Multiphoton microscopy and tissue clearing for 3D characterization of the vasculature and fibrosis remodeling in rat dystrophic skeletal muscle

Duchenne muscular dystrophy (DMD) is characterized by repeated cycles of muscle fiber necrosis/regeneration and their progressive replacement by fibrous and adipose tissues. Vascular abnormalities are also reported as pathophysiological hallmark. Traditional investigations of these morphological changes rely on two-dimensional (2D) assessments of thin tissue sections. However, they fail to capture the global spatial distribution and structural disorganization within the muscle. In this study, we developed a 3D approach combining multimodal microscopy and tissue-clearing methods to investigate the properties of microvascular and connective tissue networks in a dystrophic context. By using segmentation techniques based on deep learning models, we established a dedicated 3D image analysis workflow. We analyzed samples from healthy and dystrophic rats, quantifying key parameters for vascular and connective tissue compartments. We showed a profound spatial reorganization of the vascular network in dystrophic muscle, characterized by its embedding within connective tissue and a consequent reduction in physical interactions with muscle fibers. Our findings demonstrate that this novel imaging approach provides detailed insights into the extent of remodeling in the vascular system and connective tissue of dystrophic muscle. It holds significant potential as a powerful tool for monitoring disease progression and evaluating the impact of therapeutic interventions.