Fig. 3

Effects of Ki16425 on immune cell infiltration and microglial activation in spinal cords of EAE^low mice. Lumbar spinal cords (n = 3 per group) were obtained from the sham, EAE^low, EAE^low + Ki16425 (15 and 30 mg/kg), and Ki16425 (30 mg/kg) groups at day 19–20 post-immunization. A, B Cryosections (n = 3 per spinal cord) of lumbar spinal cord (n = 3) from each group were stained with hematoxylin and eosin dye (A) and quantified (B). C–E Lysates of lumbar spinal cords (n = 3) from each group were analyzed with real-time PCR to measure mRNA expression levels of MCP-1 (C), MIP-1α (D), and RANTES (E). F, G Cryosections (n = 3 per spinal cord) were subjected to immunofluorescent staining with anti-Iba-1 antiserum (F). Lysates were subjected to Western blot to measure Iba-1 protein expression followed by quantification (G). H, I Lumbar spinal cords (n = 3 per group) were cropped at 19–20 days after immunization to measure the level of infiltration of microglia and macrophages using flow cytometry. CD11b⁺ cells were divided into CD11b⁺/CD45^low cells (R5; microglia) and CD11b⁺/CD45^high cells (R6; macrophage) populations (H) and graphs (I) were made to show the percentages of each cell population. A total of 1 × 10⁴ events acquired within the combined gate based on FSC and SSC were used. Bars = 100 µm. Data are expressed as mean expressive value ± SEM (ANOVA test; ^#p < 0.05 versus sham group; *p < 0.05 and **p < 0.01 versus EAE^low group)