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 tight junctions (TJs), which seal the intercellular clefts, limiting the paracellular diffusion, efflux transporters, which extrude xenobiotics, and metabolizing enzymes, which may break down or convert molecules during their passage through the capillary endothelium. An ischemic insult and the subsequent restoration of blood flow, defined as reperfusion, dramatically impair the BBB integrity, resulting in increased BBB permeability, modified transport pathways, edema and tissue damage. A deeper understanding of the permeation pathways across the barrier in ischemic and postischemic brain endothelium is important for developing new medical therapies capable to exploit the barrier changes occurring during/after ischemia to permeate in the brain and 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 6 of the coculture, cells were treated with 4h of OGD by changing the culture medium with glucose-free medium and decreasing the oxygen level to below 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 normal 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 1008 Ω∙cm2 to 127 Ω∙cm2 in average after 4h of OGD treatment, with a good recovery after 48h of reperfusion (744 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 and transporters showed different trends during the OGD experiment. Most of these changes reflect what was observed in vivo in brain capillaries isolated from MCAO mice by Laser Capture Microscopy (LCM). 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.