Introduction - The blood-brain barrier (BBB) is a physical, transport and metabolic barrier which plays a key role in preventing uncontrolled exchanges between blood and brain, ensuring an optimal environment for neurons activity. This extent interface is created by the endothelial cells forming the wall of brain capillaries. The restrictive nature of the BBB is due to the presence of tight junctions, which seal the paracellular space, a low number of endocytotic vesicles and the presence of efflux transporters, resulting in a very tight layer.
Ischemic insult and the subsequent reperfusion dramatically impair the BBB integrity, resulting in increased BBB permeability, modified transport pathways, edema and tissue damage. Thus, to understand the molecular mechanisms leading to BBB breakdown during ischemia and to investigate drug transport in this condition is crucial for the development of therapies to treat this devastating disease.
Materials and Methods - Primary cultures of endothelial cells from bovine brain microvessels were cocultured with rat astrocytes in transwell inserts. At day 11, cells were treated with 4h of OGD by changing the culture medium with glucose-free medium and decreasing the oxygen level to 1% in a hypoxia workbench. To mimic the reperfusion phase, the aglycemic medium was replaced by glucose-supplemented medium and cells were further transferred in a normoxia incubator for 48h. TEER was monitored with an EVOHM and expression levels of relevant proteins were estimated using RT- PCR, immunocytochemistry was performed using CLSM.
Results - Monitoring the TEER value along the entire experimental time revealed a drastic drop in the transendothelial resistance from 1021 Ω∙cm2 to 116 Ω∙cm2 after 4h of OGD treatment, with a totally recover after 48h of reperfusion (929 Ω∙cm2). According with these results, immunocytochemistry data showed that Claudin-5 was significantly lost at the cell-cell contacts after 4h of OGD treatment whereas it reverted to form distinct continuous tight junction strands after 48h of reperfusion. Finally, RT-PCR after 4h of OGD showed a decreased mRNA expression for most of the tight junctional proteins and efflux transporters, which subsequent increased to the starting level after 48h of reperfusion. mRNA expression of receptor proteins showed different trends during the OGD experiment. To understand the functional relevance of this regulation, further experiments will be performed.
Conclusions – We have established an in vitro model of BBB in OGD condition and its characterization showed the disassembly of tight junctions at cell-cell contact with subsequent recovery during reperfusion.