We purchased the antibodies to GLUT1, neuro-filament (NF), tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) from Abcam (Cambridge, MA, USA), antibodies to occludin, claudin-5 and ZO-1 from Zymed (Invitrogen, Carlsbad, CA, USA), and antibody to α-actin from Millipore (Billerica, MA, USA). All secondary Alexa Flour antibodies were purchased from Invitrogen. D-(2-3H)-Glucose (5 mCi, 185 MBq) was purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA, USA). Cytochalasin B (CB), cycloheximide (Chx), actinomycin D (act-D), and ALC were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Primary hBECs were obtained from Dr. Persidsky, Temple University School of Medicine, and hBECs were cultured as described previously (Haorah et al. 2007a
). Briefly, all cell culture plates and glass cover slips were pre-coated with type 1 rat-tail collagen (0.09 mg/ml in double-distilled sterile water), aspirated the excess collagen, and dried the plates overnight in sterile hood. The cells were cultured on 96-well plates (20,000 cells/well) for glucose uptake and viability assays, for immunohistochemistry cells were plated on 12-well glass cover slips (40,000 cells/well), and for protein extractions cells were cultured in T 75-cm2
cells/flask). Dulbecco's modified Eagle medium (DMEM)/F-12 media containing 10 mM Hepes, 13 mM sodium bicarbonate (pH 7), 10% fetal bovine serum, penicillin, and streptomycin (100 μg/ml each, Invitrogen) was used for cell culture. Cell culture media were changed every 3 days until tight monolayers were formed in about 6–8 days.
Cortical neurons were obtained from our neural tissue core facility, where routinely isolated these cells from elective abortus specimens of human fetal brain tissue. Tissues were obtained in full compliance with the ethical guidelines of both the National Institutes of Health (NIH) and the University of Nebraska. Briefly, dissociated tissues were incubated with 0.25% trypsin for 30 min, neutralized with 10% fetal bovine serum, and further dissociated by trituration. Neurons were cultured on poly-D
-lysine pre-coated cover slips and flasks (BD Labware, Bedford, MA, USA) in Neurobasal™ medium containing 0.5 mM glutamine, 50 μg/ml each of penicillin, and streptomycin in combination with GIBCO™ B-27 supplement with antioxidants as described previously (Haorah et al. 2008b
). Purity of neurons was assessed by MAP-2 Abs (Chemicon), resulting in 100% enrichment of neurons. Neurons were plated on 12-well glass cover slips (40,000 cells/well), and cell culture media were changed every 3 days until 12–14 days.
In vitro glucose uptake
H)-Glucose uptake was performed in hBECs cultured in 96-well plates following the modified method of Takakura et al. (1991)
. Cells were exposed to 50 mM ethanol (EtOH) for 24 h with or without 10 μM cytochalasin B, an inhibitor of glucose transporter protein (CB, 10 mM stock was dissolved in dimethyl sulfoxide), or 200 μM ALC in a CO2
incubator. Cells were incubated overnight in glucose-free DMEM/F-12 media containing 2.0 μCi of D
H)-glucose (specific activity=20–30 Ci/mmol) and 2.0 mM of non-radiolabeled glucose. After washing off the excess 3
H-glucose with phosphate saline buffer (PBS), cellular protein was precipitated with 10% trichloroacetic acid (TCA) at 4°C for 15 min. Following the manufacturer's instruction, precipitated proteins were transferred onto a 96-well nitrocellulose filter using the Unifilter-96 well Harvester (PerkinElmer, Waltham, MA, USA). Using the Beckman 96-well plate reader, radioactivity was measured by β-top counter. The concentrations of EtOH (50 mM) and CB (10 μM, specific inhibitor of GLUT) were derived from dose- and time-dependent toxicity assays (5–200 mM EtOH and 0.5–100 μM CB for 24–72 h), in which 50 mM EtOH or 10 μM of CB had no cell toxicity effect (by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). The concentration of EtOH from 65 to 100 mM showed about 20% cell death after 48-h exposure. Concentration of CB higher than 20 μM was toxic to hBECs.
