Fig 1.
Transendothelial electrical resistance (TEER) and permeability of paracellular flux markers across brain endothelial cell monolayers in co-culture, during in vitro oxygen-glucose deprivation (OGD) and medium exchange treatments.
(A) Schematic overview of OGD and medium exchange protocols. At day 0, the endothelial cells were seeded on filter inserts in contact co-culture with astrocytes. At day 3 of co-culture, the cell medium (GM-) was changed to DM-TES medium. After additional 3 days (day 6), when the monolayers had developed an optimal resistance, the cells were exposed to OGD or subjected to simple medium exchange as control. After 4 h, standard culture conditions were reestablished for the OGD treated cells, in order to mimic the reperfusion phase (R). The cells were checked after 24 and 48 h of reperfusion. (B) TEER and (C-F) paracellular permeability of [14C]-mannitol, FITC-dextran 4, FITC-dextran 40 and FITC-dextran 150 across endothelial monolayers were monitored immediately before OGD/medium exchange (t0), after 4 h of OGD/medium exchange (4h OGD/m. exch.) and after 24 h and 48 h of reperfusion (24/48 h R). Bar graphs represent means normalized to t0 and error bars are +SEM. N = 12–24, n = 11–12 (B); N = 3–4, n = 3–4 (C-F). The white bars show the values at t0, the gray bars show the cells subjected to medium exchange only, while the black bars represent the cells treated with OGD. Columns were compared to t0 using one-way ANOVA and Dunnett’s multiple comparison post-test. *: p<0.05, **: p<0.01, ***: p<0.001. Each pair of columns was compared using one-way ANOVA with Bonferroni’s post-test.
Fig 2.
Transcript levels of selected junction proteins in endothelial cells co-cultured with astrocytes before, during and after oxygen-glucose deprivation (OGD) treatment or medium exchange.
Endothelial cells which had undergone OGD (black bars) or medium exchange (gray bars) were collected for mRNA isolation. After the reverse transcription of the mRNA, the resultant cDNA was quantified by RT-qPCR using primers specifically designed for bovine. The mRNA amounts of claudin-1 (A), claudin-5 (B), claudin-12 (C), occludin (D), ZO-1 (E), ZO-2 (F), tricellulin (G), marveld3 (H) and PECAM-1 (I) were normalized to reference genes (for calculation see “Materials and methods” section). Bar graphs represent means and error bars are +SEM. The white bars show the values at t0, the gray bars show the cells subjected to medium exchange, while the black bars show the OGD treated cells. Columns were compared to t0 using one-way ANOVA and Dunnett’s multiple comparison post-test. *: p<0.05, **: p<0.01, ***: p<0.001. Each pair of columns were compared using one-way ANOVA with Bonferroni’s post-test.
Fig 3.
Immunocytochemical evaluation of endothelial cell monolayer purity and determination of the cell density along the oxygen-glucose deprivation (OGD) and medium exchange protocols.
Endothelial cell monolayers co-cultured with astrocytes were treated with OGD or medium exchange and reperfused for 24/48 h. (A-G) For each condition, the cells were fixed and stained with an antibody anti-vWF (green) and counterstained with propidium iodide for the nuclei (red). Bars = 10 μm. N = 1; n = 3. (H) For each condition, the cell density expressed as number of cells per cm2 was calculated as stated in the “Materials and methods” section. Bar graphs represent means normalized to t0 and error bars are +SEM. N = 5, n = 3–4. The white bar shows the value at t0, the gray bars show the cells subjected to medium exchange, while the black bars show the OGD treated cells. Columns were compared to t0 using one-way ANOVA and Dunnett’s multiple comparison post-test. Bonferroni’s post-test was utilized to compare each pair of columns.
Fig 4.
Cellular localization of ZO-2 along the oxygen-glucose deprivation (OGD) and medium exchange protocols.
