We purchased the antibodies to GLUT1 from Abcam (Cambridge, MA), antibodies to occludin and ZO-1 from Zymed (Invitrogen, Carlsbad, CA), and antibody to α-actin from Millipore (Billerica, MA). All secondary alexa fluor antibodies were purchased from Invitrogen. D-(2-3H)-glucose (5 mCi, 185 MBq) was purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). Cytochalasin B (CB), cycloheximide (Chx), actinomycin D (Acd) and acetyl-L-carnitine (ALC) were purchased from Sigma-Aldrich (St. Louis, MO).
Primary human brain endothelial cells (hBECs) were obtained from Dr. Persidsky's Lab, Temple University School of Medicine, and hBECs were cultured as described previously . 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. For glucose uptake and viability assays, cells were cultured in 96-well plates (20,000 cells/well), for immunohistochemistry cells were plated on 12-well glass cover slips (40,000 cells/well) and for protein extractions cells were cultured in T75 cm2 flasks (1 × 106 cells/flask). 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) were used for cell culture. Cell culture media was changed every 3 days until tight monolayers were formed in about 6-8 days.
In vitro glucose uptake
Following the modified method of Takakura , D-(2-3H)-glucose uptake was performed on hBECs cultured in 96 well plates. Cells were exposed to 20 μM and 200 μM METH for 24 hr in the presence or absence of 10 μM cytochalasin B (CB, 10 mM stock was dissolved in DMSO) or 200 μM ALC in a CO2 incubator. Cells were then incubated overnight in glucose-free DMEM/F-12 media containing equimolar of D-(2-3H)-glucose (1.0 μCi) and non-radiolabeled glucose. After washing off the excess 3H-glucose with phosphate saline buffer (PBS), cellular protein was precipitated with 10% 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). Using the Beckman 96 well plate reader, radioactivity was measured by β-top counter. METH concentrations of 20 and 200 μM were derived from dose- and time-dependent toxicity assay (5, 10, 20, 50, 100, 200, 500 and 1000 μM of METH for 24-72 hrs), in which 20 μM had no cell toxicity effects while 200 μM of METH showed about 20% cell death after 48 hr exposure. ALC concentration of 200 μM was derived from dose-dependent study of 50-5000 μM on cell viability. Concentration of CB higher than 20 μM was toxic to hBECs (derived from 1 - 100 μM CB concentrations).
Cell viability assay
Cell viability on hBEC was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The assay is based on the cleavage of yellow tetrazolium salt to purple formazan crystals by metabolically active cells. Briefly, cells were cultured in 96-well microtiter plates up to 90-100% confluent. Then the cells were treated for 48 hr in the presence or absence of METH or Pyruvate, or in combination of METH+pyruvate in glucose-free media in culture. Cells were then incubated at 37°C for 45 minutes after adding 100 μl MTT (5 mg/ml MTT in 10% FBS in 1× PBS). Then 100 μl DMSO was added just after aspirating the MTT solution and the plates were incubated at room temperature for 15 min. Absorbance of the purple formazan was detected by a microtiter plate reader at 490 nm wavelength.
For immunocytochemistry, the hBECs were cultured on glass cover slips in 12 well flasks until 80-100% confluent. Cells were then treated with 20 μM and 200 μM METH with and without CB (10 μM) or ALC (200 μM) for 24 hours. For immunohistochemistry, tissue sections (8 μm thickness) were derived from chronic METH, METH+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 hr 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-occludin (1:250 dilution) and rabbit anti-ZO-1 (1:250 dilution) overnight at 4°C. After washing with PBS, cells/tissue sections were incubated for 1 hr with secondary antibody: anti-mouse-IgG Alexa Fluor 488 for GLUT1; anti-rabbit-IgG Alexa Fluor 594 for vWF; anti-rabbit-IgG Alexa Fluor 488 for occludin and ZO-1. 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) using NIS elements (Nikon, Melville, NY) 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 (Sigma) for 30 min at 4°C, centrifuged at 14000 g, and then total cell lysates protein concentrations were estimated by BCA (Thermo Scientific, Rockford, IL). We loaded 20 μg protein/lane and resolved the various molecular weight proteins by SDS-PAGE on gradient gels (Thermo Scientific) and then transferred the protein onto nitrocellulose membranes. After blocking, membranes were incubated for overnight with polyclonal antibody against mouse anti-GLUT1 protein (1:1000, Abcam, Cambridge, MA), rabbit anti-occludin antibody (1:250 dilution) and rabbit anti-ZO-1 antibody (1:250 dilution) at 4°C followed by 1 hr incubation with horse-radish 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).
