Progressive MS is associated with spinal cord axonal loss due to direct injury within lesions, along with upstream and downstream neurodegeneration. Neuromyelitis Optica (NMO) likewise affects the cord, with pathology characterized by severe axon injury, demyelination, and necrosis¹. Despite the critical contribution of spinal cord disease to clinical disability and disease progression, tools to measure spinal cord injury are limited. Diffusion tensor imaging (DTI) can serve as an imaging biomarker of spinal cord tissue injury at the tract level. Animal studies have suggested that increased radial diffusivity (RD) derived from DTI reflects demyelination, whereas decreased axial diffusivity (AD) reflects axonal injury. In our prior human studies, DTI RD demonstrated strong relationships with clinical outcomes, but surprisingly AD failed to discriminate levels of disability². We developed more sophisticated diffusion basis spectrum imaging (DBSI) to analyze both anisotropic and isotropic diffusion components. Using DBSI in animal models, reduction of axial diffusivity and increase in radial diffusivity more accurately reflected axon injury and demyelination than DTI, respectively. In this study, we re-analyze the diffusion MRI data with DBSI^{3, 4} to quantify axon loss in RRMS and NMO patients. Our results support that DBSI-derived indices may serve as outcome measure to quantitatively reflect axon loss and demyelination.This longitudinal study was approved by the Washington University Human Research Protection Office/Institutional Review Board, and all subjects provided written informed consent. Data from five RRMS and six NMO patients with MS or NMO spectrum disorder with symptoms, signs, and imaging evidence of cervical spinal cord lesions were analyzed. Data from RRMS patients included those previously scanned during the first visit and 6-month follow-up. NMO data were those scanned during the first visit. Six heathy subjects were also included.Data acquisition was performed following previously reported procedures⁵. Diffusion-weighted images (DWIs) were collected with a multi–b-value scheme (4 averages of 25 directions and maximum b-value = 600 s/mm²) at 3T (Trio; Siemens, Erlangen, Germany) with cardiac-gating, reduced field of view, single-shot spin-echo echo planar imaging sequence with voxel size of 0.9×0.9×5 mm³. Total 18 slices from cervical spinal cord segments (C1 to C6) were acquired in around 45 minutes. DTI and DBSI were computed using the in-house software developed using Matlab. Regions of interest (ROIs) for total white matter (WM) of spinal cord were determined using the FA map and b0 image. The computation was calculated for each individual segment and then averaged across all segments for analysis and comparisons. DBSI derived axon volume was defined as mean DBSI fiber fraction multiplied by white matter volume (mm³). DBSI edema volume was defined as mean DBSI non-restricted isotropic diffusion fraction multiplied by white matter volume (mm³) of the segment studied. Unpaired student t-test was employed for statistical analysis. DBSI-derived axon volume decreased significantly compared to healthy controls on first visits and remained similarly decreased at the six-month follow-up visit, suggesting permanent axonal loss had occurred in RRMS patients at their first visit, but no further loss detected after six months (Fig. 1A). NMO patients also had significant decrease in axon volumes, suggesting similar axon loss (Fig. 1B). For NMO patients, both DTI and DBSI radial diffusivity suggested demyelination of the residual axons (Fig. 2A and 2B). However the extent of demyelination suggested by DTI was overestimated due to the confounding edema (Fig. 2C). No significant decrease of radial diffusivity for RRMS patients (data not shown). There was no significant axonal injury observed in the residual white matter of RRMS or NMO patients.DBSI derived axon volume revealed axon loss that had been missed by DTI. In addition, increased DBSI-derived radial diffusivity supported the demyelination suggested by DTI. The results suggest that the multiple metrics derived by DBSI could offer an insight to the underlying pathologies responsible for the evolving neurological impairments in patients with progressive MS and NMO.