Fluvastatin prevents glutamate-induced blood-brain-barrier disruption in vitro

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Glutamate is an important excitatory amino acid in the central nervous system. Under pathological conditions glutamate levels dramatically increase. Aim of the present study was to examine whether the HMG-CoA inhibitor fluvastatin prevents glutamate-induced blood-brain-barrier (BBB) disruption. Measurements of transendothelial electrical resistance (TEER) were performed to analyze BBB integrity in an in vitro co-culture model of brain endothelial and glial cells. Myosin light chain (MLC) phosphorylation was detected by immunohistochemistry, or using the in-cell western technique. Intracellular Ca2+ and reactive oxygen species (ROS) levels were analyzed using the fluorescence dyes Ca-green or DCF. Glutamate induced a time- (1-3 h) and concentration- (0.25-1 mmol/l) dependent decrease of TEER values that was blocked by the NMDA-receptor antagonist MK801, the Ca2+ chelator BAPTA, the NAD(P)H-oxidase inhibitor apocynin and the MLC-kinase inhibitor ML-7. Furthermore we observed a concentration-dependent increase of intracellular Ca2+ and ROS after glutamate application. Glutamate caused an increase of MLC phosphorylation that was antagonized by apocynin, or BAPTA, indicating that Ca2+ and ROS signaling is involved in the activation of the contractile machinery. Fluvastatin (10-25 μmol/l) completely abolished the glutamate-induced barrier disruption and oxidative stress. The BBB-protecting effect of fluvastatin was completely lost if the cells were treated with the nitric oxide (NO) synthase inhibitor l-NMMA (300 μmol/l). In the present study we demonstrated that glutamate-induced BBB disruption involves Ca2+ signalling via NMDA receptors, which is followed by an increased ROS generation by the NAD(P)H-oxidase. This oxidative stress then activates the MLC kinase. Fluvastatin preserves barrier function in a NO-dependent way and reduces glutamate-induced oxidative stress.

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