The Effects Of Cannabidiol On Permeability In A Cell Culture Model Of The Blood-Brain Barrier Following Oxygen-Glucose Deprivation

WH Hind, SE O'Sullivan. University of Nottingham, Derby, UK

During stroke, the blood brain barrier (BBB) is subjected to a series of events, such as elevated levels of cytokines and adhesion molecules, which increase its permeability and can result in brain oedema. Cannabidiol (CBD) is beneficial in animal models of stroke, and other groups have demonstrated the ability of cannabinoids to attenuate the damage caused to the BBB in various pathologies. The aim of the present study was to establish whether CBD can modulate BBB permeability following oxygen-glucose deprivation (OGD, a model of ischaemia).

To model the BBB, co-cultures of human brain microvascular endothelial cells and human astrocytes were grown to confluence on Transwell collagen-coated inserts (Allen and Bayraktutan, 2009). BBB permeability was measured by transepithelial electrical resistance (TEER) using STX2 electrodes and an EVOM2 resistance meter. Inserts were subjected to 4 h OGD by incubating them in GasPak EZ Anaerobe pouches with glucose free RPMI medium. Reperfusion was established by returning the cells to normoxia and their specialised medium, and TEER was measured at various time-points over 28 h. CBD (100 nM, 1 μM, 10 μM), receptor antagonists, or vehicle were added to the luminal chamber before or after OGD. The levels of 9 cytokines/chemokines which are known to be altered in stroke were measured in the medium using a magnetic bead panel kit assay (Millipore).VCAM-1 levels were measured by ELISA (R&D). Statistical analysis comparing the different treatment types was conducted using one-way ANOVA with Dunnett’s post hoc test on GraphPad Prism software.

Exposing the BBB to 4 h OGD increased its permeability (P<0.001). Pre-treatment with 10 μM CBD attenuated the increased permeability (P<0.05), and 100 nM, 1 or 10 µM CBD decreased barrier permeability during reperfusion in a concentration-dependent manner (P<0.05-0.001; n = 8-11 inserts from 4-6 separate experiments). Administration of CBD post-OGD decreased barrier permeability at 8, 10 and 24 h during reperfusion in a concentration-dependent manner (P<0.05-0.01; n = 4-13 from 5 separate experiments). The receptor antagonists AM251, AM630, GW6471, SCH58261 (all at 100 nM), WAY100135 (300 μM), and capsazepine (1 μM) did not affect CBD responses, suggesting that CB1, CB2, PPARα, A2A adenosine receptor, 5HT1A and TRPV1 are not involved (n = 4-6 from 2-3 separate experiments). However, the CBD-associated decrease in barrier permeability was significantly inhibited by GW9662 (P<0.05 – P<0.001), indicating a role for PPARγ activation (n = 6 from 3 separate experiments). Detectable levels of IL-10, IL-6, MIP-1α, MIP-1β and VEGF did not differ with CBD treatment. However, VCAM-1 levels at the end of reperfusion were attenuated by CBD (P<0.05), an effect which was not sensitive to PPARγ inhibition.

In conclusion, this data demonstrates that CBD given either pre- or post-OGD decreased barrier permeability, an effect which was mediated through PPARγ.

References:

ALLEN, C. L. & BAYRAKTUTAN, U. (2009) Antioxidants attenuate hyperglycaemia-mediated brain endothelial cell dysfunction and blood-brain barrier hyperpermeability. Diabetes Obes Metab, 11, 480-90.