Nrf2 signaling increases expression of ATP-binding cassette subfamily C mRNA transcripts at the blood–brain barrier following hypoxia-reoxygenation stress

All animal experiments were approved by the University of Arizona Institutional Animal Care and Use Committee and conform to National Institutes of Health guidelines. Female Sprague–Dawley rats (200–250 g) were obtained from Envigo (Denver, CO), housed under standard 12 h light/12 h dark conditions, and provided with food and water ad libitum. Female rats were purposely selected for this study in order to correlate our results with previous data on BBB transporter changes in the setting of H/R [20]. Animals were randomly assigned to treatment groups. Animals were subjected to hypoxic (Hx) insult (i.e., 6% O₂) for 1 h as previously described [20]. Using blood-gas analysis, our laboratory has previously demonstrated that these conditions yield a severe, but recoverable, hypoxic insult [20]. Additionally, this model increases CNS expression of apoptotic markers [i.e., ratio of cleaved poly-ADP ribose polymerase (PARP) to uncleaved PARP] [20]. Rats were then euthanized or subjected to reoxygenation (i.e., 21% O₂) for 10 min, 30 min, or 1 h. These time points were selected based upon our previous work examining BBB transport mechanisms in the setting of H/R [20]. As shown in Thompson et al. [20], discrete changes in BBB transporters can be observed during reoxygenation as early as 10 min following hypoxic insult. H/R animals were compared with animals subjected to Hx only and with normoxic (Nx) controls. A subset of animals was administered sulforaphane [25 mg/kg (1.0 ml/kg), i.p.; Sigma-Aldrich, St. Louis, MO], an established Nrf2 activator, dissolved in 0.9% saline as a positive control. Following Nx, Hx, H/R or 3 h sulforaphane treatment, animals were euthanized by decapitation and prepared for microvessel isolation.

Brain microvessels were harvested as previously described by our laboratory [20]. Following anesthesia with sodium pentobarbital [64.8 mg/ml (1.0 ml/kg) i.p.], rats were decapitated and brains were removed. Meninges and choroid plexus were excised and cerebral hemispheres were homogenized in 4 ml of microvessel isolation buffer (103 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 15 mM HEPES, pH 7.4) containing protease inhibitor cocktail (Sigma-Aldrich). After homogenization, 8 ml of 26% dextran at 4 °C was added and homogenates were vortexed. Homogenates were then centrifuged (5600g; 4 °C) for 10 min and the supernatant was aspirated. Pellets were resuspended in 10 ml of microvessel isolation buffer and passed through a 70 μm filter (Becton–Dickinson, Franklin Lakes, NJ). Filtered homogenates were pelleted by centrifugation at 3000×g for 10 min. At this time, the supernatant was aspirated and the pellet, which is enriched in brain microvessels, was collected for use in further experiments.

Total RNA was extracted from brain microvessels isolated from rats subjected to Nx, Hx, and H/R using the Aurum Total RNA extraction kit (Bio-Rad, Hercules, CA). Extracted RNA was treated with amplification grade DNase I (Bio-Rad) to remove contaminating genomic DNA. The concentration of RNA in each sample was quantified spectrophotometrically by measuring UV absorbance at 260 nm. The iScript reverse transcriptase kit (Bio-Rad) was used to synthesize first-strand cDNA. Primer pairs were prepared by Integrated DNA Technologies (Coralville, IA) with sequences listed in Table 1. Each set of primers was designed with the use of Primer Express 3 software (Applied Biosystems) and validated for specificity and efficacy by using BioTaq universal rat normal tissue cDNA (BioTaq Inc., Gaithersburg, MD). Primer pairs were designed to be complementary to sequences located on two different exons separated by an intron in order to avoid amplification of genomic DNA. Quantitative PCR was performed using SYBR Green Master Mix (Bio-Rad) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The quantity of the target gene (i.e., Abcc1, Abcc2, Abcc4) was normalized to GAPDH using the comparative CT method (ΔΔCT). Results were expressed as mean ± SD of at least three separate experiments.

EMSA was performed using the LightShift Chemiluminescent EMSA kit (Pierce Biotechnology, Rockford, IL, USA) according to manufacturer’s instructions. Briefly, nuclear protein extract was isolated from brain microvessels prepared from Nx, Hx, H/R, and sulforaphane treated rats. Complementary DNA oligonucleotides 5′-CGG TCA CCG TTA CTC AGC ACT TTG-3′ and 5′-CAA AGT GCT GAG TAA CGG TGA CCG-3′ (antioxidant response element recognition sequence highlighted) were purchased from Integrated DNA Technologies, end labeled with biotin, and annealed at 95 °C for 5 min. EMSA samples were prepared using 5 μg of nuclear extract in each Nrf2/ARE binding reaction. The binding reaction was incubated at room temperature for 15 min and DNA–protein complexes were resolved on a precast 6% native polyacrylamide gel in 0.5% TBE buffer. The gel was removed from the electrophoresis unit, blotted onto a nitrocellulose membrane, and incubated with streptavidin-horseradish peroxidase for 30 min. Membranes were developed using enhanced chemiluminescence. Experiments to determine specificity of EMSA reactions for the Nrf2/ARE complex were conducted by incubating binding reactions in the presence of a rabbit monoclonal anti-Nrf2 antibody (EP1808Y; 1/20 dilution; Abcam, Cambridge, MA). Control EMSA experiments were performed by adding an excess (200×) of unlabeled probe to binding reactions.

ChIP was performed using the Imprint Chromatin Immunoprecipitation Kit (Sigma-Aldrich) according to manufacturer’s instructions. Briefly, microvessels were isolated from Nx, Hx, H/R, and sulforaphane treated rats and subsequently cross-linked in buffer containing 1% formaldehyde for 10 min at room temperature. Cross-linking was stopped by addition of glycine to a final concentration of 125 mM followed by centrifugation at 180×g for 5 min at room temperature. At this time, the microvessel pellet was resuspended in 50 μl of Nuclei Preparation Buffer and incubated on ice for 10 min. Following centrifugation at 180×g for 10 min at 4 °C, the nuclear pellet was resuspended in shearing buffer and incubated on ice for 10 min. Chromatin was sheared to 200–1000 bp by sonication on ice. Sonicated chromatin was diluted twofold in lysis buffer and 100 μl of diluted sample per immunoprecipitation reaction was used. Each sample was added to individual wells of a 96-well assay plate where each well contained 1 μg of specific rabbit monoclonal anti-Nrf2 antibody (EP1808Y) that has been previously validated in ChIP assays [21]. Assay plates were incubated for 90 min at room temperature on an orbital shaker at 75 rpm. In parallel, a no-antibody sample was run as a negative control. At this time, 40 μl of DNA release buffer was added to each well and samples were incubated in a water bath at 65 °C for 15 min. Following this step, 40 μl of reversing solution was added to each well and samples were incubated in a water bath at 65 °C for 90 min. Washes and elutions were performed in accordance with manufacturer’s instructions for the Imprint ChIP assay kit. Eluted and input DNA samples were purified using a spin column to a final volume of 50 μl. Quantitative real-time PCR was performed using 2 μl of template DNA per 25 μl of polymerase chain reaction (PCR) amplification scale as described by Hoque and colleagues [22]. Quantification of Nrf2 occupancy to the ARE within Abcc gene promoter by SYBR green real-time PCR was performed using primer sets prepared by Integrated DNA Technologies (Table 2). All measurements were performed in triplicate and results were verified in three separate chromatin preparations.