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1 NON INVASIVE MONITORING OF DISEASE PROGRESSI ON IN DUCHENNE MUSCULAR DYSTROPHY By RAVNEET SINGH VOHRA 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 2012
2 2012 Ravneet Singh Vohra
3 To my parents, brother and wife
4 ACKNOWLEDGMENTS I am grateful to several individuals for their guidance and support in my dissertation work. The greater part of this work was made possible by the guidance of my mentors and by the love and support of my family, friends and colleagues. It is with my heartfelt gratitude that I acknowledge each of them. First and foremost, I would like to thank my advisor and mentor Dr. Krista Vandenb orne, for her constant guidance and support. Also, I am grateful to Dr. Walter for his feedback and thought pr ovoking research questions. I would like to thank Dr. Martin for his help with most of the statistical analysis, constant encouragement and support throughout my doctoral education. I would also like to express my sincere thanks and gratitude to Dr. Shecht man for encouragi ng me to strive harder towards m y goals and aspirations. A special thanks to all my lab members, without whose help this dissertation would have never been possible. I would like to thank Min, Fan, Arun, and Wendy for teaching me all the n ecessary skills and techniques in the lab and for also guiding me through the PhD process. I would like to thank Dr. Mathur, Dr. Lott Dr. Forbes, Dr. Baligand and Dr. Willcocks, for helping me through out the entire proce ss. I would like to thank Jasjit, W o otaek, Arjun, Abhinandan and Ishu for helping me in completing the final stages of my dissertation I would also like to thank Dr. Day for her assistance and support I would also like to thank the staff of the Physical therapy department especially Ellen Esparolini and Christa Richmond for keeping me up to date with every deadline during my doctoral studies. Finally, I would like to express my sincere appreciation to my parents for their love and support. Last but not the least; I would l ike to thank my beloved wife Daman
5 who has been supporting me through this process. Thank you for all the sacrifices and difficulties that you have patiently endured during this process This project would never have happened without their unending support
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURE S ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 16 Struct ure and Organization ................................ ................................ ..................... 16 Dystrophin Glycoprotein Complex ................................ ................................ .... 16 Structure of Dystrophin ................................ ................................ ..................... 17 Dystrophinopathies ................................ ................................ ................................ 17 Clinical F eatures ................................ ................................ ................................ ..... 18 Pathophysiology ................................ ................................ ................................ ..... 20 Animal Models of Muscular Dystrophies ................................ ................................ 21 Approaches to Treatment of Muscular Dystrophy ................................ ................... 24 Summary ................................ ................................ ................................ ................ 27 2 NON INVASIVE TECHNIQUES FOR ASSESSMENT OF MUSCLE DYSFUNCTION ................................ ................................ ................................ ...... 28 Computed Tomography ................................ ................................ .......................... 28 Ultrasound ................................ ................................ ................................ .............. 28 Magnetic Resonance Imaging (MRI) ................................ ................................ ...... 29 Basics of Magnetic Resonance ................................ ................................ ........ 30 Properties of Atomic Nuclei ................................ ................................ .............. 30 Relaxation Times ................................ ................................ .............................. 31 Image Formation ................................ ................................ .............................. 33 MRI Applicat ions in Skeletal Muscle ................................ ................................ ....... 35 Summary ................................ ................................ ................................ ................ 39 3 OUTLINE OF EXPERIMENTS ................................ ................................ ................ 43 Experiment One ................................ ................................ ................................ ...... 43 Specific Aims ................................ ................................ ................................ .... 43 Hypotheses ................................ ................................ ................................ ...... 43 Experiment Two ................................ ................................ ................................ ...... 43 Specific Aims ................................ ................................ ................................ .... 43
7 Hypotheses ................................ ................................ ................................ ...... 44 Experiment Three ................................ ................................ ................................ ... 44 Specific Aim ................................ ................................ ................................ ...... 44 Hypothesis ................................ ................................ ................................ ........ 44 Experiment Four ................................ ................................ ................................ ..... 44 Specific Aim s ................................ ................................ ................................ .... 44 Hypotheses ................................ ................................ ................................ ...... 44 Experiment Five ................................ ................................ ................................ ...... 45 Specific Aim ................................ ................................ ................................ ...... 45 Hypotheses ................................ ................................ ................................ ...... 45 4 RELATIONSHIP BETWEEN LOWER LIMB MUSCLE CROSS SECTION AL AREA AND TORQUE PRODUCTION IN BOYS WITH DUCHENNE MUSCULAR DYSTROPHY ................................ ................................ .................... 46 Specific Aims ................................ ................................ ................................ .......... 47 Hypotheses ................................ ................................ ................................ ............. 47 Research Design an d Methods ................................ ................................ ............... 47 Participants ................................ ................................ ................................ ....... 47 Methods ................................ ................................ ................................ ............ 48 Statistical Analysis ................................ ................................ ............................ 50 Results ................................ ................................ ................................ .................... 51 Discussion ................................ ................................ ................................ .............. 53 Summary ................................ ................................ ................................ ................ 59 5 RELATIONSHIP BETWEEN LOWER EXTREMITY MUSCLES CONTRACTILE CROSS SECTIONAL AREA AND FUNCTIONAL MEASUREMENTS IN BOYS WITH DUCHENNE MUSCULAR DYSTROPHY ................................ ..................... 73 Specific Aims ................................ ................................ ................................ .......... 74 Research Desig n and Methods ................................ ................................ ............... 75 Results ................................ ................................ ................................ .................... 78 Discussion ................................ ................................ ................................ .............. 80 Summary ................................ ................................ ................................ ................ 82 6 TO MONITOR CHANGES IN T 2 WITH AGE IN HINDLIMB MUSCLES OF MDX MICE ................................ ................................ ................................ ....................... 94 Specific Aims ................................ ................................ ................................ .......... 95 Research Design and Methods ................................ ................................ ............... 95 Results ................................ ................................ ................................ .................... 96 Discussion ................................ ................................ ................................ .............. 97 Summary ................................ ................................ ................................ .............. 100 7 CHANGES IN MUSCLE T 2 AND TISSUE DAMAGE IN HINDLIMBS OF MDX MICE AFTER DOWNHILL RUNNING ................................ ................................ ... 106 Specific Aims ................................ ................................ ................................ ........ 108
8 Hypotheses ................................ ................................ ................................ ........... 108 Research Design and Methods ................................ ................................ ............. 108 Results ................................ ................................ ................................ .................. 112 Discussion ................................ ................................ ................................ ............ 114 Summary ................................ ................................ ................................ .............. 121 8 EFFECT OF UPHILL RUNNING ON MYOCARDIUM T 2 IN MDX MICE ............... 129 Methods ................................ ................................ ................................ ................ 130 Results ................................ ................................ ................................ .................. 133 Discussion ................................ ................................ ................................ ............ 133 Summary ................................ ................................ ................................ .............. 135 9 CONCLUSIONS ................................ ................................ ................................ ... 140 Overview ................................ ................................ ................................ ............... 140 Summary of Conclusions ................................ ................................ ...................... 140 LIST OF REFERENCES ................................ ................................ ............................. 143 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 163
9 LIST OF TABLES Table page 4 1 Demographics of patients. ................................ ................................ .................. 72 5 1 Subjects demographics. ................................ ................................ .................... 93 5 2 Spearman rank correlation between non contractile content, strength and functional measurements. ................................ ................................ .................. 93
10 LIST OF FIGURES Figure page 2 1 Representative three dimensional figure showing RF pulse application. ............ 40 2 2 Schematic representation of 90 degree RF pulse application and its consequence. ................................ ................................ ................................ ..... 40 2 3 Schematic diagram showing T 1 recovery curves of fat and muscle .................... 41 2 4 Schematic figure showing T 2 decay curve for muscle and damage .................... 41 2 5 Time interval between two successive 90 degree RF pulses denoted by TR. .... 42 4 1 T 1 weighted fat suppressed transaxial images of the thigh (A) and lower leg (B) obtained from an 11 year old boy with DMD. ................................ ................ 60 4 2 Scatter plots of age versus peak torque (Nm) in boys with DMD (filled circles, solid line) and controls (ope n circles, dashed line) ................................ ............. 61 4 3 Scatter plots of CSA max (cm 2 ) versus age in boys with DMD (filled circles, solid line) and controls (open circles, dashed line) ................................ ............. 64 4 4 Scatter plots of age versus specific torque (torque normalized to CSA max of muscle; Nm /cm 2 ) in boys with DMD (filled circles, solid line) and controls (open circles, dashed line) ................................ ................................ .................. 67 4 5 Group means SD of 4 timed fun ctional tasks in boys with DMD and controls. *P<0.001. ................................ ................................ ................................ ............ 70 4 6 Relationships between peak torque of the KEs (A) and PFs (B) and time to walk 9m. ................................ ................................ ................................ ............. 71 5 1 T1 weighted fat unsuppressed trans axial images of the lower leg of Ctrl (A) and DMD subject (B). ................................ ................................ ......................... 84 5 2 Comparison of percent non contractile content, non contractile CSA and contr actile CSA of lower leg muscles ................................ ................................ 85 5 3 Relationship between 30 feet walk and non contractile content (%) of lower leg muscles ................................ ................................ ................................ ......... 88 5 4 Relationship between Brookes scale and non contractile content (%) of lower leg muscles ................................ ................................ ................................ ......... 89 5 5 Specific torque of dorsiflexors (DF) and plantar flexors (PF) between DMD, Duchenne muscular dystrophy and Ctrl, control group. ................................ ...... 90
11 5 6 Non contractile and Contractile CSA comparison among different age groups in DMD. ................................ ................................ ................................ .............. 91 6 1 T 2 weighted images of mdx mouse hindlimb at different time points. ............... 101 6 2 Hindlimb muscle T 2 values and % pixels with elevated T 2 of anterior and posterior region. ................................ ................................ ................................ 102 6 3 Mean muscle T 2 values and % pixels with elevated T 2 of anterior region at different time points. ................................ ................................ ......................... 103 6 4 Mean muscle T 2 values and % pixels with elevated T 2 of anterior and posterior hindlimb muscle compartments of mdx mice. ................................ .... 104 6 5 H&E and trichrome staining of SOL and gastr in mdx and Ctrl mice at different time points. ................................ ................................ ......................... 105 7 1 Trans axial MR image of the lower hindlimb showing regions of interest (ROIs) for muscle T 2 analysis. ................................ ................................ .......... 123 7 2 Percentage of pixels with elevated T 2 and muscle T 2 ................................ ...... 124 7 3 Percentage of pixels with elevated T 2 returned to baseline values ten days after downhill running in mdx mice ................................ ................................ ... 125 7 4 Transaxial, T 2 weighted images from the lower hindlimb of an mdx mouse before and after a single bout of downhill running for 45 min ........................... 125 7 5 Three dimensional rendering of regional muscle damage in the hindlimb of an mdx mouse following downhill running. ................................ ....................... 126 7 6 Muscle T 2 values in mdx mice following a single bout of downhill running compared to horizontal running. ................................ ................................ ....... 126 7 7 Change in % muscle damage in individual muscle compartments of mdx mice following a single bout of downhill running ................................ ............... 127 7 8 EDL muscle of mdx mouse 48 hr following 1 bout of downhill treadmill running. ................................ ................................ ................................ ............ 127 7 9 Correlation between % of pixels with elevated T 2 and % of area showing ................................ ........................... 128 8 1 Custom built quadrature volume coil with 3.3 cm inner diameter. ..................... 136 8 2 Representative four and two chamber long axis images ................................ .. 137 8 3 Myocardial T 2 values in mdx mice after a single bout of uphill running compared to wild type mice.. ................................ ................................ ............ 138
12 8 4 Myocardium of mdx mouse 48 hours following a s ingle bout of uphill running 139
13 LIST OF ABBREVIATION S BMD Becker muscular dystrophy CSA cross sectional area C CSA contractile cross sectional area DMD Duchenne muscular dystrophy EDL Extensor digitorum longus muscle FID free induction decay FOV field of view GAS Gastrocnemius muscle mdx muscular dystrophy X linked MRI magnetic resonance imaging NC CSA non contractile cross sectional area SNR Signal to no ise ratio SOL Soleus muscle T 1 longitudinal relaxation rate T 2 transverse relaxation rate TA Tibilais anterior muscle
14 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 NON INVASIVE MONITORING OF DISEASE PROGRESSI ON IN DUCHENNE MUSCULAR DYSTROPHY By Ravneet Singh Vohra December 2012 Chair: Krista Vandenborne Major: Rehabilitation Science Skeletal muscle cytoskeletal proteins not only maintain the structural integrity of the myofiber but also transmit force s generated by the actomyosin complex. Muscle fibers are protected from outside the cell by a basal lamin a and inside by the actin netw ork. This structure is further bolstered by the presence of proteins such as dystrophin, utrophin and variou s trans membrane proteins Absence of any of these proteins can lead to devastating alterations in the muscles that can cause muscular dystrophy. Th e muscular dystrophies represent a class of neuromuscular diseases characterized by rapid muscle wasting. Duchenne muscular dystrophy (DMD) is one of the most common forms of muscular dystrophy affecting 1 in every 3500 newborn males. [ 1 ] Absence of the dystrophin protein makes the sarcolemmal membrane more susceptible to muscle contraction induced sarcolemmal injury. Progression of fatty tissue infiltration and muscle atrophy occurs seco ndary to sarcolemmal injury. DMD patients usually lo ose ambulation in their mid to late teens. Death usually occurs due to respiratory and cardiac muscle failure Although, the underlying cause of DMD was discovered in 1980 there is no definite treatment to cure DMD. There is an increasing
15 interest in devel oping and administration of different therapeutic agents in order to counteract muscle pathology and more so, muscle wasting in DMD patients. In order to monitor both the disease progression and effects of therapeutic agents different outcome measures used Muscle biopsies have been used as the gold standard to diagnose D uche n ne muscular dystrophy However, it becomes almost impractical to use multiple biopsies in order to monitor the disease progression. Apart fro m being invasive in nature, this technique fails to represent the heterogeneous distribution of the disease. On the other hand, non invasive techniques, such as magnetic resonance imaging (MRI) have the ability to detect and quantify the tissue pathology in three dimensions. This doctoral disserta tion presents results of five studies of non invasive monitoring of structural chan ges in skeletal muscles in DMD.
16 CHAPTER 1 INTRODUCTION Structure and Organization Skeletal muscle is composed of several myofibers which are organized into fascicles. Myofibrils are interconnected with intermedia te filament proteins called Desmin. Desmin form s a three dimensional scaffolding around the Z disks and connects the entire contractile apparatus to a subsarcolemmal cytoskeleton [ 2 ] Myofibers are divided into subunits called sarcomere s which are arranged in a series and are composed of contractile filaments. These contractile filaments are divided into two types; a) Thick filaments: contain polymers of protein myosin and b) Thin filaments: contain polymers of protein actin. Furthermor e, a ctin is linked to laminin in the extracellular matrix through a protein called dystrophin which is a component of the dystrophin associated glycoprotein (DAG) complex [ 3 ] Dystrophin Glycoprotein Complex The Dystrophin glycoprotein complex (DGC) is a large complex of membrane associated proteins that is critical for skeletal muscle fibers integrity The DGC serves as a link between the extracellular matrix and the subsarcolemmal cytoskeleton. Furthermore, DGC is thought to protect muscle cells fr om contraction induced damage [ 4 ] This complex consists of the following proteins: d ystrophin, d ystrogl ycans, s arcoglycans, s arcospan ( ), s yntrophins ( 1, 1, 2; 1 and 2), d ystrobrevin. Dystrophin binds to cytoskeletal actin and to transmembrane protein dystroglycan. The extracellular domain of dystroglycan binds to the peripheral membrane protein, dystroglycan. dystroglycan binds to l aminin 2 in the basal lamina. Thus, the DGC
17 serves as a link between the extracellular matrix and the subsarcolemmal cytoskeleton. Therefore, it is thought to protect muscle cells from contraction induced damage [ 4 ] Str ucture of Dystrophin The dystrophin gene was one of the first genes that was identified using genomic technology [ 5 ] The gene is lo cated on the human chromosome Xp21 and is one of the largest human genes identified. It is composed of 79 exons spanning more than 2.5 million base pairs, which corresponds to about 1.5% of the entire X chromosome [ 6 7 ] The dystrophin gene encodes the protein dystrophin. Full length dystrophin mRNA is about 14kb and is mainly expressed in skeletal, cardiac and smooth muscles and the brain. Analysis of the amino acid sequence of dystrophin reveals its rod shaped structure. It consists of four domains. a) An amino terminal that binds with actin b) A rod domain that consists of 24 spectrin like rod repeats and 4 hinges c) A cysteine rich domain that interacts with dystroglycan and sarcoglycan complexes and d) A carbox y terminus that interacts with s yntrophins and dystrobrevins. Four other, short er isoforms of the dystrophin protein of different molecular mass have also been reported and are 260 kDa, 140 kDa, 116 kDa, 71 kDa. These shorter isoforms lack the actin binding domain, which suggests that they may have some function, which is altogether different from that of d ystrophin. Dystrophinopathies Dystrophinopath y is a general term used to describe the disorders that occur due to lack of functional protein dystrophin. The first gene to be cloned was the dystrophin gene that is mutated in both Duc henne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD). Although both DMD and BMD are caused by mutation in the dystrophin gene, BMD is considered a less severe form of muscular dystrophy.
