Evaluation atraumatique de modèles murins de maladies musculaires génétiques. (Non-invasive evaluation of murine models for genetic muscle diseases)

...................................................................................................................................... 11 RESUMO ......................................................................................................................................... 13 RESUME .......................................................................................................................................... 15 GENERAL INTRODUCTION ............................................................................................................... 18 LIST OF PUBLICATIONS .................................................................................................................... 24 CHAPTER 1. BIBLIOGRAPHIC REVIEW: NON-INVASIVE STUDY OF GENETIC MUSCLE DISORDERS ...... 27 MUSCULAR DYSTROPHIES ................................................................................................................... 27 Vascular alterations in Duchenne muscle dystrophy ................................................................... 30 CONGENITAL MYOPATHIES .................................................................................................................. 33 ANIMAL MODELS FOR GENETIC MUSCLE DISEASES ............................................................................. 35 Animal models for Muscular Dystrophies .................................................................................... 35 Animal models for congenital myopathies ................................................................................... 38 NON-INVASIVE EVALUATION OF GENETIC MUSCLE DISEASES ................................................................. 39 NMR IN THE STUDY OF ANIMAL MODELS FOR GENETIC MUSCLE DISEASES ............................................ 41 CHAPTER 2. QUANTITATIVE T2 COMBINED WITH TEXTURE ANALYSIS OF NUCLEAR MAGNETIC RESONANCE IMAGES IDENTIFY DIFFERENT DEGREES OF MUSCLE INVOLVEMENT IN THREE MOUSE MODELS OF MUSCLE DYSTROPHY: MDX, LARGE MYD AND MDX/LARGE MYD ....................................... 44 ABSTRACT .......................................................................................................................................... 45 INTRODUCTION ................................................................................................................................... 46 MATERIALS AND METHODS ................................................................................................................. 49 Ethics Statement ........................................................................................................................... 49 Animals ........................................................................................................................................ 49 Magnetic Resonance Imaging acquisition and analysis .............................................................. 50 Histological analysis .................................................................................................................... 51 Statistic analysis........................................................................................................................... 52 RESULTS ............................................................................................................................................. 53 Muscle T2 ..................................................................................................................................... 54 Muscle texture analysis ................................................................................................................ 56 Histological analysis .................................................................................................................... 57 DISCUSSION ....................................................................................................................................... 60 CONCLUSIONS .................................................................................................................................... 65 SUPPORTING INFORMATION ................................................................................................................ 66 S2.1. T2 calculation from two images at different echo times ..................................................... 66 S2.2. Features selected for Texture Analysis ............................................................................... 67 COMPLEMENTS TO THE MANUSCRIPT ................................................................................................... 68 Validation of the 2 points T2 measurements with a multiecho sequence ..................................... 68 Post-mortem changes in the T2 values ........................................................................................ 69 CHAPTER 3. STRUCTURAL AND FUNCTIONAL ALTERATIONS OF SKELETAL MUSCLE MICROVASCULAR NETWORK IN DYSTROPHIN-DEFICIENT MDX MICE........................................................................... 72 ABSTRACT .......................................................................................................................................... 73 INTRODUCTION ................................................................................................................................... 74 MATERIALS AND METHODS ................................................................................................................. 76 Mice ............................................................................................................................................. 76 Microvascular network organisation in three dimensions ........................................................... 76 Histology ...................................................................................................................................... 77 Morphometric analysis ................................................................................................................ 77 Nuclear Magnetic Resonance analysis ........................................................................................ 78 Statistics ....................................................................................................................................... 79 RESULTS ............................................................................................................................................. 80 Normal microvascular network organisation in young-adult Flk1 GFP/+ ::mdx mouse. ................. 80 Muscle blood perfusion is modified in young-adult mdx mice. .................................................... 84 Muscle bioenergetics in young-adult mice (Table 3.1) ................................................................ 86 Alteration of microvascular network organisation in old Flk1 GFP/+ ::mdx mouse ........................ 88 Alteration of muscle perfusion in old mdx mice ........................................................................... 92 Muscle bioenergetics in 12 month-old mice (Table 3.2) .............................................................. 93 DISCUSSION ....................................................................................................................................... 94 CONCLUSION ...................................................................................................................................... 98 SUPPLEMENTARY INFORMATION .......................................................................................................... 99 S3.1. Nuclear Magnetic Resonance analysis ............................................................................... 99 CHAPTER 4. NON-INVASIVE NMR STUDY OF THE MOUSE MODEL FOR CENTRONUCLEAR MYOPATHY WITH MUTATION IN THE DYNAMIN-2 GENE ...................................................................................103 INTRODUCTION ................................................................................................................................. 103 MATERIALS AND METHODS ............................................................................................................... 105 Animals ...................................................................................................................................... 105 Nuclear Magnetic Resonance (NMR) ........................................................................................ 105 Data analysis ............................................................................................................................. 106 Histological analysis .................................................................................................................. 108 Statistical analysis ..................................................................................................................... 108 RESULTS ........................................................................................................................................... 108 Morphometrical evaluation ....................................................................................................... 108 T1 measurements ....................................................................................................................... 110 T2 measurements ....................................................................................................................... 111 Histological analysis .................................................................................................................. 113 DISCUSSION ..................................................................................................................................... 115 CHAPTER 5. PILOT FUNCTIONAL AND METABOLIC EVALUATION OF THE KI-DNM2 R465W MICE .........119 INTRODUCTION ................................................................................................................................. 119 PILOT STUDY 1. EXERCISE AS THE PARADIGM OF MUSCLE STRESS IN KI-DNM2R465W MICE ............... 120 Materials and methods ............................................................................................................... 120 Results ........................................................................................................................................ 123 Discussion .................................................................................................................................. 129 PILOT STUDY 2. PROLONGED ISCHEMIA AS THE PARADIGM OF MUSCLE STRESS ................................... 130 Materials and Methods .............................................................................................................. 130 Results ........................................................................................................................................ 131 Discusssion ................................................................................................................................ 134 PILOT STUDY 3. REGENERATION IN THE DNM2 MICE: T1, T2 AND FUNCTIONAL ANALYSIS AFTER INJURY ........................................................................................................................................................ 135 Materials and Methods .............................................................................................................. 135 Results ........................................................................................................................................ 136 Discussion .................................................................................................................................. 140 CHAPTER 6. EVALUATION OF THE POTENTIAL USE OF MICRO-COMPUTED TOMOGRAPHY IN THE STUDY OF MUSCLES FROM MURINE MODELS FOR MUSCLE DYSTROPHIES ...........................................................................................................................142 INTRODUCTION ................................................................................................................................. 142 MATERIALS AND METHODS ............................................................................................................... 143 Animals ...................................................................................................................................... 143 Muscle injury with electroporation ............................................................................................ 144 Micro-CT ................................................................................................................................... 145 Data Analysis ............................................................................................................................. 145 Statistical Analysis ..................................................................................................................... 146 RESULTS ........................................................................................................................................... 146 Phenotypical characterization of the dystrophic muscle with micro-CT ................................... 146 Evaluation of injured muscle with micro-CT ............................................................................. 147 DISCUSSION ..................................................................................................................................... 148 GENERAL CONCLUSIONS AND PERSPECTIVES .................................................................................152 BIBLIOGRAPHY ...............................................................................................................................155