Stone, Nicole
(2021)
Phytocannabinoids, neuroprotection and the blood-brain barrier.
PhD thesis, University of Nottingham.
Abstract
The blood brain barrier (BBB) is central to the neurovascular unit (NVU) where it creates a semi-permeable barrier between neuronal tissue and the vascular networks that feed the brain. In neurodegenerative conditions and ischaemic stroke, the BBB becomes compromised and as a result its permeability increases. This not only exacerbates neuronal damage at the site of injury but also causes unwanted extravasation of peripheral immune cells into the brain, fuelling the overactivation of the immune response. Endocannabinoids and phytocannabinoids have both displayed neuroprotective effects, attenuating damage in a range of models including Parkinson’s, Huntington’s, amyloid lateral sclerosis and ischaemic stroke. The current study aimed to investigate the neuroprotective properties of emerging phytocannabinoids; specifically focusing on the BBB and NVU in the context of ischaemic stroke pathophysiology.
A four-cell blood brain barrier model was constructed consisting of; human brain microvascular endothelial cells (HBMECs), astrocytes, pericytes and neurons. Cells were cultured on collagen coated transwell inserts and permeability was assessed using transepithelial resistance (TEER). A systematic review was conducted to examine work on the neuroprotective properties of minor phytocannabinoids, aside from cannabidiol (CBD) and delta 9-tetrahydrocannabinol (Δ9-THC). Following on from this, in vitro experiments were conducted using minor phytocannabinoids with the most neuroprotective potential; cannabidivarin (CBDV), cannabigerol (CBG) and cannabidiolic acid (CBDA). Inserts or monocultures (four cell model and pericyte, HBMECs and neuronal monolayers) were subjected to either a 4 h oxygen-glucose deprivation (OGD) protocol or an 8 h OGD (astrocyte monocultures), to model ischaemic stroke in vitro. Media was analysed for various chemokines and cytokines using enzyme-linked immunoassays or multiplex assays.
From the systematic review, emerging phytocannabinoids cannabidivarin (CBDV) and cannabigerol (CBG) were found to display efficacy in various neurogenerative conditions and of the limited available mechanistic data, were found to mediate some of their effects through peroxisome proliferator-activated receptor gamma (PPARy). Data showed CBDV (300 nM-10 µM) attenuated MCP-1 levels in HBMEC monolayers, as well as reducing IL-6 (30 nM, 1 µM and 10 µM; p<0.05) and VEGF (10 nM- 10 µM; p<0.01) levels in astrocyte monocultures post OGD. CBG (10 nM-3 µM; p<0.0001) also reduced levels of IL-6 secreted by astrocytes and decreased levels of DNA damage response proteins including Chk1, Chk2, H2A.X and p53 post OGD. Neither CBG, nor CBDV reduced levels of IL-6, VEG or IL-8 in pericytes compared to the vehicle control post OGD. Cannabidiolic acid (CBDA) was also investigated and was found to decrease IL-6 in pericyte monocultures which was mediated, at least in part, by 5-HT1A activation. In a four-cell model of the BBB, CBDA offset increases in permeability vs the vehicle control and offered direct protection to neurons, as shown by a lack of propidium iodide (PI) staining in CBDA treated cells, indicating live cells are present.
Data presented in this thesis show minor phytocannabinoids CBDV, CBG and CBDA provide protection against OGD mediated damage, with CBDA also offering protection against increases in permeability of the BBB post OGD. These novel data warrant further investigation into the neuroprotective properties of phytocannabinoids, particularly in ischaemic stroke.
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