Endocannabinoids modulate human blood-brain barrier permeability in vitro

Abstract

Background and purpose: Endocannabinoids alter permeability at various epithelial barriers, and cannabinoid receptors and endocannabinoid levels are elevated by stroke, with potential neuroprotective effects. We therefore explored the role of endocannabinoids in modulating blood-brain barrier (BBB) permeability in normal conditions and in an ischaemia/reperfusion model.

Experimental approach: Human brain microvascular endothelial cell and astrocyte co-cultures modelled the BBB. Ischaemia was modelled by oxygen-glucose deprivation (OGD) and permeability was measured by transepithelial electrical resistance. Endocannabinoids or endocannabinoid-like compounds were assessed for their ability to modulate baseline permeability or OGD-induced hyperpermeability. Target sites of action were investigated using receptor antagonists and subsequently identified with real-time PCR.

Key results: Anandamide (10 μM) and oleoylethanolamide (OEA, 10 μM) decreased BBB permeability (i.e. increased resistance). This was mediated by cannabinoid CB2 receptors, transient receptor potential vanilloid 1 (TRPV1) channels, calcitonin gene-regulated peptide (CGRP) receptor (anandamide only) and PPARα (OEA only). Application of OEA, palmitoylethanolamide (both PPARα mediated) or virodhamine (all 10 μM) decreased the OGD-induced increase in permeability during reperfusion. 2-Arachidonoyl glycerol, noladin ether and oleamide did not affect BBB permeability in normal or OGD conditions. N-arachidonoyl-dopamine increased permeability through a cytotoxic mechanism. PPARα and γ, CB1 receptors, TRPV1 channels and CGRP receptors were expressed in both cell types, but mRNA for CB2 receptors was only present in astrocytes.

Conclusion and implication: The endocannabinoids may play an important modulatory role in normal BBB physiology, and also afford protection to the BBB during ischaemic stroke, through a number of target sites.

Figure 1

The effects of increasing concentrations of AEA on BBB permeability measured by TEER (A) with corresponding AUC (B) (n = 9 inserts from three separate experiments). The effects of capsazepine (Cpz) (C) (n = 7 inserts from three separate experiments) or AM630 (D) (n = 7–8 inserts from three separate experiments) or CGRP (E) (n = 5–6 inserts from three separate experiments) on the effect of AEA (10 μM). The effects of dexamethasone (n = 6) and the CB₂ agonist HU308 on BBB permeability over time (F) and expressed as AUC (G). Data are given as mean ± SEM. ^***P < 0.001, ^**P < 0.01, ^*P < 0.05; AEA compared with vehicle-treated inserts; ††P < 0.01; AEA and antagonist compared with AEA alone; #P < 0.05, ##P < 0.01; HU308 compared with vehicle; one-way anova with Dunnett's (B) or Bonferroni's test (C,D,E).

Figure 2

Expression profiling of potential target sites of action in HA and HBMEC cells. Shown are the ethidium bromide-stained gels of the products obtained by RT-PCR using primers specific for PPARα, PPARγ, CB₁ receptors, CB₂ receptors, TRPV1 channels, CGRP receptors and the control gene HPRT. cDNAs generated in the presence (+) or absence (−) of reverse transcriptase on total RNA from HA or HBMEC cells were used as template for the PCRs. The 100 bp DNA ladder was used in all gels except for PPARα and PPARγ where a 10 bp ladder was used. Sizes are in base pairs.

Figure 3

The effects of AEA (n = 6) in the presence and absence of the FAAH inhibitor URB597 (n = 6) on permeability in the BBB (A), with corresponding AUC (B). Data are given as mean ± SEM. **P < 0.01, *P < 0.05 AEA versus the vehicle; ††P < 0.01 AEA and antagonist compared with AEA alone; one-way anova with Dunnett's test.

Figure 4

The effects of various concentrations of AEA either before (A) (n = 7–12 inserts from four separate experiments) or after (C) (n = 9–10 inserts from four separate experiments) 4 h OGD on permeability in the BBB, with corresponding AUC (B and D). Data are given as mean ± SEM. #, * (10 μM) † (30 μM), P < 0.05; compared with vehicle; one-way anova with Dunnett's test.

Figure 5

The effect of OEA over time on BBB permeability in the initial endocannabinoid screening (A) (n = 6 inserts from three separate experiments) as a concentration-response curve (B) (n = 4–6 inserts from three separate experiments) and in the presence of the PPARα antagonist GW6471 (C and D) (n = 5 inserts from three separate experiments). (E) The effects of NADA (10 μM) on TEER values in the BBB model (n = 7–8 inserts from four separate experiments). (F) Absorbance values for LDH assay conducted on cell culture medium obtained from the luminal (endothelial) chamber of the inserts at 48 h (n = 6 inserts from three separate experiments). Data are given as mean ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05; OEA compared to vehicles treated inserts; †††P < 0.001, ††P < 0.01; OEA and antagonist compared with OEA alone; one-way anova with Dunnett's or Bonferroni's test.

Figure 6

The effect of OEA (A and B) (n = 6 inserts from three separate experiments), PEA (C and D) (n = 5–6 inserts from three separate experiments), virodhamine (E and F) (n = 6 inserts from three separate experiments), 2-AG (G) (n = 6 inserts from three separate experiments), oleamide (H) (n = 6 inserts from three separate experiments), NADA (I) (n = 4–5 inserts from two separate experiments) and noladin ether (J) (n = 5 inserts from three separate experiments) administered before 4 h OGD on TEER. Data are given as mean ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05; compared with vehicle-treated inserts; Student's t-test.

Figure 7

The effect of OEA (A) (n = 6 inserts from three separate experiments) or PEA (B) (n = 5–6 inserts from three separate experiments) alone or in combination with GW6471 before 4 h OGD on TEER. Data are given as mean ± SEM. *P < 0.05 compared with vehicle-treated inserts; †P < 0.05 OEA and antagonist compared with OEA alone; one-way anova with Bonferroni's test.