Simvastatin, edaravone and dexamethasone protect against kainate-induced brain endothelial cell damage

Background Excitotoxicity is a central pathological pathway in many neurological diseases with blood–brain barrier (BBB) dysfunction. Kainate, an exogenous excitotoxin, induces epilepsy and BBB damage in animal models, but the direct effect of kainate on brain endothelial cells has not been studied in detail. Our aim was to examine the direct effects of kainate on cultured cells of the BBB and to test three anti-inflammatory and antioxidant drugs used in clinical practice, simvastatin, edaravone and dexamethasone, to protect against kainate-induced changes. Methods Primary rat brain endothelial cell, pericyte and astroglia cultures were used to study cell viability by impedance measurement. BBB permeability was measured on a model made from the co-culture of the three cell types. The production of nitrogen monoxide and reactive oxygen species was followed by fluorescent probes. The mRNA expression of kainate receptors and nitric oxide synthases were studied by PCR. Results Kainate damaged brain endothelial cells and made the immunostaining of junctional proteins claudin-5 and zonula occludens-1 discontinuous at the cell border indicating the opening of the barrier. The permeability of the BBB model for marker molecules fluorescein and albumin and the production of nitric oxide in brain endothelial cells were increased by kainate. Simvastatin, edaravone and dexamethasone protected against the reduced cell viability, increased permeability and the morphological changes in cellular junctions caused by kainate. Dexamethasone attenuated the elevated nitric oxide production and decreased the inducible nitric oxide synthase (NOS2/iNOS) mRNA expression increased by kainate treatment. Conclusion Kainate directly damaged cultured brain endothelial cells. Simvastatin, edaravone and dexamethasone protected the BBB model against kainate-induced changes. Our results confirmed the potential clinical usefulness of these drugs to attenuate BBB damage.

1 mm 3 pieces and enzymatically digested by 1 mg/ml collagenase type II (Roche, Switzerland), and 15 µg/ml deoxyribonuclease type I (Roche, Switzerland) in Dulbecco's modified Eagle medium/Nutrient Mixture F-12 (DMEM/F12, Gibco, Life Technologies, USA) for 55 min at 37 °C. After the digestion the cells were mixed with 20% bovine serum albumin (BSA)-DMEM and centrifuged three times (1,000 × g, 20 min) to separate the brain microvessel fraction from the myelin rich brain tissue fraction. After each centrifugation step the cell pellets were collected and pooled. The collected microvessels were further digested by 1 mg/ml collagenase-dispase (Roche, Switzerland) in DMEM/F12 containing 15 µg/ml deoxyribonuclease type I (Roche, Switzerland) for 35 min at 37 °C. Brain microvascular endothelial cell clusters were separated on a 33 % Percoll gradient (1,000 × g, 10 min) then collected and washed twice in DMEM/F12. Cells were seeded onto collagen type IV and fibronectin coated (100 µg/ml each) Petri dishes (100 mm; Corning, USA). Cells were maintained in DMEM-F12 supplemented with 15 % plasma-derived bovine serum (PDS; First Link, UK), 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite (Pan Biotech, Germany), 10 mM Hepes, 1 ng/ml basic fibroblast growth factor, 100 μg/ml heparin and 50 μg/ml gentamycin. During the first three days of culture the capillary endothelial cells were kept in culture medium containing 3 μg/ml puromycin to eliminate P-glycoprotein negative cell types [Perrière et al., 2005]. Cells were used at the first passage for experiments.
Primary rat brain pericytes were isolated by the same protocol, except that puromycin treatment was not applied. After isolation pericytes were seeded onto collagen type IV coated (100 µg/ml) Petri dishes (60 mm; VWR International, USA) and cultured in DMEM medium (Gibco, Life Technologies, USA) containing 10 % fetal bovine serum (FBS, Pan Biotech, Germany) and gentamycin (50 μg/ml). Cells were used at the third passage for experiments.
Primary rat astrocytes were obtained from 1-day-old Wistar rats. Meninges were removed then the brain tissue was mechanically dissociated with a long needle connected to a syringe.
The homogenate was filtered through a nylon mesh (40 μm, Millipore, USA) to remove larger vessels and tissue debris. Cell clusters were plated onto uncoated 75 cm 2 flasks (TPP, Switzerland) and cultured in DMEM supplemented with 10 % FBS and gentamycin (50 μg/ml) until they reached 90 % confluency. For the BBB co-culture model cells were cultured for 2 weeks (first passage) before use in appropriate collagen type IV (100 µg/ml) coated 12-well plates (Corning, USA) at a cell number of 5 × 10 4 cells/well. Confluent glia cultures included 90 % GFAP immunopositive astroglia and 10 % CD11b immunopositive microglia.
Brain endothelial cells were co-cultured with brain pericytes and astrocytes to induce the BBB characteristics [Nakagawa et al., 2009]. First pericytes (1.5 × 10 4 cells/cm 2 ) were passaged to the bottom side of the 12-well format culture inserts (Transwell clear, polyester membrane, 0.4 µm pore size, 1.12 cm 2 surface; Corning, USA) coated with collagen type IV (100 µg/ml), then brain endothelial cells (7.5 × 10 4 cells/cm 2 ) were added to the upper side coated with collagen type IV and fibronectin (100 µg/ml each). The inserts were placed into 12-well plates containing confluent astrocyte layers. Endothelial culture medium was added to both compartments. Cells were co-cultured for four days before experiments [Perrière et al., 2005].  Figure S1. Characterization of primary rat astrocytes and pericytes. Cell morphology was characterized by glial fibrillary acidic protein (GFAP, astroglia) and α-smooth muscle actin (α-SMA, pericyte) immunostaining and visualized by confocal microscopy (Leica TCS SP5, confocal laser scanning microscope; Leica Microsystems, Germany). Cells were fixed with cold acetone-methanol solution (1:1) for 10 min, washed with phosphate buffered saline (PBS) and non-specific binding sites were blocked with 3% BSA-PBS for 1 h at room temperature. Incubation with mouse anti-α-SMA (Dako, USA) and mouse anti-GFAP primary antibodies lasted overnight at 4 °C. Cells were incubated with anti-rabbit secondary antibody labeled with Cy3 or anti-mouse secondary antibody labeled with Alexa Fluor 488 (Life Technologies, USA) and H33343 dye to stain nuclei for 1 h at room temperature. Between incubations cells were washed three times with PBS. Scale bar = 25 µm. Figure S2. Effect of Triton X-100 (TX-100) on cell viability measured by impedance kinetics. Triton X-100 detergent was used at 1% concentration as a reference compound to cause cell death. The impedance of the control group (C) remained stable during the 24-h period. Figure S3. Expression of housekeeping genes (β-actin and glyceraldehyde 3-phosphate dehydrogenase -GAPDH) in rat brain cortex (BR), isolated rat brain microvessels (MV) and primary rat brain endothelial cells (BEC). The predicted length of the products is shown in Table S2. Fragments were visualized on 2% agarose gel. M: 1 kb Plus DNA ladder.