In-Vivo Experimental Methodologies for Muscular Dystrophy Research.

Muscular dystrophies (MDs) are a heterogeneous group of inherited neuromuscular disorders characterized by progressive skeletal muscle degeneration, chronic inflammation, fibrosis, and loss of muscle function resulting from mutations in genes encoding proteins essential for maintaining muscle integrity. Among these disorders, Duchenne muscular dystrophy (DMD) represents the most common and severe form, underscoring the need for robust experimental models to investigate disease mechanisms and evaluate novel therapeutic strategies. Although in vitro systems have substantially improved the understanding of molecular pathogenesis, in vivo animal models remain indispensable for reproducing the complex physiological interactions among skeletal muscle, cardiac tissue, respiratory function, immune responses, and extracellular matrix remodeling. Over the past several decades, a diverse range of animal models—including mdx, mdx52, D2-mdx, and dystrophin–utrophin double-knockout mice, dystrophin-deficient rats, zebrafish, Golden Retriever muscular dystrophy (GRMD) dogs, porcine models, rabbits, and other genome-edited animals—has been developed to mimic different genetic and pathological aspects of muscular dystrophy. These models differ in disease severity, regenerative capacity, fibrosis, cardiomyopathy, lifespan, and translational applicability, enabling investigators to select appropriate systems based on specific research objectives. Animal models have played a central role in advancing preclinical research by facilitating the evaluation of disease progression, functional impairment, pharmacokinetics, pharmacodynamics, and long-term safety of emerging therapeutic approaches. They have been extensively employed in the development of corticosteroids, anti-inflammatory and antifibrotic agents, stem cell therapies, antisense oligonucleotide-mediated exon skipping, adeno-associated virus (AAV)-mediated micro-dystrophin gene replacement, CRISPR/Cas9 genome editing, base editing, prime editing, and regenerative medicine strategies. Furthermore, integration of advanced analytical technologies—including histopathology, immunohistochemistry, molecular imaging, transcriptomics, proteomics, metabolomics, single-cell sequencing, spatial transcriptomics, and artificial intelligence-assisted image analysis—has substantially enhanced the understanding of disease pathogenesis and therapeutic responses. This review comprehensively summarizes current in vivo experimental methodologies employed in muscular dystrophy research, critically evaluates conventional and emerging animal models, discusses their applications in therapeutic development, and highlights recent technological advances that are reshaping translational neuromuscular research. Particular emphasis is placed on the strengths, limitations, and clinical relevance of each experimental platform to facilitate appropriate model selection and improve the predictive value of preclinical investigations. Collectively, these in vivo methodologies provide an essential foundation for elucidating disease mechanisms and accelerating the development of safe, effective, and precision-based therapies for muscular dystrophy.