Analyses of GLUT1 protein and mRNA levels
Primary human brain endothelial cells cultured in rat-tail collagen and fibronectin pre-coated 75-cm2 flasks were pre-incubated for 15 min with 100 ng/ml of act-D or 10 μg/ml of Chx (Sigma) prior to 16 h treatment with EtOH in the presence of act-D. Cell pellets were lyzed with ice-cold lysis buffer (50 mM Tris-Hcl, pH 7.5) containing 20% glycerol, 5 mM MgCl2, 0.5 mM DTT, and protease inhibitor cocktail for protein extracts that were used for Western blot analyses of GLUT1 protein levels. Total cellular RNA was extracted from cell pellets by RNAeasy kit (Qiagen, Valencia, CA, USA) for GLUT1 mRNA levels. RNA was reverse-transcribed with random hexamers (Promega, Madison, WI, USA). Real-time quantitative PCR was performed with cDNA using an ABI PRISM 7000 sequence detector (Applied Biosystems, Foster City, CA, USA). Thermocy-cling conditions were 95°C for 10 min, 15 s, and 60°C for 1 min. Human-specific primer pairs for amplification of GLUT1 mRNA by quantitative reverse transcription polymerase chain reaction of total RNA were forward primer 5′-GAG TGC CTG AAA CCA GAG GA-3′ and the reverse primer 5′-CTC ACA CTT GGG AGT CA-3′. Human-specific primer pairs for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were forward primer: 5′-AAG GCC ATC ACC ATC TTC CA-3′ and reverse primer: 5′-ACC CCA CTT GAT GTT GG CA-3′. Using SYBR Green I detection system, a melting temperature dissociation curve was generated for the PCR product. All PCR reagents were obtained from Applied Biosystems (Foster City, CA, USA). Gene expression was normalized to GAPDH, used as an endogenous control.
For immunocytochemistry, the hBECs were cultured on glass cover slips in 12-well flasks until 80–100% confluent. Cells were treated with 50 mM EtOH with and without CB (10 μM) or ALC (200 μM) for 24 h. Brain tissue sections (8 μm thickness) were derived from chronic EtOH, EtOH + ALC, or pair-fed control mice. Cells and tissue sections were washed with PBS; fixed in acetone–methanol (1:1 v/v) fixative; blocked the cellular antigen with 3% bovine serum albumin at room temperature for 1 h, in the presence of 0.4% Triton X-100; and incubated with respective primary antibodies such as mouse anti-GLUT1 (1:250 dilution), rabbit anti-von Willebrand factor (vWF; 1:150 dilution), rabbit anti-NF (Abcam, 1:250), sheep anti-TH (Abcam, 1:1,000 dilution), and rabbit anti-ChAT (Abcam, 1:100 dilution) for overnight at 4°C. After washing with PBS, cells and tissues were incubated for 1 h with secondary antibody: anti-mouse IgG Alexa Flour 488 for GLUT1, anti-rabbit-IgG Alexa Flour 594 for vWF, anti-rabbit IgG Alexa Flour 488 for NF, anti-sheep IgG Alexa Flour 488 for TH, and anti-rabbit IgG Alexa Flour 488 for ChAT. Cover slips were then mounted onto glass slides with immunomount containing DAPI (Invitrogen), and fluorescence microphotographs were captured by fluorescent microscopy (Eclipse TE2000-U, Nikon microscope, Melville, NY, USA) using NIS elements (Nikon, Melville, NY, USA) software. GLUT1 expression was also analyzed in brain microvessels that were surgically dissected under microscope.
The hBECs cultured in T 75-cm2 flasks were lysed with CellLytic-M buffer (Sigma) for 30 min at 4°C, centrifuged at 1,4000×g, and then total cell lysates protein concentrations were estimated by bicinchoninic acid (Thermo Scientific, Rockford, IL, USA). We loaded 20-μg protein/lane and resolved the various molecular weight proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis on gradient gels (Thermo Scientific) and then transferred the protein onto nitrocellulose membranes. After blocking, membranes were incubated for overnight with primary antibodies against mouse GLUT1 (1:1,000, Abcam, Cambridge, MA, USA), rabbit occludin (1:250), mouse claudin-5 (1:1,000), and rabbit ZO-1 (1:250) at 4°C followed by 1 h incubation with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were detected by West Pico chemiluminescence substrate (Thermo Scientific). Data were quantified as arbitrary densitometry intensity units using the Gelpro32 software package (Version 3.1, Media Cybernetics, Marlow, UK).
