PROJECT SUMMARY Skeletal muscle exemplifies structure-function relationships in biology. The organization of sarcomeres follow hierarchical ordering to form long contractile cells, bundled in extra-cellular matrix, to form larger fascicles and ultimately whole muscles. The tight relationship between structure and function allows muscle performance (and disease) to be inferred from its microstructure. For example, fiber area is directly related to isometric force production in muscle. With injury, microstructural changes in muscle fiber area (size), fibrosis (accumulation of extracellular matrix), membrane damage (permeability), and inflammation (edema) are observed, and impair muscle function. Muscle biopsy, followed by microscopic examination of the tissue (histology), is the gold standard to diagnose and monitor muscle health and disease. This tool is invasive, requiring a large bore needle and tissue removal under sterile conditions, which makes it painful and costly. Therefore, biopsy is not conducive to serial monitoring of muscle health. It is also semi-quantitative, and often difficult to extrapolate to the entire muscle, limiting its scientific and clinical value. For these reasons, there is a need for noninvasive assessment of muscle microstructure, which would facilitate the quantitative examination of muscle injury over time. Magnetic resonance imaging (MRI) has been used to noninvasively quantify changes in volume, fat distribution, and water content in muscle. Diffusion MRI (dMRI) is a version of MRI that measures anisotropic diffusion of water, which is related to tissue microstructure. Many studies have used dMRI to measure restricted diffusion in injured skeletal muscle and have theorized how microstructure relates to diffusion. However, these relationships are not explicitly tested nor carefully validated because the necessary experiments are complicated and difficult to rigorously control. To address this gap in knowledge, the purpose of this proposal is to define the relationship between restricted diffusion and muscle microstructure assessed with classic and innovative new dMRI techniques. Our central hypothesis is that dMRI measurements can be optimized to detect small but clinically relevant changes in muscle microstructure. Aim #1 will investigate a new dMRI pulse sequence and analysis technique that has enhanced sensitivity to muscle microstructure using previously established computer simulation and 3D precision engineered models. In Aim #2, we will utilize animal models of clinically meaningful muscle injuries to evaluate sensitivity of common, less common, and novel dMRI pulse sequences to detecting microstructural differences between models. In Aim #3, we will utilize a multiparametric MRI protocol to assess diagnostic, prognostic, and functional changes associated with a clinically relevant animal model of muscular dystrophy. These experiments will elucidate the relationships between microstructure and diffusion in mu...