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. 2018 Mar 1;141(4):1094–1110. doi: 10.1093/brain/awy033

Figure 1.

Figure 1

CSPGs are upregulated in the CNS of mice at peak EAE disease course. (A) Daily average EAE clinical score to Day 32. Day 0–12: pre-onset of clinical signs; Day 12: onset of clinical signs; Day 18: peak severity of clinical signs and >Day 18: chronic EAE. (B and C) Real-time PCR showing the upregulation of Tnfa (TNFα) and Tgfb1 (TGFβ) at peak EAE. For panels A–C, values are mean ± SEM of five mice. (D) Haematoxylin, eosin and Luxol fast blue staining (H&E/LFB) shows the presence of pial inflammation of the spinal cord at the onset of EAE, progressing to parenchymal lesions at both peak and chronic time points. Scale bars = 50 µm. (E) Histological severity scoring of the H&E/LFB staining, analysed by the degree and location (pial versus parenchymal) of inflammatory infiltrates. (F) Real-time PCR of the lectican CSPGs aggrecan (Acan), neurocan (Ncan), and total isoforms of versican (Vcan) (V0, V1, V2, V3). For panels E and F, values are mean ± SEM of five mice. For panels B, C, E and F, datasets passed the Shapiro-Wilk normality test and Dunnett’s ANOVA was performed, comparing immunized mice groups against naïve, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) Western blot of spinal cord samples from naïve and peak EAE mice (two mice represented/condition) of CSPGs including chondroitin sulfate 4-sulfated glycosaminoglycan chains (CSA), aggrecan, brevican, versican V1/V0, and Versican V2/V0. The presence of lower molecular weight protein cores of aggrecan, brevican, and versican V1 likely represent cleavage from matrix-metalloproteinases or a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs) within the samples. (H) Relative changes of the density of bands in peak EAE compared to the density of bands in naïve mice. Densities of the bands were normalized to β-actin as a loading control. Chondroitin sulfate A (CSA), aggrecan, brevican and versican V1 datasets passed the Shapiro-Wilk normality test, and a two-tailed Student’s t-test showed significant increases in peak EAE versus naïve mice (*P < 0.05, **P < 0.01, ***P < 0.001). Versican V2 did not pass normality, and the non-parametric two-tailed Mann-Whitney test showed a significant decrease in versican V2. Each square represents a separate mouse spinal cord. CFA = complete Freund’s adjuvant.