 87
2-8 Electromagnetic spectrum, highlighting the location of the near infrared range. ................................................................................................................. 88
4-1 Radiant efficiency reaches a steady state level between 30 minutes to 12 hours following ICG an intravenous injection. ................................................... 114
4-2 Fat suppressed T1 weighted image shows muscles of the lower leg in subjects with and without DMD, with arrows pointing to the TA (solid) and Per (dashed), highlighting intramuscular differences. ............................................. 115
4-3 Schematic representation of slice selections along the length of the lower leg. 116
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4-4 The qualitative MRI grading scale used to assess pathology within DMD muscle. ............................................................................................................. 117
4-5 Schematic study design of clinical study utilizing NIR to detect muscle damage. ........................................................................................................... 118
5-1 Two-dimensional NIR optical imaging shows an increase and recovery of fluorescent signal in muscle during reambulation following immobilization. ..... 132
5-2 MRI-T2 shows damage and recovery of soleus, but not gastrocnemius nor tibialis anterior muscles during reambulation .................................................... 133
5-3 Spectroscopic findings confirm increase in 1H2O-T2 and reveal long T2 components in the soleus of immobilized-reambulated hindlimbs .................... 134
5-4 Histological assessment confirms damage and recovery in the reambulated soleus muscle of the immobilized-reambulated hindlimbs ................................ 136
5-5 Spectrophotometric assessment confirms dye uptake into the soleus muscle at the peak of muscle damage. Absorbance, measuring EBD (5-5A) and ICG (5-5B) throughout the week of reambulation are quantified. ............................. 137
5-6 Increased radiant efficiency correlates to increased markers of damage in the soleus muscle ................................................................................................... 138
6-1 Dystrophy induced muscle pathology can be detected by NIR optical imaging, MRI, and MRS ................................................................................... 152
6-2 Increased radiant efficiency correlates with increased magnetic resonance measures in healthy and dystrophic mice ......................................................... 153
6-3 NIR optical imaging, MRI, and MRS confirm increased damage to muscle following treadmill exercising in mdx mice ........................................................ 154
6-4 Increased total radiant efficiency correlates with increased magnetic resonance measures before and after damage induced by treadmill running .. 155
6-5 gsg -/- mice treated with AAV demonstrate decreased near infrared fluorescence and lower MRI-T2 and 1H2O-T2 relaxation times following treatment .......................................................................................................... 156
6-6 Increased total radiant efficiency correlates with increased magnetic resonance measures in gsg -/- mice with and without restorative AAV therapy. 157
6-7 Representative immunofluorescence images with and without AAV delivery of γ-sarcoglycan ............................................................................................... 159
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7-1 Differences between major vasculature and surrounding muscle are able to be spatially and temporally identified ................................................................ 164
7-2 A hyperemic response is able to be quantified through NIR optical imaging. ... 165
8-1 Representative size distribution (8-1A), aggregation properties (8-1B), and fluorescence characteristics (8-1C) of ICG-PLA particles ................................. 176
8-2 Photostability at room (25°C, 8-2A) and physiologic (37°C, 8-2B) temperature of ICG-PLA particles and ICG alone. ................................................................ 177
8-3 Subcutaneous injections of PLA-ICG show prolonged maintained signal compared to Lactated Ringer’s Solution and ICG alone visually (8-3A) and quantitatively (8-3B). ......................................................................................... 178
8-4 Intramuscularly injected PLA-ICG particles maintain prolonged fluorescent signal (8-4A) at 1 (8-4B) and 28 (8-4C) days following injections. .................... 179
8-5 Ex vivo NIR optical images of excised muscles following intramuscular injections into the gastrocnemius demonstrate in vivo stability of PLA-ICG particles visually (8-5A) and quantitatively (8-5B). ............................................ 180
9-1 Qualitative MRI Scores from two representative DMD patients demonstrating differences in involvement along the length of six lower leg muscle groups. .... 197
9-2 Comprehensive degree of involvement in all slices of all subjects’ muscles .... 198
9-3 Age and function are related to MRIsingle and MRImulti scores. ........................... 199
9-4 Cross sectional analysis of upper extremity muscles in boys with DMD. .......... 200
9-5 Age and PUL function as related to MRI-T2 and MRI qualitative scores. .......... 201
9-6 Fat suppressed axial MR images of concentrically (9-6A) and eccentrically (9-6B) exercised human forearms with quantification (9-6C) of T2 relaxation times taken from the deep flexor muscles of the forearms. .............................. 202
9-7 Three dimensional absorbance reconstructions of human forearms were taken two days following eccentric (9-7A) and concentric (9-7B) exercise. ...... 203
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LIST OF ABBREVIATIONS
AAV adeno-associated virus
B0 Magnitude of static magnetic field
B1 Magnitude of excitatory radiofrequency field
BMD Becker muscular dystrophy. A form of muscular dystrophy with partial expression of the protein dystrophin. Less severe than Duchenne muscular dystrophy.
DAG Complex Dystrophin associated glycoprotein complex. A transmembrane complex of glycoproteins that link the subsarcolemmal cytosolic protein dystrophin to the extracellular matrix. This complex includes several subunits including sarcoglycans, dystroglycans, sarcospan, and syntrophins. Mutations to any of these proteins frequently lead to the limb girdle muscular dystrophies.
DMD Duchenne muscular dystrophy. The most common and severe muscular dystrophy, resulting from a lack of the protein dystrophin.
DWI Diffusion weighted imaging
ECM Extracellular matrix
FID Free induction decay
FOV Field of view
GAS Gastrocnemius muscle
gsg Gamma sarcoglycan. This is the mouse model of limb girdle muscular dystrophy, type 2C. Mice lacking gamma sarcoglycan (gsg-/-) demonstrate a severe phenotype of muscular dystrophy.
LGMD Limb girdle muscular dystrophy. This includes several forms of muscular dystrophy, identified by the dysfunctional protein of the dystrophin associated glycoprotein complex.
mdx Muscular dystrophy X-linked. This is the mouse model of Duchenne muscular dystrophy. The Dmd gene of the mouse has a premature stop codon in exon 23, resulting in an absence of the dystrophin protein.
MRI Magnetic resonance imaging
MRS Magnetic resonance spectroscopy
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NIR Near infrared
NMR Nuclear magnetic resonance
OI Optical Imaging
RF Radio frequency
SNR Signal to noise ratio
Sol Soleus muscle
STEAM Stimulated Echo acquisition mode
T1 Longitudinal relaxation rate constant
T2 Transverse relaxation rate constant
TA Tibialis anterior muscle
TE Echo time
TR Pulse repetition time
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
NEAR INFRARED OPTICAL IMAGING AND MAGNETIC RESONANCE CHARACTERIZATION OF DYSTROPHIC AND DAMAGED MUSCLE
By
Stephen Mark Chrzanowski
May 2016
Chair: Name Glenn A. Walter Co-chair: Barry J. Byrne Major: Medical Sciences – Physiology and Pharmacology The muscular dystrophies are a heterogeneous spectrum of neuromuscular
disorders that lead to rapid wasting of muscle and premature mortality. Duchenne
muscular dystrophy is the most common and one of the most devastating forms of
muscular dystrophy, leading to early loss of ambulation and death by the 3rd decade.
Current means to measure therapeutic efficacy for these diseases remain
inadequate, limited to invasive muscle biopsies and functional testing. Muscle biopsies
are inadequate because they are invasive, provide a limited sampling of this very
heterogeneous disease, and further damage already degenerative tissue. Functional
testing possesses inherent variables that remain difficult to control, such as subject
motivation and compliance. An ideal methodology of assessing therapeutic treatment must
be: highly sensitive and specific to biologic changes, inexpensive, non-invasive, minimally
exposing to harmful radiation, and comfortable for patients. Near infrared (NIR) optical
imaging (OI) and magnetic resonance imaging (MRI) and spectroscopy (MRS) may offer
potential as non-invasive modalities to quantitatively assess muscle pathology in acutely
injured and diseased muscle. Using an FDA approved near infrared fluorophore, we
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tested whether healthy and damaged muscle could be imaged and differentiated with
near NIR-OI, with confirmation provided by MRI, MRS, and histological assessment.
To assess acute muscle damage, healthy mice were cast immobilized in a
plantar flexed position for two weeks after which, mice were allowed to freely ambulate
and data were collected. Further, mdx and gsg mice were cross-sectionally compared to
age-matched unaffected mice. Next, data were collected from additional mdx mice that
were subjected to downhill treadmill running. The missing protein in gsg mice was
restored through an AAV treatment, and mice were imaged following therapy.
In the immobilization-reambulation model, damage was observed in the soleus
muscle of the immobilized leg by MRI-T2, 1H2O-T2, NIR-OI, and histologically compared
to the non-casted contralateral leg, demonstrating a peak of damage followed by
recovery. Both models of dystrophic mice demonstrated significant differences from
their control counterparts. AAV therapy in the gsg mice restored markers of muscle
damage back to baseline levels. This work supports NIR-OI as a feasible, cost effective,
non-invasive, longitudinal means to quantify muscle health.
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CHAPTER 1 MUSCLE AND THE MUSCULAR DYSTROPHIES
Skeletal Muscle
Skeletal muscle has many significant roles to the human body. Its primary role is
to provide force production, but it also plays key roles of normal maintenance of
metabolism. Unique among tissue types, skeletal muscle is impressively adaptive and
plastic, responding to anabolic, sarcopenic, and pathologic factors, allowing for
appropriate remodeling (Adams and McCue, 1998; Baldwin, 1996; Hood, 2001;
Janssen et al., 2002; Kandarian and Jackman, 2006; Kraemer et al., 2000; Roy et al.,
1991).
Growth and Repair of Skeletal Muscle
Skeletal muscle plays several roles, from providing both gross and fine motor
control to maintaining metabolic homeostasis. Importantly, muscle demonstrates
tremendous plasticity, able to either atrophy or hypertrophy, depending on the external
stimuli applied to the muscle (Hood, 2001; Kraemer et al., 2000; Lieber and Fridén,
2000; Roy et al., 1991). Weight bearing activities, even as simple as opposing gravity,
allow muscle to maintain their physiological integrity, whereas stark atrophy begins to
occur upon removal of resistance (Adams and McCue, 1998; Baldwin, 1996; Baldwin
and Haddad, 2001; Carlson et al., 1999; Dunn et al., 1999; Rittweger et al., 2005; Tesch
et al., 2004). Within days of injury, muscle has also demonstrated great capacity for
being able to regenerate itself (Ciciliot and Schiaffino, 2010; Lepper et al., 2011;
Pimorady-Esfahani et al., 1997; Tesch et al., 2004; Turner and Badylak, 2012).
