Addition of NMDA-receptor antagonist MK801 during oxygen/glucose deprivation moderately attenuates the upregulation of glucose uptake after subsequent reoxygenation in brain endothelial cells

Abstract

During stroke the blood-brain barrier (BBB) is damaged which can result in vasogenic brain edema and inflammation. The reduced blood supply leads to decreased delivery of oxygen and glucose to affected areas of the brain. Oxygen and glucose deprivation (OGD) can cause upregulation of glucose uptake of brain endothelial cells. In this letter, we investigated the influence of MK801, a non-competitive inhibitor of the NMDA-receptor, on the regulation of the glucose uptake and of the main glucose transporters glut1 and sglt1 in murine BBB cell line cerebEND during OGD. mRNA expression of glut1 was upregulated 68.7-fold after six hours OGD, which was significantly reduced by 10 µM MK801 to 28.9-fold. Sglt1 mRNA expression decreased during OGD which was further reduced by MK801. Glucose uptake was significantly increased up to 907% after six hours OGD and was still higher (210%) after the 20 hours reoxygenation phase compared to normoxia. Ten µM MK801 during OGD was able to reduce upregulated glucose uptake after OGD and reoxygenation significantly. Presence of several NMDAR subunits was proven on the mRNA level in cerebEND cells. Furthermore, it was shown that NMDAR subunit NR1 was upregulated during OGD and that this was inhibitable by MK801. In conclusion, the addition of MK801 during the OGD phase reduced significantly the glucose uptake after the subsequent reoxygenation phase in brain endothelial cells.

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Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an <em>In Vitro</em> Blood Brain Barrier Model

“…Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid-4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8 . Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.…”

“…OGD led to the disruption of the BBB function, which could be reconstituted after combined treatment with GC and proteasome inhibitor bortezomib 12 . OGD conditions led to a strong increase in glucose uptake and in the expression of glucose transporters in cerebEND, which could be attenuated by the addition of MK801, a non-competitive inhibitor of the NMDA-receptor 13 . After reaching confluence, the cells were maintained in medium containing 2% FCS.…”

Multifaceted Mechanisms of WY-14643 to Stabilize the Blood-Brain Barrier in a Model of Traumatic Brain Injury

“…Cells were maintained in an incubator at 37°C, 95% humidity and a 5% CO2/95% air atmosphere and subcultivated 1:3 or 1:20, respectively, once a week (Neuhaus et al, 2012a). Transwell experiments were accomplished as recently reported (Neuhaus et al, 2014).…”

“…Used FAM-labeled probes from Taqman ® (Applied Biosystems) were listed in Supplementary Table S1. In case of PPARα and LRP1, cDNA was preamplified using Taqman PreAmp Master mix (2x) prior qPCR analysis according to Neuhaus et al (2012a, Supplementary Part). Each sample was analyzed as triplicate.…”