Onset-treatment with HBPDS reduces the infiltration and activation of microglia/macrophages in the spinal cord of EAE mice.

<p><b>(A-F)</b> To investigate the level of infiltration and activation of microglia/macrophages on day 20–22 after immunization, lumbar spinal cord sections were analyzed by immunohistochemistry (A-D) and Western blots (E) using anti-Iba–1 antibody (A-E) and total RNA isolated from the lumbar spinal cord was analyzed by RT-PCR with primer for CD11b (F). Bands by Western blots and RT-PCR analysis were quantified as the ratio of Iba–1 (E) or CD11b (F) to GAPDH for each sample. The infiltration and activation of Iba–1 positive microglia/macrophages, the protein expression of Iba–1, and the mRNA expression of CD11b were significantly decreased by onset-treatment with HBPDS (30% ethanol extracted) in the spinal cord of EAE mice. <b>(G and H)</b> To further investigate the degree of recruitment/infiltration of microglia and macrophages, the lumbar spinal cord of mice in each group on day 20–22 after immunization was analyzed by flow cytometry. These cells were categorized into CD11b⁺/CD45^+/low cell (R10; microglia) and CD11b⁺/CD45^+/high cell (R11; macrophages) populations based on the CD45 expression levels (G) and the graph shows the percentage of each cell population (H). Onset-treatment with HBPDS (30% ethanol extracted) decreased the population of microglia and macrophages in the spinal cord with EAE mice. Data are presented as mean ± SEM. Scale bar = 100 μm. (ANOVA test; #p < 0.05, ##p < 0.01 compared to normal control mice, *p < 0.05 and **p < 0.01 compared to EAE mice).