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Animal Models for Genetic Muscle Diseases
There is no established cure for muscular dystrophies and congenital myopathies, which prompted the search for innovative therapeutic protocols, including cellular and genetic therapies. In this context, animal models for these genetic muscle diseases have an essential role in the elucidation of disease pathomechanisms and in the development of new therapeutic strategies. Several animal models for genetic muscle diseases have been described in the literature, including natural and genetically engineered models. These animals mimic the genetic, molecular and/or clinical aspects of the disease, providing information about the pathogenesis of these disorders and allowing tests for therapeutic strategies (Vainzof et al., 2008).
Animal models for Muscular Dystrophies
The Dmdmdx mouse (hereafter called simply mdx) is the most frequently studied mouse model for DMD. This mouse has a stop codon in exon 23 of the murine dystrophin gene, which leads to total absence of this protein in muscle, as observed in DMD patients (Bulfield et al., 1984; Sicinski et al., 1989). The absence of the protein dystrophin leads to an associated reduction of other DGC proteins (Ohlendieck and Campbell, 1991), with consequent progressive muscle deterioration and weakness (Pastoret and Sebille, 1995). Histological analysis shows dystrophic changes such as variation in the caliber of muscle fibers, presence of muscle fibers with centralized nuclei, clusters of degenerating and regenerating fibers, infiltration by inflammatory cells and by connective tissue (Bulfield et al., 1984). As observed in human patients, different skeletal muscles are not identically affected. Diaphragm presents accentuated and earlier dystrophic characteristics, while masseter muscle is partially spared and limb muscles like gastrocnemius have an intermediary phenotype (Muller et al., 2001; Stedman et al., 1991). Nevertheless, differently from human patients, the mdx mouse can continuously regenerate its muscles and has a mild Dog models for dystrophinopathies in general have the phenotype closer to that observed in human patients. Spontaneous mutations in the dystrophin gene were observed in Golden Retriever (Valentine et al., 1992), Rotweiller (Winand et al., 1994) and German Shorthaired Pointer dogs (Schatzberg et al., 1999). The Golden Retriever Muscular Dystrophy dog (GRMD) is the more frequently studied canine model to DMD. GRMD dogs have a mutation in the dystrophin gene, leading to absence of the protein in muscle. Histopathological evaluation shows progressive dystrophic alterations in skeletal and cardiac muscles, and the phenotype is very severe. GRMD dogs mimic the molecular, histopathological and phenotypical aspects of DMD, being frequently used in the last step of therapeutic trials before tests in human patients (Shelton and Engvall, 2005). Recently, the GRMD mutation has been transferred to Beagle dogs, a smaller canine strain widely used in research (Shimatsu et al., 2003).
Non-invasive evaluation of genetic muscle diseases
The diagnostic of genetic muscle diseases is based on clinical evaluation, quantification of muscle proteins in serum, electromyography, muscle biopsy and molecular analysis. Due to the large spectrum of genes related to muscle disorders, genetic testing can be a difficult task, especially while next generation sequencing is not widely used. In this way, imaging methods such as Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) can allow the identification of the pattern of muscle involvement, orienting the genetic testing and helping in the differential diagnosis. Muscle MRI and CT from patients with genetic muscle disorders allowed the identification of the pattern of muscle involvement in these diseases, with strong correlation with genotype. The pattern of affected and spared skeletal muscles varies among muscular dystrophies, congenital myopathies and also between other muscle pathologies, such as inflammatory myopathies (Lamminen, 1990; Mercuri et al., 2007; Quijano-Roy et al., 2011, 2012; Wattjes et al., 2010). Early and correct diagnosis can affect the management of muscular dystrophy and congenital myopathy patients, since some of these diseases may involve cardiac and pulmonary complications that require early intervention (Shieh, 2013).
NMR in the study of animal models for genetic muscle diseases
When applied to small animals, NMR faces the challenge of reducing dimensions and increasing resolution. Additionally, while in human patients with muscle disorders the muscle degeneration is followed by fat infiltration, mouse models for muscle diseases present low to zero fat infiltration in muscles (Carnwath and Shotton, 1987; McIntosh et al., 1998b). MRI analysis of intramuscular fat infiltration is therefore not as informative in mice as it is in patients, which raises the interest in other NMR approaches to non-invasively evaluate murine models, such as quantitative T1 and T2 measurements, in vivo spectroscopy and functional NMR.
It has been shown that mdx mice have increased muscle T2 values, at rest and after exercise (Mathur et al., 2011). Muscle heterogeneity in T2-weighted MRI also changes in mdx mice with the disease evolution (Pratt et al., 2013), indicating that not only T2 values but also quantitative measures of the distribution of muscle alterations can correlate with the phenotype in dystrophic mice. Muscle T2 has already been used to follow dystrophic muscle after gene therapy in a murine model for LGMD-2D (Pacak et al., 2007).
While all these studies compared dystrophic murine models to wild-type or control mice with the same background, Tardif-de-Géry and collaborators have compared muscle T2 values from two mouse models with deficiency in the protein laminin, the very severely affected dy/dy mouse and the dystrophic, but less severely affected, dy2J/dy2J mouse. Both models have increased T2 when compared to wild-type mice at least in one stage of the disease, but no difference was observed between the two dystrophic mouse strains (Tardif-de Géry et al., 2000).
Magnetic Resonance Imaging acquisition and analysis
The mice were anesthetized with intraperitoneal injection of ketamine:xylazine (2:1, 1.5-2.5 μl/g according to the lineage) and symmetrically positioned for the MRI acquisitions. The images were acquired in a 2 tesla/30 cm bore superconducting magnet (Oxford Instruments 85310HR, Abingdon, United Kingdom), interfaced to a Bruker Avance AVIII console (Bruker-Biospin, Inc., Billerica, MA, U.S.A.) running PARAVISION 5.0. A crossed-saddle radiofrequency coil projected for small animals (Papoti, 2006) was used to image the mice’s posterior limbs.
