Light sheet microscopy as a novel tool to observe satellite cell activation after eccentric contraction-induced damage at the muscle-tendon junction in a murine model

Injuries affecting skeletal muscle are frequent and debilitating in sport, age-related musculoskeletal disorders or chronic diseases. They usually occur after direct or indirect trauma and, in the latter, lesions mainly depend on an eccentric muscle contraction. After a focal injury, adult skeletal muscle undergoes regeneration thanks to the activity of a small population of muscle resident stem cells called satellite cells (SCs). SCs remain quiescent without dividing or differentiating, awaiting activating signals from damaged tissue. These signals trigger the asymmetric self-renewal in which a SC divides into one stem cell and one differentiating daughter cell. This mechanism leads to the complete differentiation in skeletal muscle fiber and functional tissue regeneration and, at the same time, it guarantees the maintenance and conservation of the stem cells niche. The aim of this study was to achieve a 3D reconstruction of murine EDL muscle (ex-vivo preparations) and to compare, at high resolution, the outcomes coming from control and damaged muscle. Fore each mouse, one EDL muscle was used as control (healthy muscle, HC) and its controlateral one was subjected to forced eccentric contractions (EC) to cause the damage as previously validated. IN particular, muscle injury was induced through stretching and exposure to a series of contractures in a solution containing a high K+ concentration. After each cycle of EC, muscles recovered in physiological Ringer-Krebs solution for 4 minutes. The extent of SCs' activation was evaluated across the entire sample by using tissue clearing and high-resolution light-sheet fluorescence microscopy. Both HC and EC muscles were cleared using the iDISCO protocol; a whole-mount immunostaining against MyoD was performed to label activated SCs. The high resolution volumetric reconstructions obtained enabled quantification of the number and spatial arrangement of this population of cells in both healthy and damaged samples. As this technique analyzes the entire sample, it removes any potential sampling bias that might be introduced by sample splicing in conventional 2D histology. Although very preliminary, this study proves the feasibility of the adopted technique in this kind of investigation, offering new additional tools for the identification/visualization of SCs’ activation after muscle injury. This can be useful to test novel therapeutic approaches to enhance tissue regeneration at the muscle-tendon injury.