Low-Intensity Physical Exercise is Associated with Improved Myelination and Reduced Microglial Activation in a Cuprizone-Induced Demyelination Model

Abstract

Demyelinating diseases like multiple sclerosis cause damage to the myelin sheath, leading to neurological problems. While the exact causes of MS are unclear, it is known that inflammatory processes and poor remyelination contribute to disease progression. Exercise has shown promise as a non-drug treatment for MS, with benefits reported for mobility, mood, and potential neuroprotection. However, the specific ways in which exercise affects remyelination and neuroinflammation in demyelinating conditions are not fully understood. This study explores the effects of low-intensity physical exercise on myelination, neuroinflammation, and neurogenesis in a cuprizone-induced demyelination model, focusing on the hippocampus, which are critical for cognitive function and interhemispheric communication. Mice subjected to cuprizone treatment underwent a low-intensity forced wheel-running exercise. The results showed that low-intensity physical exercise significantly increased the expression of myelin basic protein in the stratum lacunosum-moleculare of the hippocampus and the corpus callosum, suggesting enhanced remyelination in these regions. Additionally, cuprizone-induced demyelination led to morphological changes in microglia, activating them in the hippocampus. However, low-intensity physical exercise significantly reduced microglial activation, indicating that exercise modulated the neuroinflammatory response. Despite observing reduced microglial activation with low-intensity exercise, TNF-α levels remained elevated in the low-intensity exercise group, suggesting a complex relationship between microglial activation markers and cytokine production in this model of demyelination. This indicates that low-intensity exercise may not fully suppress the pro-inflammatory potential of microglia in the cuprizone model. Although low-intensity exercise promoted remyelination and modulated neuroinflammation in the cuprizone-induced demyelination model, it did not significantly counteract the cuprizone-induced reduction in proliferating cells and immature neurons in the subgranular zone of the dentate gyrus. These findings suggest that while the exercise regimen had beneficial effects, it did not significantly influence overall neurogenesis. This novel study investigates the region-specific effects of low-intensity exercise on myelination and neuroinflammation, with a focus on the hippocampus, which is less frequently explored in the context of demyelination models. The findings highlight the potential rehabilitative benefits of low-intensity exercise for demyelination-related neurological disorders and provide new insights into the underlying mechanisms contributing to neuroprotection.

Keywords: Cuprizone; Low-intensity exercise; Neurogenesis; Remyelination.

Fig. 1

Experimental timeline. C57BL/6 mice were fed a cuprizone diet for nine weeks to induce demyelination, followed by a five-week exercise intervention with access to low-intensity running wheels

Fig. 2

Body weight changes in Control, CPZ (cuprizone-treated), and CPZ + Ex (cuprizone-treated with exercise) groups over time. (A) The graph illustrates the changes in body weight among three groups of mice throughout the experiment. (B) The groups exhibited varying degrees of weight gain, measured as the percentage change from their initial body weights over the 9-week period. CTL group showed a consistent increase in body weight over time. In contrast, the CPZ group and CPZ + Ex group did not display significant weight gain after cuprizone administration. Tukey’s multiple comparisons test revealed that the control group had a significantly higher mean body weight than both the CPZ group and the CPZ + Ex group. However, no significant difference was observed between the CPZ and CPZ + Ex groups (p > 0.05). *: p < 0.05 for CTL vs. CPZ, **: p < 0.05 for CTL vs. CPZ + Ex. CTL vs. CPZ p < 0.05^ap < 0.05 vs. CTL; ^bp < 0.05 vs. CPZ. The error bars represent the standard deviation of the mean

Fig. 3

Immunohistochemical staining for myelin basic protein (MBP) in the hippocampus of control (CTL), cuprizone-fed (CPZ), and cuprizone-fed with exercise (CPZ + Ex) groups. SP: stratum pyramidale; SLM: stratum lacunosum-moleculare; CA3: Cornu Ammonis 3; CC: corpus callosum; PoL: polymorphic layer. Scale bar = 50 μm. Tukey’s multiple comparisons test revealed that the CPZ group had a significantly lower MBP immunoreactivity than CTL group. In addition, higher MBP immunoreactivity was found in the CPZ + Ex group than CPZ group. ^ap < 0.05 vs. CTL; ^bp < 0.05 vs. CPZ. The error bars represent the standard deviation of the mean

Fig. 4

Iba1 immunofluorescence in the hippocampus. Images show Iba1-labeled cell bodies and processes (red). SLM: stratum lacunosum-moleculare; CC: corpus callosum. Scale bar = 50 μm. Tukey’s multiple comparisons test revealed that Iba1 immunoreactivity was highest in CPZ group and lowest in CTL group. ^ap < 0.05 vs. CTL; ^bp < 0.05 vs. CPZ. The error bars represent the standard deviation of the mean

Fig. 5

Double immunofluorescence staining and analysis of microglial activation in the hippocampus. (A) Representative images of double immunofluorescence staining for Iba1 (green) and TNF-α (red) in the hippocampus, taken from different subregions (DG, CA3, and CA1) across experimental groups (CTL, CPZ, CPZ + EX). Iba1⁺TNF-α⁺ double-positive microglia appear yellow in the merged images, indicating colocalization. High-magnification orthogonal views of Iba1 and TNF-α signals within individual microglia are shown below each panel to highlight spatial colocalization. Scale bar = 25 μm. (B) Quantitative analysis of TNF-α expression in the hippocampus across experimental groups (CTL, CPZ, CPZ + EX), illustrating regional and group-specific differences in pro-inflammatory signaling. TNF-α is a key pro-inflammatory cytokine released by activated microglia. (C–E) Quantification of colocalization between Iba1 and TNF-α signals in each hippocampal subregion: dentate gyrus, CA3 (D), and CA1 (E). These measurements reflect the degree of microglial activation and pro-inflammatory status across the experimental conditions. ^ap < 0.05 vs. CTL; ^bp < 0.05 vs. CPZ. The error bars represent the standard deviation of the mean

Fig. 6

Immunofluorescence analysis of cell proliferation and neuronal markers in the hippocampal dentate gyrus. (A) Ki67 immunostaining (red) with DAPI nuclear counterstaining (blue) reveals proliferating cells in the dentate gyrus. Scale bar = 50 μm. (B) Double immunofluorescence staining for NeuN (green) and Ki67 (red) in the hippocampal subgranular zone. Scale bar = 75 μm. Insets show higher magnification views. Arrows indicate cells co-expressing Ki67 and NeuN in the merged images. Co-localization of NeuN and Ki67 indicates that a subset of proliferating cells is differentiating into mature neurons. Quantification of both panels (A and B) using Tukey’s multiple comparisons test showed significantly reduced numbers of Ki67-positive cells in the CPZ and CPZ + Ex groups compared to the CTL group (^ap < 0.05 vs. CTL). Error bars represent standard deviation of the mean

Fig. 7

DCX immunohistochemistry in the hippocampal dentate gyrus. High-magnification images show DCX-positive cell bodies in the SGZ with dendrites extending into the GCL. SGZ: subgranular zone; GCL: granular cell layer. Scale bars = 50 μm and 140 μm. Tukey’s multiple comparisons test revealed that the CPZ and CPZ + Ex group showed significant reduction of DCX immunoreactivity when compared to CTL group. However, no significant difference was observed between the CPZ and CPZ + Ex groups. ^ap < 0.05 vs. CTL. The error bars represent the standard deviation of the mean