Fig. 3

DMF promotes M2 macrophage polarization in EAN. DMF was administered orally to EAN rats (n = 6 in each group) from day 1 to day 16 or day 7 to day 16 post-immunization for preventative or therapeutic treatment, respectively. On day 16 post-immunization, sciatic nerves of each group of rats were harvested for fluorescence immunohistochemistry, and spleen mononuclear cells (MNCs) were isolated for flow cytometry, in both cases to analyze the proportion of M1 macrophages and M2 macrophages in EAN after DMF treatment paradigms. a Fluorescence photomicrographs showing M1 and M2 macrophages in the sciatic nerves of EAN rats. Tissue sections were immunofluorescence stained for markers of M1 and M2 macrophages as indicated. Scale bar is 50 μm. b Quantitation of immunohistochemistry. Counts per square millimeter of CD68⁺/CD86⁺ cells (M1 phenotype) in the sciatic nerve showed that DMF reduced the number of M1 macrophages in both DMF treatment paradigms. c Counts per square millimeter of CD68⁺/CD206⁺ cells (M2 phenotype) showed that DMF-treated groups increased the number of M2 macrophages in both DMF treatment paradigms compared to the CMC group. d Flow cytometry results of spleen MNCs showed the same pattern of M1/M2 phenotype shift as a result of both DMF-preventative and DMF-therapeutic treatment paradigms. e Quantitation of flow cytometry. Percentage of M1 phenotype in spleen MNCs was lower in both DMF-treated groups. f Percentage of M2 macrophages in spleen MNCs was greater in both DMF-treated groups. Comparisons to CMC group were performed using the Mann–Whitney U test; quantitation shows means ± SEM (*p < 0.05 for comparison between CMC and DMF-treated groups; n = 4–5). CMC control group receiving vehicle, DMF-P preventative DMF treatment, DMF-T therapeutic DMF treatment. The experiment was repeated 3 times with similar results