Alterations of the Endocannabinoid System in Post-Ischemic Endothelial Cells of the Blood Brain Barrier
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Issue Date
8/15/2019
Authors
Thurston, Andrew B. '19
Degree
MS in Pharmaceutical Sciences
Advisor
Zheng, HaiAn
Committee Members
Voigt, Jeffrey M.
Musteata, F. Marcel
LaRocca, Timothy
Musteata, F. Marcel
LaRocca, Timothy
Journal Title
Journal ISSN
Volume Title
Abstract
The continued prominence of ischemia-based conditions (stroke, TBI, and CTE) in the US population and lack of an efficacious medicinal therapy, has led to the exploration of the emerging field of medicinal cannabis for a potential remedy. The purpose of this study is to confirm the existence of the endocannabinoid system (ECS) in endothelial cells of the Blood Brain Barrier and explore the effects of ischemia on the cannabinoid receptor expression and the capacity of the endocannabinoids as protective agents.
Using RT-qPCR and Western blot the cannabinoid receptor expression (C\302\254B\302\2541/CB2) under normal conditions and 0, 2, 8, and 24 hrs after a 4-hour oxygen-glucose deprivation (OGD) incubation were quantified. Endocannabinoids (eCBs), AEA and 2-AG, in mixture or solo were administered under normal conditions and 0, 2, 8, and 24 hrs after OGD to evaluate their effects on membrane integrity, cell activity, an ion regulation via TEER analysis, MTT assay, and an intracellular calcium stain. RT-qPCR and Western blot confirmed the presence of the cannabinoid receptors at the genetic and protein level and that their expression relationship under normal conditions mimicked that of other cells related to the CNS. The CB1 receptor displayed a downward trend following OGD incubation while the CB2 receptor displayed an upward trend with a noted dip at 2 hours following OGD. A mixture of the endocannabinoids, under normal conditions, resulted in a dose-dependent increase in monolayer membrane integrity with reduced permeability. Following OGD incubation 2-AG was found to be a more potent agent than AEA in supporting barrier integrity. Additionally, 2-AG also attenuated decreased cell activity at 0 and 2 hrs after OGD and 2 hrs after OGD for 10 and 25 ng/mL doses, respectively. Lastly, AEA at 10, 25, and 50 ng/mL doses increased intracellular calcium, while 2-AG generally had no impact on calcium levels compare to control.
This study confirmed the presence of the ECS on endothelial cells of the BBB along with the alteration of the cannabinoid receptor expression as a result of ischemic-mimicking conditions (OGD). Our data indicates that 2-AG acts as a protective agent, while AEA displayed disruptive properties, indicating an important role of the eCBs and ECS in barrier integration and validity, especially following ischemic stress.
Citation
Thurston AB. Alterations of the endocannabinoid system in post-ischemic endothelial cells of the blood brain barrier [thesis]. Ann Arbor (MI): Proquest LLC; 2019. 67 p.
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