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 progressive muscle fiber degeneration and replacement by fibrous and adipose tissues, alongside significant vascular abnormalities. Traditional two-dimensional (2D) histological assessments provide limited insight into the complex three-dimensional (3D) spatial organization and structural disorganization within dystrophic muscle. Here, we present a novel 3D approach combining multiphoton microscopy (second harmonic generation [SHG] and two-photon excited fluorescence [TPEF]) with tissue-clearing methods to comprehensively characterize microvascular and connective tissue remodeling in dystrophic skeletal muscle. We established a dedicated 3D image analysis workflow utilizing deep-learning-based segmentation techniques to quantify key parameters in both vascular and fibrotic compartments in healthy and dystrophic rat muscle samples. Our findings reveal a profound spatial reorganization of the vascular network in dystrophic muscle, marked by its embedding within an expanded connective tissue and a significant reduction in physical interactions with muscle fibers. This advanced imaging and analysis pipeline provides detailed insights into the extent of vascular and fibrotic remodeling in dystrophic muscle, and represents a powerful tool for monitoring disease progression and evaluating the efficacy of therapeutic interventions.