(A) Schematics of treatment strategies for EAE. Female, 8–10 week-old C57BL/6 mice were immunized with MOG35-55 and pre-treated with PBS or PM (nasopharyngeal inhalation, 5.0 mg/kg/d) daily, starting at day –30 (before immunization) until 30 p.i. All mice were sacrificed and their tissues (brain, spinal cord, draining lymph nodes, and spleen) were harvested for Q-PCR, flow cytometry, ELISA, or immunohistochemistry analysis at day 30 p.i. (B) EAE development was evaluated daily by two researchers blindly, following to a 0–5 scale. (C) Distribution of disease status at the end points of experiment (day 30 p.i.). (D) Thoracic spinal cord sections were assayed for inflammation by H&E and demyelination by Luxol fast blue (LFB), and CNS pathology was scored on a 0–3 scale. (E) Absolute number of CNS mononuclear cells (MNCs) in cell suspension of each mouse (brain and spinal cord) was counted. (F) Representative images of spinal cord sections of PBS- and PM-treated EAE mice in the dorsal funiculus. Quantitative analysis of MBP, IBA1, A2B5, and GFAP expression was assessed by using Image-Pro. The measured areas included 8–10 fields and covered virtually all the white matter of the spinal cord. Dorsal column at the thoracic spinal cord is shown as representative images. (G) Effects of PM treatment on the various inﬂammatory cells in the CNS. MNCs from spinal cords and brains were isolated at day 30 p.i., stimulated with MOG35–55 (10 μg/mL) for 24 h, and analyzed by flow cytometry. Cells were gated as CD45+CD11b+ (microglia and infiltrating macrophages) and CD45+CD11b- (other infiltrating immune cells), and their subsets were further defined. One representative of three independent experiments is shown. Symbols represent mean ± SD; n = 4–5 mice in each group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, compared to PBS-treated group, two-way ANOVA comparison with Multiple t’ tests. Scale bar = 40 µm in D, Scale bar = 10 µm in F.