Following damage to muscle, a cascade of cytokines and growth factors are released,
recruiting semi-pluripotent satellite cells and inflammatory cells to the injured muscle
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fibers (Tidball, 1995, 2005, 2011). Maintenance of muscle health remains critical to
homeostatic maintenance of overall health.
Satellite cells are the semi-pluripotent stem cells of muscle, capable of dividing
into new muscle cells as well as self-regenerating their own populations (Aziz et al.,
2012; Ferrari et al., 1998; Schultz, 1985; Schultz et al., 1985). Recruitment of satellite
cells allows for either fusion with already existing populations of existing damaged fibers
or with each other to form new fibers (Schultz et al., 1985). Mature muscle fibers exist
in a post-mitotic stage of development and do not divide further. Growth and repair, by
way of satellite cells, occurs by the introduction of new myonuclei to the already existing
myofibers (Tedesco et al., 2010). Satellite cells primarily reside between the
sarcolemma and the basal lamina, predominantly exist in a quiescent state in healthy
adult muscle (Aziz et al., 2012; Schultz, 1985). Because satellite cells have the unique
capability to create both new myonuclei and replenish their own population, when
satellite cell populations are depleted, muscle is unable to appropriately regenerate
itself (Boldrin et al., 2015; Fry et al., 2015). Satellite cells are biochemically
characterized by being positive for M-cadherin, Pax7, Myf5, and nCAM-1 (Péault et al.,
2007; Relaix et al., 2005). Upon fusion and activation with existing myofibers, embryonic
myosin is expressed, allowing researchers to identify growing and repairing myofibers
(DiMario et al., 1991; Murry et al., 1996). Further, the repaired fibers exhibit centrally
located nuclei, allowing for histological identification of fiber growth and repair.
Traditionally, the ‘exhaustion’ of satellite cells has been though to be through
mechanisms such as a loss of telomeres (Decary et al., 1997, 2000; Heslop et al., 2000;
Mouly et al., 2005; Renault et al., 2000; Sacco et al., 2010), but more recently, it has
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
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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
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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
collection two days later. The second subject cohort is composed of boys (ages 10-15)
with confirmed DMD. This group will undergo imaging (MRI, MRS, and optical imaging) at a
single time point to assess the status of their muscle without exercise testing. For this
second cohort, a lower age limit of 10 years was selected, since upper extremity
muscles are affected at a later age than lower extremity muscles, with hand weakness
starting at the age of 10 years (Jones H et al. 2003).
Exercise testing
The first study arm (Figure 4-5) consists of healthy subjects who will undergo
eccentric and concentric forearm contractions in opposite arms on an isokinetic
dynamometer (Biodex Corp., Shirley, NY). Eccentric exercises have been shown to cause
temporary reversible damage to muscle by T2, serum creatine kinase, and delayed onset of
soreness (DOMS), whereas concentric exercises are known to cause minimal muscle
damage (Foley et al., 1999; Sesto et al., 2008). Eccentric loading of the wrist musculature
is achieved through slowly performing 6 sets of 10 repetitions of 120% of the 1 concentric
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repetition maximum with 90 seconds between sets. This exercise protocol has previously
been shown to result in increases in various indices of acute muscle damage after 48
hours (Davies et al. 2011), including T2 (+64%), creatine kinase (CK) serum levels, and
rating of delayed onset muscle soreness (DOMS) (Cleary et al., 2002). In addition, a
parallel concentric protocol is performed by the contralateral forearm. Concentric
exercise is known to result in only minimal muscle damage (Clarkson and Hubal, 2002;
Clarkson et al., 1986). Therefore, the concentric protocol serves as a control for other
potential effects of exercise on the measurements. The order of testing and the arm that
performs each protocol is randomized.
Magnetic resonance imaging and spectroscopy
Prior to scheduled testing, subjects are asked to refrain from any unnecessary
vigorous physical activity, and DMD subjects are asked to use a wheelchair or equivalent
mobility device when travelling to avoid excessive walking. All human MRI are performed in
a 3T whole body magnet (Philips Achieva Quasar Dual 3T) in the McKnight Brain Institute
at the University of Florida. When appropriate, a parent or staff member accompany
subjects into the testing room, and subjects lay in a supine position without sedation. Fat
suppressed and non-suppressed T1 weighted images are acquired to quantify the muscle
contractile area (fat-free muscle cross sectional area [CSA]) and maximal cross-
sectional area (CSAmax) of the forearm muscles. CSA and CSAmax are then normalized
to body surface area (BSA) to account for growth and differences in body size. Additionally,
T2 weighted imaging is performed to calculate T2 relaxation times based on mono-
exponential decay curve fittings to examine the distribution of affected versus unaffected
tissue. The total affected tissue volume (percentage of pixels with T2 values > 2 standard
deviations above control values) are recorded.
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Indocyanine green enhanced near infrared optical imaging
In order to determine whether NIR optical imaging can detect damaged and
dystrophic muscle, subjects with and without DMD will undergo the same imaging
procedures as the unaffected subjects. Following the MR procedures, all subjects have
NIR optical images taken of their forearms. All optical images are be acquired on a CTLM
Model 1020 scanner (Imaging Diagnostics System Inc., Ft. Lauderdale, FL). Dynamic
image acquisition will occur both 5 minutes before and after intravenous injection of IC-
Green (0.5 mg/kg; IC-Green, Akorn Inc., Buffalo Grove, IL) to assess enhancement kinetics.
The targeted region will be the belly of the wrist flexor muscles (Hillman et al., 2001;
Miyakawa et al., 2009). Dually MR and NIR visible fidicial markers on the skin of the
forearm are used to register MRI to NIR optical images. Optical image signal
enhancement kinetics are determined on a pixel-by-pixel basis based on changes in
signal intensity in the reconstructed images. The rate of tissue enhancement as well as
the final enhancement level at 5 min are used to create a rate of enhancement, area
under the curve, and a delayed enhancement map.
Blood draws and questionnaire
Following MR and NIR optical image acquisitions, blood is intravenously drawn for
analysis of muscle damage markers, such as creatine kinase. Additionally, a
questionnaire to determine the subjective rating of pain intensity is administered. This
questionnaire includes a visual analog scale ranging from “0 = no pain” to “10 = most
pain imaginable.” The change in this reported pain level is used as a construct for
DOMS. CK activity is determined in duplicate 0.02-ml aliquots at 37°C by using
standard photometric techniques and a Sigma diagnostic test kit (CK-10, Sigma
Diagnostics, St. Louis, MO).
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Figure 4-1. Radiant efficiency reaches a steady state level between 30 minutes to 12
hours following ICG an intravenous injection.
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Figure 4-2. Fat suppressed T1 weighted image shows muscles of the lower leg in
subjects with and without DMD, with arrows pointing to the TA (solid) and Per (dashed), highlighting intramuscular differences.
DMD
Control
Proximal Middle Distal
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Figure 4-3. Schematic representation of slice selections along the length of the lower leg.
Proximal
Distal
Mid-Proximal
Middle
Mid-Distal
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Figure 4-4. The qualitative MRI grading scale used to assess pathology within DMD
muscle.
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Figure 4-5. Schematic study design of clinical study utilizing NIR to detect muscle
damage.
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CHAPTER 5 NEAR INFRARED OPTICAL IMAGING IN A PRE-CLINICAL MODEL OF ACUTE
MUSCLE DAMAGE
Introduction
Techniques to Assess Muscle Damage
Muscle damage is an important and unavoidable outcome of many pathological
states such as muscular dystrophies, inflammatory myopathies, and physical trauma.
Several pre-clinical models have been developed to induce acute muscle damage,
including eccentric loading (Armstrong et al., 1983; Clarkson and Hubal, 2002; Clarkson
et al., 1986; Proske and Morgan, 2001), immobilization-reloading (Frimel et al., 2005b),
and myotoxin injection (Gutiérrez and Ownby, 2003; Lomonte and Gutiérrez, 1989;
Lomonte et al., 1993, 2003). In particular, eccentric loading of muscle has
demonstrated ability to robustly disrupt sarcolemmal integrity in a well controlled
manner (Armstrong et al., 1983; Childers et al., 2002; Clarkson et al., 1986; Lovering
and De Deyne, 2004; Proske and Morgan, 2001). A compromised sarcolemma
releases muscle enzymes such as creatine kinase, while concurrently passively taking
up large serum proteins and markers such as Evan’s blue dye (Hamer et al., 2002) and
small inorganic dyes such as procion orange (Barton-Davis et al., 1999; Greelish et al.,
1999; Nguyen and Tidball, 2003; Palacio et al., 2002; Spencer and Mellgren, 2002;
Tidball and Wehling-Henricks, 2007; Villalta et al., 2011) into damaged muscle.
Muscle pathology has been measured by a number of techniques, all of which
possess inherent limitations. These include including muscle biopsy, serology,
functional measures, and imaging methods. Muscle biopsy, while the most direct
measure of pathology, has limited capacity to be considered a longitudinal measure of
muscle pathology in clinical trials, due to the necessity of repeated sample collections.
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Serology and functional testing, while providing a proxy to the overall state of muscle
health, fail to sensitively localize pathology, instead providing information regarding the
general health of all muscles in the body and are complicated by changes in lean body
mass typically associated with myopathy. MRI has evolved as a noninvasive method to
detect and quantify muscle pathology (Dunn and Zaim-Wadghiri, 1999; Frimel et al.,
2005a, 2005b; Kobayashi et al., 2008; Vohra et al., 2015; Walter et al., 2005), but has
several limitations, such as cost, speed of operations, contraindications for patients with
metallic implants, claustrophobia, and compliance issues (Brockmann et al., 2007; Dunn
and Zaim-Wadghiri, 1999; Lovering and De Deyne, 2004). An attractive alternative
would be the use of clinically approved fluorescent optical contrast agents to image
muscle damage in vivo similar to those currently used for traditional histological
measurements (Baudy et al., 2011; Inage et al., 2015; Kossodo et al., 2010).