18 DMD is the most common form of m uscular dystrophy where as BMD is a less c ommon form [ 8 ] DMD/BMD is a severe X linked degenerative disorder that occurs with an incidence of 1 in 3500 children and 1 in 30,000 children respectively [ 1 ] The term DMD is coined after the French neurologist Duchenne de Boulogne, who described the disease approximately 150 years back. H e also devised a harpoon like needle and a procedure (muscle biopsy) to study and demonstrate the severity and progressive nature of the disease. Twenty five years ago, the genetic defect underlying DMD was mapped to chromosome Xp21 in humans [ 9 10 ] Since the gene is the largest gene identified the size of the gene itself could account for its relati vely high frequency of mutation [ 11 ] The large DMD gene can accommodate prod uction of several dystrophin isoforms. Mutations/deletions in the dystrophin gene prevent the production of dystrophin leading to its absence in muscle fibers [ 12 ] However, in BMD, the nature of mutations is such that synthesis occurs of a truncated and partly functional dystrophin protein. In DMD patients, vario us mutations in the DMD gene, such as missense, nonsense, deletion, insertion or duplication have been identified. When the reading frame has been disrupted by a mutation (out of frame), dystrophin is not expressed and this results in a phenotype of DMD. O n the contrary, if the reading frame is maintained, a truncated but still functional dystrophin is expressed. This result in a milder form called Becker muscular dystrophy. In a DMD gene, there are two hot spots for mutation s : around exons 3 7 and 45 55. C linical Features Boys who have DMD exhibit a progressive loss of muscle function. Although the muscles are affected at birth, the clinical symptoms of proximal muscle weakness
19 usually manifest between 3 to 5 years of age. The boys usually start walking at about 18 months of age, compared to their siblings. Toe walking is commonly seen in patients with DMD. Initially the appearance of the lower limbs may be normal but, by the age of 3 to 4 years, pseudo hypertrophy of the calf muscle can be seen. DMD patient has difficulty performing daily activities like, running, ju mping and hopping even during early stages of life As the disease progresses, the affected boys develop a lumbar lordosis and a Trendelenburg gait. They fall more often and have trouble when ris ing up. When rising up from the They get into a knee elbow position, extend their elbows and knees, bring their hands and feet as close as possible and place one hand a time on their knees They then place their hands on their thighs and move proximally in steps, climbing up their legs. Boys with DMD are at increased risk f or certain cognitive concerns. However, unlike their muscle weakness, which is progressive in nature, their cognitive s kills do not deteriorate over time. Furthermore, muscle biopsy of patients with DMD demonstrates necrotic or degenerating muscle fibers, which are often observed in clusters. These necrotic fibers are surrounded by macropahages. Small immature fibers with central nuclei are observed which reflects muscle regeneration. This regenerative capacity later on gets exhausted and muscle fibers are gradually replaced by connective and adipose tissue. Unlike DMD, BMD patients show much more heterogenous clinical mani festations. Initially it was reported that the mean age of onset for BMD was 11 years but this may vary [ 13 ] Patients with BMD stay ambulatory un ti l 20 years of age and mean age at death is around the mid 40s [ 13 ] With the improvement in diagnostic
20 techniques, it has become well known that a number of patients can remain ambulatory until 40 years or beyond and they can survive up to 60 years of age. Clinically, hypertrophy of the calf muscle is a characteristic feature of juvenile BMD patients [ 14 ] Calf muscle hypertrophy tends to vanish with aging. There is symmetrical weakness of muscles of the pelvic girdle and thighs. Moreover, it is well known that cardiac function is afflicted in the patients with BMD. Patients with BMD have nor mal developmental milestones and can carry out strenuous exercises. Pathophysiology DMD is characterized by p rogressive muscle fibers degeneration and replacement by fibro fatty tissue. The degenerative process becomes more evident with time. During late teenage years, there is a predominance of fibro fatty tissue with only small amount s of muscle tissue left Furthermore, the ability of muscle fibers to endure eccentric contraction appears to be reduced in DMD [ 4 15 ] The abs ence of dystrophin could compromise the muscle membra ne integrity particularly after sustained contractions. Membrane fragility results in the accumulation of cytoplasmic proteins that are normally not present in muscle fibers, such as albumin and immunogl obulins. Furthermore, m uscle biopsies from DMD patients reveal that there is an increased amount of intracellular levels of calcium and hypercontracted muscle fibers [ 16 ] Increase in intracellular levels of calcium for prolonged period s of time activates proteases, especially calpains. Consequently, there is additional damage to the muscle membrane escalating calcium entry. Additionally, it has been documented that CD4+ cells promote the pathology of the dystrophic muscle. [ 17 ] Therefore, depletion of these cells or macrophages significantly reduces the pathology in dystrophic muscle. In order to void the infl ammatory effects, corticosteroids, like prednisolone and deflazacort, have
21 been used. These drugs slow down the disease progression, prolong ambulation, and forestall development of scoliosis [ 18 19 ] In additio n, decreased production of nitric oxide (NO) has been shown to escalate the pathophysiology in DMD. [ 20 ] In skeletal muscles, nNOS is bound to the DGC complex through a protein called syntrophin. [ 21 ] A bsence of dystrophin leads to disruption of DGC complex. The DGC complex is r esponsible for recruitment of neuronal nitric oxide synthase (nNOS) to the sarcolemma to produce a signa ling molecule called nitric oxide (NO). [ 22 23 ] The refore, absence of dystrophin leads to removal of nNOS from the sarcolemma, which in turn attenuates NO production. Attenuation of NO p roduction leads to functional ischemia due to impaired vascular relaxation in DMD. [ 24 26 ] In order to study potential therapeutic agents various anima l models have been developed in laboratories. Since many experimental studies in dystrophy use animal models in the laboratory setting a thorough explanation of different animal models is warranted. A nimal Models of Muscular D ystrophies With more underst anding of underlying genetic causes of various diseases it has be come easier to develop knockout animal models. These knockout animal models have been helpful in providing insights about the pathophysiology of various diseases. The genetic models to study muscular dystrophy cover a wide range of species. The dystrophin gene is highly conserved among various species. Homologues of DMD have been identified in both vertebrates and invertebrates. There is a mounting pressure for different ther apies to be developed, including gene and drug therapies to treat DMD. In order to study potential therapies several animal models of muscular dystrophy have been used in laboratory settings. Caenorhabiditis elegans (C. elegans) is the first multicellular organisms in which the genome has been sequenced
22 completely [ 27 ] It was discovered that approximately 65% of human disease genes have their counter parts in C. elegans [ 28 ] C. elegans move by contraction of longitudinal striated muscles which express a dystrophin homologue called dys1 [ 29 ] This model has an advantage over other models in that they are readily available and can be grown in laboratory settings This mak es them ideal for genetic and pharmacological studies. The second model is the Drosophilla melanogaster or the fruitfly This model is one of the most commonly used animal models to study neuromuscular development and function. Drosophilla muscle cells share many molecular, cellular and physiological features of vertebrate muscles. About 75% of all known human diseases have fly homologues [ 30 ] Furthermore, the drosophila model of dystrophinopathies has been helpful in providing new insights into the function of the d y strop hin dystroglycan complex. Insights gained from drosophila genetic system could therefore be applied to vertebrate systems. The third model to study DMD is the murine model, the mdx mouse. This model is one of the most commonly used animal models for Duchen ne muscular dystrophy. Loss of dystrophin occurs as result of a point mutation in exon 23 of the dystrophin gene [ 31 ] Although, the mdx mouse has proved to be a valuable model for understanding the underlying mechanisms responsible for muscular d ystrophy it has been criticized, since the model presents a mild phenotype with the mice living nearly a normal life span. Progression of the disease is thus much milder as compared to humans [ 32 ] This has been attributed to the presence of 1) a dystrophin homologue, Utrophin, 2) better calcium homeostasis and 3) presence of revertant fibers. Subsequent to the discovery of the mdx mouse a number of other mutant mice have been created. m dx 2 cv mdx 3 cv mdx 4 cv and mdx 5 cv were produced
23 following administration of a mutagen to C57BL6 mice [ 33 ] Later on it was discovered that mdx 2 cv and mdx 3 cv resulted fro m mutation in intron 42 and 65 respectively [ 34 35 ] On the other hand mdx 4 cv and mdx 5 cv resulted from mutation in exon 53 and 10 respectively. Furthermore, in order to look at the efficacies of therapeutic strategies laboratories have been interested in a more severe model of DMD. As a result, mice lacking both dystrophin and utrophin (utrn / / mdx ) were genetically engineered. These mice show more similar progression to Duchenne mu scular dystrophy. For example they have a reduced life s pan ( between 4 and 20 weeks), suffer from severe muscle weakness with joint contractures, grow th retardation, kyphosis, and show signs of cardiomyopathy. This model suggests that both dystrophin and utrophin play complementary roles. Finally, t he c anine model includes the gold en retriever (GRMD), Rottweiler German shorthaired pointe [ 36 ] Labrador retriever, Cocker spaniel, and Tibetian spaniel. These models have been studied both at a clinical and molecular level. The Golden retriever is the m ost commonly studied canine model of muscular dystrophy. Several studies have defined clinical signs of GRMD [ 37 38 ] suggesting that the pathogenesis of GRMD is similar to DMD. There is a high mortality rate in GRMD dogs due to selective muscle degeneration. Surviving dogs show signs of muscle degeneration followed by muscle regeneration and an inflammatory response [ 39 ] GRMD dogs develop a stiff gait and atrophy of trunk and temporalis muscles occur. Affected dogs di splay hyperextension of carpal joints and flexion at tibio tarsal joints. Furthermore, because of phyrangeal muscle invo lvement they show signs of excessive drooling.
24 Limitations of canine models : Although the GRMD model provides valuable insight about the pat hology of muscular dystrophy, for practical reasons like availability and high cost of maintenance it is unlikely to replace the mdx model H owever, dogs can be used as a translational model between human subjects and the murine model. They can be used to assess the feasibility of applying technologies to human subjects that have been validated in mouse model. Approach es to Treatment of Muscular Dystrophy In 1987, the underlying genetic defect causing DMD was identified. However, the specific mechanism of myofibre damage is still not clearly defined [ 40 ] and there is still no effective tre atment for DMD. Therapeutic approaches for DMD fall into two primary c ategories gene therapy and pharmacological therapy Restoration of dystrophin : The fundamental cause of pathology is the absence of dystrophin. Therefore, the bulk of therapeutic approa ches are aimed at restoration of a functional version of dystrophin protein. Various experimental approaches that have been undertaken to achieve this goal include gene repair [ 41 42 ] modification of splicing [ 43 44 ] and gene transfer [ 45 46 ] Stop mutations account for about 5% to 15 % of all DMD cases. These mutations occur when deletion of substitution of individual DNA bases leads to the formation of premature stop codon. Such signals terminate the translation of protein, which is then rapidly de graded. Administration of aminoglycoside antibiotic gentamicin leads to read through of the stop mutation in the mdx mouse and production of sufficient amount of dystrophin protein. [ 47 ] A similar study was performed on the DMD patients. [ 48 ] Al though there was no clear clinical benefit, the DMD patients demonstrated reduced levels of serum creatine kinase. Treatment of the patients harboring nonsense mutation with gentamycin promotes production of dystrophin gene,
25 however, the lack of potency an d the need for intravenous and intramuscular gentamycin have limited the clinical usefulness of this approach. [ 49 ] On the other hand, Ataluren, a small molecule appears to read thro ugh at much lower doses and has less toxic side effects than gentamycin. [ 50 ] Muscle growth: Increasing the muscle mass is yet another aspect that have gained en ormous focus from scientists around the world. Myostatin is a member of transforming growth factor inhibitor of skeletal muscle growth. [ 51 ] The deletion of myostatin in mice induces an extensive growth of skeletal muscle mass. This increase has been attributed both to hypertrophy and hyperplasia. [ 51 52 ] The double muscling phenotype of a few cattle and sheep breeds is caused by mutations in their myostatin gen es. [ 53 54 ] Therefore, the most important function of myostatin is the regulation of skeletal muscle growth. Furthermore, myostatin appears to initiate signaling by directly binding to its serine/threonine kinase receptor. [ 55 56 ] Downstream of the receptors, Smads are first demonstrated to be mediators of signals for myostatin. [ 55 56 ] Anti infl ammatory strategies : The ultimate treatment of DMD includes repairing the defective gene through various therapeutic agents. Considering the large size of dystrophin protein it is a daunting task to introduce dystrophin protein through various vectors. Fur thermore, gene therapy may be able to target individual or group of muscles however to treat DMD a more systemic approach is required. Pharmacological approaches involving systemic delivery are more likely to enhance muscle function globally and thereby in crease patient survival. Corticosteroids are routinely utilized in clinics for DMD, but there are varieties of alternative compounds that are being tested,
26 either in mdx mice or in limited human clinical trials. Currently, Prednisone and deflazacort are the sole available therapies for DMD. An improvement in functional muscle strength and a sustained slowing of disease progression for up to three years has been observed with oral prednisone [ 57 ] Another study showed that DMD children responded well to an intermittent low dosage regime of prednis one [ 58 ] Both prednisone and deflazacort are associated with adverse side effects like weight gain. However, these side effects appear to be less severe with Prednisone. Furthermore, different immunosuppressive drugs have demonstrated benefits in animal models, resulting in increasing recognition for treating inflammation in DMD. Various anti inflammatory drugs l ike cyclosporine [ 59 60 ] pentoxyfylline [ 61 62 ] Oxatomide have s hown to improve muscle strength in animal models. However, a clinical trial with oxatomide in DMD has showed only minor benefits. Another alternative approach that has been undertaken is to reduce muscle necrosis. This approach involves targeting tumor nec rotic factor (TNF alpha), a key pro inflammatory cytokine with neutralizing antibody infliximab [ 63 ] Furthermore the protective effect of TNF alp ha blockade has been bolstered by other studies as well [ 64 ] In addition, i t has been documented that high levels of reactive oxygen species can damage skeletal muscle s [ 65 ] On the other hand a ntioxidants such as Coenzyme Q1 0 and green tea extract have been shown to prevent the damage caused by reactive oxygen species [ 66 67 ] Furth ermore, ab sence of dystrophin causes damage to sarcolemmal damage Thi s leads to increase in calcium ions influx thus initiating a series of cellular events including activation of proteases leading to further muscle damage. Studies have shown that protease inhibitors help in reducing the pathological conditions seen in mdx muscle [ 68 7 0 ]
27 Although there are many exciting new discoveries in the therapeutic approaches however they are still in experimental stage and most of them have been developed and tested in the mdx mouse. Scientist face number of concerns associated with translating the studies from small animals to DMD patients including ( 1) Muscle size : In order to effectivel y deliver gene in DMD patients a grid of injections will be required spaced not further than 1 2 cm apart. On the other viral and non viral vectors have been administered via arterial [ 71 72 ] and intravenous route [ 73 74 ] ( 2) Immunological response: M ost of the vectors that show promising results in murine models are based on the viruses that commonly infect man. As a consequence humans tend to develop immune response to previous trials on any treatment involving these vectors [ 75 ] Summary The muscular dystrophies represent a heterogeneous and devastating group of neuromuscular disorders. DMD is one of the most common of the muscular dystrophies affecting male children. The underlying cause of DMD is absence of the dystrophin protein. Recent discoveries have opened new avenues in both gene and drug cased treatment strategies. T he ultimate treatment for DMD lies in the systemic introduc tion of dystrophin gene in muscles of DMD boys. However, it is difficult to predict when this treatment will be available. In the meantime, focus has been on the novel pharmacological drugs to improve muscle function. However, current drug selections for D MD patients are primarily limited to corticosteroids. Different novel treatments have show n promising re sults in animal models of DMD and are progressing to wards clinical trials.
