Fig. 4

Cuprizone induces early BBB dysfunction and edema. a Experimental procedure to measure BBB permeability by quantifying extravasation of the tracers Evans blue (EB) and sodium fluorescein (NaFl), and edema (brain water). Tracers are i.v. injected, and mice are flushed with PBS after 4 h. The dissected brain is lyophilized (lyo). Brain water content (edema) is calculated from the weight difference of wet to lyophilized tissue. Tracers are extracted, and fluorescence intensity quantified. b Brain water (g/g dry brain) in mice fed cuprizone for 3 days to 5 weeks as indicated, and untreated controls (N = 4–5). Significance to control was evaluated by 1way ANOVA with Tukey’s post test (***P < 0.001). c, d Extravasated EB (N = 4–6) and NaFl (N = 4) after cuprizone administration for 3 days to 5 weeks. Significance to control was evaluated by 1way ANOVA with Tukey’s post test (**P < 0.01, ***P < 0.001). e Water content (g/g dry tissue) in dissected corpus callosum (CC) or cortex (Ctx) of mice fed cuprizone for 5 days or controls (N = 4–5). Significance was determined by unpaired Student’s t-test for each brain region (***P < 0.001). Extravasated (f) EB (N = 4) and (g) NaFl (N = 5) in dissected CC or Ctx of mice fed cuprizone for 5 days normalized to untreated controls. Significance to controls was evaluated by Student’s t-test (*P < 0.05, ***P < 0.001). Quantitative RT-PCR analysis on dissected CC or Ctx from mice that received cuprizone for 5 days (N = 4) or control mice, evaluating cellular markers (h) and inflammatory mediators (i). Values of individual mice are shown as fold differences to the mean of N = 4–5 controls (set to 1) according to color code