This study investigates the cellular architecture and dynamics of evolving neuroinflammatory lesions in multiple sclerosis (MS) using single-cell spatial expression profiling in mouse and human models. By analyzing mouse experimental autoimmune encephalomyelitis (EAE) and human MS spinal cord tissue, the researchers reveal the spatial and temporal progression of disease. They find that active EAE lesions propagate centrifugally, with disease-associated (DA)-glia dynamically induced and resolved over time. Spatial preferences of glial states drive lesion compartmentalization, and DA-glia are found to be distributed differently in human MS lesions compared to healthy tissue. The study also identifies new lesion areas and sub-compartments, demonstrating the complexity of MS pathology. In human MS samples, ISS reveals distinct pathological compartments, including areas with increased DA-glia and glial activation. The study highlights the role of DA-glia in MS pathology, showing that they are not confined to lesion areas but are dynamically induced and resolved. The findings suggest that DA-glia may play a role in both disease progression and resolution. The study also identifies spatial preferences of homeostatic and DA-glia in human spinal cord samples, aligning with observations in mice. The results provide a detailed understanding of the cellular dynamics underlying MS, including the evolution of lesions and the role of DA-glia in disease progression. The study underscores the importance of spatial mapping in understanding MS pathology and highlights the potential of ISS in revealing novel insights into MS and other diseases.This study investigates the cellular architecture and dynamics of evolving neuroinflammatory lesions in multiple sclerosis (MS) using single-cell spatial expression profiling in mouse and human models. By analyzing mouse experimental autoimmune encephalomyelitis (EAE) and human MS spinal cord tissue, the researchers reveal the spatial and temporal progression of disease. They find that active EAE lesions propagate centrifugally, with disease-associated (DA)-glia dynamically induced and resolved over time. Spatial preferences of glial states drive lesion compartmentalization, and DA-glia are found to be distributed differently in human MS lesions compared to healthy tissue. The study also identifies new lesion areas and sub-compartments, demonstrating the complexity of MS pathology. In human MS samples, ISS reveals distinct pathological compartments, including areas with increased DA-glia and glial activation. The study highlights the role of DA-glia in MS pathology, showing that they are not confined to lesion areas but are dynamically induced and resolved. The findings suggest that DA-glia may play a role in both disease progression and resolution. The study also identifies spatial preferences of homeostatic and DA-glia in human spinal cord samples, aligning with observations in mice. The results provide a detailed understanding of the cellular dynamics underlying MS, including the evolution of lesions and the role of DA-glia in disease progression. The study underscores the importance of spatial mapping in understanding MS pathology and highlights the potential of ISS in revealing novel insights into MS and other diseases.