28 CHAPTER 2 NON INVASIVE TECHNIQUES FOR ASSESSMENT OF MUSCLE DYSFUNCTION Throughout the present work on both boys with DMD and animal model s of DMD, emphasis has been laid on characterizing the lower extremity muscles using non invasive techniques. Traditionally, invasive techniques such as muscle biopsy have been u sed in order to study skeletal muscle properties as well to study pathological processes in DMD. However, different non invasive techniques have gained momentum in assessment of muscle dysfunction. Computed To mography CT is a cross sectional technique th at uses ionizing radiations. In CT, an X ray beam is projected through the patient at different orientations and attenuation of X rays is measured using gas ionizing chambers. Unlike radiography, CT has superior contrast resolution and it can distinguish b etween bone, tendons and ligaments, muscle, and fluid. Since CT can differentiate between normal and pathological muscle it can be used is assessment of DMD progression. In 1977, Doherty et al used CT to scan skeletal muscle in DMD patients. Muscle CT sca ns can be used to assess nature and extent of lesions. Although CT has a clear edge over radiography but the spatial resolution in other modalities give clearer and artifact free images. Besides computed tomography (CT), other techniques such as Ultrasound (US), magnetic resonance imaging (MRI), as well as EMG, are frequently used in the diagnosis of neuromuscular disorders Ultrasound Ultrasound has been used in medical practice since the early 1950s. Wild and colleagues [ 76 ] discovered the ability o f high frequency ultrasonic waves to visualize living tissues After they showed its application to visualize the living tissue, use of
29 ultrasound rapidly expanded leading to its widespread use especially in the field of medicine because of its non invasiv eness nature. In 1980, it was first discovered that affected muscles showed a different appearance on ultrasound compared to healthy muscles. Subsequently, several studies have established high sensitivities and specificities of ultrasound in the detection of neuromuscular disorders [ 77 ] [ 78 79 ] Ultrasound uses longitudinal sound waves with the frequency of 2 12 MHz above the range of human hearing. U ltrasound techniques have been vastly improve d, resulting in display of muscle tissue with resolutions up to 0.1 mm. Pathological changes, which disrupt normal muscle architecture, will increase the number, and alter the orientation of reflective surfaces within a muscle, leading to an increase in th e ultrasound echo. As well as an increase in echo from abnormal muscle, ultrasound may show a reduction in the intensity of the reflection from underlying bone and may be used to measure muscle bulk, atrophy and hypertrophy [ 80 ] DMD patients demonstrate normal muscle thickness with in creased muscle echo intensity [ 80 81 ] Further, within the muscle the echo intensity has a homogenous i.e. fine granular distribution and the proximal muscles have the highest echo intensities. Moreover, these echo intensities can become so high that the bone echo is diminished or absent in the later cases. The severity of the ultrasound findings has been reported t o relate to age and clinical severity of the disease [ 82 ] M agnetic Resonance Imaging (MRI) MRI forms an integral component of present work and is used to monitor the changes occurring in DMD patients as well as mdx mouse. Magnetic resonance imaging (M RI) is an imaging technique, used to produce high quality images of the inside of the human body, involving magnetic fields and radiofrequency (RF)
30 electromagnetic waves. Although, the MR was discovered in 1946 its clinical application was recognized in 19 77. Since then, MRI has been considered a useful tool. MRI can produce images with excellent contrast between soft tissues and high spatial resolution in every direction. Furthermore, the radiation has low energy and appears to be safe under normal operat ing conditions. Basics of Magnetic Resonance The key co mponents of any magnet include a static magnetic field, which is sustained by a liquid helium cooled, super conducting electromagnet. The field strength (B) is measured in the unit Tesla (T), which is equivalent to 10,000 Gauss (G). The field strength of the magnet is the strongest at the convergence of the magnetic fi eld lines i.e. in the middle of the magnet bore. Furthermore, the s ignal strength is directly related to the field strength. The stronger the B 0 is, greater the signal strength will be. The majority of the work presented in this dissertation was collected at 3.0 T and 4.7T, Electromagnetic gradient coils, which are used for spa tial encoding of the signal, and Radio frequency (RF) coil, which is used to excite and receive the NMR signal Properties of Atomic Nuclei The n ucleus accommoda tes most of the elemen tal mass of the atom and consist s of neurons and protons. Nuclei with an odd number of neutrons or protons possess spin angular momentum. The nuclei of certain elements, such as Hydrogen and Fluorine, have these properties. Every one of these nuclei with an odd number of protons or neutro ns can be used for imaging in MR. Spin It is the fundamental property of the protons that forms the core of MRI. Two or more particles with spins having contrasting signs can pair up to eliminate the signs of spin In nuclear magnetic resonance, it is u npaired nuclear spins that are of
31 importance. When a particle is placed in a magnetic field of strength B, it can absorb a photon, of frequency. This frequency depends on the gyro magnetic ratio, of the particle and is denoted by equation: = B where is the gyromagnetic ratio. For hydrogen, = 42.58 MHz / T. Radio Frequency ( RF ) pulse RF pulse of a specific frequency is delivered to the patient Before the RF pulse is transmitted, all the spins are aligned with the axis of external magnetic field B 0 By convention, we apply RF pulse in the X direction and for this reason the vector ends up along Y axis (Fig ure 2 1) When RF pulse is transmitted along the X axis the protons start to precess about the axis of that magnetic field at frequency o The protons precess about the axis of B 0 RF pulse also has a magnetic component that is in the direction of X axis. As soon as RF pulse is switched off, there are two basic phenomenon that occur namely, 1) T 1 relaxation and 2) T 2 relaxation. Relaxation Times Longitudinal ( T 1 ) relaxation time : The term relaxation means that the spins are relaxing back to their lowest energy state or back to equilibrium state. Right after the excitation, there is no longitudinal magnetization as it has been tipped into the transverse plane (Fig ure 2 2) As soon as the RF pulse is turned off, the protons give up all their excess energy and re align themselves with axis of B 0 T 1 is called longitudinal relaxation time because it refers to the time it takes for the spins to re align along the longitudinal axis. It is also called spin lattice relaxation time because it refers to the time it takes for a spin to give off excess energy to the surroundings in order to return to their equilibrium state. After the RF pulse is turned of f:
32 The M xy component of the vector decreases and the M z component recovers along the z axis (Fig ure 2 2 ) The recovery of longitudinal magnetization (M Z ) can be given by the formula: M Z (t) = M o (1 e t/T1 ) (2 1) where T 1 is the exponential time constant that is required for longitudinal component to recover to 63% of its equilibrium alignment. Transverse ( T 2 ) relaxation time : After the protons have been tipped into the transverse plane, the longitudinal magnetization vect or recovers and the transverse vector M xy decays. This decay rate is characterized by T 2 relaxation or spin spin interaction. It includes exchange of energy among neighboring spi ns and is given by the formula: M xy (t) = M o e t/T2 (2 2) T 1 recovery a nd T 2 decay are two independent processes and T 2 decay occurs 5 10 times more rapidly than T 1 recovery. This is because of a phenomenon called dephasing. Dephasing is a phenomenon by which the spins, which are in phase after a 90 o pulse gets out of phase. Two underlying causes are responsible for dephasing ( A ) Interaction between Individual spins: When two spins are in close proximity to each other, magnetic field of one proton affects the proton next to it. Furthermore, the proton aligned with B 0 creates s lightly higher magnetic field for its neighboring proton. On the contrary, the proton that points against B 0 experiences slightly less magnetic field. This interaction is inherent property of every tissue and is measured by T 2 Therefore, the spin spin i nteraction is the first cause of dephasing. ( B) External magnetic field inhomogeneity: Because of external magnetic field inhomogeneity, each spin is
33 exposed to slightly different magnetic field and therefore the protons precess at different frequencies. Unlike T 1 and T 2 which are inherent properties of the tissue, TR (the repetition time) and TE (echo time) are under operators control. By apropriate setting of 1 or T 2 ( Figure 2 3, 2 4) TR ( the repetition time ) Th e time interval between application of two RF pulses is called TR ( Repitition time) (Fig ure 2 5 ) After application of a 90 o pulse, the transverse component of the magnetization vector M xy decays much faster than the longitud i nal component M Z Between the application of two RF pulses the following processes take place: (A) Immediately before a 90 o RF pulse is applied, the magnetization vector points along z axis. (B) Immediately after a 90 o RF pulse, the magnetization vector is tipped in the xy plane. (C) After time t = TR another 90 o RF pulse is applied. By this time some of the magnetization vector has recovered in z direction. However, most of the magnetization vector is lost in transverse direction. Therefore the magnitude of the magnetization vector al ong z axis is given by : M z (TR) = M o (1 e TR/T1 ) (2 3) (A) At time t = TR the magnetization vector (M z ) is less than the original vector M o This is because the second RF pulse is applied before complete recovery of the M z component of the mag netization vector. (B) Again after second TR the longitudnal magnetization vector will be flipped into xy plane. However this time longitudnal magnetization vector will be equal to the preceding vector. Image Formation The signal received contains information from the entire part of the body imaged. The fundamental process used to define the location of the sources of MR signal and hence identify the specific body part imaged, is by application of magnetic field
34 gradient s. In a homogeneous magnetic field, water protons resonate at the same frequency, regardless of location. If a second magnetic field is now superimposed upon the main magnetic field, a predictable variation is observed in the magnetic field along a predete rmined axis. The resulting magnetic field is highest at one end of the gradient and lowest at the other; between are intermediate values along the axis of the gradient. As a result of application of gradient field, protons at one end of the gradient spin s lower and protons at the other end spin faster. Gradients therefore create temporary inhomogeneties in the main magnetic field to obtain spatial information. These signals from the protons can now be measured and used to construct images. Depending on the function gradients are referred to as the slice select gradient (G x ) the frequency encoding gradient (G y ), and the phase encoding gradient (G z ). The three basic steps in an imaging process are discussed below: Slice selection This process involves use of G x during application of an excitation or refocusing pulse. Application of a gradient causes tipping of protons only in a specific slice A slice is selected by simultaneous application of a selective RF pulse and gradient alon g the z axis such that alignment of protons in a specific width of tissue is disturbed. At the end of the brief RF pulse, specific spins that are exposed to a specific gradient in a sample are excited. Magnetization vectors representing out of phase locati ons remain undisturbed. Although the signal is obtained from the entire slice, the slice image is not yet seen because of lack of in plane spatial information about the slice. In order to gather that information the signals are phase and frequency encoded. Further depending on the frequency bandwidth, slice thickness can be established
35 Phase encoding This step involves application of magnetic field gradients and therefore image construction in the y direction. In order to get spatial information in the y direction, a gra dient is applied in the same direction. The phase encoding gradient is turned on before application of the frequency encoding gradient (G x ). It is usually applied right after the RF pulse or just before the G x gradient or anywhere in betw een. Because the gradient brings about a change in the phase of proton spins this process of Frequency encoding Frequency encoding conventionally defines application of magnetic field gradients and hence ima ge construction in the x direction. This process occurs after the application of G y and it is during this cycle that the signal is recorded as a function of time. The different frequencies are summed to form a signal that is detected by the receiver coil. MRI Applications in Skeletal Muscle T he availability of magnetic resonance imaging (MRI) scanners offers a variety of methodologies to monitor musculoskeletal structure and function. MR is considered one of the best imaging modality to perc eive soft tissu e abnormalities. It provides three dimensional high resolution information which can be used to measure muscle size i.e muscle cross sectional area as well as muscle volume, monitor muscle damage, and intramuscular fat. In addition, i t is used to evaluate the presence or absence of muscle edema, its extent and also potential sites for muscle biopsy [ 83 84 ] T 1 weighted, T 2 weighted and Short Tau Inversion recovery (STIR) are t he most common form of imaging technique used for skeletal muscle imaging. T 1 weighted image s are used to detect contrast between s keletal muscles and fat, therefore can provide valuable information regarding fat ty tissue infiltration in the skeletal mus cles. On the other hand,
36 both STIR and T 2 weighted images are used in detecting edema and are therefore referred to as fluid sensitive sequences. Skeletal muscle function depends on the morphological properties of the muscle [ 85 87 ] Muscle size measurements include anatomical and physiological cross sectional area (CSA), muscle thickness and length, and muscle volume. Methods like limb circumferenc e, ultrasonography and dual X Ray absorptiometry (DEXA) have been used to assess muscle size. These measurements however, do not clearly differentiate between contractile and non contractile tissue. Consequently, inclusion of non contractile tissue like in tramuscular fat and connective tissue in muscle size measurements tend to overestimate muscle size. MRI can differentiate between contractile and non contractile tissues within a muscle and can image the entire length of more than one muscle at the same ti me [ 88 90 ] Numerous studies have used MRI as a standard technique to assess skeletal muscle size following disease processes [ 89 91 ] progression of muscle atrophy following disuse [ 92 ] and effects of interventions [ 93 ] Specifically, i n DMD patients T 1 weighted scans are normal during early stages of life [ 94 ] Subsequently they may show a characteristic pattern of muscle involvement that evolves over time. Furthermore, d uring the ambulant stages [ 94 95 ] children with DMD exhibit abnormal signals in the gluteus maximus and adductor magnus, followed by quadriceps, rectus femoris and biceps femoris, with selective prese rvation of the sartorius, gracilis, semitendinosus and semimembranosus [ 90 96 ] In the lower leg, peroneals, gastrocnemius and soleus are more severely affected than the other muscle groups [ 97 ] However, use of T 2 and short T 1 invers ion recovery imaging will show
37 edema and/or inflammation in other muscles that are spared from fatty infiltration and have normal signal intensity on T 1 weighted imaging. These findings are of interest because they suggest that an inflammatory component plays a significant role in the early phases of muscle damage, before the muscle is replaced by fat or fibrotic tissue. The transver se relaxation time constant (T 2 ) is a quantitative measure of a basic biophysical property that leads to signal contrast on MRI. Furthermore, m uscle T 2 relaxation properties are sensitive to muscle contraction and therefore possess widespread applications in studying muscle characteristics. Following the first study by Fleckenstein in 1988, which reported that there is increase in signal intensity of skeletal muscle T 2 after a bout of exercise [ 98 ] numerous s tudies have successfully demonstrated positive correlations between elevated T 2 times following exercise with integrated EMG patterns [ 99 ] Furthermore, exercise induced contrast in muscles has been detected by using T 2 maps [ 86 ] Consequently, if a muscle shows more contrast, it reflects more u se of the muscle and vice versa. [ 99 ] [ 100 ] [ 86 ] This phenomenon of muscle T 2 has be en used to infer muscle activity and the extent of their contribution. Thus, T 2 changes are sensitive to the activity status of the muscle and therefore hold tremendous potential in identifying muscle activation patterns in both normal and diseased conditi ons. Although the exact mechanism for increase in T 2 has not been elucidated, studies indicate that the mechanisms of change in muscle T 2 following rest to work transitions are a consequence of increase in T 2 relaxation times of muscle water [ 101 ] [ 102 ] This is probably a result of osmotically driven shifts of water into
38 intramyocellular spaces secondary to accumulation of end products of muscle metabolis m and/or intracellular acidosis [ 103 ] [ 104 ] [ 102 ] The increase in T 2 values after normal activity is transient and has been shown to resolve within minutes to a couple of hours [ 98 ] [ 105 ] However, persistence of T 2 values for longer periods may indicate muscle damage [ 106 ] This is commonly observed following e ccentric exercise protocols [ 107 ] Several studies have reported that eccentric exercise induced muscle injury is associated with marked elevations in T 2 values in both healthy and patient populations [ 86 ] [ 103 ] [ 108 ] Elevated T 2 values fo llowing strenuous exercises correlate with markers of muscle damage including ser um creatine kinase activity muscle soreness and maximal isometric force [ 109 ] [ 110 ] Repeated MRI measurements of skeletal muscle for as long as t wo to three months following eccentric exercise continue to show elevated T 2 relaxation times; suggesting of a long lasting change in the muscle Intriguingly, long lasting elevated T 2 relaxation times reflective of muscle damage have also been reported following reloading after hind limb immo bilization, spaceflight and disease processes in both humans and animals [ 111 ] [ 20 112 114 ] Muscle damage following reloading is attributed to mechanical disruption of muscle fibers and inflammation in muscle [ 115 ] Frimel et al have shown strong correlations between the elevated T 2 relaxation times and histological markers of muscle damage [ 112 ] High resolution MRI has the ability to differentiate between soft tissues within the muscle using dis tinct MRI techniques. Therefore, MRI has served as a no n invasive measure to quantify and monitor disease progression within the skeletal muscle s
39 conditions [ 116 ] [ 117 118 ] Furthermore, b ecaus e of its non invasive nature MRI it can be applied repeatedly in the same subject for longitudinal studies. Furthermore, elevated T 2 values can suggest different pathological processes occurring in muscle s including damage [ 119 ] [ 120 ] [ 112 ] edema [ 103 ] [ 102 ] fibrosis and fat [ 90 121 ] investigators are now making attempts to discriminate between these physiological processes utilizing a variety of advanced MR techniques including DIXON imaging, diffusion weighted imaging, magnetization transfer contrast and spectroscopy. Thus, the scope of MR in studying skeletal muscle is on a constant rise. Summary Ever since MRI was proposed to scan three dimensional biological structures in 1972, the use of MRI to monitor changes in biological tissues has grown by leaps and bounds. This chapter reviews the fundamental concepts of MRI and compares different non invasive techniques. MRI has become the tool of choice because of its non invasive nature. Furthermore, use of T 1 and T 2 parameters are discussed to monitor physiol ogical changes occurring in skeletal muscles.
40 Fig ure 2 1 Representative three dimensional figure showing RF pulse application. Fig ure 2 2 Schematic representation of 90 degree RF pulse application and its consequence
41 Fig ure 2 3 Schematic diagram showing T 1 recovery curves of f at and muscle Fig ure 2 4 Schematic figure showing T 2 decay curve for muscle and damage
42 Figure 2 5. Time interva l between two successive 90 degree RF pulses denoted by TR.
43 CHAPTER 3 OUTLINE OF EXPERIMEN TS The overall objective of this dissertation was to monitor the pathological variations occurring in the skeletal muscles of DMD patients and the mdx mouse, a murine model of DMD. An outline of the aims and hypotheses related to each experiment is given below. Experiment One Specific Aim s 1. Quantify the CSA and torque production of primary lower extremity muscles in ambulatory boys with DMD 2. E xamine the relationship of age with muscle strength, CSA, and specific torque in boys with DMD and compare these relationships with controls 3. Examine the relationship of lower limb muscle strength with functional ability in boys with DMD. Hypotheses 1. Boys with DMD hav e larger CSA of all l ower limb muscles bu t lower torque production com pared with healthy controls. 2. Increase in muscle CSA and torque production with age is lower in boys with DMD compared with controls. 3. Torque production of the lower extremity muscles is related to performance on functional tasks such as walking and transfers in boys with DMD. Experiment Two Specific Aim s 1. To assess contractile and non contractile content in individual muscles of the lower leg in DMD and age matched healthy controls. 2. To evaluate the relationship between the proportion of non contractile content in leg muscles and functional ability.
44 Hypotheses 1. The non contractile content in the lower leg muscles of boys with DMD is higher than in age matched healthy controls. 2. The non contractile content in leg muscles is positively correl ated to functional ability in boys with DMD. Experiment Three Specific Aim To monitor the progression of dystrophic alterations with age in the hindlimb muscles of mdx mice using Transverse rel axation (T 2 ) time constant. Hypothesis Transverse relaxation (T 2 ) can be used to assess pathological changes occurring in the hindlimb muscles of mdx mice with age. Experiment Four Specific Aim s 1. To monitor the response of muscle T 2 over a time course of 48 hours after a single bout of downhill running in mdx mice to wild type (control) mice 2. To compare the effects of horizontal vs. downhill running on muscle damage assessed by T 2 MRI in mdx mice 3. T o compare the effects of downhill running on the lower anterior vs. posterior hindlimb muscles of mdx mice. Hypotheses 1. After a single bout of downhill running muscles of mdx mice demo nstrate a greater increase in T 2 than control mice. 2. A single bout of dow nhill running instigates a greater increase in T 2 than horizontal running in mdx mice. 3. The anterior compartment muscles of mdx mice display greater increase in T 2 than the posterior compartment after a bout of downhill running.
45 Experiment Five Specific A im To monitor the response of muscle T 2 on cardiac muscle after a single bout of uphill running in mdx mice to wild type (control) mice. Hypotheses 1. Myocardium of mdx mice is more susceptible to activity induced muscle injury than wild type mice. 2. T 2 is a sensitive marker of myocardial damage in mdx mice.
46 CHAPTER 4 RELATIONSHIP BETWEEN LOWER LIMB MUSCLE CROSS SECTIONAL AREA AND TORQUE PRODUCTION IN BOYS WITH DUCHENNE MUSCULAR DYSTROPHY 1 Boys with DMD experience profound muscle weakness and functional limitations as early as ages 3 to 5 years of age. The progressive decline in muscle function is first observed in the proximal muscle groups (e.g., shoulder girdle, hips), with the distal muscle groups being affected at a later age. Manual muscl e testing (MMT) has been used to look at the age related changes in strength in DMD children [ 122 123 ] and this method has also been used in clinical trials. However, the MMT has been criticized for having limited reliability, accuracy and sensitivity; therefore other muscle testing meth ods such as dynamometry are preferred for assessing muscle strength. A hallmark of muscle quality is the amount of force produced per unit of muscle mass [ 124 ] A recent study on boys with DMD showed only low to moderate correlations between regional lean body mass measured using Dual energy X ray absorptiometery (DXA) and muscle strength [ 125 126 ] However, the main limitation was that DXA could not be used to quantify the size of the muscle. The cross sectional area (CSA) of individual muscles or muscle groups can be qua ntified using MRI and this technique has been used repeatedly to examine muscle atrophy after disuse [ 87 ] spinal cord injury [ 127 ] and in other chronic conditions [ 128 ] 1 Sunita Mathur, Donovan J Lott, Claudia Senesac, Sean A Germain, Ravneet S Vohra, H Lee Sweeney, Glenn A Walter, Krista vandenborne Age related differences in lower limb muscle cross sectional area and torque production in boys with Duchenne muscular dystrophy. Arch Phys Med Rehabil. 2010 Jul;91(7):1051 8 ( Used with permission from the American congress of Rehabilitation Medicine).
47 Specific Aims The primary objectives of this study are: 1. To quantify the CSA and torque production of primary lower extremity muscles in ambulatory boys with DMD. 2. To examine relationship of age with muscle strength, CSA and sp ecific torque in boys with DMD and compare these relationships with those in healthy age matched controls. 3. To examine the relationship of lower limb muscle strength with functional ability in boys with DMD. Hypotheses Our hypotheses are as follows: 1. All l ower limb muscles in boys with DMD have larger CSA but lower torque production compared with healthy controls. 2. Torque production of the lower extremity muscles is related to performance on timed functional tasks in boys with DMD. Research Design and Metho ds Participants Twenty two boys with DMD and 10 healthy boys from the general population (controls) aged 5 to 14 years old volunteered to participate in the study. A report confirming the diagnosis of DMD using molecular genetic testing ( e.g. polymerase chain reaction amplification) and/or immunohistochemical staining of muscle biopsy was and were being treated with corticosteroids (either prednisone or de flazacort). The study was approved by the institutional review board at the University of Florida. Written were measured in light clothing with shoes off.
