The Effects Of Anandamide On Permeability In A Cell Culture Model Of The Blood-Brain Barrier

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

The blood-brain barrier (BBB) is a selective barrier which is vital for maintaining a precisely regulated microenvironment for reliable neuronal signalling. Other groups have demonstrated the ability of various cannabinoids (the endogenous cannabinoids 2-arachidonoylglycerol and anandamide (AEA); and the synthetic cannabinoids arachidonyl-2’-chloroethylamide, CP55940 and WIN55212-2) to attenuate damage caused to the BBB in a variety of pathologies (chronic head injury, multiple sclerosis and HIV infection). The role that AEA plays in regulating BBB permeability has yet to be investigated using human cells or in the context of ischaemic stroke. Therefore, the aim of the present study was to establish whether AEA modulates BBB permeability in these conditions.

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. Resistance readings were taken at various intervals over 96 h, and all readings were compared to their baseline value. AEA and/or antagonist were added to the luminal (i.e. endothelial) cells at 0 and again at 48 h when the medium was replaced.

In a separate series of experiments, inserts were subjected to 4 h oxygen-glucose deprivation (OGD, to model ischaemia) 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 (with or without AEA), and TEER was measured at various time-points over 28 h. Statistical analysis comparing the different treatment types was conducted using one-way ANOVA with Dunnett’s post hoc test on GraphPad Prism software.

Administration of AEA at 10 µM, but not 100 nM, 1 µM or vehicle, resulted in an acute decrease in BBB permeability compared to baseline (0 h) at 2, 50 and 52 h (i.e. immediately after AEA administration; n = 9 from 3 experiments; P<0.05-0.001). Receptor involvement was probed, and AM251 (1 μM), GW6471 (100 nM) and GW9662 (100 nM) did not inhibit the response to AEA, suggesting that CB1, PPARα and PPARγ are not involved. However, the AEA-induced decrease in permeability was significantly inhibited by AM630 (1 μM) (P<0.05-0.01) and capsazepine (1 μM) (P<0.05-0.01), indicating a role for CB2 and TRPV1 receptor activation. The decrease in permeability was also inhibited by URB597 (1 μM), indicating a role for the metabolites of AEA (P<0.05-0.01) (n = 4-6 from 2-3 separate experiments for all antagonist studies). Exposing the BBB to 4 h OGD increased its permeability (P<0.001), but administration of AEA (100 nM, 1, 10 or 30 μM) following OGD did not further alter permeability (n = 7-12 from 3 separate experiments).

This study demonstrates that luminal administration of AEA results in an acute decrease in permeability of the BBB in vitro, mediated by CB2, TRPV1 and metabolites of AEA. However, AEA does not affect BBB permeability following OGD.