THE BBB PERMEABILITY: HOW MUCH BLOOD FLOW-INDUCED SHEAR STRESS AFFECT IT

The blood-brain barrier (BBB) is the well-known physiological wall that allow the selective influx and efflux of molecules between the brain parenchyma and the blood circulation. The protagonists of this mechanism are the endothelial cells, assisted by the presence of pericytes and astrocytes. On the other hand, cerebral blood flow is also strictly regulated and its increase can induce shear stress. Indeed, in the past years, neurodegenerative disorders were associated to blood pressure variability. In the present research we evaluated the shear stress effect on rat brain endothelial cell line (RBE4), a widely used BBB in vitro model, in order to investigate the putative role of increasing flow in tight junction dislocation. To mimic blood flow in our in vitro model, we used the LiveBox2 (LB2) instrument (IVTech S.r.l., Lucca, Italy) that allow to set-up a millifluidic flow, ranging from 50 to 500 μl/min. Briefly, the RBE4 cells were gently seeded on the cover slip of LB2 chamber system, and allowed to growth at least for 24 h. The day after, in order to induce the medium flow on the chamber system, the system was connected to the pump and the appropriate flow rate was set-up. The system was left for 3 days at 37°C, 5% CO2 in humidified atmosphere, then the system was opened and the cells were fixed in cold methanol for 20 min. at 4°C. Immunofluorescent staining for zonula occludens-1 (ZO-1) was performed in order to evaluate the tight junction dislocation. Our results clearly demonstrated that shear stress affect the ZO-1 localization starting from 100 μl/min flow rate. Such a deleterious effect can be hypothesized as a possible mechanism that induces an increase in barrier permeability and therefore allows the entry of harmful substances that alter the brain parenchyma.