Ishola Azeez O, Pillai Athira, Ahn Taeyong, Hsu Chih-Wei, Lee Brendan, Haelterman Nele, Wythe Joshua D
Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22903, USA.
Department of Integrative Physiology; Optical Imaging and Vital Microscopy Core, Advanced Technology Cores; Department of Education, Innovation and Technology, Baylor College of Medicine, Houston, TX 77030, USA.
bioRxiv. 2025 Jun 27:2025.06.26.661802. doi: 10.1101/2025.06.26.661802.
The vasculature is an essential regulator of tissue oxygenation, metabolism, and immune surveillance under developmental and homeostatic conditions, as well as in regeneration and disease. Within the musculoskeletal system, blood vessels are involved in bone development and resorption, and they also mediate the inflammatory processes that contribute to diseases affecting the joints, including osteoarthritis. Historically, visualization of vascular networks in joints has been limited to conventional histological approaches due to technical limitations and the inherit structure of the skeletal muscle and bones. Recent advances in optical tissue clearing technologies and 3D fluorescence imaging approaches enabling three-dimensional analysis in whole, intact tissues offer an entrée to interrogate musculoskeletal development and disease at a cellular resolution. However, these techniques were not originally developed to image hard mineralized tissues, such as the femur and tibias. To circumvent these challenges, we have optimized tissue decalcification conditions and systematically compared aqueous- and solvent-based tissue clearing approaches to establish an optimal pipeline for clearing and imaging of mineralized tissues with superior resolution of the vasculature. Collectively, this work shows that optical clearing and lightsheet microscopy presents a viable method for generating high-resolution images of the murine hindlimb vasculature, with potential applications in aging and disease modeling.
在发育、稳态条件下以及再生和疾病过程中，脉管系统是组织氧合、代谢和免疫监视的重要调节因子。在肌肉骨骼系统中，血管参与骨骼发育和吸收，还介导促成影响关节疾病（包括骨关节炎）的炎症过程。从历史上看，由于技术限制以及骨骼肌和骨骼的固有结构，关节血管网络的可视化一直局限于传统组织学方法。光学组织透明技术和3D荧光成像方法的最新进展能够在完整的全组织中进行三维分析，为以细胞分辨率研究肌肉骨骼发育和疾病提供了途径。然而，这些技术最初并非用于对诸如股骨和胫骨等坚硬矿化组织进行成像。为了克服这些挑战，我们优化了组织脱钙条件，并系统地比较了基于水和溶剂的组织透明方法，以建立一种用于矿化组织透明化和成像的最佳流程，从而实现对脉管系统的高分辨率成像。总体而言，这项工作表明光学透明化和光片显微镜技术是生成小鼠后肢脉管系统高分辨率图像的可行方法，在衰老和疾病建模方面具有潜在应用价值。