Near Infrared Imaging and Indocyanine Green
Fluorescent optical imaging is a widely used technique in pre-clinical models of
disease to detect pathology by fluorescent dyes, proteins, and conjugates (Frangioni,
2003; Tan and Jiang, 2008). By utilizing optical imaging in the NIR range (700-1,000
nm), two primary advantages exist over traditional fluorophores that operate at shorter
wavelengths: deeper photon penetration within tissues and minimal tissue
autofluorescence (Frangioni, 2003; Weissleder, 2001; Weissleder and Ntziachristos,
2003). When imaging in the NIR range, penetration of signal can reach up to 30-40 cm
of tissue depth, overcoming some of the scattering limitations that other fluorescent
imaging techniques at smaller wavelengths encounter, overcoming the limitation of only
being able to image superficial surface structures (Ntziachristos et al., 2002, 2005).
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The first, and still only FDA approved NIR fluorescent contrast agent is ICG, a
775 Dalton di-sulfonated fluorescent dye with a very well characterized safety profile,
demonstrating minimal toxicity in humans (Alford et al., 2009). ICG is rapidly bound to
albumin within the circulation, thus acts as a blood pooling NIR fluorescent agent,
highlighting vasculature (Chen et al., 1999; Desmettre et al., 2000; Kobayashi et al.,
2014; Raabe et al., 2003). Additionally, ICG passively accumulates in tumors through
the enhanced permeation and retention (EPR) effect in a similar manner to gadolinium,
as used for contrast enhanced MRI (Corlu et al., 2007; Ntziachristos et al., 2000). The
EPR effect is a phenomena by which certain molecules preferentially are uptaken into
surrounding tissue. This is most frequently due to pores and fenestrations in the target
tissue or vascular endothelium supplying such tissue, as often observed during
inflammatory states and cancer (Fang et al., 2011; Maeda, 2012; Maeda et al., 2000;
Radermacher et al., 2009).
ICG enhanced NIR optical imaging has been used for several other clinical
purposes, such as imaging of the vasculature of the retina (Chen et al., 1999;
Desmettre et al., 2000; Herbort et al., 1998; Mueller et al., 2002), breast cancer tumors
(Gurfinkel et al., 2000; Ntziachristos et al., 2000; Troyan et al., 2009; Verbeek et al.,
2014; Zelken and Tufaro, 2015), cerebral vasculature and tumors (Haglund et al., 1996;
Raabe et al., 2003), gastrointestinal vessels (Borotto et al., 1999), and cardiac
vasculature and myocardial perfusion (Nakayama et al., 2002; Taggart et al., 2003).
Despite its widespread use in other organs, there is only one recent occurrence in the
literature as method to image muscle pathology is a recent development (Inage et al.,
2015). With this in mind, we hypothesized that ICG will behave similar to EBD (Hamer
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et al., 2002), and accumulate in damaged muscle fibers, allowing for quantification of
muscle damage in a longitudinal and in vivo manner. Importantly, because there are no
FDA approved MRI blood pooling imaging contrast agents approved for use in pediatric
studies, ICG could fulfill an important role in children as a NIR optical imaging contrast
agent.
Results
Animal Procedures
Throughout the duration of experiments, all mice maintained body weight within
10% of pre-immobilization weights and three needed recasting because of abrasive
lesions developed on the skin. In these rare cases, topical antibiotics were applied to
address abrasive lesions, all with resolution. One mouse unexpectedly expired
following data collection in the MR scanner and was not used for data analysis. The
single hindlimb casting procedures were otherwise well tolerated through the duration of
experiments.
Near Infrared Imaging of Mouse Hindlimbs
When comparing pre-immobilization and day 0 reambulated hindlimbs, no
difference was observed between immobilized and non-immobilized hindlimbs.
Throughout the reambulation phase, radiant efficiency in the immobilized-reambulated
hindlimb significantly peaked by day 2 and was 3.86 fold higher than pre-casted values,
followed by a return back to baseline by day 7 (Figure 5-1). Interestingly, the
contralateral hindlimb also demonstrated an increase (2.45 fold) in total radiant
efficiency between day 2 reambulation and pre-casted values, but did not reach
significance. NIR images are also presented in the left panel of Figure 5-1, allowing for
qualitative demonstration of the immobilized (right) vs. non-immobilized (left) hindlimbs.
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The immobilized-reambulated hindlimbs at days 2 and 3 of reambulation were
significantly different than contralateral hindlimbs, pre-immobilized hindlimbs, and day 0
of the immobilized-reambulated limbs.
Magnetic Resonance Imaging and Spectroscopy Confirm Muscle Damage Following Cast Immobilization and Reambulation
Pre-immobilization hindlimbs demonstrated homogenous contrast in the all their
muscles on T2 weighted MRIs in both hindlimbs (Figure 5-2). The soleus muscle of the
immobilized-reambulated hindlimb demonstrated the greatest T2 changes, peaking at
two days following the cast removal (1.41 fold change increase in T2), and returning
values comparable to baseline by day 5 of the reambulation phase (Figure 5-2A).
Interestingly, both the gastrocnemii and tibialis anterior muscles of the immobilized-
reambulated hindlimbs demonstrated a subtle, yet significant difference (1.13 and 1.14
fold changes, respectively) from their contralateral non-immobilized limbs at the
initiation of reambulation, but this was not significant from pre-immobilization measures
(Figure 5-2B and 5-2C).
Further confirmation of the damage and recovery of the soleus muscle is
provided by 1H2O-T2 spectroscopic analysis (Figure 5-3). Representative mono-
exponential T2 curves demonstrate differences in decay rates of 1H2O-T2 signal between
the immobilized-reambulated and control hindlimbs (Figure 5-3A). Following multiple
exponential decomposition by non-negative least squares (NNLS) analysis (Bryant et
al., 2014), a representative characteristic long T2 component is shown in Figure 5-3B.
Similar to MRI-T2 measures, 1H2O-T2 values demonstrated a similar trend of damage
peaking at the second day of reambulation with a 1.28 fold change compared to pre-
casted values, followed by an eventual return to baseline (Figure 5-3C). Within the
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entire cohort of mice, long T2 components (T2 > 80ms) were present in half of mice at
the peak of muscle damage (day 2) during reambulation (Table 5-1). The occurrence of
long T2 components in both hindlimbs is summarized in Table 5-1. The only
demonstration of a long T2 component in the non-immobilized limbs occurred in 10% of
mice at the second day of reambulation.
Histology of Healthy and Damaged Muscle
Appearance of EBD accumulation at both the microscopic and macroscopic
levels within the soleus muscles confirmed the well-established time course of damage
and recovery during reloading following immobilization. Figure 5-4A qualitatively
demonstrates that EBD is minimally taken up into soleus muscle fibers immediately
following cast removal. Quantitative demonstration of dye uptake into the immobilized-
reambulated and contralateral control solei are demonstrated in Figure 5-4B. The
percentage of EBD positive fibers in the Gas and TA was not significantly elevated and
was comparable to baseline values throughout the reambulation period (data not
shown). By the second day of reambulation, fibers of the immobilized-reambulated
soleus appeared with 47.1 ± 15.6% of the fibers being EBD positive in a checkerboard
patter. EBD uptake into the contralateral soleus was less with 15.1 ± 6.3% of the being
EBD positive, but this was not significantly different than day 0 of soleus muscle the
same control limb. At the end of the reambulation week, EBD signal is again less
visible with only 5.6 ± 2% and 1.6 ± 1.8% of the fibers being EBD positive for the
immobilized-reambulated and control hindlimbs, respectively.
H&E stained sections of the immobilized and reambulated soleus at various time
points throughout the week demonstrated the well characterized histopathological
features of muscle damage and inflammation, most noticeably at the second day of
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reambulation (Frimel et al., 2005b). At the peak of damage (day 2), widened extra
cellular spaces, massive macrophage infiltration, a decreased density of muscle fibers,
and variability in fiber size were all observed. A final observation made was that
throughout the week of reambulation, there was an absence of centrally nucleated
myofibers, as centrally nucleated fibers do not typically occur until greater than ten days
following insult to muscle (Frimel et al., 2005b).
Spectrophotometric Quantification of ICG and EBD
Muscle lysates were analyzed by a spectrophotometer to quantify both EBD and
ICG accumulation within each lower leg muscle throughout the week of reambulation
(Figure 5-5). Absorbance, normalized to muscle weight, was determined at 620 nm
(EBD; Figure 5-5A) and 780 nm (ICG ; Figure 5-5B) and demonstrated significant peaks
in signal at the second day of reambulation for both dyes (EBD: 1.72 fold change and
ICG: 1.87 fold change), followed by a return back to baseline by the end of the week of
reambulation (EBD: 1.21 fold change and ICG: 1.20 fold change). Absorbance of the
gastrocnemii and tibialis anterior lysates were comparable to background noise levels
(data not shown), indicating minimal dye uptake per tissue weight into these two muscle
groups.
Correlation Between MRI and Near Infrared Optical Imaging
To determine if a correlation exists between muscle damage measures and total
radiant efficiency, 1H2O-T2, MRI-T2, and spectrophotometric absorbance values were
compared to total radiant efficiency at the peak of muscle damage. Figure 5-6 shows
the linear relationships between radiant efficiency and 1H2O-T2 (5-6A, r2 = 0.72) and
MRI-T2 (5-6B, r2 = 0.57) in the immobilized-reambulated and control muscles. Table 5-2
quantitatively shows the significance of linear regression correlations between NIR
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optical imaging radiant efficiency compared to 1H2O-T2, MRI-T2, and optical density 780
nm / mg tissue. Significant correlations (Table 5-2) are demonstrated only when
comparing the solei measures, and neither the gastrocnemii, nor tibialis anterior
muscles demonstrate any significant correlations to the total radiant efficiency.
