Fig. 8.

Impairment of BBB by OPN and its response to anti-OPN therapy in vitro. a Schematic depicting the isolation and culture of primary porcine or mouse brain microvascular endothelial cells (PBMEC or MBMEC) followed by transendothelial electrical resistance (TEER) measurement across the endothelial monolayer or qRT-PCR experiment. Immunostaining of transwell inserts representing well-developed tight and adherens junctions of the PBMEC monolayer including CLDN5 (red) and VECAD (green). b Representative graph for continuous TEER values of the PBMEC monolayer treated with recombinant murine OPN (0.5 µg/mL) or vehicle (Control). c 12, 24 and 48 h TEER values of OPN-treated PBMEC normalised to their respective time-point control; n = 3 independent experiments, d Activation of OPN signaling was assessed by qRT-PCR on MBMEC treated 24 h with recombinant murine OPN and normalised to their untreated control. n = 4 independent experiments. e Neutralisation effect of the anti-OPN antibody (3 µg/mL) was assessed by qRT-PCR on MBMECs treated with recombinant murine OPN for 24 h n = 3–4 independent experiments. f Schematic depicting the experimental paradigm for oxygen-glucose deprivation (OGD, 1% O₂, glucose-free basal medium) performed on MBMEC followed by qRT-PCR, staining of the cells, TEER measurement or permeability assay across the endothelial monolayer. Control condition cells were cultured in 19.5% O₂ and 5.6 mM glucose containing basal medium. g Representative images of osteopontin (OPN, green) in endothelial monolayer 24 h post-OGD treatment. CD31 (white) was used as endothelial marker and DAPI (blue) to reveal nuclei. h Representative graph for continuous TEER values of the MBMEC monolayer in control normoxic conditions (treated with isotype control) and for OGD conditions—isotype control or anti-OPN antibody treated. i Quantification of 24 h TEER values of MBMEC treated with isotype control or anti-OPN antibody in control or OGD conditions; n = 4 independent experiment. j Permeability (pe) index values of fluorescent tracers of different molecular weight through MBMEC monolayer subjected to OGD and treated with isotype control or anti-OPN antibody. Results were normalized to pe index values obtained with inserts from each respective control condition (dashed line); n = 4 independent experiments. k BBB disruption by 24 h OGD and effect of the anti-OPN antibody treatment were assessed by qRT–PCR on MBMECs. Results were normalized to each respective control culture condition (dashed line); n = 4 independent experiments, *P < 0.05; **P < 0.01, ***P < 0.001 and ns P > 0.05 by two-tailed, paired t test (c–e, i–k). Scale bars: 10 µm (a, g)