254 Scientific Programme 22. European Stroke Conference © 2013 S. Karger AG, Basel 14 Translational stroke research Cannabidiol Ameliorates The Increased Permeability Of A Cell Culture Model Of The Blood- Brain Barrier Following Oxygen-Glucose Deprivation W.H. Hind1, T.J. England2, S.E. O’Sullivan3 University of Nottingham, Derby, UNITED KINGDOM1, University of Nottingham, Derby, UNITED KINGDOM2, University of Nottingham, Derby, UNITED KINGDOM3 The permeability of the blood-brain barrier (BBB) increases during stroke, and cannabidiol (CBD; which has an excellent tolerability profile in humans) is beneficial in animal stroke models. The aim of this study was to establish whether CBD modulates BBB permeability following oxygen-glucose deprivation (OGD). Co-cultures of human brain microvascular endothelial cells and human astrocytes were grown on transwell inserts, and BBB permeability was measured by transepithelial electrical resistance (TEER). Inserts were subjected to 4h OGD by incubating them in GasPak EZ Anaerobe pouches with glucose free RPMI medium. Following reperfusion (return to normal conditions), TEER was mea-sured over 28h. CBD, various receptor antagonists, or vehicle were added pre- or post-OGD. Levels of lactate dehydrogenase (LDH), VCAM-1 and 9 cytokines/chemokines known to be altered in stroke were measured. Statistical analysis was conducted using t-test or one-way ANOVA with Dunnett’s post hoc test. Exposing the BBB to 4h OGD increases barrier permeability (P<0.001). CBD given pre-OGD attenu-ated this increase (P<0.05) and decreased permeability during the reperfusion period (P<0.05-0.001). Administration of CBD post-OGD also decreased barrier permeability during reperfusion (P<0.05- 0.01). Antagonists of CB1, CB2, TRPV1, PPAR-alpha, A2A adenosine receptor and 5HT1A did not affect the response to CBD. However, CBD’s effect was inhibited by GW9662 (P<0.05-0.001), indicating a role for PPAR-gamma activation. LDH levels were raised following 4h OGD and 28h reperfusion, and this was inhibited by CBD (non-PPAR-gamma mediated). Detectable levels of IL-6, IL-10, MIP-1alpha, MIP-1beta and VEGF did not differ with CBD treatment. VCAM-1 levels were attenuated by CBD (P<0.05) (non-PPAR-gamma mediated). In conclusion, CBD-mediated protection of the BBB may represent an as yet unrecognised facet of neuroprotection that CBD affords in experimental stroke. 13 Vascular biology Hyperglycaemia promotes cerebral barrier dysfunction through activation of protein kinase C-beta B . Shao1, U . Bayraktutan2 The University of Nottingham, Nottingham, UNITED KINGDOM1, The University of Notting-ham, Nottingham, UNITED KINGDOM2 Background: Brain oedema emerging from the disruption of the blood-brain barrier (BBB) con-tinues to be the leading cause of mortality within the first week after an ischaemic stroke. As the incidence of brain oedema is higher in stroke patients with diabetes, it is thought that hyperglycae-mia (HG) plays a prominent role in the pathogenesis of this defect. The current study investigat-ed whether protein kinase C (PKC) and associated downstream mechanisms are involved in HG-evoked BBB damage. Methods: The activities of total PKC (Peptaq assay), NADPH oxidase (lucigenin assay) and matrix metalloproteinase-2 (MMP-2; gelatin zymography) were measured in human brain microvascular endothelial cells exposed to normoglycaemia (5.5 mM) or HG (25 mM) using the specific assays indicated in parentheses. The integrity and function of the in vitro models of human BBB were as-sessed by assessments of transendothelial electrical resistance and paracellular flux of permeability markers, respectively. Occludin protein expression was studied by immunoblotting. Results: HG significantly compromised the BBB integrity and enhanced total PKC activity to which increases in PKC-alpha, PKC-beta and PKC-betaII isoforms contributed the most. Elevations in NADPH oxidase and MMP-2 activities and decreases in occludin levels contributed to barrier dys-function. Selective inhibition of PKC-beta isoform prevented the changes observed in occludin ex-pression and the aforementioned enzyme activities and thus effectively preserved barrier integrity. Similarly, apocynin, a specific NADPH oxidase inhibitor, also effectively neutralised the effects of HG on barrier integrity, MMP-2 activity and occludin expression. Conclusion: HG promotes cerebral barrier dysfunction through activation of PKC-beta and conse-quent stimulations of oxidative stress and tight junction dissolution.
Karger_ESC London_2013
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