Spectroscopy was not performed in the gastrocnemii or tibialis anterior, so correlations
could not be drawn for spectroscopy from these muscles. Finally, in order to determine
the robustness of each imaging modality, Cohen’s D effect sizes were calculated to
determine the magnitude of difference between day 0 and day 2 of the immobilized–
reambulated hindlimbs. The effect sizes for MRI-T2, 1H2O-T2, and NIR optical imaging
were 1.79, 1.39, and 1.57, suggesting comparable magnitudes of differences between
each of the imaging modalities.
Discussion
The main purpose of this study was to assess the ability of an FDA approved NIR
fluorescent contrast agent (ICG) and NIR optical imaging to noninvasively image muscle
in a well-characterized model of acute muscle damage and recovery. Muscle damage
and recovery in the soleus muscle of immobilized-reambulated mouse hindlimbs was
visualized and quantified using ICG enhanced NIR optical imaging, with further
supporting confirmation provided by MRI-T2, 1H2O-T2, histology, and spectrophotometric
assessments. The time course of muscle damage and recovery following immobilization
and free reambulation using both imaging modalities agreed with histological and
biochemical analysis of the extracted tissues. This study demonstrates the ability of
ICG enhanced NIR optical imaging to two dimensionally visualize and quantify muscle
damage in vivo.
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Near Infrared Optical Imaging as a Novel Method to Assess Muscle Damage
By utilizing a NIR optical imaging enhanced with an FDA approved contrast
agent (ICG) to assess muscle health, it is anticipated that this foundational work will be
able to quickly translate to clinical application. We chose to use ICG as a NIR
fluorescent blood-pooling agent because ICG behaves similarly to Evan’s blue dye
(Hamer et al., 2002) in that it binds to serum albumin and it was hypothesized that ICG
would accumulate in damaged muscle cells with compromised sarcolemmal
membranes. We exploited that optical imaging in the NIR range allows for deep tissue
imaging and minimal tissue autofluorescence, allowing for imaging of deep muscle
(Frangioni, 2003). Additionally, NIR optical imaging has the advantage that it can
compliment current MR techniques (Ntziachristos et al., 2000), with the additional
advantage that data acquisition can be achieved in a much more cost efficient manner
and shorter time. While NIR optical imaging of ICG has been demonstrated clinically in
other tissues of the body, the primary use of NIR optics has been to perform NIR
spectroscopic (NIRS) analyses to assess changes in perfusion status has been
performed in muscle, though the ability to image muscle damage in a rat model using
an ICG conjugate has been demonstrated (Inage et al., 2015).
In this study, we sought to quantify and visualize muscle damage through using
an established model of hindlimb immobilization followed by reambulation (Frimel et al.,
2005b). This model of hindlimb immobilization was chosen to test the ability of NIR
optical imaging to detect and quantify muscle damage for this study as the technique
demonstrates specific damage to the soleus, in the deepest of the lower hindlimb
muscles (3.54 ± 0.43 mm below the surface of the posterior skin). Furthermore, the
time course of muscle damage and recover is well characterized, and the uncasted,
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contralateral hindlimb allows for an internal control for each mouse (Frimel et al.,
2005b). NIR optical images were used to visualize muscle damage and was confirmed
by MRI-T2, mean 1H2O-T2, histology, and ICG/EBD accumulation in isolated muscles.
Importantly, a return to baseline levels of all measures of muscle damage was
observed, indicating that NIR optical imaging is can be used to image both damage and
recovery of muscle. By comparing the effect sizes of MRI-T2 (d = 1.79), 1H2O-T2 (d =
1.39) and NIR optical imaging (d = 1.57), we determined that NIR imaging was similar in
its ability to detecting muscle damage as MRI. Additionally, we found a significant linear
relationship between NIR optical imaging and 1H2O-T2 and MRI-T2, further solidifying
confirming NIR optical imaging as a capable non-invasive modality to detect muscle
damage. Even though MRI is frequently used in pediatric populations, no MRI blood
pooling contrast agents are currently clinically indicated for imaging muscle damage.
ICG, with long standing FDA approval and a safety record, could adequately fulfill this
void in the clinic arena in conjunction with NIR optical imaging (Frangioni, 2003).
From of the breadth of information that can be revealed through MR
spectroscopic analysis of muscle, we utilized this to attempt to better understand the
generation of fluorescent signal from ICG within the damaged soleus muscle (Araujo et
al., 2014; Bryant et al., 2014; Fan and Does, 2008; Frimel et al., 2005b; Hollingsworth,
2014; Walter et al., 2005). Because of significant correlations between NIR optical
imaging measures to MRI-T2, 1H2O-T2, and tissue accumulation of EBD/ICG in the
soleus, it can be hypothesized that the NIR optical imaging is pre-dominantly due to
induced pathology within the soleus rather than either the gastrocnemius or tibialis
anterior. We chose to investigate the multi-component decay of 1H2O-T2 signals,
129
allowing for differentiation between intracellular (20-40 ms), extracellular (80-120 ms),
and protein associated (<10ms) 1H2O-T2 contributions, in an attempt to elucidate what
fluid compartment ICG may end up associated with (Ababneh et al., 2005; Araujo et al.,
2014; Bryant et al., 2014; Gambarota et al., 2001). Demonstration of a long 1H2O-T2
component has been observed during edematous and inflammatory states within
muscle, indicating a large contribution of extracellular fluid within the muscle (Bryant et
al., 2014; Fan and Does, 2008). Interestingly, at the second day of reambulation
following immobilization, the long 1H2O-T2 component was present in half of the
immobilized hindlimbs. This concurrently occurred with an increased NIR optical
imaging signal, suggesting that immobilization followed by reambulation induces muscle
edema, allowing for pooling of ICG in the damaged muscles. Histological results were
consistent with previously reported data, as we observed expanded interstitial space
and infiltrating cells in only the immobilized-reambulated soleus (Bryant et al., 2014;
Frimel et al., 2005b). Lastly, biochemical analysis of tissue EBD and ICG accumulation
at the peak of muscle damage confirmed that soleus ICG content was 12.8 and 5.2 fold
higher than the gastrocnemius and tibialis anterior, respectively. The disproportionate
uptake of ICG into the soleus indicates that the immobilized-reambulated soleus
(Figures 5-5B and 5-6B) most likely is the primary contributor to fluorescent signal as
seen in Figure 5-1.
It is important to consider that MR and NIR optical imaging assess different
properties of tissue, with MR assessing inherent magnetic properties of tissue and NIR
optical imaging assessing vascular perfusion and membrane stability. NIR optical
images taken within minutes after injection (Figure 4-1) show changes in major
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vasculature and may significantly alter the NIR signal, as previously shown in muscle
(Mancini et al., 1994; Možina, 2011). By waiting an hour after injections, we ensured
that the fluorescent signal observed was predominately from muscle uptake rather than
vascular contributions. As previously described, contralateral non-immobilized limbs
experience an overload of stimuli, which may explain the insignificant, but observable
increase in total radiant efficiency observed at day two of the non-immobilized hindlimb
in Figure 5-1 (Caron et al., 2009). For these reasons, it is suggested that NIR optical
imaging complement, rather supplement MR technology, providing additional
information in a cost and time efficient manner.
Limitations to Experiments
ICG, as a non-targeted contrast agent demonstrates both advantages and
disadvantages in this study. An advantage is that it can be used to quantitatively
assess muscle damage and recovery. Because it is not specific to the pathology
induced by the immobilization-reambulation technique used in this manuscript, it can
theoretically be applied to other pathologies and diseases affecting muscle. Though
ICG-albumin uptake is nonspecific, with future modifications, it may provide a platform
for targeting specific cell moieties to add further diagnostic and therapeutic value (Kraft
and Ho, 2014; Sheng et al., 2014). Another limitation is the lack of spatial sensitivity
while using contrast enhanced NIR optical imaging. Though NIR optical imaging was
deemed comparably sensitive to MR techniques to detect muscle damage by
magnitude of effect size assessment, additional technology development should be
pursued to increase spatial sensitivity of the technology. Due to the limit of TE sampling
in the STEAM MRS acquisitions, it is quite possible that we were only sensitive to large
differences in 1H2O-T2 fractions and potentially, with greater TE sampling and signal-to-
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noise, a long component may have been easier to resolve in damaged muscle (Bryant
et al., 2014).
Summary
I demonstrated the feasibility of using a novel technology (NIR optical imaging)
with an FDA approved fluorescent contrast dye (ICG) to tomographically assess and
image acute muscle damage and recovery in a well-characterized model of muscle
damage in mice. I have also optimized each of the imaging modalities (MRI, MRS, and
NIR Imaging) to quantify and visualize the muscle damage. Because of the cost
effectiveness, lack of ionizing radiation or radioactive substrates and longitudinal
capabilities, NIR optical imaging can be used for a diverse range of purposes (Baudy et
al., 2011; Inage et al., 2015; Možina, 2011; van de Ven et al., 2010; Verbeek et al.,
2014). By using a clinically approved contrast dye with NIR optical imaging, a
multipurpose, non-invasive, and safe imaging technology, it is anticipated that this
technology can be expeditiously applied to other diseases of the muscle, both in
animals and humans.
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Figure 5-1. Two-dimensional NIR optical imaging shows an increase and recovery of
fluorescent signal in muscle during reambulation following immobilization.
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Figure 5-2. MRI-T2 shows damage and recovery of soleus, but not gastrocnemius nor
tibialis anterior muscles during reambulation. Representative MR images are shown along the left panel for each of the days of reambulation. Soleus (5-2A), gastrocnemius (5-2B), and tibialis anterior (5-2C) MRI-T2 relaxation times are shown before immobilization and throughout the week of reambulation.
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Figure 5-3. Spectroscopic findings confirm increase in 1H2O-T2 and reveal long T2
components in the soleus of immobilized-reambulated hindlimbs. Characteristic mono-exponential T2 decay curves are shown (5-3A), as well as a representative characteristic long T2 component (5-3B). 1H2O-T2 relaxation times before immobilization and the week of reambulation are shown (5-3C).
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Table 5-1. Frequency of long 1H2O-T2 component in damaged hindlimbs of immobilized-reambulated mice.