Four scans were performed in each mouse, with the same geometry (4 slices, 1.5 mm slice thickness, 4 mm inter-slice distance and spatial resolution 0.176X0.176 mm2/pixel): two scans for anatomical images with the same parameters (repetition time – TR=1800 ms, echo time – TE=52.5 ms), with and without fat suppression for qualitative evaluation of possible fat infiltration in the muscles (with fat suppression:16 averages; without fat suppression: 4 averages); and two scans for the calculation of the T2 maps, each one using a different echo time (to avoid the contribution of stimulated echoes): TE1=12.1 ms and TE2=40 ms (TR=1500 ms, 1 average when TE1=12.1 ms, 4 averages when TE2=40 ms, spatial resolution: 0.176X0.176 mm2/pixel). The T2 value for each pixel was calculated using the Bloch equation for the spin-spin relaxation time (Supporting Information S2.1), and T2 maps were generated using a routine developed in the MATLAB software (The MathWorks, Inc., Natick, Massachusetts, USA). The total acquisition time was 51 minutes. The examination time never exceeded 1 hour and 30 minutes.
In the T2 maps, two slices were selected for analysis: one positioned at the lower leg and one at the thigh. One slice was considered representative of thigh and leg muscles since injured fibers would present anomalies along all its length. Nevertheless, this limits the analysis to the muscles observed in a defined anatomical position.
Table of contents :
ABSTRACT
RESUMO
RESUME
GENERAL INTRODUCTION
LIST OF PUBLICATIONS
CHAPTER 1. BIBLIOGRAPHIC REVIEW: NON-INVASIVE STUDY OF GENETIC MUSCLE DISORDERS
MUSCULAR DYSTROPHIES
Vascular alterations in Duchenne muscle dystrophy
CONGENITAL MYOPATHIES
ANIMAL MODELS FOR GENETIC MUSCLE DISEASES
Animal models for Muscular Dystrophies
Animal models for congenital myopathies
NON-INVASIVE EVALUATION OF GENETIC MUSCLE DISEASES
NMR IN THE STUDY OF ANIMAL MODELS FOR GENETIC MUSCLE DISEASES
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, LARGEMYD AND MDX/LARGEMYD
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Ethics Statement
Animals
Magnetic Resonance Imaging acquisition and analysis
Histological analysis
Statistic analysis
RESULTS
Muscle T2
Muscle texture analysis
Histological analysis
DISCUSSION
CONCLUSIONS
SUPPORTING INFORMATION
S2.1. T2 calculation from two images at different echo times
S2.2. Features selected for Texture Analysis
COMPLEMENTS TO THE MANUSCRIPT
Validation of the 2 points T2 measurements with a multiecho sequence
Post-mortem changes in the T2 values
CHAPTER 3. STRUCTURAL AND FUNCTIONAL ALTERATIONS OF SKELETAL MUSCLE MICROVASCULAR NETWORK IN DYSTROPHIN-DEFICIENT MDX MICE
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Mice
Microvascular network organisation in three dimensions
Histology
Morphometric analysis
Nuclear Magnetic Resonance analysis
Statistics
RESULTS
Normal microvascular network organisation in young-adult Flk1GFP/+::mdx mouse.
Muscle blood perfusion is modified in young-adult mdx mice.
Muscle bioenergetics in young-adult mice (Table 3.1)
Alteration of microvascular network organisation in old Flk1GFP/+::mdx mouse
Alteration of muscle perfusion in old mdx mice
Muscle bioenergetics in 12 month-old mice (Table 3.2)
DISCUSSION
CONCLUSION
SUPPLEMENTARY INFORMATION
S3.1. Nuclear Magnetic Resonance analysis
CHAPTER 4. NON-INVASIVE NMR STUDY OF THE MOUSE MODEL FOR CENTRONUCLEAR MYOPATHY WITH MUTATION IN THE DYNAMIN-2 GENE
INTRODUCTION
MATERIALS AND METHODS
Animals
Nuclear Magnetic Resonance (NMR)
Data analysis
Histological analysis
Statistical analysis
RESULTS
Morphometrical evaluation
T1 measurements
T2 measurements
Histological analysis
DISCUSSION
CHAPTER 5. PILOT FUNCTIONAL AND METABOLIC EVALUATION OF THE KI-DNM2R465W MICE
INTRODUCTION
PILOT STUDY 1. EXERCISE AS THE PARADIGM OF MUSCLE STRESS IN KI-DNM2R465W MICE
Materials and methods
Results
Discussion
PILOT STUDY 2. PROLONGED ISCHEMIA AS THE PARADIGM OF MUSCLE STRESS
Materials and Methods
Results
Discusssion
PILOT STUDY 3. REGENERATION IN THE DNM2 MICE: T1, T2 AND FUNCTIONAL ANALYSIS AFTER INJURY
Materials and Methods
Results
Discussion
CHAPTER 6. EVALUATION OF THE POTENTIAL USE OF MICRO-COMPUTED TOMOGRAPHY IN THE STUDY OF MUSCLES FROM MURINE MODELS FOR MUSCLE DYSTROPHIES
INTRODUCTION
MATERIALS AND METHODS
Animals
Muscle injury with electroporation
Micro-CT
Data Analysis
Statistical Analysis
RESULTS
Phenotypical characterization of the dystrophic muscle with micro-CT
Evaluation of injured muscle with micro-CT
DISCUSSION
GENERAL CONCLUSIONS AND PERSPECTIVES
BIBLIOGRAPHY