48 Methods Isometric muscle strength testing : Isometric peak torque of the KEs, PFs, and DFs of the right leg was measured using a Biodex dynamometer 2 For KE testing, the knee and hip joints were placed at 90 of flexion. For PFs and DFs, the knee was placed between 0 to 10 of flexion and the ankle was placed in neutral (for PFs) or 30 of plantarflexion (for DFs). The subject was instructed to push or pull as hard as possible for 5 seconds, followed by a 2 m inute rest. Five trials were performed for each muscle group, and the highest torque value was used for analysis (peak torque). One subject with DMD was unable to perform the dorsiflexion contraction. This protocol was tested for between day reliability (2 month interval) by the same tester, in a subset of children with DMD (n=6) and controls (n=10). ICCs were calculated for both groups of subjects. High reliability was found for all 3 muscle groups (KEs: ICC=.89 in DMD and .99 in controls, PFs: ICC=.88 in DMD and .98 in controls, and DFs: ICC=.87 in DMD and .95 in controls P<.001 for all ICCs). Because the body size of boys between the ages of 5 and 14 years varies considerably, peak torque was normalized to BSA. BSA is widely used as a biometric unit to no rmalize physiologic parameters. Hence for each of the functional muscle groups, torque normalized to BSA (ie, normalized torque; units=N*m/m 2 ) was reported. BSA (m 2 ) was calculated using the equatio n developed by Gehan and George [ 129 ] and modified by Mosteller [ 130 ] 2 Biodex Medical Systems, 20 Ramsay Rd, Shirley, NY 11967 4704.
49 Muscle CSA : MRI was performed on either a 1.5T (Signa) 3 or 3.0T (Achieva) 4 whole body s canner. Subjects were placed in a supine position with their lower leg positioned in either a lower extremity quadrature coil (1.5T) or an 8 channel sensitivity encoding, receive only extremity coil (3.0T) for lower leg imaging or a flexible surface coil for thigh imaging. Thigh imaging was not completed in 2 of the control subjects and 2 of the boys with DMD. Three dimensional transaxial fat suppressed gradient echo images were acquired with the following parameters: repetition time=24ms, echo time=1.8ms, flip angle=20, and optimized field of view (calf: 12 14cm 2 thigh: 18cm 2 ). Individual muscles (quadriceps, soleus, medial, and lateral gastrocnemius and tibialis anterior) were manually outlined using OsiriX 5 an open source software ( Figure 4 1) [ 131 ] To determine the CSA max of a muscle, the axial image with the largest CSA in the series was identified and the mean of 3 consecutive images (ie, the slice with the greatest CSA and the immediate proximal and distal slices) was used to obtain the valu e for CSA max The CSA max of the triceps surae muscle group was taken as the sum of the individual CSA max of the soleus, medial, and lateral gastrocnemius muscles. The CSA max of the quadriceps was taken as the sum of the CSA max of the 3 vasti together and t he rectus femoris. Specific torque : The force generating capacity of the muscle per unit area (ie, specific torque; units=Nm/cm 2 ) was calculated by dividing the isometric peak torque by the CSA max measured by MRI. KE peak torque was divided by quadriceps CSA max 3 Signa; GE Medical Systems, 3000 N Grandview Blvd, Waukesha, WI 53188. 4 Achieva; Phillips Medical Systems, 3000 Minteman Rd, Andover, MA 01810 1099. 5 OsiriX Imging Software, available from: http:www.osirix viewer.com/
50 while the PF torque was divided by the triceps surae CSA max and DF torque by the tibialis anterior CSA max Functional ability : Subjects performed 4 timed functional tasks, in the foll ow ing order: time to rise from the floor, rise from a chair, walk 9m and ascend 4 stairs [ 132 ] The subject was given rest breaks between tasks to minimize the effects of fatigue. Subjects performed each task 3 times, and the best time was used for analysis. The functional ability of the DMD subjects was ranked using the Vignos Lower Extremity Functional Scale [ 133 ] This scale ranges from grade 1 (able to walk and climb stairs independently) to grade 10 (confined to bed). Main outcome measures : Maximal muscle cross sectional area (CSA max ) assessed by MRI of the quadriceps, plantar flexors and dorsiflexors, peak isometric torque from dynamometry, and timed functional tests. Subjects will perform 4 timed functional tasks: time to rise from floor, rise from chair, walk 9m and ascend 4 stairs [ 132 ] Statistical Analysis 1. To examine differences in muscle strength, muscle CSA max and specific torque between boys with DMD and controls, independent sample t tests was used after 2. Analysis of covariance was used to test for differences between groups in the slopes of the relationships between age and muscle strength as well as muscle CSA max and specific torque. The level of significance for between group comparisons is set at an alpha of 0.05 and adjusted for multiple comparisons using the modified Bonferroni correction. 3. To examine the relationships between functional test times and muscle strength, bivariate li near correlations was made using Pearson product moment correlations (alpha=0.05).
51 Results Boys with DMD were shorter in stature and had a higher body mass index than controls (Figure 4 7 ); however, BSA was similar between groups. No significant difference s were noted for age between the 2 groups. The lower extremity Vignos score ranged from grade 1 (independence in walking and climbing stairs) to grade 4 (walks unassisted and rises from chair but cannot climb stairs) in boys with DMD. Muscle Strength : Peak torques of the PFs, DFs, and KEs were significantly lower in boys with DMD compared with controls (PFs: 31.313.8Nm vs 84.952.1Nm; DFs: 10.93.2Nm vs 24.612.3Nm; KEs: 20.57.9Nm vs 79.546.6Nm in boys with DMD and controls, respectively; P<0.001). The s lopes of peak torque versus age ( Figure 4 2) for all three muscle groups were also significantly different between boys with DMD and controls (P<0.001). The difference in slopes between the DMD and control groups are the result of a considerable gain in mu scle strength as a function of age in the control group, which was largely absent in boys with DMD. In the control group, peak torque was 3 to 4 times greater in all muscle groups across the age range. In boys with DMD, the PFs and DFs showed only a slight increase in peak torque with age, while KE peak torque did not vary with age. The difference in peak torque between groups was also examined after normalization to body size, using BSA, which incorporates the age, height, and weight of the child. [ 129 130 ] Normalized peak torque of the KE and PF was 3 times highe r in controls than boys with DMD (KE: 64.227.3Nm/m 2 vs 20.97.8Nm/m 2 P<0.0021; PFs: 68.630.2Nm/m 2 vs 20.9 7.8Nm/m2, P<0.001). Normalized peak torque of the DFs was twice as high in controls as DMD subjects (DFs: 20.4 6.4Nm/m 2 vs 10.82.6Nm/m 2 respect ively; P<0.001). As expected, normalized torque in controls
52 showed an increase with age (slopes=0.08 8.6), while a negative slope was found in the boys with DMD in all 3 muscle groups (slopes=.22 to 1.4). Maximal CSA : The average CSA max of the triceps s urae muscle group was about 60% higher in boys with DMD compared with controls (39.113.6cm 2 vs 24.59.3cm 2 ; P<0.002). In contrast, no significant difference was found between groups in the CSA max of the tibialis anterior muscle (3.81.1cm 2 vs 4.01.5cm 2 P<0.78). Furthermore, the slopes of lines expressing muscle CSA max versus age for the triceps surae and tibialis anterior were not significantly different between boys with DMD and controls (P<0. 44 and 0.31, respectively; Figure 4 3 A and B). However, ther e was a significant difference in slope for the CSA max of the quadriceps muscle group between boys with DMD and controls (P<0.006; Figure 4 3 C). The CSA max of the quadriceps appeared larger in the boys with DMD under the age of 10 years compared with cont rols. However, in subjects aged 11 years and older, boys with DMD tended to have a smaller CSA max than controls (see Figure 4 3 C). Specific torque : Specific torque (ie, peak torque normalized to muscle CSA max ) was significantly different between groups for the PFs (0.880.41Nm/cm 2 in DMD vs 3.261.08Nm/cm 2 in controls; P<.001), DFs (2.950.99Nm/cm 2 in DMD vs 6.031.52Nm/ cm 2 in controls, P<0.001), and KEs (0.690.21Nm/cm 2 in DMD vs 2.76.77Nm/cm2, P<0.001). The specific torque of the KEs and PFs was appr oximately 4 times as high in the controls compared with boys with DMD. In contrast, a 2 fold difference was found in the DFs. The change in specific torque with age is shown in Figure 4 4 A significant difference in slope was observed between the boys wit h DMD and controls for specific torque of the PFs (P<0.001) and DFs (P<0.006). For both
53 muscle groups, the boys with DMD showed a decline in specific torque with age, whereas controls showed an increase with age. No difference was found between groups for the slope of KE specific torque versus age (P<0.78). Functional ability : Boys with DMD required significantly more time to com plete all functional tasks com pared with controls (Fig ure 4 5) (P<0.001). There was a progressive increase in the time required to complete functional tasks with age in the boys with DMD, whereas the controls were relatively consistent in their times to complete all 4 tasks. Out of the sample of 22 boys with DMD, 5 were not able to rise from supine (ages 8.8 14y) and 3 were unable to climb 4 stairs (ages 8.8 14y). In boys with DMD, significant relationships were observed between time to walk 9m and KE peak t orque (r=0.57, P<0.005) (Figure 4 6 A), as well as PF peak t orque, (r=0.46) (P<0.031) (Fig ure 4 6 B). Furthermore, torque norma lized to CSA max of the KEs was correlated to both the time to walk 9m and time to rise from a chair (r=.51 and r=0.56, respectively; P<.05, not shown). Similar correlations were seen for PF normalized torque with these 2 functional tasks (r=0.45 and r=0.48 respectively; P<0.05, not shown). Interestingly, no significant relationships were observed between the functional tasks with DF strength. Discussion The findings of this study provide new insights into the age related changes in CSA max and specific torq ue production of important lower extremity muscle groups in ambulatory boys with DMD. We found approximately 60% greater CSA max of the triceps surae muscles (soleus, gastrocnemius) of ambulatory boys with DMD compared with controls. The increase in triceps surae CSA max was observed across all ages, and in fact the slope between muscle CSA max and age was similar in boys with DMD and
5 4 controls. In contrast, the age related increase in the tibialis anterior CSA max appeared to be unaffected by dystrophy. The qua driceps muscle showed a distinct pattern with larger muscles in young boys with DMD, whereas boys older than 11 years demonstrated relatively smaller CSAs than controls. Torque production was impaired in all 3 muscle groups, including the DFs in boys with DMD, both when expressed as absolute values as well as after normalization to BSA or to muscle CSA max (ie, specific torque). Specific torque showed a modest decline with age in boys with DMD, while healthy boys showed large gains in specific torque between the ages of 5 and 14 years. The torque production of the KEs and PFs, but not the DFs, was correlated to performance on functional tasks such as walking and rising from a chair. Muscle weakness was observed in all three muscle groups in boys with DMD, alt hough KE strength deficits were observed at an earlier age. KE torque in DMD was on average 71% of controls at 5 to 7 years old and declined to 15% of controls in subjects over 11 years old. This difference was primarily the result of a 4 fold increase in KE torque of the controls with age. McDonald et al [ 132 ] reported a sharp decline in KE strength with age in boys with DMD, both by isometric testing and the MMT. In their study, boys aged 4 to 6 years had an MMT grade of 4, which corresponded to an isometric strength of 40% to 50% of control values [ 132 ] The MMT grade continued to decline with age, and isometric strength was reported to be less than 5% of controls by the age of 8 years [ 132 ] In comparison, we found that although the relative difference between controls and boys with DMD increased with age, there was little change in absolute torque production of the KE in DMD.
55 Ou r findings showed that peak torque of the PF and DF were about two thirds of controls at ages 5 to 9 years, whereas McDonald et al [ 132 ] reported an earlier loss o f DF strength com pared with PFs using the MMT. This may be attributed to differences in the position of the ankle joint in testing the DFs between studies. In the MMT, the ankle is placed at or near full DF, whereas in our study, the DFs were tested near their optimal angle for force generation (30 plantarflexion) [ 134 135 ] Between the ages of 5 and 14, we observed a 3 fold increase in PF and DF muscle strength in controls. Only a small increase in abs olute torque was observed among boys with DMD over the same age. Similarly, Beenakker et al [ 122 ] reported that the greater disparity in lower extremity muscle strength in older boys with DMD was primarily the result of an increase in strength among the controls. Another important factor to consider in m uscle strength changes with age in our subjects is that all subjects in our study were taking corticosteroids, whereas the previous study by McDonald et al [ 132 ] did not indicate the use of corticosteroids. However, at the time at which they collected longitudinal data (1982 1992), corticosteroids were not part of the standard clinical care for patients with DMD. Because corticosteroids have been shown to stabilize muscle force [ 136 ] this intervention may have contributed to an improved preservation of muscle torque in our subjects compared with the previous study. This theory is substantiated by a recent study by Parreira et al [ 137 ] in which the progressive loss in muscle strength in boys with DMD who had initiated corticosteroids was found to be less than that reported in an older study by Scott et al [ 138 ] in which the cohort of boys with DMD were not taking corticosteroids.
56 Previous MRI studies ha ve focused on examining fatty tissue infiltration, and in particular the distribution of muscle involvement in a variety of muscular dystr ophies and inherited myopathies. [ 139 142 ] We used MRI to measure the total muscle CSA max including both muscle and fat tissue, although we also observed differential fatty tissue infiltration across the lower extremity muscles ( Figure 4 1), intramuscular fat infiltration was not quantified in the current study. Although advanced MR techniques such as proton spectroscopy can be used to quantify lipid content of muscle, [ 143 ] this study was conducted to specifically measure differences in CSA max with age and to examine the relationship between muscle size and muscle strength in boys with DMD. We found that PF CSA max was larger in the boys with DMD relative to contro ls across all ages. In contrast, DF CSA max was not different between groups and showed a similar increase in size with age in both controls and DMD. For the quadriceps, however, the slope of line relating CSA max to age was lower in boys with DMD compared w ith controls. From the scatter plot it appears that young boys with DMD (under 10y) had a higher quadriceps CSA max than controls, whereas older boys with DMD (over age 10) had a lower CSA max than controls. This difference between DMD and controls must be c onsidered in light of our limited sample of young controls for quadriceps CSA max because the 2 control subjects who were under 7 years old did not participate in this measurement. Peak torque normalized to muscle CSA (ie specific torque) provides an index of muscle quality. Our findings clearly showed that increases in muscle size were not reflected by proportional increases in muscle strength in boys with DMD, consistent with a decline in muscle quality. This is similar to recent findings using DXA, which show low correlations between muscle strength and regional lean body mass in boys with DMD
57 between 5 and 13 years of age.15 In our study, specific torque was impaired in all 3 muscle groups, with the average KE and PF m uscles showing 4 times higher values in controls compared with boys with DMD. However, absolute specific torque only showed a modest decline with age in boys with DMD, while in controls; large age related gains in specific torque were noted. Interestingly, even though age related in creases in the DF CSA max mirrored those of controls, the average specific torque in controls was 2 fold higher than that of boys with DMD. In addition, the relationship between DF specific torque and age was significantly diffe rent in ambulatory boys with DMD compared with controls. These data demonstrate that while muscle growth (increase in CSA max ) may appear normal in the tibialis anterior muscle, there is a loss of muscle quality. In healthy boys, there is a sharp rise in sp ecific torque around the onset of puberty (age 13 15y ears ) [ 144 ] This rapid increase in force generating capacity may be related to increases in anabolic hormone levels [ 145 ] and stature [ 146 ] Oral corticosteroid use has been shown to decelerate growth rate in boys and sup press adrenal function [ 147 ] The subjects with DMD in this study were of shorter stature than controls, whi ch may also have contributed to a smaller gain in specific torque with age. Interestingly, we noted that there were differences in the specific torques of the different muscle groups in healthy subjects. There was a 2 fold difference between PFs and DFs in normalized peak torque: approximately 3Nm/cm 2 in PF and approximately 6Nm/cm 2 in DF. Specific tension of the DFs has been reported to be higher than PFs in healthy adults and may be partially attributed to differences in muscle architecture [ 148 ] We found that the ability to rise from the floor and climb stairs was lost in 50% of the boys with DMD over age 10, although their absolute lower limb muscle strength was
58 similar to younger boys who were still able to complete these tasks. This is most likely because of an increase in body stature with little increase in absolute muscle strength to compensate, as indicated by the lower muscle torques normalized to BSA in boys with DMD. Furthermore, we did not evaluate the strength of the upper limbs and trunk and their contribution to the ability of the boys with DMD to perform functional tasks. These muscle groups also play an important role in functional ability in boys with DMD, especially as leg muscle strength declines and compensatory movements are required to maintain independent mobility. Relationships between muscle strength and ability to walk and perform sit to stand transfers were observed in the KEs and PFs. Previous studies have also found relation ships between lower limb muscle strength and ambulation and transfers. [ 122 149 ] Furthermore, PF strength has been implicated as a key mu scle in maintaining gait in boys with DMD [ 150 ] Interestingly, we did not observe any relationships between DF muscle strength and functional ability, although a previous study [ 151 ] found that ankle DF muscle strength of less than grade 4 was a key prognostic factor in determining time to wheelchair dependence in boys with DMD. Because all subjects in our study were ambulatory, DF torqu e may not have played a role in their current walking ability. Although we observed differential fatty tissue infiltration across the lower extremity muscles ( Figure 4 1) in boys with DMD, fibrosis and intramuscular fat infiltration was not quantified in t he current study. All magnetic resonance images in this study were acquired using a standard T 1 weighted imaging sequence, with fat suppression, to facilitate segmentation of the muscle boundaries and quantification of the muscle CSA. Advanced MR technique s such as volume localized spectroscopy and
59 3 point Dixon can be used to measure the intramuscular lipid composition ; [ 88 ] however, the quantification of fibrosis in skeletal muscle presents significant challenges because of extremely short T 2 of collage n. MRI of human skeletal muscle also suffers from partial volume filling because of limited spatial resolution, adding to the complexity of quantifying non contractile tissue in muscular dystrophies. Summary Using MRI in combination with dynamometry allowe d us to measure the muscle CSA max and specific torque production of primary lower extremity muscles in children with DMD. Age related changes in muscle CSA and specific torque production in lower extremity muscles showed distinctly different patterns in t he KE, PF, and DF muscles of boys with DMD. The distal triceps surae muscles showed a larger CSA max by approximately 60% in ambulatory boys with DMD compared with controls across ages, while the tibialis anterior muscle showed age appropriate increases in CSA max In the quadriceps muscle, CSA max tended to be higher than controls in younger boys with DMD but lower in older boys with DMD over 10 to 11 years of age. However, all muscles showed significant deficits in specific torque, with a 4 fold difference i n the PF and KE, and 2 fold difference in the DFs. Specific torque showed a modest decline with age in boys with DMD, while in controls, large age related gains in specific torque were noted. Future clinical trials for therapeutic strategies in DMD should consider examining multiple muscles to evaluate the efficacy of treatment, as well as the inclusion of physiologic measures such as muscle CSA and specific torque. This study also underlines the importance of including age matched healthy controls in evalu ating boys with DMD.
