Abstract
Ischemic stroke has been shown to induce breakdown of the blood-brain barrier, although these changes are not fully characterized. Oxygen-glucose deprivation (OGD) has been used to investigate the effects of ischemia in cultured brain capillary endothelial cells, however this involves a change of medium which in itself may affect the cells. The aim of the present study was to investigate the effect of OGD and simple medium exchange followed by 48 h of reperfusion on barrier properties of primary bovine endothelial cells co-cultured with rat astrocytes. Barrier properties were evaluated by transendothelial electrical resistance measurements, passive permeability of flux markers, RT-qPCR and immunocytochemistry. Both OGD and simple medium exchange caused an increase in endothelial monolayer permeability. This correlated with reduced transcript levels of a number of tight junction and tight junction-associated proteins (claudin-1, claudin-5, occludin, ZO-1, tricellulin, marveld3 and PECAM-1), as well as with altered transcript level of several transporters and receptors (GLUT-1, HB-EGF, InsR, TfR, two members of the low density lipoprotein receptor family, LDLR and LRP-1, and the efflux transporter BCRP). In contrast, effects induced specifically by OGD were transient de-localization of claudin-5 from the junction zone, increased InsR localization at the plasma membrane and transient downregulation of MRP-1 and P-gp transcript levels. In conclusion, OGD caused changes in claudin-5 and InsR localization, as well as in MRP-1 and P-gp transcript levels. Our results however also indicated that medium exchange alone caused changes in functional barrier properties and expression levels of wide range of proteins.
Highlights
The aim of the present study was to investigate the changes in barrier properties as well as in the localization and transcript level of selected junction proteins, transporters and receptors induced by experimental oxygen-glucose deprivation (OGD) and reperfusion, and to compare them with the changes observed upon medium exchange in a high-resistance brain endothelial/astrocyte co-culture cell model
Rabbit polyclonal anti-Claudin-5: ab15106, mouse monoclonal anti-Insulin Receptor-β [C18C4]: ab69508, rabbit polyclonal anti-heparin-binding EGF-like growth factor (HB-EGF): ab92620, rabbit polyclonal anti-VWF: ab6994 were from Abcam (Cambrige, United Kingdom), rabbit polyclonal anti-Glut-1: PA11063 and rabbit polyclonal anti-ZO-2: PA5-17155 were from ThermoFisher (Slangerup, Denmark), mouse monoclonal anti-P-gp: NB600-1036 was from Novus Biologics (England, United Kingdom)
After 24 h of reperfusion, the Transendothelial electrical resistance (TEER) increased to 691 ± 67 O cm2 in the cell treated with OGD and to 581 ± 57 O cm2 in the cells subjected to medium exchange, and after 48 h of reperfusion it returned to the initial levels in both experimental set up (928 ± 89 O cm2 in the OGD-treated cells and 929 ± 92 O cm2 in the cells that had undergone medium exchange)
Summary
The aim of the present study was to investigate the effect of OGD and simple medium exchange followed by 48 h of reperfusion on barrier properties of primary bovine endothelial cells co-cultured with rat astrocytes. The aim of the present study was to investigate the changes in barrier properties as well as in the localization and transcript level of selected junction proteins, transporters and receptors induced by experimental OGD and reperfusion, and to compare them with the changes observed upon medium exchange in a high-resistance brain endothelial/astrocyte co-culture cell model
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