Reambulation Day IMM (% with long component)
Non-IMM (% with long component)
Pre-immobilized 0 0
0 0 20
1 0 20
2 10 50
3 0 30
5 0 20
7 0 0
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Figure 5-4. Histological assessment confirms damage and recovery in the reambulated
soleus muscle of the immobilized-reambulated hindlimb at the second day of reambulation. Representative immunofluorescence and H&E images are shown (5-4A) as well as quantification of EBD positive fibers throughout the week of reambulation (5-4B).
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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.
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Table 5-2. NIR optical imaging radiant efficiency measures were correlated to 1H2O-T2, MRI-T2, and Optical Density 780 nm / mg tissue, and r2 values (with associated p values in parentheses).
1H2O-T2 MRI-T2 Absorbance 780 nm / mg
tissue
So
l
0.72 (<0.001) 0.57 (<0.001) 0.46 (0.002)
Ga
s
N/A 0.21 (0.051) 0.001 (0.156)
TA
N/A 0.12 (0.167) <0.001 (0.999)
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CHAPTER 6 QUANTIFICATION OF MUSCLE PATHOLOGY IN MDX AND GSG -/- MICE
Introduction
Muscular Dystrophies Render Muscle More Susceptible to Damage
Phenotypically, the muscular dystrophies are defined a common clinical
presentation of progressive, degenerative, and irreversible muscle weakness (Amato
and Griggs, 2011; Flanigan, 2012). With the advent of modern sequencing
technologies, over 50 genetically identifiable forms of muscular dystrophy have been
identified, based on the genetic mutation causing pathology to the muscle (Amato and
Griggs, 2011; Kang PB and Griggs RC, 2015). The most common of the muscular
dystrophies is DMD, with an incidence of 1 in 5,000 live male births (Greenberg et al.,
1988; Mah et al., 2014; Mendell et al., 2012). DMD is caused by a mutation in the
dystrophin gene, which encodes for the dystrophin protein. Another form of muscular
dystrophy is LGMD-2C, which results from mutations in the SGCG gene, which encodes
for production of the γ-sarcoglycan protein. Dystrophin connects the intracellular
contractile actin to the DAG complex, stabilizing the sarcolemmal membrane during
muscle contractions (Ervasti and Campbell, 1991; Hoffman et al., 1987). γ-sarcoglycan
is one of several sarcolemmal transmembrane glycoproteins that forms the DAG
complex, and when absent, leads to increased susceptibility to injury in the
sarcolemma, as seen in LGMD-2C (Amato and Griggs, 2011; Barton, 2006).
Mdx and Gsg -/- Mouse Models
Currently, no cures for the muscular dystrophies exist, though many promising
therapies have shown promise in preclinical and early clinical trials. Many therapies
have been developed through extensive use of protein knockout mice, which, similar to
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their human counterparts, lack proteins specific to their disease. Two commonly
studied dystrophic mouse models are the DMD (mdx) and LGMD-2C (gsg -/-) null mouse
strains (Hack et al., 1998; Hoffman et al., 1987; McNally et al., 1996a, 1996b; Sicinski et
al., 1989). These models have been effectively used to study the natural progression of
both diseases, as well as develop a variety of therapies to mitigate pathologic insult
from the diseases, such as pharmacological interventions (Anderson et al., 1996;
Barton et al., 2005; Durham et al., 2006), exon skipping (Echigoya et al., 2015;
Goyenvalle et al., 2015; Matsuo et al., 1991), viral delivery (Barton, 2010; Hayashita-
Kinoh et al., 2015), and RNA restoring therapies (Barton-Davis et al., 1999; Welch et al.,
2007). Interestingly, mdx exhibit a characteristic progression of disease, initially
experiences a large assault of inflammatory cascades, followed by a plateau of recovery
due to their ability to upregulate utrophin, a homolog of dystrophin (McDonald et al.,
2015; Vohra et al., 2015). The gsg -/- mice demonstrate a more severe phenotype,
demonstrating decreased growth, premature death, and severely dystrophic muscle as
the mice age (Hack et al., 1998). The ability to sensitively demonstrate mitigation of
disease, or worsening of pathology is a critical role of biomarkers. Therefore, a pressing
need exists for sensitive biomarkers to detect changes in disease progression,
additional pathologic insult in dystrophic muscle, and response to potential therapies in
the muscular dystrophies, both in preclinical and clinical models.
Techniques to Assess Muscle Damage Due to Dystrophies
Assessment of the ability to quantify inducible damage and therapeutic
intervention in dystrophic muscle is limited to several modalities, including histological
markers of muscle damage such as Evan’s Blue Dye (Frimel et al., 2005b; Hamer et al.,
2002) and Procion orange dye (Consolino and Brooks, 2004) and ex vivo muscle
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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
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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.
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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.
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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.
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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
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.
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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.
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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).
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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).
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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
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through rAAV therapy, but this study is not without limitations. First, ICG is a non-
specific contrast agent, which has both advantages and disadvantages. It may be
advantageous to use because it can ubiquitously be applied for several different
applications within pathologies that affect muscle. For the same reasons, this may be
viewed as a disadvantage, as it is taken up non-specifically anywhere where there may
be a compromised membrane. Another advantage of ICG is that it is an FDA approved
contrast agent, allowed to be used in pediatric populations, to which there are none
currently available for use in MR studies. Although NIR optical imaging was deemed to
be comparable to MRI and MRS by effect size measurements, the development of
technology to provide better spatial resolution (i.e., assessment in different planes)
would provide much benefit to NIR imaging. Future studies warrant longitudinal
investigations of muscular dystrophies and using other disease modifying agents to
determine if NIR optical imaging can similarly detect amelioration of disease both in pre-
clinical and clinical models.
Summary
In summary, we demonstrate the utilization of NIR optical imaging with an FDA
approved NIR fluorophore, ICG, to spatially assess and quantify pathology resulting
from two different muscular dystrophies in mice, worsening of muscle pathology through
eccentric loading, as well as correction of disease through an AAV restorative therapy.
By its comparable demonstration of disease detection to MRI and MRS, NIR optical
imaging serve as a multipurpose, non-invasive, safe imaging technology that can be
applied to other disorders of muscle in both animals and humans, available for rapid
translation in clinical trials.
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Figure 6-1. Dystrophy induced muscle pathology can be detected by NIR optical
imaging, MRI, and MRS. NIR optical imaging quantification (6-1A) is shown between healthy control (6-1B), mdx (6-1C), and gsg -/- (6-1D) mice. Additionally, differences by MRI-T2 (6-1E) and 1H2O- T2 (6-1F) are shown with representative healthy control (6-1G), mdx (6-1H), and gsg -/- (6-1I) mice.
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Figure 6-2. Increased radiant efficiency correlates with increased magnetic resonance
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Figure 6-3. NIR optical imaging, MRI, and MRS confirm increased damage to muscle
following treadmill exercising in mdx mice. Quantitative differences for mouse forelimbs and hindlimbs before and after treadmill running are shown by way of NIR optical imaging (6-3A), MRI-T2 (6-3B) and 1H2O- T2 (6-3C). Representative NIR optical images (6-3D) and MR images (6-3E) are also shown.
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Figure 6-4. Increased total radiant efficiency correlates with increased magnetic
resonance measures before and after damage induced by treadmill running. Correlations comparing radiant efficiency to MRI-T2 (6-4A) and 1H2O-T2 (6-4B) are shown.
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Figure 6-5. gsg -/- mice treated with AAV demonstrate decreased near infrared
fluorescence and lower MRI-T2 and 1H2O-T2 relaxation times following treatment. NIR optical imaging was quantitatively assessed for the hindlimbs (6-5A) and forelimbs (6-5B). Representative NIR optical images are shown for baseline control gsg -/- (6-5C), to-be-treated gsg -/- mice (6-5D) as well as control gsg -/- (6-5E) and AAV treated gsg -/- (6-5F) mice. MRI-T2 and 1H2O-T2 data were quantified for both cohorts before and 6 weeks after intervention. In a parallel fashion to 6-5C-F, MR images of baseline control gsg -/- (6-5I), to-be-treated gsg -/- mice (6-5J) as well as control gsg -/- (6-5K) and AAV treated gsg -/- (6-5L) mice. Note that the dashed gray box indicated in figures 6-5A, 6-5B, 6-5G, and 6-5H indicate the 95th percentile for control values of each of the measures.
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Figure 6-6. Increased total radiant efficiency correlates with increased magnetic
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Table 6-1. Effect size magnitude demonstrates comparable differences between NIR optical imaging and MR measures.
NIR optical imaging MRI-T2 1H2O-T2
Control vs. mdx 2.57 3.63 2.93
Control vs. gsg -/- 4.14 3.15 1.89
Treadmill induced damage 1.88 0.73 1.96
AAV delivery of γ-sarcoglycan 3.30 1.51 1.59
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Figure 6-7. Representative immunofluorescence images with and without AAV delivery
of γ-sarcoglycan. Shown are stains for γ-sarcoglycan, wheat germ agglutinin, DAPI, and combined images.
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CHAPTER 7 NIR OPTICAL IMAGING CAN DETECT CHANGES IN MAJOR VASCULATURE
Introduction
One area that has received increased attention in the muscular dystrophy world
are the vascular defects of dystrophic muscle (Thomas, 2013). Prior to the discovery of
dystrophin, it was noted that small random groups of muscle fibers appeared to be
necrotic, and it was proposed that this focal insult was due to pathologies in local
microvasculature (Cazzato and Walton, 1968; Engel, 1967). This theory was supported
through early experiments, that caused localized ischemia in muscle (Hathaway et al.,
1970; Mendell et al., 1971). Support for this theory diminished as dystrophic muscle
microvasculature was found to be comparable to control muscle (Jerusalem et al., 1974;
Leinonen et al., 1979; Musch et al., 1975). Upon discovery of the co-localization of
neuronal nitric oxide synthase µ (nNOSµ) and dystrophin to the subsarcolemmal
surface, studies investigating the importance of vascular defects in dystrophic muscle
were reinvigorated (Brenman et al., 1995; Chang et al., 1996; Lai et al., 2009). nNOSµ
produces nitric oxide (NO), which acts as a local paracrine vasodilatory signal (Nathan,
1992). When dystrophin is deficient in dystrophic muscle, nNOSµ concurrently is
mislocalized, leading to a state of functional ischemia in dystrophic muscle. Through
restoration of nNOSµ through a dystrophin mini-gene that contains the nNOSµ binding
sites, exercise tolerance was improved (Lai et al., 2009; Zhang et al., 2013). Similarly,
when treated with phosphodiesterase-5 inhibitors, which mitigate the degradation of
NO, and allow vasodilation to occur, damage to dystrophic muscle is lessened
(Kobayashi et al., 2008).
