Figure 2: Surface permeability of BBB spheroid to high molecular weight dextran and intact tight junctions are modulated by VEGF.

(a) Fluorescence images showing the expression of tight junction markers, claudin 5 and occludin (white). Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar, 100 μm (× 20 objective). (b) Magnified fluorescence images showing claudin 5 and occluding expression. Scale bar, 100 μm (× 60 objective). (c) Fluorescence images showing decreased expression of tight junction marker (ZO-1: green) with increasing VEGF-A concentration (at 5, 20 and 50 ng ml⁻¹) in primary HBMEC (pre-labelled with CellTracker Red dye) and (d) immortalized hCMEC/D3 ECs. Cell nuclei were labelled with Hoechst dye (blue). Scale bar: 50 μm in lower-magnification images; 10 μm in magnified images. (e) Dextran permeability assay showing that VEGF-A (at 25, 50 and 100 ng ml⁻¹) increased spheroid permeability to TRITC-Dextran (155 kDa; red; 10 mg ml⁻¹) using spheroids established using primary HBMEC ECs. The image panels above the graph depict a representation of how permeability was assessed. The white dotted line marks the area within the core of the spheroid, where the mean fluorescence intensity was quantified. Scale bar, 50 μm; n=8. (f) Dextran permeability study (as in e) using spheroids established using immortalized hCMEC/D3 ECs. n_spheroid=3–5. Both graphs show mean TRITC fluorescence intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars (**P<0.01; *P<0.05). Statistical analyses were performed using the one-way ANOVA and Dunnett’s multiple comparison test.