SUMMARY Axonal damage occurs early in multiple sclerosis and is considered the pathologic substrate of progressive disability. The spatiotemporal dynamics of axonal loss in relation to acute and chronic demyelination are not well characterized and likely vary between patients, lesion types, and within the normal-appearing white matter (NAWM). With the emergence of promising therapies targeting remyelination, noninvasive imaging markers with greater specificity to axonal pathology are needed to improve our understanding of disease progression in MS. Histopathological analyses of MS tissue have confirmed significant reductions in axon density within lesions and NAWM accompanied by a range of morphological alterations in axonal structure, including the appearance of ovoids, swelling, thinning, and transection. We have demonstrated the imaging correlates of axonal swelling and loss in the corpus callosum of MS patients using high-gradient diffusion MRI, leveraging the 300 mT/m gradient strengths on the Connectome scanner for the in vivo microscopic assessment of axonal structure. Our goal is to validate these cross-sectional imaging findings through longitudinal investigation and systematic comparison against histopathology to gain a better understanding of the spatial and temporal evolution of axonal degeneration in MS and the pathogenic factors influencing disease progression. We hypothesize that chronic demyelination leads to axonal swelling and eventual dropout that can be detected as increased axonal size and decreased density by high-gradient diffusion MRI, and that the degree of axonal morphologic change throughout the brain reflects progressive axonal dysfunction and manifests as progressive clinical disability. We will pursue a longitudinal imaging study to determine the relationship between demyelination and progressive axonal structural pathology in MS lesions and NAWM throughout the whole brain. We will evaluate the relative influence of demyelination and axonal damage on the development of progressive physical disability and cognitive dysfunction in MS. In collaboration with investigators at the Cleveland Clinic, we will calibrate and refine diffusion MRI measures of axon diameter and density in MS lesions and NAWM against axon diameter and density measurements from histopathology in postmortem MS brain tissue. The prevalence of axonal loss in postmortem samples, and the absence of a reliable biomarker for axonal loss in living patients, reinforces the need for a validated imaging correlate of axonal degeneration that would allow us to track changes in axonal structure in real time rather than at their end stage on autopsy specimens. High-gradient dMRI techniques that map axon diameter distributions in MS patients will enable us to corroborate the changes in axon diameter observed on pathology. The data generated from this study will advance our understanding of the role of axonal damage in the pathogensis of MS and facilitate the deve...