60 Fig ure 4 1 T 1 weighted fat suppressed transaxial images of the thigh (A) and lower leg (B) obtained from an 11 year old boy with DMD. The rectus femoris and vasti muscles are outlined on the thigh, and the TA, soleus, MG, and LG are outlined on the lower leg. Note the presence of fatty infiltration to a greater extent in the thigh musculature than in the calf. Abbreviations: LG, lateral gastrocnemius; MG, medial gastrocnemius; RF, rectus femoris; TA, tibialis anterior.
61 F igure 4 2. Scatter plots of age versus peak torque (Nm) in boys with DMD (filled circles, solid line) and controls (open circles, dashed line ) (A) PFs (n = 22 DMD, n = 10 control), (B) DFs (n = 21 DMD, n = 10 control), and (C) KEs (n = 22 DMD, n = 10 control). Please note that in A, 2 subjects with DMD had overlapping values for plantar flexion peak torque (age 6.2y, 33.2Nm and 33.4Nm); in B, 1 subject with DMD was unable to perform the dorsiflexion torque measurement.
64 Figure 4 3. Scatter plots of CSA max (c m 2 ) versus age in boys with DMD (filled circles, solid line) and controls (open circles, dashed line )
67 Fig ure 4 4. Scatter plots of age versus specific torque (torque normalized to CSA max of muscle; Nm/cm 2 ) in boys with DMD (filled circles, solid line) and controls (open circles, dashed line)
70 Fig ure 4 5. Group means SD of 4 timed functional tasks in boys with DMD and controls. *P<0.001.
71 Fig ure 4 6. Relationships between peak torque of the KEs (A) and PFs (B) and time to walk 9m.
72 Table 4 1. Demographics of patients. Characteristics DMD (n=22) Controls (n=10) P Age (y) 9.62.7 9.73.0 0.48 Height (m) 1.20 0.13 1.400.18 0.002* Weight (kg) 31.311.2 33.111.6 0.67 BMI (kg/m2) 20.75.3 16.31.7 0.002* BSA (m2) 1.020.23 1.150.25 1.6 Vignos score (median) 1.5(1 4) 1 NA
73 CHAPTER 5 RELATIONSHIP BETWEEN LOWER EXTREMITY MUSCLES CONTRACTILE CROSS SECTIONAL AREA AND FUNCTIONAL MEASUREMENTS IN BOYS WITH DUCHENNE MUSCULAR DYSTROPHY Muscular dystrophy includes a large group of genetic disorders that result in loss of muscle fibers, leading to progressive skeletal muscle weakness. Duchenne muscular dystrophy (DMD) is one of the most common forms of muscular dystrophy affecting approxim ately 1 in 3500 newborn males. DMD is caused by a mutation in the dystrophin gene leading to absence or non functional structural protein, dystrophin (dys). [ 12 ] Absence of dys trophin protein affects sarcolemmal integrity leading to progressive fatty tissue infiltration and muscle atrophy. Loss of muscle strength and ske letal functional muscle mass profoundly leads to an increase d risk of disability, morbidity and ultimately quality of life. [ 152 153 ] During the initial stages of the disease inflammatory changes and repair are seen which are followed by irrever sible fat ty tissue infiltration. [ 154 ] Progressive fatty tissue infilt ration and muscle weakness lead to loss of ambulation between the age of 10 and 15 years. Disease progression and the loss of functional muscle mass ultimately leads to pr emature death due to cardiopulmonary complications in early twenties. [ 155 ] Selective muscle involvement is an important feature of DMD with some muscles displaying hypertrophy and others muscle atrophy In DMD patients, there is a significant loss of skeletal muscle mass [ 91 118 141 156 ] especially in proximal muscles including pelvis and thigh muscles. Furthermore, extensor muscles are we aker than flexor muscles [ 132 ] used in DMD to describe selective enlargement of the posterior compartment muscles especiall y the calf muscles assuming that a large component of the muscle is occupied by non contractile
74 material Other studies have shown selective sparing of the gracillis (Gr), sartorius (Sar), semimembranosus (SM) muscles [ 118 141 ] and tibial is posterior (TP) [ 97 157 ] In fact, there are reports of t rue hypertrophy in Gr and Sar muscles [ 141 ] Assessing changes in muscle cross sectional area is an important measure in muscular dystrophy patients as muscle size is an importan t determinant of muscle strength [ 91 141 ] b ut differentiating between contractile and non contractile component holds even more significance [ 90 ] Magnetic resonance imaging (M RI) has been used to monitor alterations in skeletal muscle in conditions like obesity [ 158 ] sarcopenia [ 159 ] cachexia [ 160 ] Furthermore, MRI has also been used to monitor disease progression in various myopathies [ 161 162 ] MRI methods, such as T 1 and T 2 weighted imaging, and three point DIXON imaging, have been utilized to estimate intramuscular fat ty infiltration in DMD patients. [ 156 161 ] Recently, we demonstrated the differences in age related changes in lower extremity muscles CSA and specific torque production in boys with DMD compared to controls [ 91 ] Furthermore, we evaluated the contractile and non contractile tissue in thigh muscles of boys with DMD. [ 90 ] However, to our knowledge no study has quantified the amount of contractile and non contractile tissue in lower extremity muscles which is the focus of thi s chapter Specific Aims To assess the non contractile and contractile content of individual muscles of lower extremity in boys with DMD and age matched healthy controls. 2 ) To evaluate the relationship between non contractile content and functional abil ity of boys with DMD. 3) To evaluate age related changes in contractile and non contractile content of lower leg muscles in boys with DMD.
75 Research Design and Methods Subjects : A cohort of thirty one boys with DMD (DMD group) and thirteen healthy boys from the general population (CONT group) volunteered to participate in this observational cross sectional study. Ambulatory status and mean (SEM) physical characteristics of all su bjects are shown in Table 5 1. A report confirming the diagnosis of DMD using molecular genetic testing (e.g. PCR amplification) and/or immunohistochemical staining from muscle biopsy was obtained from each of the DMD subjects. In the DMD group, twenty nin e boys were ambulatory and three boys were non ambulatory. All DMD subjects were being treated with corticosteroids (either Prednisone or Deflazacort). CONT subjects were relatively sedentary, in that they did not participate in sport specific training 2 o r more times per week. This study was approved by the Institutional Review Board at the University of Florida. Written informed from each participant. MRI acquisition : MRI was performed on 3.0T (Achieva ) 1 whole body scanner. Subjects were placed in a supine position with their lower leg positioned in either a lower extremity quadrature coil (1.5T) or an 8 channel sensitivity encoding, receive only extremity coil (3.0T) for lower leg imaging. Three dimensional transaxial gradient echo images were acquired with the following parameters: repetition time=24ms, echo time=1.8ms, flip angle=20, and optimized field of view 12 14cm 2 Isometric muscle strength testing : Isometric peak torque of the KEs, PFs, and DFs of the right leg was measured using a Biodex 2 dynamometer. For KE testing, the 1 Achieva; Philips Medical Systems, 3000 Minuteman Rd, Andover, MA 01810 1099. 2 Biodex Medical Systems, 20 Ramsay Rd, Shirley, NY 11967 4704
76 knee and hip joints were placed at 90 of flexion. For PFs and DFs, the knee was placed between 0 to 10 of flexion and the ankle was placed in neutral (for PFs) or 30 of plantar flexion (for DFs). The subject was instructed to push or pull as hard as possible for 5 seconds, followed by a 2 minute rest. Five trials were performed for each muscle group, and the highest torque value was used for a nalysis (peak torque). This protocol was tested for between day reliability (2 month interval) by the same tester, in a subset of children with DMD (n=6) and controls (n=10). ICCs were calculated for both groups of subjects. High reliability was found for all 3 muscle groups (KEs: ICC=0.89 in DMD and 0.99 in controls, PFs: ICC=0.88 in DMD and 0.98 in controls, and DFs: ICC=0.87 in DMD and 0.95 in controls; P=. 001 for all ICCs). Functional abilities : Subjects were asked to perform timed functional tasks. Th ese tasks included the time to walk 30 feet (30 feet Walk), time to rise from floor (Supine up), time to rise from a chair (Chair Up), the pediatric timed up and go (TUG) and ascend four stair s (4 stairs) [ 132 ] Subjects were asked to perform each tests three times and the fastest time was recorded for analysis. The functional ability was also ranked using Brookes Lower Functional scale (Brooke et al., 1981). This s cale is ranged from grade 1 (able to walk and climb stairs independently) to grade 10 (confined to bed). MRI data analysis : Medical Image Processing, Analysis and Visualization (MIPAV) 3 software (version 4.2.1; National Institutes of Health, MD) was used to analyze the MR images. We selected a single image corresp onding to the most proximal slice in which Flexor h allucis longus (FHL) could be visually con firmed. 3 MIPAV imaging software, available from: http://mipav.cit. nih.gov/.
77 Furthermore, we one immed iate proximal and distal slice was used for data analysis. We identified 5 individual muscles on the image: tibialis anterior (TA), extensor digitorum longus (EDL), peroneals (PER), medial gastrocnemius (MG) and soleus (SOL). Representative MR images for D MD and Ctrl subjects are shown in Fig ure 5 1. Contractile cross sectional area (C CSA) and non contractile cross sectional area (NC CSA) in the lower leg muscles was calculated using a modified image analysis technique as prev iously described [ 159 163 ] The first step of the analysis was to correct for image heterogeneity caused by sub optimal radiofrequency coil uniformity, or gradient driven eddy currents, using a well established non parametric non uniform intensit y normalization (N3) algorithm [ 164 ] This step was essential for subsequent analyses that assume homogenous signal intensity across the images. Optimized image cor rection parameters (N3) were determined (end tolerance 0.0001; maximum iterations 100, signal threshold 1; field distance 33.33 mm; subsampling factor 2; Kernel full width half maximum of 0.15; Wiener filter noise 0.01), and the same parameters were applie d to all images. To separate contractile and non contractile tissues in the pixel number signal intensity histogram with minimal bias, we implemented the Maximum Entropy method Furthermore, t o minimize manual tracing errors, three trials were averaged, and the values were applied to all muscles of interest. After tracing all muscles of interests, following parameters were calculated 1) total number of pixels with in ROI; 2) the number of pixel s with a signal intensity lower than the threshold value (contractile tissue) and 3) the number of pixels with a value higher than the threshold value (non contractile tissue). The proportion of contractile and non contractile content of each
78 muscle was ca lculated as : Contractile content (%) = (CC)/[(CC)+(non CC)] x 100 Non contractile content (%) = (non CC)/[(CC)+(non CC)] x 100 The contractile and non contractile CSA was calculated as: Contractile CSA (cm 2 ) = (CC) x (FOV/matrix size) 2 Non contractile CS A (cm 2 ) = (non CC) x (FOV/matrix size) 2 Where CC = contractile tissue pixel number; non CC = non contractile tissue pixel number; FOV= field of view. Intra class correlation coefficient in individual muscles ranged from 0.93 to 1.00 for non contractile c ontent. Statistical analysis : All values are reported as means and standard error of the parametric Mann Whitney tests were used to compare % non contractile content, contractil e CSA and non rank correlation tests were used to determine the relationships between two independent variables. The level of significance was set at p<0.05. Results Subject demographics : Boys with D MD were shorter in height and had higher body mass index compared to controls (Table 5 1); however no significant difference was found between age and weight of DMD and CTRL groups. Furthermore, the mean Brookes scale was 2.20.4 in DMD boys indicating th at most patients in this group were ambulatory, except for three subjects who were non ambulatory. Contractile and non contractile content of lower leg muscles : Non contractile content (%) (%NCC) Contractile CSA (cm 2 ) (C CSA) and non contractile CSA (cm 2 ) (NC CSA) of lower le g muscles of DMD and Ctrl are shown in Figure 5 2 Both the % NCC and NC CSA of the Per, MG and SOL differed signific antly between
79 DMD boys and Ctrl (p<0.05). On the other hand, no difference was found in the % NCC and NC CSA of the TA and EDL betw een DMD and Ctrl group Relationships between non contractile c ontent of lower leg muscles and functional abilities : relationship between the % NCC of different muscle s, streng th measurements, timed functional tests and B able 5 2). (A) We found significant correlation between % NCC of Per (p<0.05), MG (p<0.01), SOL (p<0.01) and plantar flexor peak torque. (B) Furthermore, significant correlations w ere found between % NCC of al l muscles and 30 feet walk (rs=0.45 0.61, p<0.001) as well as the Brookes scale (0.43 0.62, p<0.001) Figure 5 3 and 5 4 (C) Significant correlations were also found between %NC C of EDL (p<0.05), Per (p<0.05) and dorsi flexor peak torque, however we did not find a significant correlation between % NCC of TA and dorsi flexor peak torque. In additi on, we found that specific torque of DF and PF was significantly reduced in DMD as compared to Ctrl group ( Figure 5 5 ). Age r ela ted changes in contractile and non contractile CSA : Muscles of the lower limb of DMD boys were evaluated for age related changes in contract ile and non contractile CSA ( Figure 5 6, 5 7 ). All DMD subjects were divided into three sub groups i.e 5 8 years, 8 10 years and 10 years and above (10 years <) An age dependent progressive increase in bot h NC CSA and C CSA was observed (A) NC CSA of all the lower leg muscles was significantly different between 5 8 and 8 10 years old DMD boys (p<0.01). (B) Between 8 10 and 10 years < groups NC CSA was significantly different in Per, MG, SOL (p<0.001). (C) CCSA Per and MG differed significantly between group 5 8 and 10 years < (p<0.01).
80 Discussion The main findings of this study were (1) DMD boys had a significant increased proportion of % NCC and NC CSA in lower leg muscles compared to age matched controls espe cially in Per, MG and SOL. (2) T h e proportion of % NC C of leg muscles correlated with functional ability as assessed by the 30 foot walk and B rookes scale, however muscle % NC C did not correlate with strength measurements assessed using d ynamometer muscle testing (3) NC CSA of all the lower leg muscles increased with age in DMD boys (4) C CSA of Per and MG differed significantly between 5 8 year and 10 years < age group in DMD boys. (5) T he DF and PF specific torque production was significantly reduced in DMD boys as compared to healthy boys. Assessment of non contractile content in lower leg muscles of DMD boys : Although, all the lower leg mu scles (TA, EDL, Per, MG, SOL) in the DMD group had increased proportion of % NC C, it was significantly greater in Per, MG and SOL muscles compared to Ctrl group. Our findings indicate that TA and EDL muscles were relatively spared with %NCC less than 10%. Similarly, Arai et al [ 165 ] reported that the TA and TP were relatively preserved up to 14 years of age. Similarly Torriani et a l [ 97 ] found that TA, E DL and TP were relatively spared in DMD patients. Furthermore, we have shown t hat the %NC C ranged from 20% 40% in Per, 18% 42% in MG and 11% 22% in SOL mu scle whereas in healthy boys % NC C ranged f rom 1 8% in all muscles These findings are in accordance with previous studies [ 166 ] in which the authors reported a percentage of 18 38% increase in fatty fibrous tissue in gastrocnemius muscle whereas this component never exceeded 8% in normal sub jects. Contractile, Non contractile CSA and specific torque production in DMD boys : Amount of force generated per unit muscle mass (specific force) is an important
81 determinant of muscle quality. Our results showed that, NC CSA of Per, MG (DMD, 92% of Ctrl) and SOL (DMD 82% of Ctrl) were significantly greater in DMD than Ctrl In contrast, C CSA of lower leg muscles was not significantly different between DMD and Ctrl subjects A similar pattern of muscle involvement has been previously reported in DMD patients [ 9 7 157 167 ] It is valuable to differentiate and quantify both C CSA and NC CSA because patients having more muscle mass preserved may resp ond well to various therapies. A study by van Deutekom et al [ 167 ] showed that muscles with less fatty tissue infiltration responded well to antisense oligonucleotide treatment. Pseudo hypertrophy of the calf muscles is one of the hallmark signs of DMD. It is assumed to be caused by rep lacement or infiltration o f muscles by collagenous and/or fatty tissue. Although, the most commonly affected muscles are the calf muscles pseudo hypertrophy has been reported to occur in temporalis muscle as well. [ 168 ] Increase in both C CSA and NC CSA can lead to an increase in overall size of the muscle which can be measured by assessing calf circumference [ 169 ] and CSA [ 91 ] Our result s indicate that there is an increase in both C CSA and NC CSA in the lower leg muscles in DMD patients compared to healthy boys. However, the increase in NC CSA is more pronounced especially in Per, MG and SOL muscles. Similar to our results, Jones et al [ 170 ] reported true hypertrophy in the calf muscles of DMD boys using needle biopsy. Furthermore, they reported that despite the presence of hypertrophied fibers it was insufficient to cause muscle enlargement. Age related changes in C CSA and NC CSA in lower leg muscles : We have reported that NC CSA increases significantly with age in all the examined lower leg
82 muscles. However, only C CSA of Per and MG (42% and 45% respectively) differed significantly between 5 8 and 10 < years Muscle contractile area and specific torque : Our study showed a significant decrease in DF and PF specific force in DMD patients (43% and 62% r e spectively) as com pared to Ctrl Furthermore, both PF and DF specific torque stron gly correlated with age in Ctrl subjects whereas this relationship was absent in DMD patients Similar results have been reported in animal studies affirming that specific force (P 0 ) of EDL an d SOL muscles differ significantly between mdx and C57Bl mice [ 171 ] Limitations : There were some limitations to our study. Firstly, even after correcting the images with N3 inhomogeneity correction we were unable to do so completely. We did not include lateral gastrocnemius because of presence of B 1 inhomogeneity. Secondly, because of limited spatial resolution MR images suffer from partial volume filling leading to underestimation of the contractile area. Summary We have shown that T 1 weighted images can be used to quantify contractile and non contractile tissue content in lower leg muscles. The amount of non contractile content assessed by MRI was significantly greater in lower leg muscles of boys with DMD especially Per, MG and SOL. Moreover, there was a significant correlation between amount of non contractile tissue present and functional measurements such as 30 foot walk and Brookes scale. We have also shown that C CSA did not differ significantly between DMD and Ctrl group. In ad dition, we have shown that there is increase in C CSA was significantly greater in DMD boys especially in
83 an provide critical information, which can be used to assess the disease progression in DMD boys.
84 Figure 5 1. T1 weighted fat unsuppressed trans axial images of the lower leg of Ctrl (A) and DMD subject (B).
85 Figure 5 2. Comparison of percent non contractile content, non contractile CSA and contractile CSA of lower leg muscles. A) Comparison of non contractile content of lower leg muscles between DMD and Ctrl boys. TA, tibilais anterior; EDL, extensor digitorum longus; Per, peroneals; MG, meadi al gastrocnemius; SOL, soleus. *p<0.05. B) Comparison of non contractile cross sectional area of lower leg muscles between DMD and Ctrl boys. C) Comparison of contractile cross sectional area of lower leg muscles between DMD and Ctrl boys. TA, tibilais ant erior; EDL, extensor digitorum longus; Per, peroneals; MG, medial gastrocnemius; SOL, soleus. *p<0.05, # p=0.07.
