Effect of mouse cytokines on bend-3 mono-culture barrier and bEnd-3/HFA co-culture barrier. a) Cumulative effect of mouse cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) applied to apical + basal sides of mouse brain endothelial mono-cultures. Resistance was recorded daily (7 d). Significant increase in the resistance of mouse brain endothelium was observed in a rank order of IFN-γ > TNF-α > IL-1β compared with control. 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 mouse cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) of mouse brain endothelial solute permeability. Solute permeability was measured at 30', 1 h, 2 h, 3 h, 4 h and 6 h after 3 d of treatment. TNF-α and IFN-γ treated cultures showed lesser permeability than control or IL-1b treated cultures. The solute permeability of mouse brain endothelium in this experiment was in a rank order of IFN-γ ≈ TNF-α > IL-1β ≈ Con. 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. c) Effect of mouse cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) on contact dependent bEnd-3/HFA co-culture system. Resistance was recorded daily. Significant increase in mouse brain endothelial barrier was observed with IFN-γ > IL-1β ≥ TNF-α compared to controls. 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. d) Effect of mouse cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) on contact independent bEnd-3/HFA co-culture system. Resistance was recorded daily. Significant increase in the resistance of brain endothelium was observed with IFN-γ > IL-1β ≥ TNF-α compared with control. 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.

Effect of mouse cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) on mouse brain endothelial metabolism. TNF-α (20 ng/ml) and IFN-γ (1000 U/ml)) significantly decreased mouse brain endothelial cell metabolism by 4 d but not IL-1β (20 ng/ml).

Effect of human cytokines on HBMEC-3 mono-culture barrier and HBMEC-3/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 human brain endothelial (HBMEC-3) mono-cultures. Resistance was recorded daily. No significant difference in the resistance of cytokine treated HBMEC-3 barrier to that of untreated HBMEC-3 barrier was noted in this experiment. 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 HBMEC-3/HFA co-culture barrier. Human cytokines were added to both apical and basal sides of the co-culture system and TEER recorded daily. Co-cultures treated with cytokines showed slightly lesser barrier integrity than untreated controls. The rank order of this experiment is Con> IL-1β ≈TNF-α> IFN-γ *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 HCMEC-D3 metabolism. TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) significantly decreased mouse brain endothelial cell metabolism by 3 d.

Effect of human cytokines on human astrocytes in contact-independent mouse brain endothelial co-culture barrier. Astrocytes were treated with human cytokines (TNF-α (20 ng/ml), IL-1β (20 ng/ml) and IFN-γ (1000 U/ml)) in a contact independent bEnd-3/HFA co-culture system. Resistance was recorded daily. hIFN-γ treated co-cultures from 5 d- 7 d showed decreased barrier compared to other treatment and control conditions. Inset shows the mode of co-culture system 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.

Effect of human cytokines on HCMEC-D3 mono-culture barrier and HCMEC-D3/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 human brain endothelial (HCMEC-D3) 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 ≈ Con ≈ IL-1β > TNF-α 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 HCMEC-D3/HFA contact dependent co-culture barrier. Human cytokines were added to both apical and basal sides of the contact dependent co-culture system and TEER recorded daily. Co-cultures treated with TNF-α showed a higher loss in barrier integrity than other conditions. The rank order of this experiment is Con≈IL-1β>IFN-γ> TNF-α *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 (1000U/ml)) on HCMEC-D3/HFA contact independent co-culture barrier. Human cytokines were added to both apical and basal chamber of the co-culture system and TEER recorded daily. Co-cultures treated with cytokines showed lesser barrier integrity than untreated controls. The rank order of this experiment is Con> IL-1β ≈IFN-γ > TNF-α *p < 0.05 was considered to be statistically significant, **p < 0.01 very significant, and ***p < 0.001 highly significant. d) Effect of human cytokines (TNF-α (20ng/ml), IL-1β (20ng/ml) and IFN-γ (1000U/ml)) on HCMEC-D3 metabolism. TNF-α (20ng/ml) and IFN-γ (1000U/ml)) significantly decreased mouse brain endothelial cell metabolism by 3 d but not IL-1β (20ng/ml).

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.