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). The ECIS system provides real-time monitoring of changes in TEER. In brief, hBECs at 20000 cells/well were plated on collagen type I coated 8W10E electrode arrays (Applied Biophysics). Once tight cell monolayers were formed, stable TEER value was monitored for 1 hr prior to treatment of cell monolayers with 20 μM and 200 μM METH in the presence or absence of CB or ALC followed by 10 hr recording of TEER at 400 hz with 10 min intervals. Confluent cell monolayers demonstrated baseline TEER readings of 1100 to 1200 Ω.cm2.
In vivo glucose transport assay
Five-week old male C56/BL-6J mice purchased from Jackson Laboratory (Bar Harbor, ME) were maintained in sterile cages under pathogen-free conditions in accordance with institutional ethical guidelines for care of laboratory animals, National Institutes of Health (NIH) guidelines, and the Institutional Animal Care Use Committee. On the basis of weight matched, initially mice were grouped into control, METH and METH+ALC and they were fed the normal Lieber-DeCarli liquid-diet (Dyets, Inc. Bethlehem, PA) for 5-6 weeks. ALC was mixed in the liquid diet (1.0 mg/mL) and METH (15 mg/kg body weight) was administered daily by i.p injection for 5-6 weeks. After week 5 pair feeding of liquid-diet and monitoring the body weights, the control, METH and METH+ALC mice were anesthetized with ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight), and equimolar of 2-3H-glucose (2 μCi) and unlabelled glucose in 100 μl of saline were infused through the right common carotid artery. After 1 hr, mice were euthanized and then microvessels were removed and tissues were dissected from different brain regions. Known tissue weights were homogenized with 100 μl of Krebs-Ringer phosphate-HEPES (KRPH) 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 2-3H-glucose in the samples were detected by liquid scintillation counter (Beckman) along with a standard curve of 2-3H-glucose that was run in parallel. Results were extrapolated from the standard curve and data were expressed as counts per minutes (cpm) per milligram 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 METH on BBB permeability was examined in acute and chronic animal model following an established method . In acute studies, mice were anesthetized, infused with 100 μl of 200 μM of ALC or 20 μM of CB or 200 μM of METH via the common carotid artery. ALC was infused 30 min prior to METH infusion. After 1 hr of CB and METH infusion, NaFI/EB mixture was infused into the CCA and waited for another 30 min before euthanizing the animals. In chronic studies, the controls, METH (15 mg/kg body weight) and METH+ALC (ALC, 1.0 mg/mL) were given for 5-6 weeks as in glucose uptake assay. Daily i.p infusion of CB (20 μM) or CB+METH lasted for 7 days only because mice that received the CB+METH injection were getting weak by then. Mixture of NaFl/EB dyes was then infused directly into the right carotid artery as in acute studies. After decapitating, brains were removed, dissected, weighed and homogenized in 600 μl 7.5% (w/v) trichloroacetic acid (TCA). Resulting suspensions were divided into two aliquots (300 μl each). One aliquot was neutralized with 50 μl 5 N NaOH and measured by fluorimetry on a GENios microplate reader (excitation 485 nm, emission 535 nm) for NaFI determination. The other aliquot was centrifuged 10 min at 10000 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.
Values are expressed as the mean ± SEM. Within an individual experiment, each data point was determined from three to five replicates. Statistical analysis of the data used GraphPad Prism V5 (Sorrento Valley, CA). Comparisons between samples were performed by one-way ANOVA with Dunnett's post-hoc test. Differences were considered significant at P values ≤ 0.05.