Longitudinal magnetic resonance imaging and spectroscopy in a mouse model of cuprizone-induced demyelination

The cuprizone (CPZ) lesioned mouse is a widely used model of demyelination and remyelination, but most studies rely on histopathological analysis at terminal timepoints, limiting understanding of disease dynamics. Here, we present a longitudinal multimodal magnetic resonance imaging and spectroscopy (MRI/MRS) study of CPZ-induced pathology, pooling control arms from three independent experiments (n = 40). Mice were imaged at baseline, exposed to 0.2% CPZ in food for 5 weeks, and repeatedly imaged at days 24, 35, 49, 63 and 77 after the start of CPZ treatment, spanning the expected phases of demyelination and remyelination. Imaging and analysis methods included multi-parameter mapping (MPM), diffusion tensor imaging (DTI), tensor-based morphometry (TBM), and single-voxel MRS in the corpus callosum. Histological analysis (MBP, silver, GFAP, Iba1) was performed at selected timepoints (Day 24, 35, 42 and 77 from start of CPZ) for validation. An additional cohort of CPZ-lesioned mice (n = 18) was imaged ex vivo using a different higher resolution MRI protocol and compared with non-CPZ controls (n = 19). MPM-derived MTsatδ and R₁ reductions indicated changes consistent with demyelination in the corpus callosum and deep cerebellar nuclei by Day 24, expanding to cortex and hippocampus by Day 35. Only partial recovery was observed by Day 77, consistent with histological evidence. TBM revealed dynamic volumetric alterations, including hippocampal and cerebellar expansion alongside cortical and subcortical shrinkage, persisting beyond CPZ cessation. DTI demonstrated early (Days 24-35) decreases in FA and MD, followed by complex trajectories consistent with microstructural disruption and partial repair. MRS detected early increases in GABA, glutamine, taurine, and glutathione, with corresponding decreases in NAA, while inositol showed a biphasic decrease-increase profile, likely reflecting acute astrocytic dysfunction followed by gliosis - neuroinflammatory processes that were corroborated by immunohistochemistry. Together, these results demonstrate that multimodal MRI/MRS sensitively captures widespread, dynamic, and only partially reversible pathology in CPZ-treated mice. Longitudinal imaging provides a non-invasive, translational approach to characterising demyelination, gliosis, and remyelination, offering a powerful complement to histology for preclinical studies and longitudinal therapeutic screening.