88 Figure 5 3 Relationship between 30 feet walk and non contractile content (%) of lower leg muscles. (A)T ibialis anterior (TA), (B) peroneals (Per), (C) soleus (SOL), (D) m edial gastrocnemius (MG) and (E) extensor digitorum longus (EDL) in boys with DMD.
89 Figure 5 4 Relationship between Brookes scale and non contractile content (%) of lower leg muscles. (A) Tibialis anterior (TA), (B) peroneals (Per), (C) soleus (SOL), (D) medial gastrocnemius (MG), and (E) extensor digitorum longus (EDL) in boys with DMD.
90 Figure 5 5 Specific torque of dorsiflexors (DF) and plantar flexors (PF) between DMD, Duchenne m uscular dystrophy and Ctrl, control group. p<0.005.
91 Figure 5 6 Non contractile and Contractile CSA comparison among different age groups in DMD. Non contractile CSA (A) and Contractile CSA (B) of lower leg muscles of DMD subjects. Significant difference between 5 8 years and > 10 years (p<0.001), # Significant difference between 8 10 years and > 10 years (p<0.001). A B
92 Figure 5 7. A) Non contractile CSA and Contractile CSA comparison among different age groups in DMD.
93 Ta ble 5 1. Subjects demographics. Values are mean SEM. DMD, Duchenne muscular dystrophy group; Ctrl, control group; BMI, body mass index; BSA, body surface area. Subject Demographics DMD Ctrl Number of subjects 31 13 Age (years) 8.83 +/ 0.44 9.4 +/ 0.81 Height (cm) 124.09 +/ 2.39 138.3 +/ 4.31 Weight (Kg) 29.88 +/ 2.04** 31.7 +/ 2.80 BMI (kg/m sq) 18.84 +/ 0.70* 16.2 +/ 0.66 ** p<0.01 versus Ctrl group *p<0.05 versus Ctrl group Table 5 2 Spearman rank correlation between non contractile content, strength and functional measurements. Functional measurements n TA EDL PER MG SOL Plantar flexor peak torque 32 NA NA 0.32* 0.47** 0.41** Dorsi flexor peak torque 32 0.17 0.32* 0.31* NA NA 30 feet walk 29 0.45** 0.56** 0.48** 0.61** 0.52** Brooks scale 32 0.43** 0.62** 0.57** 0.61** 0.54** F astest supine up 26 0.138 0.12 0.101 0.201 0.01 Chair up 29 0.191 0.023 0.012 0.203 0.039 TUG 29 0.213 0.143 0.248 0.445* 0.262 4 stairs 29 0.052 0.231 0.212 0.019 0.147 **Correlation is significant at 0.01 level (2 tailed) Correlation is significant at 0.05 level (2 tailed)
94 CHAPTER 6 TO MONITOR CHANGES IN T 2 WITH AGE IN HINDLIMB MUSCLES OF MDX MICE While the contractile and non contractile cross sectional area can be estimated using T 1 weighted images another potentially valuable MR marker of disease progression in DMD is transverse relaxation time (T 2 ). T 2 has been shown to be dependent on a number of compositional changes relevant to DMD, such as fibr osis, muscle damage and edema. In order to examine the potential of T 2 to monitor disease progression the mdx mouse model can be utilized. Animal models have t he advantage that they enable a more direct histological confirmation of our specific measures. The mouse model, the DMD mdx ( mdx 10ScSn) mouse, is the most commonly used animal model of DMD. mdx mice, similar to DMD patients lack dystrophin and share some biochemical and histopathological features. The result of many histological studies has confirmed that hind limb muscles and diaphragm muscle of mdx mice show progressive weakness and muscle deterioration with age. Despite these similarities, the phenotyp e of mdx mice is considered less severe than DMD patients. However, the phenotype exhibited by old mdx mice closely resembles the myopathy of DMD. In addition t he mdx mouse is readily available and offers an economic way for testing possible therapeutic interventions. Different invasive techniques have been incorporated to measure the efficacy of an intervention to reduce the underlying pathology in DMD. However, the ability to non invasively measure the pathological features of muscular dystrophy may be valuable for studying the response of different experimental therapeutic strategies. The transverse relaxation time constant (T 2 ) is a quantitative measure of a basic biophysical property that leads to signal contrast on MRI. Changes in the T 2 of skeletal muscle have been
95 observed during both acute physiological responses in healthy muscle and under pathophysiological conditions such as muscle damage and inflammation [ 117 118 ] Specific Aim s To monitor the progression of dystrophic changes in the hind limb muscles of mdx mice based on changes in the t ransverse relaxation time constant (T 2 ). Research Design and Methods Animals : C57BL/10ScSn DMD mdx ( mdx ; n=18 ) and wild type C57BL/10ScSn (n=12 ) male mice were included in the study. mdx and wild type mice were divided equally in three groups: young, adult and old. M ice were obtained from Jackson laboratories (Bar Harbor, ME) and were housed in an AAALAC approved facility with 12 hour light:dark cy cle (72 o F, 42% humidity and free access to food and water ) The protocol. Magnetic resonance imaging : Magnetic resonance imaging (MRI) was performed in a 4.7 T, horizontal bore magnet (Bruker Avance). The animals were anesthetized using an oxygen and isoflurane mixture (3% isoflurane) and maintained under 0.5 1% isoflurane for the duration of the MR procedure. Respiratory rate of the mouse was monitored for the duration of t he scan. The lower hindlimbs of the mouse were inserted up to the knee into a 2.0 cm internal diameter, solenoid 1 H coil (200 MHz). T 2 weighted images were acquired with the following parameters: Multiple slice, single spin echo images repetition time (TR ) =2000ms, echo time (TE)=14ms and 40ms, FOV 10 20mm, slice thickness 0.5 1mm, acquisition matrix = 128 x 256 and two signal averages [ 112 ] Diffusion weighting was fixed at both TEs (diffusion weighting 3 mm 2 /s at both 14 and 40 ms). Hahn spin echoes were implemented to avoid the
96 contribution of stimulated echoes in the T2 measurement. Signal to noise ratios were 25:1 at TE = 14 ms and 9:1 at TE= 40 ms. MRI m uscle T 2 Analysis : Muscle T 2 values of the anteri or and posterior muscle compartment were computed from a T 2 map, created from two echo times (TEs; 14 and 40 ms) using in house s oftware as described previously. [ 112 ] The T 2 relaxation time of each pixel wa s calculated using a single exponential decay with the following equation: T 2 = (26 ms)/ ln (SI 14 /SI 40 ), where SI 14 and SI 40 are the pixel intensities at 14 ms and 40 ms respectively. [ 172 ] The percent of muscle damage detected by MRI was defined as the percentage of pixels in a region of interest, which had T 2 values over two standard deviations above the mean muscle T 2 found in control mice (> 29ms) up to a maximum value of 100 ms. Statistical Analysis : All statistical analysis was performed using SPSS for mac (version 20.0) 1 Results were expressed as mean standard error of mean. Research hypothesis was tested at an alpha level of 0.05. One way analysis of variance (ANOVA) was performed to compare T 2 between mdx and control group at different time points. Furthermore, one way analysis of variance (ANOVA) for repeated measurements was performed to monitor the changes in hindlimb muscles of young mdx mice. Results Assessment of damage in hindlimb muscle s mdx mice: Muscle T 2 and the percentage of pixels with elevated T 2 in the lower hindlimb muscles were compar ed between mdx and control mice using T 2 maps (Figure 6 1) As shown in Figure 6 2, (1) mdx mice had higher mean T 2 values than control mice in both the young and adult age 1 IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp
97 groups (p<0.05). (2) However, mean T 2 values in old mdx mice did not differ from age matched control group (3 ) T he percentage of pixels with elevated T 2 in mdx mice was significantly different from age matched controls in all three groups: you ng, adult and old age groups. (4 ) In young mdx mice, there was a three fold increase in the percentage of pixels with elevated T 2 compared to adult and old mdx mice. As shown in Figure 6 3 mdx mice showed asynchronous, cyclical changes in the T 2 values of both ante rior and posterior compartment hind limb muscles with age We measured mean muscle T 2 and the percent pixels with elevated T 2 in both anterior and posterior compartment. There was a significant increase in the mean muscle T 2 from the 5 week to the 7 week time point. At 10 week s the mean muscle T 2 was significantly reduced Furthermore, T 2 and % pixels with elevated T 2 of both anterior and posterior compartment were significantly greater than other time points ( Figure 6 4). In vitro Histological analysis: I n vitro histological analysis of hindlimb muscles of mdx mice 6 months of age showed a large number of centrally nucleated fibers compared to age matched controls ( Figure 6 5 ) Presence of centrally nucleated fibers was also noted at 96 w eeks of age. In addition muscles revealed excessive collagenous tissue accumulation in mdx mice at 96 week of age. Discussion The hind limb muscles are the most commonly studied muscles in mdx mice. They include the tibialis anterior (TA) and extensor digitorum longus (EDL) in the anterior compartment, and the gastrocnemius (GAS) and the soleus (SOL) in the posterior compartment. In dystrophin deficient mice pathological changes observed include muscle fiber damage, inflammation and fibrosis in the later stages. Age related
98 studies are important to assist in optimal targeting of various pathological processes. Age dependent p athological changes have been well documented in dystrophin deficient muscles using various histopathological techniques [ 173 174 ] However, no one has looked at time specific responses in mdx mice using a non invasive measure such as T 2 weighted MRI. In our study, we found that T 2 is a sensitive biomarker that allows us to follow the longitudinal changes occurring in the skeletal muscles of mdx mice. We found that, with age both mean muscle T 2 values and percentage of pixels with elevated T 2 show ed asynchronous and cyclical changes in both the anterior and posterior compartments hindlimb muscles of mdx mice especially in young mdx mice i.e up to 6 months of age We found that the mean muscle T 2 and the percentage pixels with elevated T 2 peak s peaked at about 7 8 weeks. This finding has been corroborated in the literature by various studies using histological param e ters. [ 175 176 ] Furthermore, we observed a decrease in the mean muscle T 2 and percent pixels with elevated T 2 in old mdx mice aged 96 week. Depending on the age of the mice, disease progression can be roughly divided into three phases. First, muscles of young mice undergo repeated cycl es of degeneration/regeneration. [ 177 178 ] It has been suggested that this phase occurs due to increased exploratory behavior and locomotor activity of young mice. Second, musc les of adult mdx mice despite being bigger and heavier than control mice have reduced specific force. The skeletal muscles do not show extensive fibrosis, except for in the diaphragm. [ 179 ] At this stage the extent of involvement of the muscle group s also
99 varies and is related to position or use of a muscle group in the body. In the t hird phase older mdx mice show a progressive dystrophic phenotype. M uscle necrosis and regeneration is most pronounced between 3 8 weeks of age [ 176 180 ] Evidence fro m the present study further support this pattern of pathology. The TA typically fi rs t manifests muscle necrosis around 21 days after birth and this has been demonstrated to be more pronounced than in the quadriceps [ 181 182 ] Another study observed more necrosis in soleus than EDL at 24 days with greater cumulative muscle damage in soleus (~86%) than EDL (~36%) muscle at 34 days [ 183 ] The precise reason for the acute onset of dystropathology at this age is unknown but speculations include 1) increase in locomotor activity 2) downregulation of various genes including utrophin [ 184 ] and genes involved in creatine synthesis [ 185 ] 3) and downregulation of proteins involved in excitation contraction coupling mechanisms [ 186 187 ] Increase in locomotor activity during early phase of life has a temporal relationship with the histological abnormalities in mdx mice. Furth ermore, it has been shown that locomotor activity varies considerably with age in mdx mice reducing significantly at 9 week post natal tim e period. [ 188 ] Moreover, levels of utrophin protein have been shown to be greatest in the fetal stages and decrease significantly at 2 week post natal time point and stay at lower levels throughout ad ult life [ 184 ] Furthermor e, Bertocchini et al. [ 186 ] found an increase in RyR3 levels at the 2 3 week time point, which corresponds to a period where skeletal muscles undergo morphological and biochemical change s. Expression of RyR3 start s t o decrease at the 3 week time point as the levels of other proteins such as RyR1 start increasing steadily.
100 Activity induced changes in muscle T 2 have been well documented. For example, after a single bout of exercise, the a ctivated muscles show an increase in T 2 which may last up to an hour. The exact mechanism is still not clear but changes in T 2 have been attributed to redistribution of water molecules in muscle cells. Furthermore, eccentric exercise has been shown to inc rease muscle T 2 which remains elevated un till at least 7 days after the initial bout. Our results show that T 2 in hindlimb muscles of mdx mice change with age, which may be related to the increase in voluntary activity. Furthermore, any change in biophysical properties such as upregulation or downregulation of different proteins may also cause changes in muscle T 2 Age relate d s tudies are critical because there are a number of pathological changes occurring in mdx muscles and treatment strategies may be more effective in certain time windows For example, therapies improving membrane stability (gene repair, drug therapy) shoul d be targeted with in the time period when there is excessive damage in the muscles. On the other hand, therapies involving antifibrotic treatment should be given after 6 months of age when early signs of collagen accumulation are noticed. Summary MRI is non invasive tool that has the ability to examine several muscles simultaneously. We used T 2 weighted MRI to distinguish muscles of mdx and control mice. Furthermore, we used MRI to monitor asynchronous and cyclical changes occurring in hindlimb muscles o f young mdx mice. The mean muscle T 2 and percent pixels with elevated T 2 peaked between 7 8 weeks and they remain ed fairly leveled after 6 months of age.
101 Figure 6 1 T 2 weighted images of mdx mouse hindlimb at different time points.
102 Figure 6 2. Hindlimb muscle T 2 values and % pixels with elevated T 2 of a nterior and posterior region. Mean muscle T 2 values and % pixels with elevated T 2 of anterior region (A and C) Mean muscle T 2 values and % pixels with elevated T 2 of anterior region and (B and D) p osterior region of md x and age matched control mice.
103 Figure 6 3 Mean muscle T 2 values and % pixels with elevated T 2 of anterior region at different time points. M ean muscle T 2 (A) and % pixels with elevated T 2 (B) in anterior and posterior compartment of hindlimb muscles of mdx mice at different time points. represents significant difference from 5 week time point, # represents significant difference from 10 week time point and (p<0.05).
104 Figure 6 4 Mean mus cle T 2 values and % pixels with elevated T 2 of anterior and posterior hindlimb muscle compartments of mdx mice. M ean muscle T 2 (A) and % pixels with elevated T 2 (B) in anterior and posterior compartment of hindlimb muscles of mdx mice at different time points. Represents significant difference from 7 week time point (p<0.05).
105 Figure 6 5 Figure showing H&E and trichrome staining of SOL and g astr in mdx and Ctrl mice at different time points.
106 CHAPTER 7 CHANGES IN MU SCLE T 2 AND TISSUE DAMAGE IN HINDLIMBS OF MDX MICE AFTER DOWNHILL RUNNING 1 The absence of dystrophin compromises the entire dystrophin glycoprotein complex, leading to an increased vulnerability to muscle damage during contractions. Furthermore, exercise induced muscle damage occurs after high force, unaccustomed exercise, particularly after eccentric (i.e., lengthening) muscle contracti ons. The initial mechanical damage is followed by an inflammatory response and subsequent reparative and regenerative process within the muscle. Models of eccentric loading commonly employed in animal models, such as direct electrical stimulation of an excised muscle under passive stretch [ 4 189 ] or electrical stimulation of intact muscles under lengthening conditions [ 190 191 ] can be used to produce marked damage to individual muscles. However, these models are invasive and require termination of the animal to examine muscle structure, and therefore they cannot be used for longitudinal studies. In contrast, downhill treadmill running has been used as a physiologically relevant model of eccentric loading in mdx mice [ 192 193 ] other rodent models and humans. Downhill running has several advantages over eccentric contractions produced from electrical stimulation of muscles. First, it incorporates a physiological load and voluntary muscle contractions, which may translate into mo re relevant findings for studies involving human subjects. Second, downhill running has been used to accelerate muscle weakness and fibrosis in the limb muscles of the mdx mouse to more closely mirror the muscle pathology seen in patients 1 Sunita Mathur, Ravneet S Vohra, Sean A Germain, Sean Forbes, Nahan D Bryant, Krista Vandenborne, Glenn A Walter. Changes in muscle T2 and tissue dam age after downhill running in mdx mice. Muscle Nerve. 2011 Jun;43(6):878 86.
107 with DMD [ 61 194 ] Finally, this method can be used for repeated bouts of muscle damage in the same animal, allowing for longitudinal studies of muscle damage and repa ir [ 195 ] Downhill treadmill running has previously been shown to produce muscle damage in the gastrocnemius and soleus muscles, as well as the tibial is anterior and extensor digitorum longus (EDL) of mdx mice [ 192 ] A limitation of this earlier work is that the change in mu scle damage after downhill run ning was compar ed between mdx mice that under went downhill running to a group of mdx mice that did not run. This comparison has inherent limita tions, because mdx mice have damaged muscle fibers even under conditions of normal cage activity, and there is high variability in muscle degeneration between animals [ 194 ] I deally, the response of dystro phic muscle to d ownhill running should be exam ined in the same animal both pre and postexercis e. This can be achieved using a noninvasive approach such as MRI The transverse relaxation time constant (T 2 ) is a quantitative measu re of a basic biophysical prop erty that leads to signal contrast on MRI. Changes in the T 2 of skeletal muscle have been ob served during both acute physiological responses in healthy muscle and under pathophysio logical condi tions. For example, acute and transient increases in muscle T 2 lasting approximately 1 hour have been observed in activate d muscles after a bout of exer cis e [ 196 197 ] Although the exact mechanisms that account for these changes have not been fully elucidated, T 2 changes have been attributed to a redistribution of water molecules in the muscle cells [ 198 ] Furthermore, after bouts of eccentric exercise, a later phase of increased muscle T 2 occurs after the initial increase, and it peaks approximately 2 7 days after the initial exercise bout [ 109 197 ] Chronic
108 changes in muscle T 2 have also been detected in resting muscles of individuals with incomplete spin al cord injury [ 199 ] stroke [ 200 ] DMD [ 118 ] and dermatomyositis [ 201 ] all condit ions associated with muscle damage and inflammation. Specific Aims 4. To monitor the response of muscle T 2 over a time course of 48 hours after a single bout of downhill running in mdx mice to wild type (control) mice 5. To compare the effects of horizontal vs. downhill running on muscle damage assessed by T 2 MRI in mdx mice 6. T o compare the effects of downhill running on the lower anterior vs. posterior hindlimb muscles of mdx mice. Hypotheses Our hypotheses are as follows 1) After a single bout of downhill running muscles of mdx mice will show greater increase in T 2 than control mice. 2) A single bout of downhill running will cause greater increase in T 2 than horizontal running. 3) The anterior compartment muscles will show greater increase in T 2 than the posterior compartment after a bout of downhill running. Research Design and Methods Both C57BL/10ScSn DMD mdx ( mdx ; n = 27) and wild type C57BL/10ScSn (controls n = 6) adult male mice (aged 5 15 months) will be included in the stud y. Wild type mice and breeding pairs from the mdx colony were obtained from Jackson Laboratories (Bar Harbor, Maine) and thereafter maintained in house. Animals were housed in an AAALAC approved facility with a 12 hour light:dark cycle (72 o F, 42% humidity) and free access to food and water. The institutional animal care and use committee of University of Florida approved the experimental protocol.
