PROJECT SUMMARY Our goal is to develop and assess the clinical potential of quantitative imaging biomarkers of masticatory myofascial pain syndrome (MMPS). MMPS afflicts nearly 10% of Americans. Many treatments have been proposed. However, the lack of a reliable non-invasive tool for evaluating changes in myofiber microstructure has been a major hindrance in assessing existing treatment methods and developing new therapies for myofascial pain. We have recently developed methods to measure and analyze Diffusion Tensor MR Imaging (DTI) data with varying diffusion times, DTI(t): from the time-dependent diffusion signal transverse to fibers, we extract anatomical maps of myofiber diameter and sarcolemma permeability to water molecules, and from the time- dependent diffusion signal along the myofibers, our preliminary results reveal sensitivity to the sarcomere length. This innovative non-invasive quantitative approach, referred to as the random permeable barrier model (RPBM), is based on effective medium theory, and has been validated using Monte Carlo simulations and animal models, as well as tested in human skeletal muscle studies. The proposed biomarkers are the RPBM parameters for myofiber diameter, sarcolemma permeability and sarcomere length scale. These biomarkers are highly favorable biophysical parameters of interest to study myofiber integrity in myofascial pain syndrome (MPS): Indeed, sarcomeres have been shown to be abnormally contracted/shortened and myofibers enlarged in myofascial trigger points observed in MPS. Treatments of MPS including botulinum toxin A treatment may lead to reduction of myofiber diameter. Furthermore, study of the entire muscle rather than local biopsy is needed to understand the initiation of myofascial trigger points, which may occur when exceeding a given threshold in case of muscle overuse, trauma or psychological stress. In the R61 phase, we will develop and optimize the RPBM method to measure myofiber changes associated with MMPS, with the following three aims: in Aim 1 we will establish an optimal 15-minute long MRI protocol and processing pipeline to robustly estimate RPBM parameters. Aim2 is to establish an optimal MRI protocol that includes the optimized 15-min DTI(t) and a 15-min protocol for conventional quantitative MRI measures including T1, T2, and fat fraction, and evaluate their repeatability and reproducibility. Aim 3 is to assess the diagnostic performance of the RPBM parameters. In the R33 phase with a clinical trial, we will determine the RPBM parameters and their combinations that best assess and predict treatment response in MMPS. Once completed, the same RPBM method can be directly applied to assessing other musculoskeletal areas, such as the shoulder, the upper/lower back and the pelvis.