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These discoveries support the importance of appropriate vasculature
maintenance in dystrophic muscle. Several mechanisms exist to measure changes in
blood flow, such as ultrasound and near infrared spectrometry (Ahmad et al., 2011). For
our interests, we study ICG enhanced NIR optical imaging of vasculature. Because ICG
rapidly binds to albumin, it provides contrast of vascular compartments. It is rapidly
cleared out of circulation via the liver, and passively taken up into cells with
compromised membranes, as we demonstrated occurs in muscle in previous sections.
Previously, contrast enhanced NIR optical imaging has been used to asses
vasculature in a variety of settings, including imaging of the vasculature of the retina
(Chen et al., 1999; Desmettre et al., 2000; Herbort et al., 1998; Mueller et al., 2002),
breast cancer tumors (Gurfinkel et al., 2000; Ntziachristos et al., 2000; Troyan et al.,
2009; Verbeek et al., 2014; Zelken and Tufaro, 2015), cerebral vasculature and tumors
(Haglund et al., 1996; Raabe et al., 2003), gastrointestinal vessels (Borotto et al., 1999),
and cardiac vasculature and myocardial perfusion (Nakayama et al., 2002; Taggart et
al., 2003). As it has been used to image vasculature in a number of different tissues,
we hoped to expand on this knowledge by imaging vasculature in the leg. It was our
intention to demonstrate contrast enhanced NIR optical imaging to visualize, and
quantitate changes in blood flow in muscle. To first demonstrate proof of principle, we
chose a robust model of perturbations of blood flow, assessing if we can detect and
quantify the inducible hyperemic response in hindlimb of mice (Joannides et al., 1995).
In experiments assessing damage to muscle, all NIR optical imaging data was collected
greater than 1 hour following ICG injection, but we hypothesize that by imaging ICG
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enhancement immediately following delivery, we will be able to measure vascular flow
and muscle perfusion.
Results
Proof of principle experiments demonstrated the capability to image the
vasculature of the lower hindlimb of mice. The major lower hindlimb’s major vasculature
was able to be quantitatively visualized in a time dependent manner, and distinguished
from the surrounding muscle (Figure 7-1A). Differences between the femoral artery and
surrounding muscle are able to be quantified (Figure 7-1A) and visualized at several
representative timepoints 1 second, 15 seconds, and 180 seconds after ICG injection
(Figures 7-1B, 7-1C, and 7-1D). Immediately following injections, fluorescence from ICG
rapidly peaked as ICG disseminated throughout the vasculature of the body.
Fluorescence precipitously declined at several timepoints (Figures 7-1B to 7-1D)
following injection, and quickly fluorescence of muscle and vasculature became similar.
Following removal of a blood pressure cuff, a characteristic hyperemic response
was observed by ICG enhanced NIR optical imaging (Figure 7-2). Following removal of
a blood pressure cuff, a hyperemic response can be observed in the cuff-and-released
limb as compared to the contralateral control hindlimb (Figure 7-2A). Representative
images 300s (Figure 7-2B) and 2000s after injection (Figure 7-2C) are shown. When
comparing the control limb that did not undergo blood pressure cuff occlusion to the
variable experimental limb, similar initial fluorescence values were observed in both
hindlimbs, but the cuff-and-release limb maintained higher fluorescence longer than the
contralateral control limb (Figure 7-2).
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Discussion and Summary
Those not yet fully completed, these preliminary experiments present proof of
principle findings that ICG enhanced NIR optical imaging can quantitatively image
normal and modified vascular flow within muscles in a preclinical model. Differentiation
between surrounding muscle, and the primary vasculature of the lower leg are
distinguished (Figure 7-1). Further, a characteristic hyperemic response is observed,
with greater blood flow in the cuff-and-release hindlimb as compared to contralateral
control limbs (Figure 7-2). Recently, contrast enhanced NIR optical imaging recently has
developed as an effective way for several clinical applications, such as perfusion
studies (Desmettre et al., 2000; Kobayashi et al., 2014; Raabe et al., 2003) and tumor
identification (Corlu et al., 2007; Ntziachristos et al., 2000; Zelken and Tufaro, 2015).
These experiments lay the foundational work necessary to develop further experiments
in studying the vasculature, and changes to vasculature in both healthy and dystrophic
muscle.
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Figure 7-1. Differences between major vasculature and surrounding muscle are able to
be spatially and temporally identified. Quantitation of muscle and vessel fluorescent intensity is quantified (7-1A). Further, representative images from 1 second, 15 seconds, and 180 seconds after ICG injection are shown (7-1B, 7-1C, and 7-1D, representatively).
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Figure 7-2. A hyperemic response is able to be quantified through NIR optical imaging.
Quantitation of the hyperemic response observed is presented (7-2A), as are representative NIR images 300 and 2000 seconds after cuff release (7-2B and 7-2C, representatively.
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CHAPTER 8 POTENTIAL OF NEAR INFRARED RESPONSIVE PARTICLES AND
QUANTIFICATION OF DRUG DELIVERY
Introduction
The utilization of contrast sensitive biocompatible particles has great potential to
aid the understanding of the delivery of therapeutic agents to targets. In muscle,
contrast enhanced NIR imaging without particles has revealed great insight into
pathology within muscle, in a rat model of muscle damage (Inage et al., 2015) and a
mouse model of muscular dystrophy (Baudy et al., 2011; Huynh et al., 2013).
However, tracking of the delivery of therapeutic treatments to disease models is only
possible through later analysis of tissue, and real time assessment is not available
through current methods. Delivery and biodistribution of particles carrying antisense
oligonucleotides (AONs) that were concurrently NIR fluorescently responsive has been
performed in the mdx mouse model (Falzarano et al., 2014a, 2014b). As more
efficacious strategies to treat the muscular dystrophies continue to progress, we intend
to boost the efficacy of therapeutics through theranostic vehicles, capable of delivering
therapeutic drugs and providing real time in vivo tracking of the delivery of these
particles.
As with all systemically delivery therapeutic agents, the ability to track local
delivery of agents in a highly efficacious manner is critical to confirm positive delivery of
therapy to target tissue. In dystrophic muscle, this is a particular challenge due to
muscle’s perfusion defects and fibrotic deposition, as well as the required systemic
delivery of therapy. As much excitement surrounds different genetic therapies,
efficacious manners to deliver drugs are continually investigated. In DMD, AONs have
emerged as a promising therapeutic option to induce functional dystrophin and are
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currently undergoing clinical trials (Bishop et al., 2015; Flanigan et al., 2014; Hoffman,
2014; Koo and Wood, 2013; Mendell et al., 2013, 2016; Sazani et al., 2014). While
naked AONs are reasonably stable in circulation, their bioavailability is limited by poor
cell trafficking and endosomal entrapment, requiring repeated and high doses to render
clinical efficacy (Järver et al., 2012). With a pressing need to discover a capable vehicle
to transport and protect AONs, it is our intention to develop an optimal drug delivery
system, capable of being tracked in vivo in real time, as well as delivering therapeutic
agents. Particle mediated delivery of AONs may resolve these issues, resulting in
improved therapeutic outcomes.
Particle based drug delivery systems have been investigated, balancing positive
benefits with negative consequences of different modalities. Previously, liposomes,
polymers, and cell-penetrating peptides have all utilized as vehicles to deliver drugs,
with each having their own benefits and shortcomings. Such shortcomings include a
lack of tropism to hone in effective specific targeting and difficulty controlling release of
the packaged contents. Several issues to consider in the synthesis and delivery of drug
conjugated particles include: (1) ability to be systemically distributed, (2) consistent
tissue targeting, (3) adequate in vivo stability of the drug-particle complex, (4) optimizing
the intracellular uptake, (5) ability to track and determine the biodistribution in vivo. The
lack of noninvasive tools to monitor and ensure the drug delivery to the target tissue
under in vivo conditions increases the uncertainty of clinical effectiveness. An approach
that allows for non-invasive tracking and monitoring of drug delivery to dystrophic
muscle sites in vivo using biocompatible biodegradable particles would help to relate
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drug delivery with therapeutic outcome and help in developing effective treatment
options.
In the effort to design optimal particles, poly-lactic acid (PLA), a biocompatible
polymer, was chosen to compose the particles. Previously, polymeric particles such as
those made of poly(methylmethacrylate) (PMMA) have been successfully employed as
carrier for delivering AONs in mdx mice (Falzarano et al., 2013; Rimessi et al., 2009).
As we additionally sought to track particles in real time, we conjugated ICG, an FDA
approved NIR fluorescent dye, to our PLA particles. Silica particles have been
developed with adsorbed ICG to provide an in vivo means to optically image the
distribution of such particles (Lee et al., 2009). ICG is an ideal NIR fluorescent contrast
agent to conjugate to biocompatible nanoparticles because of its negligible side effects
and low toxicity (Lutty, 1978). ICG is a blood-pool NIR contrast agent, clinically
approved for use in retinal angiography (Yannuzzi, 2011), blood flow measurements (El-
Desoky et al., 1999; Keiding et al., 1998), guiding biopsies (Motomura et al., 1999),
perfusion studies (Guenette et al., 2011), and lymphatic mapping (Rasmussen et al.,
2009). Despite multiple applications, there are inherent challenges regarding ICG
stability in solution and biological systems, including dependence of physical and optical
properties of ICG on pH, temperature, and exposure to light (Björnsson et al., 1982;
Holzer et al., 1998). Additionally, ICG has demonstrated optical instability in
physiologically relevant solutions (Desmettre et al., 2000; El-Desoky et al., 1999;
Landsman et al., 1976; Muckle, 1976). While beneficial when interested in vascular
properties, a rapid clearance from circulation (2-4 minute half life), is disadvantageous
when trying to assess uptake into damaged tissue (Wolfe and Csaky, 2004). The
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encapsulation of ICG into biocompatible particles offers great potential to maintain the
optical properties of ICG in an in vivo setting.
