This study investigates the cellular dynamics and architecture of evolving neuroinflammatory lesions in multiple sclerosis (MS) using single-cell spatial expression profiling (ISS) in mouse experimental autoimmune encephalomyelitis (EAE) and human post-mortem spinal cord samples. The authors found that active EAE lesions propagate centrifugally, with lesions in the lumbar spinal cord being the most inflamed and complex, while lesions in cranial regions are simpler. They identified three distinct compartments within complex lesions: the lesion core, lesion rim, and peri-lesion. The study also revealed that disease-associated (DA) glial states, such as DA-microglia and DA-oligodendrocytes, are dynamically induced and resolved over the disease course, and their spatial distribution is influenced by the inflammatory environment. In human MS spinal cords, ISS confirmed the differential distribution of homeostatic and DA-glia, enabled the deconvolution of active and inactive lesions into sub-compartments, and identified new lesion areas. The findings highlight the intricate cellular dynamics underlying MS and provide a spatial resource for further research.This study investigates the cellular dynamics and architecture of evolving neuroinflammatory lesions in multiple sclerosis (MS) using single-cell spatial expression profiling (ISS) in mouse experimental autoimmune encephalomyelitis (EAE) and human post-mortem spinal cord samples. The authors found that active EAE lesions propagate centrifugally, with lesions in the lumbar spinal cord being the most inflamed and complex, while lesions in cranial regions are simpler. They identified three distinct compartments within complex lesions: the lesion core, lesion rim, and peri-lesion. The study also revealed that disease-associated (DA) glial states, such as DA-microglia and DA-oligodendrocytes, are dynamically induced and resolved over the disease course, and their spatial distribution is influenced by the inflammatory environment. In human MS spinal cords, ISS confirmed the differential distribution of homeostatic and DA-glia, enabled the deconvolution of active and inactive lesions into sub-compartments, and identified new lesion areas. The findings highlight the intricate cellular dynamics underlying MS and provide a spatial resource for further research.