Endothelial cells in co-culture with astrocytes treated with OGD or medium exchange and reperfused for 24/48 h, were fixed and stained with an antibody anti-ZO-2. Fig A shows the localization of ZO-2 before the treatments (t0), Fig B shows the localization of ZO-2 after 4 h from medium exchange, and Fig C shows the localization of ZO-2, 4 h after OGD. Bars = 10 μm. N = 1; n = 3. (D) For each condition, the intracellular signal intensity was estimated using ImageJ as described in the “Materials and methods” section. Bar graphs represent means normalized to t0 and error bars are +SEM. (N = 9–12, n = 3–4). The white bar shows the value at t0, the gray bars show the cells subjected to medium exchange, while the black bars show the OGD treated cells. Columns were compared to t0 using one-way ANOVA and Dunnett’s multiple comparison post-test. *: p<0.05, ***: p<0.001. Bonferroni’s post-test was utilized to compare each pair of columns. #: p<0.05.
Fig 5.
Claudin-5 subcellular localization along the oxygen-glucose deprivation (OGD) and medium exchange.
Figs A-C show antibody staining of Claudin-5 under the different treatments. Bars = 10 μm. N = 1; n = 3. (D) For each condition, the intracellular signal intensity was estimated using ImageJ as described in the “Materials and methods” section. Bar graphs represent means normalized to t0 and error bars are +SEM. (N = 9–12, n = 3–4). The white bar shows the value at t0, the gray bars show the cells subjected to medium exchange, while the black bars show the OGD treated cells. Columns were compared to t0 using one-way ANOVA and Dunnett’s multiple comparison post-test. **: p<0.01. Bonferroni’s post-test was utilized to compare each pair of columns. ##: p<0.01.
Fig 6.
Transcript levels of membrane transporter GLUT-1 and the receptor HB-EGF, InsR, TfR, LDLR and LRP-1 in endothelial cell monolayers before, during and after oxygen-glucose deprivation (OGD) treatment or medium exchange protocol.
The transcript level of GLUT-1 (A) and different receptor proteins (B-F) in endothelial cells that had undergone OGD (black bars) or medium exchange (gray bars) were evaluated by RT-qPCR and normalized to reference genes. Primers specifically designed for bovine orthologues were utilized. Bar graphs represent means and error bars are +SEM. N = 3, n = 3–4. Columns were compared to t0 using one-way ANOVA and Dunnett’s multiple comparison post-test. *: p<0.05, **: p<0.01, ***: p<0.001. Bonferroni’s post-test was utilized to compare each pair of columns.
Fig 7.
Immunocytochemistry analysis of GLUT-1, HB-EGF and InsR protein localization before, during and after oxygen-glucose deprivation (OGD) and medium exchange treatment.
Endothelial cells co-cultured with astrocytes were subjected to OGD for 4 h or to medium exchange, followed by 24/48 h of reperfusion. For each condition, a filter insert was collected, fixed and stained with an antibody against GLUT-1 (A-G), HB-EGF (H-N) or InsR (O-U) (green), and counterstained with propidium iodide for visualizing the cell nuclei (red). Bars = 10 μm. N = 1, n = 3.
Fig 8.
Transcript level of BCRP, MRP-1 and P-gp and cell localization of P-gp in endothelial cells before, during and after 4 h of oxygen-glucose deprivation (OGD) or medium exchange.
The mRNA levels of BCRP (A), MRP-1 (B) and P-gp (C) at the different time points of the OGD (black bars) and medium exchange (gray bars) protocol were measured by RT-qPCR and normalized to reference genes. Primers specifically designed for bovine orthologues were utilized. Bar graphs represent means and error bars are +SEM. N = 3, n = 3–4. Columns were compared to t0 using one-way ANOVA and Dunnett’s multiple comparison post-test. *: p<0.05, **: p<0.01, ***: p<0.001. Bonferroni’s post-test was utilized to compare each pair of columns. #: p<0.05. Figs D-J show antibody staining of P-gp (green), and cell nuclei staining with propidium iodide (red) under the different treatments. Figs D’-J’ show exclusively the P-gp staining from Figs D-J. Bars = 10 μm. N = 1; n = 3.