Diffusion magnetic resonance imaging (MRI) enables the ability to probe both tissue microstructure and structural connectivity of the central nervous system. However, there are no validated methods to model and interrogate the pathways that connect the brain and spinal cord, which inhibits our ability to fully characterize and understand the complete damage that may occur in neurological disorders. For example, disease progression in patients with multiple sclerosis (MS) is known to stem from axonal damage in both the brain and spinal cord, yet, coordinated medical image analysis of both structures simultaneously has not been shown. Thus, the overall goal of the proposed research is to develop and optimize simultaneous tissue microstructural mapping of the brain and spinal cord for clinical assessment of MS using magnetic resonance imaging (MRI), specifically interrogating the microstructure and connectivity of motor pathways of the central nervous system. The critical challenges to this goal are (1) quantifying tissue microstructure of the brain and spinal cord in unison has not been performed, (2) clinical MRI lacks specificity for microstructural tissue integrity, and (3) there are few methods available that allow mapping of MS lesions and pathological abnormalities in relation to critical fiber pathways. To address this, in Aim 1 we will develop a cohesive acquisition and image processing pipeline, minimizing artifacts and maximizing reproducibility, in order to facilitate a unified analysis of the central nervous system. In Aim 2, we will utilize diffusion MRI modeling and fiber tractography to characterize tissue microstructure and connectivity from the cortex to the spinal cord. Modeling will enable quantification of highly specific pathophysiological indices of edema, axonal swelling, demyelination, and axonal loss, whereas tractography will facilitate feature localization to specific white matter pathways and along specific pathways. Finally, evaluate microstructure and connectivity of the motor pathways to interrogate pathology in MS, quantifying radiological biomarkers over space and time that may contribute to impairment in this disease. The overall impact of this proposal will be quantitative biomarkers for disease burden that may improve the value of imaging the brain and spinal cord together as it relates to understanding pathology in vivo.