Cannabidiol protects an in vitro model of the blood–brain barrier from oxygen-glucose deprivation via PPARγ and 5-HT1A receptors
William H Hind, Timothy J England and Saoirse E O’Sullivan
British Journal of Pharmacology, 2016, 173, 815–825.
Doi : 10.1111/bph.13368
BACKGROUND AND PURPOSE
In vivo and in vitro studies have demonstrated a protective effect of cannabidiol (CBD) in reducing infarct size in stroke models and against epithelial barrier damage in numerous disease models. We aimed to investigate whether CBD also affects blood–brain barrier (BBB) permeability following ischaemia.
Human brain microvascular endothelial cell (HBMEC) and human astrocyte co-cultures modelled the BBB. Ischaemia was modelled by oxygen–glucose deprivation (OGD) and permeability was measured by transepithelial electrical resistance.
CBD (10 μM) prevented the increase in permeability caused by 4 h OGD. CBD was most effective when administered before the OGD, but protective effects were observed up to 2 h into reperfusion. This protective effect was inhibited by a PPARγ antagonist and partly reduced by a 5-HT1A receptor antagonist, but was unaffected by antagonists of cannabinoid CB1 or CB2 receptors, TRPV1 channels or adenosine A2A receptors. CBD also reduced cell damage, asmeasured by LDH release and by markers of cellular adhesion, such as the adhesion molecule VCAM-1. In HBMEC monocultures, CBD decreased VCAM-1 and increased VEGF levels, effects which were inhibited by PPARγ antagonism.
CONCLUSIONS AND IMPLICATIONS
These data suggest that preventing permeability changes at the BBB could represent an as yet unrecognized mechanism of CBD-induced neuroprotection in ischaemic stroke, a mechanism mediated by activation of PPARγ and 5-HT1A receptors.
The blood–brain barrier (BBB) is formed by brain endothelial cells that line the cerebral micro-vasculature, the capillary basement membranes and the endfeet of astrocytes. Tight junctions restrict the paracellular pathway for diffusion of hydrophilic solutes, allowing the body to control which substances can gain access to the brain (Abbott et al., 2006).
Cerebral reperfusion following ischaemia initiates a cascade of events including inflammation, protease activation and oxidative and nitrosative stress, all of which increase BBB permeability (Lo et al., 2003). Increased BBB permeability aggravates haemorrhagic transformation and vasogenic oedema, and uncontrolled cerebral oedema represents the leading cause of patient mortality within the first week
following an ischaemic stroke (Hacke et al., 1996).
Cannabidiol (CBD) is the second most abundant plantderived cannabinoid. CBD has multiple pharmacological targets, behaving as an agonist of TRPV1 channels, PPARγ, the adenosine A2A and 5-HT1A receptors and antagonizing the novel endothelial receptor, GPR55, and μ and δ opioid receptors. CBD is a low-affinity ligand which can modulate cannabinoid CB1 receptor activity (Laprairie et al., 2015; see also Petitet et al., 1998; McPartland et al., 2015) and has weak antagonistic activity toward signalling mediated at CB2 receptors by the cannabinoid ligands, CP 55,940 and WIN 55212-2 (Bisogno et al., 2001; McPartland et al., 2015). A CBD/THC combination (1:1 ratio, Sativex/Nabiximols, GW Pharmaceuticals, UK) is currently licensed internationally in more than 20 countries for the treatment of spasticity in multiple sclerosis, and a product containing only CBD (Epidiolex, GW Pharmaceuticals, UK) has entered an expanded access programme in children with intractable epilepsies. CBD has also received orphan designation status in treating newborn children with neonatal hypoxic-ischaemic encephalopathy.
The protective qualities of CBD in ischaemic stroke using rodent in vivo models have been shown in numerous studies. CBD reduces infarct size (when given either before or after middle cerebral artery occlusion) without the development of tolerance, increases cerebral blood flow (CBF), improves motor behaviour and increases survival (Hayakawa et al., 2010). The mechanism of action includes the ability of CBD to ameliorate glutamate neurotoxicity, behave as an antioxidant and anti-inflammatory agent and to attenuate adhesion molecule expression, neutrophils and the transendothelial migration of monocytes. The protective effects of CBD in vivo are inhibited by 5-HT1A receptor antagonists, with no
role for TRPV1 channels, CB1 or CB2 receptors (Hayakawa et al., 2010).We recently conducted a meta-analysis to examine the effects of exogenous application of endocannabinoids, phytocannabinoids and synthetic cannabinoids on infarct volume, functional outcome and survival. In 21 studies, involving 188 animals, treatment with CBD showed a highly significant reduction in infarct volume (P < 0.00001), with a standardized mean difference of 1.20 (England et al., 2015).
At the BBB, in vivo studies have shown that CBD decreased BBB permeability in a mouse model of multiple sclerosis (Mecha et al., 2013) and reduced LPS-induced BBB disruption in mice (Fernandez-Ruiz et al., 2013). CBD also modulated the permeability of several other epithelial barriers. For example,
blood–retinal barrier permeability is increased in diabetic rats in vivo and this was prevented by regular treatment with CBD (El-Remessy et al., 2006). CBD also decreased in vitro permeability in human coronary artery endothelial cells exposed to high glucose conditions (Rajesh et al., 2007). In gut epithelial cells, CBD enhanced the speed of recovery of either chemical-induced or cytokine-induced permeability activity via activation of CB1 receptors (Alhamoruni et al., 2010; Alhamoruni et al., 2012). A recent study from our group found that endocannabinoid-like molecules (oleoylethanolamide, palmitoyl-ethanolamide and virodhamine) decreased the oxygen–glucose deprivation (OGD)-induced increase in permeability using a in vitro model of the human BBB, showing that endocannabinoids can affect BBB permeability in an ischaemic setting (Hind et al., 2015).
The potential effects that CBD has on BBB permeability are yet to be assessed in the context of ischaemia, and we hypothesized that activity at the BBB could represent an as yet unrecognized mechanism of CBD-mediated protection in ischaemic stroke. To address this, we examined the effects of CBD in an in vitro BBB model, where the potential mechanisms of action were also probed.