109 MRI was be conducted prior to treadmill running in both mdx and control mice to determine baseline muscle T 2 values of the lower hindlimb muscles. The methods of the MRI protocol and T 2 analysis are outlined in what follows. After the initial MRI, each mouse underwent one of two treadmill running protocols: a bout of downhill running or a bout of horizontal grad e running. Specifically, mice were run either on a downhill slope (14 degrees decline) or a horizontal (0% grade) motorized treadmill at a speed of 8 10 m/min, for 45 minutes. To ensure the mice ran for the entire duration of the protocol, they were observ ed continuously. If necessary, a short burst of compressed air or the use of a brush at the base of the tail was used to encourage running. From a total of 18 mdx mice screened, 11 were able to complete the downhill running protocol with some encouragement needed during the first 5 10 minutes of running and near the end of the 45 mi nute period. All control mice (6 of 6) were able to complete the downhill running protocol without encouragement. All mdx mice (9 of 9) were able to complete the horizontal runni ng protocol without external stimulation. MRI of the lower hindlimbs will be conducted immediately post exercise (approximately 20 minutes after the exercise bout), and at 24 and 48 hours post exercise In 5 of the mdx mice that ran downhill, a 10 day post exercise follow up MRI scan was also conducted. Lower hindlimb muscles were extracte d in a subset of mdx (n = 8) and control mice (n = 5) at the end of the study for histological confirmation of muscle damage. Magnetic resonance imaging : MRI was performed in a 4.7 T horizontal bore magnet (Bruker Avance, ParaVision 3.02; B ruker). The animal was anesthe tized using an oxygen and isoflurane mixture (3% isoflurane) and m aintained under 0.5 1% isoflur ane for the duration of the MR procedure. The respiratory rate of the mouse was
110 monitored for the duration of the scan (SA Instruments ) 2 The lower hindlimbs of each mouse were inserted up to the knee into a custom built solenoid 1H coil (200 MHz) with a 2.0 cm internal diameter. Multiple slice, diffusion controlled single spin echo images were acquired with the following parameters: repetition time (TR) = 2000 ms; echo time (TE) = 14 ms and 40 ms; field of view (FOV) = 10 20 mm; slice thickness = 1 mm; acquisition matrix = 128 X 256; and with two signal aver ages, as previously described [ 112 ] Diffusion weighting was fixed at both TEs (diffusion weighting 3 mm2/s at both 14 and 40 ms). Hahn spin echoes were implemented to avoid the contribution of stimulated e choes in the T2 measurement. Signal to noise ratios were 25:1 at TE = 14 ms and 9:1 at TE = 40 ms. Muscle T 2 analysis from MRI : Muscle T 2 values of the entire lower hindlimb (excluding bone), as well as of individual muscle compartments (anter ior, posteri or, and medial; Figure 7 1), were computed from a T 2 map, created from two TEs (14 and 40 ms) using in house softwa re as previously described [ 20 112 ] T 2 relaxation time wa s calculated using single expo nential decay wit h the following equation: T 2 = (26 ms) / ln (SI 14 / SI 40 ), where SI 14 and SI 40 are the pixel intensities at 14 and 40 ms, respectively [ 172 ] The percentage of muscle damage detected by MRI was defined as the percentage of pixels in a region of interest that had T 2 values ove r 2 standard devia tions above the mean muscle T2 found in control mice (>29 ms) up to a maximum value of 100 ms. Histologica l confirmation of muscle damage: Histo logical confirm ation of muscle damage was con ducted in a subset of mdx and control mice from e ach of the 2 SA instruments, Inc., Stony Brook, New york.
111 pr otocols: downhill running (n = 4 mdx n = 5 control) and horizontal grade running (n = 4 mdx only). Tw enty four hours after the exer cise bout, mice were injected with EBD (0.10 mL per 10 g of body weight) via intraperito neal injec tion. Forty e ight hours after exercise (i.e., 24 hours postinjection), the following muscles of the lower hindlimb were harvested: tibialis anterior (TA); extensor digitorum longus (EDL); flexor digitorum longu s (FDL); soleus; and gastrocne mius. Muscles were coated wit h OCT, frozen in iso pentane cooled with liquid nitrogen, and stored at 80 0 C. Each muscle was sectioned at the mid belly region, at 10 m thickness, and mounted on slides using a fluo rescence mounting medium with 4 ,6 diamidino 2 phenyl indole (DAPI; VectaShield, Vec tor Labs). Slides were viewed using fluorescence microscopy, optimized for EBD (Texas red filter). Images of the entire muscle cro ss section were captured at 10 X mag nification using a light micro scope (Leica Microsystems, Bannockburn, Illi nois) attached to a digital camera with slightly overlapping fields to obt ain images from the entire mus cle. The cross s ection of the muscle was recon structed in Adobe Photoshop from the digital images. EBD positive fibers were outlined using NIH Image J 3 and the t otal area occupied by EBD posi tive fibers was calculated as a percentage of the total muscle area. Statistical analysis : Data were analyzed using SPSS, version 11.0 4 The descriptive data are presented using mean and standard e rror of the mean (S EM). To com pare the differences in muscle T 2 and percent muscle damage b etween mdx and control mice af ter a single bout of downhill running (study objec tive 1), independent 3 Image J available from http://rsbweb.nih. gov/ij/ 4 SPSS Inc, 233 S. Wacker Drive, 11 th floor, Chicago, Illinois 60606.
112 sample t tests were used to make comparisons at each time point between groups (p= 0.05, adjus ted for multiple compari sons using a modified Bonferroni correcti on). Paired t tests were used to compare differences in muscle T 2 and percent damage from pre exercise values to post exercise time points (immediately post exercise, 24 and 48 hours post exercise), in each of the mdx and control groups ( alpha = 0.05, adjusted for multiple comparisons). A comparison of the response of muscle T 2 and percent damage in mdx mice be tween downhill running and hori zontal running (study objective 2) was conducted using independent sample t tests at each time point (P < 0.05). To examine study objective 3, the response of each muscle compartment to one bout of downhill treadmill running in mdx mice was compared using a two way, repeated measures anal ysis o f variance (ANOVA) and the Tukey post hoc tests (main factors: time point and muscle, P < 0.05). Finally, the Spearman rank correlation was used to examine th e relationship between the per centage of muscle cross sectional area positive for EBD and the perc entage of pixels with elevated T 2 Results Downhill running in mdx compared with control mice : Muscle T 2 and the percentage of pixels with elevated T 2 in the lower hindlimb muscles was compared between mdx and control mice after a single bout of downhill r unning over a period of 48 hours. As shown in Figure 7 2 A, mdx mice had a higher muscle T 2 compared with control mice both pre exercise and at each time point after downhill running (P < 0.001). A twofold increase in the number of pixels with elevated T 2 w as detected immediately post exercise in mdx mice (14.5 6 2.9% of pi xels with elevated T 2 post exer cise vs. 7.1 6 1.5% pre exercise) and remained ele vated vs. baseline va lues at 24 and 48 hours post ex ercise (P < 0.001; Figure 7 2B). Muscle T 2 was higher immediately post exercise
113 in the mdx mice (P < 0.05; Figure 7 2A), but not at 24 or 48 hours. The per centage of pixels with elevated T 2 consistently returned to baseline values 10 days after downhill running in mdx mice ( Figure 7 3). Immediatel y after downhill running and 24 and 48 hours later, the control mice did not show any difference in their muscle T 2 or per centage of elevated pixels com pared with their pre exercise values. An example of transaxial T 2 weighted images from an mdx mouse afte r downhill running is shown in Figure 7 4 with follow up MRI taken 10 days after the exercise bout. A distinct hyperintense region is observed on the T 2 weighted images of the medial muscle compartment. Figure 7 5 shows a projection from a three dimension al rendering of the medial compartment wit h elevated muscle T 2 after down hill running. Downhill vs. H orizontal running in mdx mice : The response of muscle T 2 and percentage of muscle damage was compared in two independent groups of mdx mice that u nderwent either a bout of down hill or horizontal treadmill running. As shown in Figure 7 6 muscle T 2 increased only in the group of mdx mice immediately after downhill running, whereas no change in T 2 was observed in those that underwent horizontal treadmill running. The percentage of pixels with elevated T 2 showed an increase in the horizontally run mice immediately postexercise (5.0 6 1.0% at baseline vs. 6.8 6 1.5% immediately postexercise, P < 0.05 ), but remained elevated only in the downhill running group at 24 and 48 hours after the exercise bout. Comparison between muscle compartments in mdx mice after downhill running : As mdx mice showed an increase in muscle damage of their lower hind limb mus cles after downhill running, a further ex amination of re sponse in the three muscle com partments (anterior, medial, and posterior) was conducted. As sh own in Figure 7 7
114 the medial mus cle compartment had a significantly higher per centage of pixels wi th elev ated T 2 at baseline com pared with the anterior and posterior muscle groups. Furthermore, the medial compartment showed a significant increase in the percentage of elevated pixels at each time point after exercise compared with pre exercise values (P < 0.05 ). However, a significant change was not observed in the anterior and posterior muscle compartments. Histological : Individual muscles from the lower hind limb of mdx and control mice were extracted follow ing the 48 hour MRI scan and examined for EBD uptake ( Figure 7 8) indicative of muscle damage. The percent of the total muscle cross sectional area positive for EBD fibers was compared to the percent of pixels with elevated T 2 from the corresponding muscle compartment (i.e. tibialis anterior and extensor digitorum longus muscles were compared to the anterior muscle compartment; gastrocnemius and soleus muscles to the posterior muscle compartment and flexor digitorum muscle to t he medial muscle compartment). Bivariate correlational analysis demonstrated a strong relationship between the percent of muscle cross sectional area that was EBD positive and the percent of pixels with elevated T 2 ( r = 0.79, p < 0.001 ; see Figure 7 9 ). D iscussion The findings of our study demonstrate that increases in muscle T 2 can be visualized using MRI following downhill running in mdx mice. Furthermore, T 2 elevation was evident in mdx following downhill running but not control mice after downhill running or mdx mice after horizontal (flat) treadmill running, which did not experience muscle Dye positive f ibers) and T 2 elevation was found in this study. However, a limitation of
115 this study was that we were not able to determine the relative contributions of muscle fiber damage, edema and/or inflammation to the measured elevated muscle T 2 Furthermore, MRI al lowed for individual muscle compartments to be examined simultaneously in the same animal. The medial muscle compartment had greater changes in muscle T 2 following downhill running in mdx mice and therefore may be more prone to muscle damage than the anter ior and posterior muscle compartments. The effects of two standardized, exercise based perturbations resulted in different responses in mdx mice. Downhill treadmill running consistently caused muscle T 2 elevation above baseline in mdx mice (measured as th e percent of pixels with elevated T 2 ), whereas horizontal running resulted in no change in muscle T 2 in mdx mice. These physiologically relevant protocols may prove useful in pre clinical testing of the effects of therapeutic interventions, which are aimed at improving the integrity of dystrophic muscle. Activity based outcomes such as treadmill running have been identified as relevant endpoints for translational research as they parallel the outcome measures used in clinical trials. The addition of MRI as sessment following a known exercise perturbation would allow for the direct visualization of tissue damage and inflammation. [ 194 ] Muscle T 2 was higher in the mdx mice compared to controls at baseline, and is likely due to regions of muscle degeneration and damage in the dystrophic mice. [ 114 202 203 ] Elevated muscle T 2 has been observed in the pelvic an d thigh muscles, particularly the gluteus maximum, of children with DMD. [ 118 ] However, the high T 2 values in the muscles of children with DMD were attributed to fatty infiltration, which does not occur in young mdx mice. [ 204 ] In other animal models of DMD that are
116 characterized by large amounts of fatty tissue replacement similar to the human condition (e.g. golden retriever dog model), ch anges in muscle T 2 have been used to visualize both the corrective [ 205 ] and inflammatory [ 206 ] aspects of therapeutic intervention. In our study, we found that mdx mice showed an additional increase in the percent of pixels with elevated T 2 from baseline val ues following a bout of downhill running, which remained elevated up to 48 hours. On the other hand, following the same exercise protocol in control mice, the percent of elevated pixels did not show an immediate (20 min post) or prolonged increase. An acut e elevation of muscle T 2 observed immediately after exercise has been attributed to changes in intra and extramyocellular water content [ 98 207 ] and redistribution of water molecules within muscle [ 198 208 ] and typically dissipates within one hour post exercise. [ 109 ] T 2 elevation that lasts longer than 24 hours post exercise is most likely due to the inflammatory response and increased membrane permeability, resulting f rom the initial muscle damage. [ 112 195 ] In our study, the control mice did not show an acute increase in muscle T 2 which may indicate that their level of exercise was not intense enough to cause a prolonged shift in water compartmentalization. [ 209 ] This is consistent with previous findings of Kobyashi et al. [ 20 ] who reported that mdx mice have an accumulation of muscle water con t ent in their hindlimbs after mild treadmill exercise, which is not seen in wild type mice. Vilquin et al. [ 193 ] also showed that wild type mice that ran downhill for 5 minutes did not experience muscle damage as measured by an increase in plasma creatine kinase ( CK ) whereas mdx mice did. However using a more intense protocol, Lynch et a l. [ 210 ] reported histological signs of muscle damage in the
117 soleus and EDL muscles wild type mice (60 min at 16% decline). Similarly, Ca rter et al. [ 211 ] reported a large increase in plasma CK in wild type mice that ran downhill until exhaustion. Therefore, the amount of muscle damage that occurs in controls is likely dependent on the intensity of the running protocol and this was not achieved with the current protocol. We found that the percent of pixels with el evated T 2 48 hours after downhill running in mdx mice was associated with a histological measurement of increased sarcolemma from membrane damage. [ 212 ] Muscle T 2 elevation occurring 48 hours following exercise is likely due to multiple causes including fiber damage, inflammato ry responses, and subsequent edema. Marqueste et al. [ 195 ] reported that increased plasma markers of muscle damage (creatine kinase and lactate dehyd rogenase) were correlated to increased muscle T 2 3 to 17 days after downhill running in rabbits. However the authors also noted an increase in muscle edema, which could also contribute to elevated T 2 [ 195 ] The process of muscle damage is associated with an inflammatory response as well as edema within the muscle, [ 195 ] both of which can independently result in increased T 2 [ 201 213 ] Future studies using other imaging methods, suc h as those that enable multiexponential analysis, along with histological analysis of inflammation and edema may be able to further elucidate the mechanisms accounting for increases in muscle T 2 In comparison to downhill running, mdx mice that ran on a h orizontal grade treadmill only demonstrated an acute increase in the percentage of pixels with an elevated T 2 which then returned to baseline levels by 24 hours post exercise. This
118 indicates that although horizontal running caused significant muscle activ ation in mdx mice, it did not result in muscle damage to the same extent as downhill running. This result is consistent with the well established finding that downhill running an eccentrically biased activity [ 214 ] is more damaging than horizontal running which is predominately concentric. The comparison between horizontal and downhill running in mdx mice is an interesting model by which to compare the effects of dystrophin muscle damage in untreated mdx mice. Previous studies on downhill running in mice and rats have shown that the tibialis anterior muscle is damaged to a lesser extent than the gastrocnemius and soleus muscles, [ 192 195 ] which was attributed to the plantarflexor m uscles undergoing a longer period of eccentric work than the dorsiflexors during downhill running. [ 214 ] We found no difference between the anterior and posterior muscle compartments in T 2 elevation following downhill running in mdx mice. However muscles of the medial compartment of the lower hindlimb showed a higher muscle T 2 at baseline than both the anterior or posterior compartment muscles. There was also an increase in muscle T 2 in the medial compartment following downhill running, which was not observed in other two muscle compartments. These findings imply that the medial compartment muscles may be more s usceptible to contraction induced muscle damage both during regular cage activity (i.e. baseline) and during exercise. The medial compartment muscles consist of the long toe flexors located between the tibia and fibula (flexor digitorum longus and flexor h allucis longus), tibialis posterior and the peroneals. We have not identified other studies that have examined the response of these muscles to downhill
119 running in rodents. It is plausible that one or more of these muscles undergoes greater strain or eccen tric activity during downhill running than the others, resulting in greater muscle damage and T 2 elevation. Furthermore, these muscles are primarily composed of fast twitch fibers in rodents [ 215 ] which also increases their susceptibility to muscle damage. The functional role of these muscle s during downhill running and their susceptibility to muscle damage in mdx mice warrants further investigation. We utilized two measures from T 2 weighted imaging to examine changes in signal intensity after exercise: mean T 2 relaxation time (ms) from a region of interest and the percentage of pixels exceeding a pre defined threshold. We found that the threshold method used in this study (% of pixels between 30 and 100 ms) was more sensitive for detecting differences in T 2 between mdx and control mice after downhill running than the mean T 2 of the ROI. Threshold methods for pixel intensity from MRI have previously been used to examine dystrophic muscle [ 114 ] and muscle activation following exercise. [ 216 ] This method differs from the calculation of the mean T 2 from a specified region, as it identifies the pr oportion of pixels that exceed a given threshold. In our study the threshold was set two standard deviations above the mean T 2 of uninjured, control muscle, theref ore was used to identify muscle, which had particularly high signal intensity on T 2 weighted imaging. About 60% of mdx mice in our study were able to complete the downhill running protocol with encouragement and 100% of control mice were able to run downhill and did not require any external encouragement. Previous reports have indicated that mdx mice have a greater fatigue response to treadmill or free wheel running than wild type mice. [ 20 217 ] Vilquin et al. [ 193 ] reported that mdx mice aged 9 to14 months were
120 unable to run for more than 5 minutes without external stimulation on a more intense downhill running protocol than used in our study ( 16 o declin e at 10m/min). They also the mice died one hour post exercise. We did not have any fatalities from running in our study, although the protocol was much longer in le ngth (45 minutes) and the mice were of a similar age. We also found that all of the mdx mice were able to complete the horizontal running protocol, even though downhill running is less energy consuming than horizontal or uphill activity. Therefore, this di fference may not have only to do with muscle fatigue but also a protective response to muscle damage. The tolerance of mdx mice to downhill running has important implications when planning studies to examine the response to intervention. Depending on the a ge of the animal and the intensity of the protocol a proportion of mice may not be able to complete the protocol, therefore a The limitations of this study must also be consi dered when interpreting the results. First, we did not include a group of control mice in the horizontal running protocol to compare with the mdx group. However, since there was no prolonged T 2 elevation observed in the mdx mice following horizontal runnin g, the comparison to controls may not have provided any additional information regarding susceptibility to muscle damage. Second, the T 2 measurements were made using single exponential analysis from two echo times, which has previously been employed by our group [ 112 213 ] and others. [ 216 ] Although the experiment may have benef itted from multi exponential analysis in order to further differentiate muscle damage from inflammation, it is challenging to obtain images at multiple TEs during in vivo
121 experiments. Therefore this approach was beyond the scope of this study. Finally, the correlation between EBD positive fibers and percent of pixels with elevated T 2 is limited by the fact that perfect registration between the histological cross section and trans axial MR image was not obtained, although the mid belly of the muscle was samp led for both measures. However, we observed macroscopically that bundles damaged muscle fibers, positive for EBD, ran continuously along the entire length of a muscle and were therefore captured on multiple cross sections. This has also been shown using lo ngitudinal muscle sections of EBD positive fibers in mdx mice by Straub et al. [ 218 ] Similarly, areas of elevated muscle T 2 were observed in the same spatial location on multiple trans axial images of the muscle. Although the cross sections were not exactly registered, the correlation between histology and MRI findings of muscle damage based on using the mid belly region of the muscle seems acceptable based on the distribution of damaged fibers along the entire length of a muscle. Summary The protocol for downhill running used in this study provides a stimulus to induce muscle damage in the lower hindlimbs of mdx mice, which can be noninvasiv ely visualized using T 2 weighted MRI. Using MRI to examine T 2 changes following exercise allows for repeated and longitudinal measurements to be made from multiple muscles in a single animal. The ability to examine several muscles simultaneously is particu larly important since not all muscles respond in the same way to downhill running. Therefore MRI may be used to complement histological studies by determining which muscles experience the most damage and should be examined further. An important aspect of t he downhill running protocol used in this study was its ability to differentiate mdx mice that experience muscle T 2 changes from downhill run wild type mice and mdx mice run
122 on a horizontal grade treadmill, which do not experience elevated T 2 after running. In addition, this protocol allowed for the examination of acute muscle damage following a known and timed perturbation event, in the presence of asynchronous bouts muscle damage and recovery that are typically observed in dystrophic mice.
