Animals and inhalation exposure protocol
Twelve-week-old male Apo E−/− mice (strain B6.129P2-Apoetm1Unc N11, on a C57Bl6 background, backcrossed for 10 generations; Taconic, Oxnard, CA) were placed on a high fat diet (TD88137 Custom Research Diet, Harlan Teklad, Madison, WI; 21.2% fat content by weight, 1.5 g/kg cholesterol content) beginning 30 days prior to initiation of exposure protocol or normal rodent chow. Mice were then randomly grouped to be exposed by whole-body inhalation to a mixture of whole gasoline engine exhaust and diesel engine exhaust (MVE: 30 μg PM/m3 gasoline engine emissions + 70 μg PM/m3 diesel engine, n = 20) or filtered-air (controls, n = 20) for 6 h/d for a period of 30 days. In a separate study, 12-week old male C57Bl6 wildtype mice (Jackson Labs, Bar Harbor, Maine) fed a standard mouse chow diet, were exposed by the same methods to either filtered air (n = 8) or MVE (n = 8). MVE was created by combining exhaust from a 1996 GM gasoline engine and a Yanmar diesel generator system, as previously reported [42, 59, 60]. Mice were housed in standard shoebox cages within an Association for Assessment and Accreditation of Laboratory Animal Care International-approved rodent housing facility (2 m3 exposure chambers) for the entirety of the study, which maintained constant temperature (20–24°C) and humidity (30–60% relative humidity). Mice had access to chow and water ad libitum throughout the study period, except during daily exposures when chow was removed. All procedures were approved by the Lovelace Respiratory Research Institute’s Animal Care and Use Committee and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).
Upon completion of the designated exposure period, animals were sacrificed 14–16 hours after their last exposure, and tissues were collected. Mice were anesthetized with Euthasol (390 mg pentobarbital sodium, 50 mg phenytoin sodium/ ml; diluted 1:10 and administered at a dose 0.1 ml per 30 g mouse) and euthanized by exsanguination. For all animals but those on the Na-F (see below) leg of the study, the brain tissue was carefully dissected from the skull, meninges were removed, and were either  embedded in OCT (VWR Scientific, West Chester, PA) (n = 6 FA, n = 6 MVE) and frozen on dry ice or  immediately snap frozen in liquid nitrogen for protein assays (n = 8 FA, n = 8 MVE). Tissue was stored at −80 ºC until assayed.
Changes in BBB permeability were assessed using the fluorescent tracer, sodium fluorescein (Na-F) in a subset of mice on study (n = 6 MVE, n = 6 FA exposed), as previously described . Briefly, Apo E−/− mice exposed to either filtered air or mixed vehicular engine exhaust were injected intraperitoneally with 100 μl of 2% Na-F in 1x PBS 30 min prior to the end of their final exposure on day 30. Mice were anesthetized 1 hr post exposure and transcardially perfused with sterile saline until colorless perfusion was visualized. The brains were isolated, and the meninges, cerebellum, and brain stem were gently dissected away, split in half by a mid-sagittal cut and one-half of the cerebrum was embedded and frozen in OCT and sectioned at 10 μm. The other half of the cerebrum was weighed and homogenized in 10x vol of 50% TCA. The homogenate was then centrifuged at 13,000xg for 10 min at RT and the supernatant neutralized with 5 mol/L NaOH (1:0.8). Na-F fluorescence was measured at ex/em wavelengths of 440/525 nm on a fluorometer and fluorescent dye content was calculated using external standards (10 to 200 ng/ml). Data is expressed as amount of tracer per gram of tissue.
In situ zymography
MMP activity was analyzed on frozen serial brain sections (10 μm thick) of the cerebrum, which were incubated with 150 μl of 10 μg/ml dye quenched (DQ)-gelatin (EnzChek, Molecular Probes, Invitrogen, Carlsbad, CA) and 1 μg/ml DAPI (nuclei stain, Invitrogen) in 1% UltraPure™ low melting point agarose (Invitrogen) cover-slipped, chilled for 5 min at 4ºC, and then incubated for 6 h in a dark, humid chamber at 37ºC. Some slides were co-incubated with a specific gelatinase inhibitor (MMP-2, -9 inhibitor IV, Chemicon, Millipore, Temecula, CA). Slides were analyzed using fluorescent microscopy and densitometry was calculated using white/black images and quantified using Image J software (NIH, Bethesda, MD; performed on 6 sections per sample, 3 regions per section, 6 samples per group). Background fluorescence (fluorescence present in total image outside of the vessel) was subtracted from each section before statistical comparison between groups.
Brain sections (10 μm) were prepared for either occludin or claudin-5, and vonWillebrand factor (vWF) double immunofluorescence. Brain sections were incubated with 10% normal goat serum for 30 min at room temperature, washed in PBS, and incubated with 300 μl per section of the appropriate primary antibody (anti-rabbit or anti-sheep occludin, claudin-5: 1:500 dilution, Abcam, Cambridge, MA) and anti-goat vWF (1:1000 dilution, Abcam) diluted in rinse wash buffer [1 part 5% blocking solution (0.5 ml Normal Rabbit Serum in 10 ml 3% w/v Bovine Serum Albumin) and 4 parts Phosphate Buffered Saline (PBS)] with Hoescht nuclear stain (1 μl/ml; 300 μl/section) for 1 hr at RT. Slides were then rinsed 3 times with PBS. The slides were then incubated in 300 μl per section of a mixture of secondary antibodies Alexa Fluor 488 (anti-rabbit) and Alexa Flour 594 (anti-goat or anti-sheep) (1:1000 dilution, Vector Laboratories, Biovalley, Marne la Vallée, France) in the dark for 1 hr at room temperature. Slides were then rinsed 3 times in PBS, and cover-slipped with Aqueous Gel Mount (Sigma Aldrich, St. Louis, MO). Slides were imaged by fluorescent microscopy at 10x, 40x, and 100x using the appropriate excitation/emission filters, digitally recorded, and analyzed by image densitometry using Image J software (NIH). Double immunofluorescence was quantified by merging Alexa 488 (fluorescein isothiocyanate) and Alexa 594 (Cy3) signals into Red-Green-Blue (RGB) images. Colocalization was determined by quantifying total fluorescence of overlayed signals from minimum of three slides, two sections each, three regions from each section (n = 4 per group).
