Figure 6

Effect of human cytokines in ECV-304 mono-culture barrier and ECV-304/HFA co-culture barrier. a) Effect of human cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) applied to apical + basal sides of ECV-304 mono-cultures. Resistance was recorded daily. Significant increase in the resistance of human brain endothelium treated with cytokines in a rank order of IFN-γ ≈ TNF-α ≈> IL-1β > Con was observed. Inset shows the mode of culture and cytokine treatment. Bars indicate standard error. Repeated measured ANOVA with Dunnett's post-hoc test. *p < 0.05 was considered to be statistically significant, **p < 0.01 very significant, and ***p < 0.001 highly significant. b) Effect of human cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) on contact independent ECV-304/HFA co-culture system. Resistance was recorded daily. After 5 d barrier was pronouncedly lost in all conditions. TEER readings obtained until 5 d were plotted to observe the effect of human cytokines on species matched co-culture barrier. A rank order of Con>TNF-α>IL-1β ≈ IFN-γ was observed. Inset shows the mode of contact dependent system used and cytokine addition. Bars indicate standard error. Repeated measures ANOVA with Dunnett's post-hoc test. *p < 0.05 was considered to be statistically significant, **p < 0.01 very significant, and ***p < 0.001 highly significant. c) Effect of human cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) on in vitro cell metabolism. TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) significantly decreased ECV-304 metabolism by 3 d. Bars indicate standard error. One way ANOVA with Dunnett's post-test. *p < 0.05 was considered to be statistically significant, **p < 0.01 very significant, and ***p < 0.001 highly significant.