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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月21日

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  • Ibrahim Hassani,
  • Mireille Ledevin,
  • Chantal Thorin,
  • Tony Fiore,
  • Marie-Anne Colle,
  • Karl Rouger,
  • Laurence Dubreil

摘要

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.