High-Speed-Ventral-Plane Videography Identifies Specific Gait Pattern Changes in Cuprizone-Induced Demyelination in Mice

Gait disturbances are among the most prominent motor symptoms in multiple sclerosis (MS), yet their functional characterization in preclinical models remains limited. In this study, we used high-speed ventral plane videography (DigiGait™) to analyze locomotor behavior during 5 weeks of cuprizone-induced demyelination in 10 male C57BL/6 mice. Gait analysis revealed significant alterations in stride time (left front paw from 0.303 ± 0.01 s to 0.257 ± 0.007 s; <i>p</i> = 0.003), paw angle (right fore paw from −13.78 ± 0.928° to 5.456 ± 2.146°; <i>p</i> = 0.003), and midline distance (right hind paw from 1.889 ± 0.099 cm to 1.216 ± 0.096 cm; <i>p</i> = 0.013), particularly in the hind limbs. These behavioral impairments correlated with histopathological findings of reduced myelination and elevated microglial activation in motor-relevant brain regions, including the corpus callosum, caudate-putamen, and motor cortex. Notably, specific gait parameters showed strong correlations with the degree of demyelination, supporting their relevance as functional biomarkers. Our data demonstrate that high-resolution gait analysis provides a sensitive, non-invasive tool to monitor functional deficits in demyelinating models and may aid in evaluating therapeutic efficacy in future studies.