123 Figure 7 1. Trans axial MR image of the lower hindlimb showing regions of interest (ROIs) for muscle T 2 analysis. Muscles in each region include: tibialis anterior and extensor digitorum longus in the anterior compartment, flexor hallucis and flexor digitorum in the medial compartment and gastrocnemius, soleus and plantaris in the posterior compartment.
124 Figure 7 2 P ercentage of pixels with elevated T 2 and muscle T 2 A) Percentage of pixels with elevated T 2 (% damaged muscle; mean SEM) in the lower hindlimb muscles of mdx and control mice before and after downhill running B) Muscle T 2 of the lower hindlimb muscles of mdx and control mice before and after a single bout of downhill running. The mdx mice had higher T 2 values at all timepoints (pre and post exercise) than controls (p < 0.001; denoted by *). The mdx mice showed a significant increase in muscle T 2 immedia tely post was observed in T 2 values of control mice following a bout of downhill running.
125 Figure 7 3 Percentage of pixels with elevated T 2 (% damaged muscle; mean SEM ) returned to baseline values ten days after downhill running in mdx mice (n = 5). The mdx mice showed a significant increase in % damaged muscle immediately post exercise, 24 hr and 48 hrs post exercise, compared to pre exercise values (p < 0.05, denoted by *), however no significant difference was seen 10 days after downhill running compared to baseline. Figure 7 4 Transaxial, T 2 weighted images from the lower hindlimb of an mdx mouse before and after a single bout of downhill running for 45 min ( 14% grade, 8m/min). Note specific area of hyperintensity in the medial region, between the tibia and fibula, which is recovered after 10 days.
126 Figure 7 5 Three dimensional render ing of regional muscle damage in the hindlimb of an mdx mouse following downhill running. Figure 7 6 Muscle T 2 values in mdx mice following a single bout of downhill running compared to horizontal running. Muscle T 2 showed a significant increase immediately following downhill running (compared to pre exercise, p < 0.05, denoted by *) but not following horizontal grade running.
127 Figure 7 7 Change in % muscle damage in individual muscle compartments of mdx mice f ollowing a single bout of downhill running. The medial muscle compartment (flexor hallucis/digitorum longus muscles) had a higher % of damaged muscle at baseline than the anterior and posterior muscle compartments (p < 0.05; denoted by *) and also showed s ignificant increases in % damage from pre exercise values, immediately post exercise and at 24 Figure 7 8 EDL muscle of mdx mouse 48 hr following 1 bout of downhill treadmill running.
128 Figure 7 9 Correlation between % of pixels with elevated T 2 and % of area showing
129 CHAPTER 8 EFFECT OF UPHILL RUNNING ON MYOCARDIUM T 2 IN MDX MICE Duchenne muscular dystrophy patients exhibit progressive skeletal and cardiac muscle patholo gy. Skeletal muscles of DMD patients display signs of weakness at approximately 5 years of age. Furthermore, loss of diaphragm function leads to respiratory failure and this account for m ore than 75% of deaths in DMD patients. As a result, research has bee n more focused on understanding the manifestation of D MD in skeletal muscles. I mprovement in ventilator support techniques over the past decade has lead to prolongation in the life expectancy of DMD children. Moreover, novel experiment strategies have show n promise in restoring skeletal muscle function. On the other hand, these strategies often leave the dystrophic cardiac muscle essentially untreated. Clinically apparent cardiomyopathy usually occurs after 10 years of age and almost all the patients are af fected by the age of 18 years [ 219 ] As a result of increased life expectancy complications arising fr om cardiac involvement dominate the later part of life in DMD children. With the improvement in strategies involving skeletal and respiratory systems, it is estimated that approximately 20% of DMD patients die of heart failure. [ 220 221 ] Research on DMD has vastly benefited fr om animal models, particularly the murine model, the mdx mouse which result s from a nonsense mutation exon 23 of t he dystrophin gene [ 222 ] Mdx mice display histological signs of skeletal muscle pathology as early as 3 weeks of age which becomes more prominent between 3 rd and 8 th week of life. On th e other hand, mdx mice do not demonstrate any signs of cardiac pathology till later stages of life [ 223 ] Novel gene and cell therapies have been utilized to increase
130 levels of dystrophin and thereby restoring both skeletal and cardiac function in mdx mouse. [ 224 227 ] Cardiac stress testing has been widely used to identify cardiac abnormalities that are undetectable at rest. [ 227 228 ] Several methods of per forming stress testing have been used in the animal models including administration of a low dose dobutamine [ 229 ] However, uphill running is a more physiological relevant model of cardiac stress test. This model has been utilized to study changes i n both skeletal [ 230 ] and cardiac muscles. [ 231 ] Furthermore, uphill running has several advantages over other methods, such as, it incorporates the physiological load on the heart and it can be used repeatedly during longitudinal studies of damage and repair. [ 195 ] Magnetic resonance imaging (MRI) has been proposed as a non invasive method of monitoring tissue structure in DMD patients. The measure of transverse relaxat ion (T 2 ) of tissue has been well documented and it has been used to detect various soft tissue pathologies. T 2 is believed to increase with fatty tissue accumulation and increase in water content and edema whereas it decreases with collagen accumulation or fibrosis. Because T 2 relaxation time changes with the disease progression it may be used to monitor disease progression and response to therapy. T 2 weighted imaging has been demonstrated to be a valuable tool in cardiac imaging of DMD patients. [ 232 233 ] Methods Both C57BL/10ScSn DMD mdx ( mdx ; n = 5) an d wild type C57BL/10ScSn (controls n = 5) adult male mice (aged 3 4 months) were included in the study. Wild type mice and breeding pairs from the mdx colony were obtained from Jackson Laboratories (Bar Harbor, Maine) and thereafter maintained in house. An imals were housed in an
131 AAALAC approved facility with a 12 hour light: dark cycle (72 o F, 42% humidity) and free access to food and water. The institutional animal care and use committee of University of Florida approved the experimental protocol MRI was c onducted prior to treadmill running in both mdx and control mice to determine baseline muscle T 2 values of the lower hindlimb muscles. After the initial MRI, each mouse underwent a bout of uphill running. Specifically, mice were run on a uphill slope (14 d egrees decline) motorized treadmill at a speed of 8 13 m/min, for up to one hour. To ensure the mice ran for the entire duration of the protocol, they were observed continuously. If necessary, a short burst of compressed air or a brush was applied at the base of the tail was used to encourage running. C ardiac magnetic resonance (CMR) was conducted at 24 hours post exercise Magnetic resonance i maging : A 4.7T Oxford Magnet with an Agilent/Varian operating system was used to acquire gated T 2 weighted single spin echo (SE) images of the left ventricle in the short axis view (TR 750 ms; TE 12.5 ms and TE 30 ms; field of view, 25X25 mm 2 ; slice thickness, 1.0 mm; acquisition matrix size, 256 X 128; averages, 8) with a custom built quadrature volum e coil ( 3.3 cm inner diameter). The animal was anesthe tized using an oxygen and isoflurane mixture (3% isoflurane) and m aintained under 0.5 1% isoflur ane for the duration of the MR procedure. The respiratory rate of the mouse was monitored for the duration of the scan (SA Instruments, Inc., Stony Brook, New York). Diffusion weighting was fixed at both TEs (diffusion weighting 3 mm 2 /s at both 14 and 40 ms). Cardiac MR T 2 analysis : Short axis slices from the mid papillary region were used to calculate mean T 2 Mean T 2 was calculated using the average signal intensity at
132 each TE by manually tracing the myocardium. T 2 relaxation time wa s calculated using a single expo nential decay wit h the following equation: T 2 = (26 ms) / ln (SI12.5 / SI30), where SI12.5 and SI30 are the pixel intensities at 12.5 and 30 ms, respectively [ 172 ] Histological confirmation of myocardial membrane damage : Twenty four hours after the exercise bout, mice were injecte d with EBD (0.10 mL per 10 g of body weight) via intraperitoneal injection. Forty eight hours after the exercise bout (i.e 24 hours post injection), heart was excised, coated with OCT, frozen in isopentane cooled with liquid nitrogen and stored at 80 o C. F rozen heart sections were sectioned at 10 microns in a cryostat and mounted on slides using a fluorescence mounting medium with 4 6 diamidino 2 phenylind ole (DAPI; VectaShield, Vec tor Labs). Slides were viewed using fluorescence microscopy, optimized fo r EBD (Texas red filter). Images of the entire muscle cross section were cap tured at 10 X mag nification using a light micro scope (Leica Microsystems ) 1 attached to a digita l camera with slightly overlap ping fields to obt ain images from the entire mus cle. Th e cross s ection of the muscle was recon structed in Adobe Pho toshop from the digital images. Statistical analysis : Data were analyzed using SPSS, version 20.0 2 The descriptive data are presented using mean and standard error of the mean (SEM). To compare t he differences in myocardium T 2 between mdx and control mice after a single bout of downhill running, independent sample t tests were used to make comparisons at each time point between groups (alpha = 0.05, adjusted for multiple comparisons using a modifi ed Bonferroni correction). Paired t tests were used to compare differences in 1 Leica Microsystems, Bannockburn, Illinois. 2 IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp
133 muscle T 2 and percent damage from pre exercise values to post exercise time points, in each of the mdx and control groups (alpha = 0.05, adju sted for multiple comparisons). Results Myocardium T 2 was compared between mdx and control mice after a single bout of uphill running. As shown in Figure 8.3, muscle T 2 significantly differed between control and mdx mice post exercise. A t the pre exercise time point the muscle T 2 did n ot differ significantly between the two groups. Each control mouse completed one hour of uphill running (800 meters), while there was considerable variability in the amount of time and distance run by mdx mice (33 21 minutes; 329227 meters), with only one mouse completing one hour. Discussion This study was the first to examine the myocardium after uphill run ning in dystrophy using MRI T 2 The main finding of this study was that uphill running results in an increase in T 2 in the myocardium of dystrophic mi ce, but not in wild type mice. As a result, this protocol may be valuable to non invasively test interventions aimed at improving sarcolemmal integrity in dystrophic myocardium using murine models. MRI T 2 : T 2 has been well established to be sensitive to muscle damage, as Blue positive fibers (chapter 7) and presence of central nuclei (chapter 6). Keeping this in account, we used T 2 weighted images as an index of myocardial injury in mdx mice. The measure of transverse relaxation time (T 2 ) of tissue has been well characterized. T 2 weighted MR images of skeletal muscles have been shown to be sensitive to both acute injury and e xercise induced contrast enhancement. Furthermore, elevated T 2 may be due to an increase in tissue fluid content or decreased muscle integrity. In recent years,
134 T 2 weighted imaging has been utilized in cardiac imaging of acute myocardial process such as my ocardial infarct [ 234 ] and myocarditis. [ 235 236 ] T 2 has also shown promise in monitoring cardiac involvement in boys wi th Duchenne muscular dystrophy [ 232 ] Disease progression in Dystrophy: Both DMD patients and mdx mice have complete loss of dystrophin from skeletal muscle fibers as well as cardiomyocyte. Unlike skeletal muscles of mdx mice, the heart does not show any abnormal functional pathological signs until 9 11 months. [ 237 ] In this study we utilized mean T 2 of myocardi um to quantify myocardial damage in mdx mice after a single bout of uphill running. MR measurements of T 2 in myocardium were significantly higher in mdx mice compared to control mice after a single bout of uphill running. The el evated T 2 of cardiac muscle after a single bout of uphill running were suggestive of an increase in myocardial damage. Furthermore, t his was supported by presence of EBD positive fibers. Model to test interventions: Uphill running has previously been used in murine model to test the sarcolemmal integrity of muscle fibers in myocardium. For example Michele et al. [ 231 ] used a n uphill running protocol similar to this study and found that muscle s lackin g normal dystroglycan function were more susceptible to damage. Furthermore, they found an increase in the number of EBD positive fibers in cardiac myocytes after an acute bout of uphill running. Uphill running has been used as a physiological relevant mod el for cardiac stress testing. Accumulation of EBD has been shown to be one the earliest indicator of cardiac disease. By 3 months of age there is an increase in accumulation of dye as compared to controls. However, the extent of damage remains low because of resealing mechanisms that take place t horough our
135 lifetime. [ 238 239 ] Furthermore, accumulation of fibrotic tissue has been reported to occur in 6 months old mdx mice. Therefore mdx mice aged between 3 5 months may be preferential for a therapy that improves the membrane stability, where as older mdx mice may be valuable for anti fibrotic studies. Summary The uphill running protocol used in this s tudy is sufficient to induce myocardium damage in mdx mice. T 2 weighted MRI and histological studies can be used to visualize myocardium damage. This protocol allowed us to examine acute myocardium da mage after a timed perturbation which may provide valua ble information regarding pot ential therapeutic agents.
136 Figure 8 1 Custom built quadrature volume coil with 3.3 cm inner diameter.
137 Figure 8 2 Representative four and two chamber long axis images (A) Four chamber and (B) two chamber image used to position scans in the short axis view. Short axis SE images were acquired at TE=12.5 ms (C) and TE=30 ms (D) to estimate myocardial T 2 from the mid papillary region of the left ventricle.
138 Figure 8 3 Myocardial T 2 values in mdx mice after a single bout of uphill running compared to wild type mice. Myocardial T 2 showed significant increase in mdx mice compared to wild type after a single bout of uphill running (*p<0.05).
139 Figure 8 4 Myocardium of mdx mouse 48 hours following a single bout of uphill running.
140 CHAPTER 9 CONCLUSIONS Overview The muscular dystrophies are a group of heterogeneous genetic disorders characterized by muscle wasting. During the last decade, new avenues have opened up both in gene and drug based therapi es. In order to monitor the disease progression and response to a therapy a reliable and quantitative non invasive method is required. In the present work I have used MRI as a non invasive technique in order to monitor the pathological changes occurring in DMD. Summary of Conclusions Relationship between lower limb muscle cross sectional area and torque production in boys with Duchenne muscular dystrophy : MRI along with dynamometry was implemented to measure CSA max and specific torque production of lower extremity muscles in children with DMD. Age related changes in muscle CSA and specific torque production in lower extremity muscles displayed different patterns in the knee extensor, plantar flexor, and dorsi flexor muscles of boys with DMD. The distal tr iceps surae muscles showed a larger CSA max by approximately 60% in ambulatory boys with DMD compared with controls across ages, while the tibialis anterior muscle showed age appropriate increases in CSA max Furthermore, in the quadriceps muscle, CSA max ten ded to be higher than controls in younger boys with DMD but lower in older boys with DMD over 10 to 11 years of age. All muscles showed significant deficits in specific torque, with a 4 fold difference in the PF and KE, and 2 fold difference in the DFs.
141 R elationship between lower extremity muscle contractile cross sectional area and function al measurements in boys with DMD: The amount of non contractile content, measured by MRI was greater in dystrophic peroneal, medial gastrocnemius and soleus muscles. The increase in non contractile tissue lead to deterioration in timed functional tasks such as 30 i ncrease in contractile cross sectional area was seen in the peroneal and medial plantar flexor muscles showed a 2 fold difference in specific torque. To monitor changes in T 2 with age in hindlimb muscles of mdx mice: MRI is non invasive tool that has the ability to examine several muscle simul taneously. We used T 2 weighted MRI to distinguish muscles of mdx and control mice and evaluate time dependent changes Using T 2 MRI we found asynchronous and cyclical changes in both the anterior and posterior hindlimb muscles of young mdx mice. The mean muscle T 2 and percent pixels with elevated T 2 peaked betwee n 7 8 weeks. Changes in muscle T 2 a nd tissue damage in hindlimb muscles of mdx mice after downhill running: The protocol for downhill running used in this study provides a stimulus to induce muscle damage in the lower hindlimbs of mdx mice, which can be noninvasively visualized using T 2 weighted MRI. The ability to examine several muscles simultaneously is particularly important since not all muscles respond in the same way to downhill running. Therefore MRI may be used to complement histological studies by determining which muscles expe rience the most damage and should be examined further. Using the downhill running protocol we were able to differentiate between mdx mice and wild type mice with mdx mice experiencing extensive muscle T 2 changes
142 following downhill running. The increase in T 2 in mdx mice was observe d for an extended period, consistent with the presence of muscle damage. mdx mice that ra n on a horizontal grade treadmill did not experience elevated T 2 after running. Effect of uphill running on myocardium T 2 in mdx mice: T he uphill running protocol used in this study is sufficient to induce myocardium damage in mdx mi ce. T 2 weighted MRI in combination with histological studies can be used to visualize myocardium damage. This protocol allowed us to examine acute myocardium dama ge after a timed perturbation.
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163 BIOGRAPHICAL SKETCH Ravneet Singh Vohra was born in Amritsar, India. He completed his high school and senior secondary education from St. Francis School, Amritsar and Khalsa College, Amritsar, respectively. He received his undergraduate degree in physical therapy from Guru Nanak Dev University, Amritsar. He joined the doctoral program in rehabilitation sciences at the Unive rsity of Florida in fall of 2006 He received his PhD from the University of Florida in t he fall of 2012.