Fig. 3

Myeloid-specific ikkβ gene deletion inhibits the recruitment/infiltration of residential microglia and of peripheral macrophages in spinal cord lesion during EAE. a-n Spinal cord sections were obtained from WT and LysM-Cre/Ikkβ ^F/F mice (n = 5) at 15–18 days after immunization, stained with H&E dye (a-d), and immunostained with anti-Iba-1 (e-h) and anti-CD68 antibodies (i-l) to investigate the degree of cellular recruitment/infiltration. Representative photographs show ventrolateral white matter of the lumbar spinal cord (a-l). The degree of recruitment/infiltration was quantified and the data are expressed as mean score ± SEM (m and n). Bars = 100 μm. o Spinal cords from each group (n = 5) at 15–18 days after immunization were analyzed with real-time RT-PCR. Data are expressed as mean fold induction ± SEM. p and q Spinal cords were dissected from each group (n = 5) at 15–18 days after immunization to investigate the degree of recruitment/infiltration of macrophages with flow cytometry. Tissues were dissociated, and cells were incubated with APC-conjugated anti-CD11b and PE-conjugated anti-CD45 antibodies. CD11b⁺ cells were divided into CD11b⁺/CD45^+(high) cells (R2; macrophage) and CD11b⁺/CD45^+(low) cells (R3; microglia) populations based on CD45 expression levels (p), and the percentages of each population are denoted in the graph (q). Mean ± SEM values from 3 independent experiments are shown in the graph. r-t Total RNA was isolated from the lumbar spinal cord of each group (n = 3) at day 15–18 post-immunization to measure the level of the mRNA expression of IL-1β (r), TNF-α (s), and iNOS (t) with real-time RT-PCR. The mRNA level of each gene is presented as the fold induction ± SEM compared with the levels measured in the normal control mice. (ANOVA test; *p < 0.05 and **p < 0.01 versus WT EAE mice; #p < 0.05 and ##p < 0.01 versus normal control mice)