Onset-treatment with HBPDS helps to maintain the integrity of the BBB in EAE mice.

<p><b>(A-L)</b> To assess the level of disruption of the BBB at day 20–22 after immunization, lumbar spinal cord sections was immunostained with anti-GFAP antibody (A-D), lumbar spinal cord lysate was analyzed by Western blot with anti-GFAP antibody (E), total RNA isolated from the lumbar spinal cord was analyzed by RT-PCR with primers for GFAP (F) and adhesive molecules (ICAM–1 and VCAM–1; G-I), and the total RNA was analyzed by real time-PCR with primers for junctional molecules (claudin–3, claudin–5, and zona occludens–1; J-L). Bands by Western blots and RT-PCR analysis were quantified as the ratio of GFAP to GAPDH for each sample (E and F). The mRNA expression level of each gene by real time-PCR is presented as the fold induction compared with the levels measured in normal mice (J-L). Onset-treatment with HBPDS (30% ethanol extracted) reduced the increased GFAP-immunoreactivity (A-D), the increased expression of protein and mRNA for GFAP (E and F), and the increased expression of mRNA for ICAM–1 and VCAM–1 (G-I), but increased the reduced expression of mRNA for claudin–3, claudin–5, and zona occludens–1 in the spinal cord of EAE mice (J-L). Data are represented as mean ± SEM. Scale bar = 100 μm. (ANOVA test; #p < 0.05 and ##p < 0.01 compared to normal control mice, *p < 0.05 and **p < 0.01 compared to EAE mice).