Purpose <p>The cuprizone (CPZ) mouse model for multiple sclerosis enables researchers to investigate the underlying mechanisms of demyelination and remyelination, as well as the effect of therapeutic interventions thereon. Over the last five decades, research revealed detailed analyses of brain tissue derived from CPZ-fed animals at different times of de- and re-myelination. Yet, longitudinal analysis of the effects of a CPZ diet on locomotor performance, myelination and neuroinflammation over a two-month recovery period are still unexplored.</p> Procedures <p>In this study, we comprehensively examined behavioural and neuropathological changes in the CPZ mouse model for a follow-up period of two months. We combined a longitudinal PET imaging approach with a more traditional approach of obtaining tissue and performing immunohistochemistry and behavioural tests in cohorts.</p> Results <p>We found that mice fed with 0.2% CPZ for 5&#xa0;weeks showed transient motor deficits which recovered quickly after cuprizone was removed from the diet. Similarly, remyelination also promptly restored myelin content after CPZ toxic insult, as seen by PET imaging with [¹¹C]MeDAS and myelin histological staining. Remarkably, five weeks of CPZ feeding led to persistent glial activation, especially in the corpus callosum, for at least two months. This effect was measured both with [¹¹C]PK11195 PET and with immunohistochemical staining for microglia and astrocytes.</p> Conclusions <p>Taken together, this longitudinal study reveals that a five-week CPZ diet leads to transient motor and loss of myelin, which recover quickly after CPZ is removed. In contrast, neuroinflammation persists for at least two months following exposure and is mainly driven by astrocyte activation. The study warrants deeper analysis of the sustained neuroinflammatory response in the context of demyelinating diseases.</p>