With the progress of new therapeutic strategies and investigations to mitigate the
pathologies of the muscular dystrophies, ICG enhanced NIR imaging provides a
minimally invasive longitudinal modality to monitor drug delivery and therapeutic
response in vivo. Development, characterization, and application of biocompatible poly-
lactic acid particles with encapsulated ICG are necessary to better understand the
capabilities of these particles. Through development of biocompatible particles that
encapsulated ICG that were tested in vitro and in vivo, we have laid the groundwork
necessary to accomplish this.
Results
Synthesis and Characterization of ICG-PLA Particles
First, physical and optical properties of ICG-PLA particles were characterized.
Size distribution was determined by digital light scattering, and found to range from 40-
100 nm (Figure 8-1A). Quantum yield (Φ) is the ratio of emitted to absorbed photons in
fluorophores and was determined to be 0.032, which is better than the reported range
for ICG dye in aqueous solutions (0.027 to 0.01) (Larush and Magdassi, 2011; Russin et
al., 2010). Corresponding scanning electron microscope (SEM) images demonstrated
size and morphologic homogeneity amongst the ICG loaded particles (Figure 8-1B).
Fluorescence of the ICG-PLA particles was also determined, and the peak of
fluorescence in ICG-PLA particles was found to be comparable to ICG in solution at 815
nm, with a small (~10 nm) red shift compared to the ICG monomers (Figure 8-1C),
comparable to previous studies (Gomes et al., 2006; Ranjan et al., 2011; Saxena et al.,
2004). Furthermore, the encapsulation efficiency of ICG in the PLA particles was
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determined to be ~ 70%, which is comparable to the ranges reported for electrostatically
assembled mesocapsules and micelles (Kim et al., 2010, 2010). The zeta potential of
the ICG - PLA particles ranges from -30 to -38 mV (SD) and +26 to +37mv range (SD)
for particles prepared in polyvinyl alcohol (PVA) and didodecyldimethylammonium
(DDAB) surfactants, respectively.
Photostability at Room and Physiological Temperatures
To assess the effect of temperature and time on the photostability of ICG and
ICG-PLA particles, samples were left at either 23°C (Figure 8-2A) or 37°C (Figure 8-2B)
for up to four weeks. Total radiant efficiency was recorded throughout the month, and it
was demonstrated that the ICG-PLA particles retained much of initial fluorescence at
room (90%) and physiological (83%) temperatures, respectively. Comparatively, the
fluorescence from ICG dye alone rapidly decayed at both temperatures.
In Vivo Contrast Enhanced NIR Optical Imaging of PLA-ICG via Subcutaneous Injections:
Intrascapular subcutaneous injections of ICG-PLA particles, ICG dye alone, or
lactated ringer’s buffer (LRB) were given to mice (Figure 8-3). As expected, the ICG
dye alone caused an initial increase in signal, but quickly returned back to baseline
values. Furthermore, fluorescent signal was maintained only the ICG-PLA particle
injected mice over the course of 10 days. The injection of lactated ringer’s solution
demonstrated no change in signal from baseline, serving as the negative control.
Representative images immediately following injections, as well as two days after
demonstrate the lack of fluorescent signal in mice that received LRB, the quick
deterioration of fluorescent in the ICG cohort, and maintenance of signal in mice that
received ICG-PLA particles. Besides day 0 measurements, all ICG-PLA particles are
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significantly elevated as compared to ICG alone measures. Additionally, ICG-PLA
particles become significantly lower than day 0 values after 4 days.
In Vivo Contrast Enhanced NIR Optical Imaging of PLA-ICG via Intramuscular Injections
Intramuscular injections of ICG-PLA particles into gastrocnemii demonstrate
maintenance of fluorescent signal for up to 2 weeks (Figure 8-4). Similar to the
These investigations of the upper extremities provide foundational data to further
understand the progression of DMD beyond the traditionally studied skeletal muscles of
the lower extremity. While loss of function in the lower extremity is more visibly
apparent as boys transition to wheelchairs, maintenance of function the arms is
arguably more critical for males to retain independence throughout their lives
(Alemdaroğlu et al., 2015; Janssen et al., 2014b; Pane et al., 2014). Preservation of arm
function, even after loss of ambulation, provides males the ability to live their lives in as
independent of a state as possible, allowing them to achieve activities of daily living.
To the muscular dystrophy community, clinical trials remain a beacon of hope for
the potential of a cure. Participation in such trials remains a major hurdle for many
individuals because of study design requirements, and rigid inclusion and exclusion
criteria (Mercuri and Muntoni, 2013; Ricotti et al., 2015). In many studies, adequate
ambulatory function is a requirement to allow participation in trials, and because of this,
many males with DMD are simply not eligible to participate and potentially benefit from
such trials. Therefore, developing a better understanding of how DMD affects the upper
extremities was a major goal of this study. Proximal muscles, such as the deltoids,
were shown to be affected greater from the disease than the distal forearm muscles, in
agreement with the described proximal to distal time course of DMD (Figure 9-4)
(Bushby et al., 2010a; Fischmann et al., 2012; Willcocks et al., 2014). Further, as
males aged, MR measures showed more significant markers of damage (Figures 9-5A
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and 9-5B). When comparing functional measures of the upper extremity to MR
measures, they again significantly correlated with each other (Figures 9-5C and 9-5D),
suggesting that MR may be an appropriate proxy to assess the state of muscle health in
DMD.
This study is our first investigation of utilizing MRI to assess muscle in the upper
extremity in DMD and is not without limitations. As the study is still being optimized, not
all subjects underwent PUL functional testing, and therefore some MR data does not
have a corresponding functional measure. Furthermore, the sequences and scans
being utilized have undergone optimization, and we hope that with further data
collection, standard operating procedures will be able to be established.
Summary of Upper Extremity Findings
In summary, this preliminary study assesses the state of muscle health in the
upper extremity, demonstrating proximal versus distal differences in the amount of
pathology caused by DMD. Furthermore, both age and function demonstrate significant
correlations to the MR measures performed in this study, suggesting that MRI may be
an adequate proxy measure of disease progression in the upper extremities of males
with DMD.
Differences Between Concentric and Eccentric Lower Arm Exercises
Introduction
The ultimate goal of our studies is to translate NIR optical imaging from
conceptual preclinical studies to practical human studies, demonstrating the ability of
NIR optical imaging in humans to detect damaged and diseased muscle. The clinical
study discussed in the former half of this chapter served to demonstrate the ability of
MRI to detect muscle that has been damaged as a result of natural disease in humans.
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In a parallel manner, it is the intention of the experiments described below to
demonstrate that NIR optical imaging can longitudinally, quantitatively, and repeatedly
assess the state of muscle health.
Optical imaging in the NIR range in humans is relatively unexplored, and to date,
has been limited primarily to research of the breast (Altinoğlu et al., 2008; Jiang et al.,
2000; Poellinger et al., 2011; Schneider et al., 2011), exercising muscle (Boushel and
Piantadosi, 2000; Brizidine et al., 2013; Guenette et al., 2008; Hamaoka et al., 2007),
brain (Wolf et al., 2007), and joints (Yuan et al., 2007). A redistribution of tissue water
and blood after exercising results in optical signal changes, which can be detected with
clinical NIR optical imaging. It is our intention to utilize these intrinsic properties of the
body to investigate changes in muscle permeability caused by an exercise routine in a
healthy population and in the natural progression of DMD affected children. Although this
study is still in progress, preliminary results are reported in this dissertation.
Results
To assess temporary and reversible exercise-induced muscle damage resulting
from forearm exercise in healthy subjects, a detailed MR characterization of both
forearms in all subjects is performed. Each subject serves as his own control as
concentric exercise causes minimal muscle damage compared to eccentric exercises.
Fat suppressed T2 weighted images of concentrically (Figure 9-6A) and eccentrically
(Figure 9-6B) exercised forearms are shown. Muscles targeted to be damaged through
our exercise protocol are outlined with a dashed red line in Figure 9-6B. Quantitative
MRI analysis revealed that eccentric contractions increase muscle T2 by ~5% (p<0.05)
when compared to the concentrically exercised forearm (Figure 9-6C).
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Similar to the developmental stage of the MR data, the NIR optical imaging
demonstrate that the concentrically exercised forearm (Figure 9-7A) demonstrate less
signal than the eccentric exercised forearm (Figure 9-7B). The optical images show
increased blood content of the forearm due to elevated blood flow and fluid retention
within the eccentrically exercised forearm when compared to the concentrically loaded
forearm.
Discussion
Early studies have shown MR and optical measurements are sensitive to
eccentrically induced, acute muscle damage in unaffected control subjects (Cermak et
al., 2012; Fulford et al., 2014; Sesto et al., 2008). In addition, pilot studies in the arms
of DMD boys indicate that unlike the upper arm and the shoulder the forearm is
relatively preserved when considering the degree of fatty tissue deposition determined
by MRI (Alemdaroğlu et al., 2015; Bushby et al., 2010a; Hudak et al., 1996). This is
advantageous for optical imaging, due to the presence of chronic muscle damage not
being obscured by the highly scattering lipids (Cerussi et al., 2001). The DMD forearm
is ideal for our imaging applications for the following reasons: 1) early disease
involvement based on quantitative T2 measures, 2) low levels of fatty tissue deposition
to minimize confounding light scattering results resulting from light scatter by lipid and
atrophic muscle, 3) its anatomical location allows it to be easily inserted to the CTLM
optical imaging device for imaging even while seated in a wheelchair.
Elevated T2 values have been commonly observed following eccentric
contractions grossly in larger muscles (Fulford et al., 2014; Sesto et al., 2008), these
results illustrate that MRI possesses adequate sensitivity and spatial resolution to image
acute T2 changes in the much smaller wrist flexor muscles of the forearm after eccentric
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exercise, as we observe in this study (Figure 9-6). Quantitative changes in muscle T2
and volume of affected tissue (calculated from T2 imaging maps) are expected to
correlate to changes observed with NIR-OI of the forearm. Building off of the MR
findings, NIR optical imaging also demonstrates the capability to distinguish
eccentrically from concentrically exercised forearms (Figure 9-7). During the
sarcolemmal damaging contractions that the eccentric exercises induce onto muscle,
edematous inflammation occurs, leading to a detectable signal by NIR optical imaging.