In vivo glucose transport assay
Five-week-old C57BL/6J male mice from Jackson Laboratory (Bar Harbor, ME, USA) were housed in University of Nebraska Medical Center animal facility following NIH Guide for Care and Use of Laboratory Animals. Mice were acclimated to Lieber–DeCarli control and 28% calorie (4% vol/vol) ethanol liquid diets from Dyets Inc. for 5 days prior to 8- to 9-week weight-match pair-feeding regimens. Pair feeding was based on the amount of liquid diets consumed by ethanol group animals. In mouse model, 4% ethanol is considered as moderately high alcohol concentration; thus, in this study, the effects of ethanol on glucose transport and BBB function referred to high-dose chronic effects. ALC (1.0 mg/ml) was given daily in liquid diets mixture for 8 to 9 weeks. Experimental conditions consisted of control liquid diets, EtOH liquid diets, ALC in EtOH liquid diets, and ALC in control liquid diets. After week 8 of liquid-diet feeding, D-(2-3H)-glucose (4 μCi) and 4.0 mM non-radiolabeled glucose in 500 μl of saline were cavaged orally for control, EtOH, and EtOH + ALC animals. After 1 h, mice were euthanized, microvessels were removed carefully, and tissues were dissected from different brain regions. Known tissue weights were homogenized with 100 μl of Krebs–Ringer phosphate HEPES buffer, centrifuged at 12,000 rpm for 15 min, and 20 μl of supernatants from each condition was mixed with 4 ml of scintillation fluid. The levels of 3H-glucose in the samples were detected by liquid scintillation counter (Beckman) along with a standard curve of 3H-glucose that was run in parallel. Results were extrapolated from the standard curve, and data were expressed as nanocurie per gram tissue weight.
In vivo BBB permeability assay
Using the sodium fluorescein (NaFl) and Evans Blue (EB) tracer dye mixtures (5 μM each), the effect of EtOH on BBB permeability was examined in acute and chronic animal model following an established method (Hawkins and Egleton 2006
). In acute studies, mice were anesthetized, infused 100 μlof200 μM of ALC or 650 mM of EtOH through the common carotid artery for 1 h, and followed by infusion of NaFl/EB mixtures for another 30 min. ALC was infused at 30 min prior to EtOH infusion. In chronic studies, the conditions of control, EtOH, and EtOH + ALC were same as that of glucose uptake assay. Mixtures of NaFl/EB dyes were directly infused into the right carotid artery. Animals were decapitated; brains were removed, dissected, weighed, and homogenized in 600 μl 7.5% (w
) TCA. Resulting suspensions were divided into two aliquots (300 μl each). One aliquot was neutralized with 50 μl of 5 N NaOH and measured by flourimetry on a GENios microplate reader (excitation 485 nm, emission 535 nm) for NaFl determination. The other aliquot was centrifuged for 10 min at 10,000 rpm 4°C, and the supernatant was measured by absorbance spectroscopy at 620 nm for EB determination. Standard curve was constructed by serial dilutions of a standard EB/NaFl solution in 7.5% TCA.
To determine the integrity of BBB function, changes in trans-endothelial electrical resistance (TEER) across the BBB were analyzed by a highly sensitive 1600R ECIS system (Applied Biophysics, Troy, NY, USA). The ECIS system provides real-time monitoring of changes in TEER. In brief, hBECs at 20,000 cells/well were plated on type I rat-tail collagen-coated 8W10E electrode arrays (Applied Biophysics). Once tight cell monolayers were formed, stable TEER readings were monitored for 2 h prior to treatment of cell monolayers with 50 and 100 mM EtOH in the presence or absence of CB or ALC. We then monitored the TEER readings for another 10 h at 400 Hz and with 10-min intervals. Confluent cell monolayers demonstrated baseline TEER readings of 1,100 to 1,200 Ω/cm2.
Values are expressed as the mean ± SD. Within an individual experiment, each data point was determined from three to five replicates. Statistical analysis of the data was performed by using GraphPad Prism V5 (Sorrento Valley, CA, USA). Comparisons between samples were performed by one-way ANOVA with Dunnett's post hoc test. Differences were considered significant at P values ≤0.05.