This is visible by the elevation of hyperintensities in the eccentrically loaded forearms as
compared to the contralateral concentrically exercised forearms. Though this study is
incomplete, the data collected thus far is encouraging. Moving forward, the study simply
needs to be performed. Because of several hardware issues with the CTLM System,
recruitment and enrollment into the study has been delayed, but is again underway.
Summary Concentric and Eccentric Lower Arm Exercises
This study provides preliminary data that supports the ability of NIR optical imaging as a
feasible technology to assess the state of muscle health in muscle that has been
damaged by eccentric exercising and from the natural progression of DMD. Differences
between eccentric and concentrically exercised forearm muscles suggest that the
protocols implemented in these studies are appropriately designed to test the
capabilities of NIR optical imaging to assess muscle health.
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Figure 9-1. Qualitative MRI Scores from two representative DMD patients
demonstrating differences in involvement along the length of six lower leg muscle groups. X axes are labeled with P (proximal), MP (mid-proximal), M (middle), MD (mid-distal), and D (distal).
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Figure 9-2. Comprehensive degree of involvement in all slices of all subjects’ muscles
(A: Peroneus, B: Extensor Digitorum Longus, C: Tibialis Anterior, D: Soleus, E: Medial Gastrocnemius, F: Lateral Gastrocnemius), ranging from 0 (white) to 5 (black). A diagonal line over a point indicates that the data was deemed unreliable due to a low SNR or the muscle of interest was not present at the selected slice.
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Figure 9-3. Age and function are related to MRIsingle and MRImulti scores. Correlations
between age and ScoreMulti and ScoreSingle are shown in 9-3A, and 9-3B, respectively. Comparison to Vignos functional scores and ScoreMulti and ScoreSingle are shown in 9-3C and 9-3D, respectively.
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Figure 9-4. Cross sectional analysis of upper extremity muscles in boys with DMD.
Quantitative MRI-T2 measures demonstrate significant differences between control and DMD subjects in the deltoid, biceps, and triceps, but not the forearm muscles (9-4A). Qualitative MRI scores are shown for DMD subjects, and the only significant difference was between the anterior forearm and deltoid muscles (9-4B). Note that all control subjects scored ‘0’ for their qualitative MRI scores, and are not shown.
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Figure 9-5. Age and PUL function as related to MRI-T2 and MRI qualitative scores.
Correlations between age and MRI-T2 and MRI qualitative scores are shown in 9-5A, and 9-5B, respectively. Comparison to PUL functional scores and MRI-T2 and MRI qualitative scores are shown in 9-5C and 9-5D, respectively.
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Figure 9-6. Fat suppressed axial MR images of concentrically (9-6A) and eccentrically
(9-6B) exercised human forearms with quantification (9-6C) of T2 relaxation times taken from the deep flexor muscles of the forearms.
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Figure 9-7. Three dimensional absorbance reconstructions of human forearms were
taken two days following eccentric (9-7A) and concentric (9-7B) exercise. Hyperintensities in the eccentric (9-7A) arms indicate elevated fluid retention as compared to the contralateral concentrically exercised (9-7B) forearms.
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CHAPTER 10 CONCLUSION
Overview
The muscular dystrophies are a collection of progressive and irreversible muscle
wasting disorders with no current curative therapy. Modern treatments include non-
curative interventions, such as mechanisms to increase muscle mass, correct blood
flow perturbations, minimize inflammation and fibrosis, and correct calcium handling.
Investigations that may ultimately provide a cure to the different muscular dystrophies
include protein, transcriptome, and genome restoring remedies. These therapies and
developments to treat and mitigate the pathologies have been developed from basal
understandings of muscle physiology and growth and repair mechanisms. Though
clinical trials offer great hope to the muscular dystrophy family, all have experienced
setbacks and failures thus far. Inadequate measures of muscle health, namely biopsies
and functional tests, effectively null any positive benefits that drugs in clinical trials may
possess, necessitating the development of other outcome measures. Such outcome
measures should be non-invasive, objective requiring minimal subject involvement,
safe, repeatable, and quantifiable.
NIR optical imaging and MRI offer the ability to non-invasively, longitudinally, and
objectively quantify the state of muscle health in complimentary manners. In this
dissertation, I have presented a collection of non-invasive techniques that assess and
monitor basic processes in healthy, exercised damaged, disease damaged, and
disease treated muscle. Both technologies possess their own advantages and
limitations, but in combination, reveal complimentary information regarding the state of
muscle health. This allowed me to quantitatively track progression of disease, or
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conversely, regression of pathology resulting from therapeutic intervention. Importantly,
these measures are longitudinal, non-invasive, objective, and quantifiable. Confirmatory
support of these measures is provided by histology and tissue spectrophotometry. All
together, these non-invasive imaging techniques hold great promise to fulfill the need
for a non-invasive imaging method to monitor and quantify cellular damage, muscle
perfusion, and drug delivery to accelerate testing of drug efficacy in clinical trials for
muscular dystrophy and potentially other muscle disorders.
Summary of Experiments
Capabilities of ICG Enhanced NIR Optical Imaging in Preclinical Models
ICG enhanced NIR optical imaging detected damaged and dystrophic muscle in
several preclinical models. In an acute model of muscle damage, caused by
immobilization followed by reambulation to murine hindlimbs, a characteristic
timecourse of muscle damage and recovery was observed by ICG enhanced NIR
optical imaging. Further confirmatory measures were performed using MRI, MRS,
histology, and tissue spectroscopy. A second round of experiments demonstrating the
ability of ICG enhanced NIR optical imaging to cross sectionally detect muscle that has
been damaged due to two different muscular dystrophies was performed. Additional
insult to muscle, by way of downhill treadmill running demonstrated that exacerbation of
damage to dystrophic muscle is able to be measured. Therapeutic treatment of
dystrophic muscle, through intramuscular administration of AAVs containing the missing
gene of interest, was able to be quantified using ICG enhanced NIR optical imaging.
Similar to the immobilization-reambulation experiments, MRI, MRS, histology, and
tissue spectrophotometry confirmed the NIR optical imaging findings. In all, these
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preclinical studies demonstrate the capabilities of ICG enhanced optical imaging as a
potent modality to assess the state of static and dynamic muscle health in relevant
mouse models.
Potential of Near Infrared Responsive Particles
First, preliminary experiments have demonstrated the capabilities of ICG
enhanced NIR optical imaging to quantify baseline blood flow of the major vasculature
of the mouse hindlimbs, as well as perturbations to blood flow. Furthermore, through
conjugation of ICG to biocompatible PLA particles, we have laid the foundational work
to track the delivery of disease modifying therapies to dystrophic muscle. Through our
findings in these sets of experiments, I demonstrated the prolongation of physical
stability and optical fluorescence of ICG in in vitro and in vivo settings. As these
experiments are still in the developmental stages, my future steps include the
incorporation of disease correcting drugs within the NIR responsive ICG loaded PLA
particles.
Clinical Application of MRI and NIR Optical Imaging
Translation of preclinical findings to the clinical arena is the ultimate goal of all scientific
endeavors. In the first clinical investigation performed, I demonstrated the ability of MRI
to differentiate disease pathology along the lengths of several lower leg muscles of boys
affected by DMD. Through use of a multi-slice evaluation of the lower legs of boys with
DMD, I showed that muscles are not affected equally, disease involvement is more
severe near the myotendinous junctions, individuals are affected uniquely, and that
qualitative MRI grades correlate to age and function. Next, I assessed progression of
disease in the upper extremities of males with DMD, identifying a proximal to distal
pattern of disease progression that correlates to age and function. Though still in
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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.
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BIOGRAPHICAL SKETCH
The impetus for Steve’s motivation to research the muscular dystrophies began at a
young age, when he began to volunteer at summer Muscular Dystrophy Association (MDA)
camps for individuals with neuromuscular disorders. During the era that Steve began
working with the MDA, much noise, ruckus, and publicity was raised to help raise awareness
for the muscular dystrophies. Though many knew what the muscular dystrophies were
because of his efforts, something was clearly still lacking – a cure. Having seen many
friends’ lives prematurely because of the muscular dystrophies, Steve observed that clinical
management was the best that clinicians could provide to this population. A clear calling to
do research stemmed from this realization.
Unsure of the optimal route to pursue his interests, Steve serendipitously ventured to
study Biomedical Engineering at the University of Cincinnati. The years at the University of
Cincinnati have molded Steve’s personal and professional life in many ways. Through the
Co-op Program at the University of Cincinnati, Steve was introduced to Dr. Christy Holland,
who still remains a close confidante to this day. In her research lab, Dr. Holland took Steve
under her wing, wisely providing appropriate motivation and encouragement, to help Steve
co-author two papers. More importantly, Dr. Holland planted the seed of excitement in Steve
of the possibilities of research, introducing him to a network of positive influences that have
proved vital to his successes down the road. She introduced Steve to many MD-PhD’s,
including Drs. Kate Hitchcock, Chip Shaw, Patrick Kee, and Shaoling Huang, who have been
successful both personally and professionally, showing Steve opportunities that he did not
know existed. With great guidance and input from Dr. Kate Hitchcock, Steve sought
application to MD-PhD programs, and despite many curveballs, found a future home at the
University of Florida.
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At the University of Florida, Steve completed the first two years of medical school and
completed the USMLE 1, at which time, his pre-doctoral graduate training was to commence.
By joining the combined lab of Drs. Glenn Walter and Krista Vandenborne, Steve sought to
be trained in a well-rounded, translational lab studying the muscular dystrophies. With Dr.
Walter as his primary mentor, Steve joined the Department of Physiology and Functional
Genomics and soon after, received two T-32 Training Grants (Neuromuscular Plasticity and
Hypertension) to perform his research. His current work, which has formed the bulk of this
dissertation, has been focused on developing non-invasive biomarkers using near infrared
optical imaging and magnetic resonance imaging.
Steve’s long-term interest involves investigating the development and quantification
of novel therapeutic treatments of the spectrum of muscular dystrophies as a physician-
scientist. Beyond the lab life, Steve enjoys music, beer brewing, traveling, exercising, and