Dihydroethidium (DHE) staining
To visualize ROS levels in the brain of study animals, sections of brains (embedded in O.C.T. and cryosectioned at 10 μm) were immediately processed through DHE staining. Slides were washed in PBS for 30 s, and rinsed 100 μl of 10 μM DHE. Slides were cover-slipped and then incubated at 37°C for 1 hr. Ethidium staining was visualized by fluorescent microscopy at 63x, digitally recorded, and analyzed by image densitometry (color images converted to white/black) using Image J software. Superoxide signal specificity was confirmed by incubating selected sections with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD, 50 U/ml) for 30 min at 37°C.
Western blot analysis
Protein levels of claudin and occludin were measured in cerebral microvessels (n = 4), and iNOS and IL-1β from the parenchyma (temporal lobe) (n = 5), from the brains of separate group of study animals via Western blot. Cerebral microvessels (arterioles and venules, targeted in the size range of less than 100 μm) were dissected from the superior surface of the cerebrum of thawed mouse brains, microscopically, on an ice-block in ice-cold HEPES-PSS. Importantly, TJ proteins claudin and occludin are heterogeneously expressed in endothelial cells of brain microvessels . Vessels from 2 animals in each group were pooled for a total n value of 8 per group (n = 4 pooled samples FA, n = 4 pooled samples MVE). Protein was isolated using a RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 5 μg/ml Aprotinin, 5 μg/ml Leupeptin, 1% Triton x-100, 1% Sodium deoxycholate, 0.1% SDS) for homogenization and quantified using a BCA assay (Pierce, Thermo Scientific, Rockford, IL). 5 μg of protein was loaded into each lane (n = 3–5 for each group), and subsequently run through SDS-PAGE electrophoresis under reducing conditions. After membrane transfer, membranes were blocked overnight at 4ºC in 5% blotto [5% weight/vol powdered milk: 100 ml 1X TBS (Biorad): 5% Tween vol/vol (Sigma Aldrich)]. Membranes were incubated in rabbit polyclonal anti-mouse MMP-9, claudin-5, occluding or iNOS (1:3000; Abcam), and beta-actin primary antibody (1:2000, Abcam) for 1 hour at RT. Anti-rabbit antibody conjugated to HRP (1:2000 Abcam) was used for the secondary antibody for 1 hour at RT. Bands were visualized with chemiluminescence using ECL Plus (GE Healthcare, Amersham Biosciences, Piscataway, NJ) and imaged on the FLA-5100 (Fujifilm, USA) digital image scanner; densitometry was performed utilizing Image J software (NIH).
BBB co-culture model
Primary endothelial and glial cells from mouse were isolated and cultured as previously described . Briefly, for brain endothelial cells (BEC)s, brain tissues were digested enzymatically (1 g.L-1 collagenase/dispase, 20 U.mL-1 DNAse I, 0.147 mg.L-1 TCLK in HBSS, 1 h at 37°C). A 20% BSA gradient was used for isolation of capillaries. After a second enzymatic digestion, cells were plated in 75-cm2 coated culture flasks in EBM medium completed by the EGM-2 MV SingleQuots kit (Lonza, Basel, Switzerland). Cultures were maintained at 37°C in a humidified 5% CO2 atmosphere for 5–6 days before being trypsinized and frozen. For BBB modelling, glial cells were seeded at a density of 5,700 cells.cm-2 on transwell plates in a glial-specific basal medium. BECs were plated on the upper side of a coated polyester transwell membrane (pore size 0.4 μm, Costar) in a BEC-specific medium. Microplates were then incubated at 37°C in a humidified 5% CO2 atmosphere for 10–12 days before treatment with serum from MVE or FA-exposed Apo E−/− mice or C57Bl6 mice. Experiments were performed in triplicate. Upper and lower chambers will be referred to as apical and basal compartments, respectively.
BBB permeability assay
500 μL of diluted serum (1/20) from MVE- and FA-exposed C57Bl6 mice was applied to the BBB co-culture (on the apical compartment). After 24 hr, transwells with HBMEC monolayers were transferred to new plates and a T buffer (150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl2, 0.2 mM MgCl2, 6 mM NaHCO3, 2.8 mM glucose and 5 mM Hepes) was added (1.5 ml) to the basolateral compartment and 0.5 mL to the apical compartment (A), which also contained 0.37 × 1010
μq/mL of [14
C]-labeled sucrose. After 60 min incubation at 37°C, supernatants from both the A and B compartments were collected and the amount of tracer that passed through the endothelial monolayer was determined by scintillation counting. The Papp value was calculated as follows:
Where dQ/dT is the amount of compound transported per time-point, A is the membrane surface area and C0 the initial donor concentration. The mass balance (R) was calculated as:
Where A and D are the amounts of compounds in the acceptor and donor chambers and D0 is the amount introduced at t = 0. Mass balances of sucrose were between 80 and 120%. Monolayers were validated for sucrose permeability from A to B and B to A below 8 ×10-6 cm s-1 as reported previously .
P-glycoprotein transport activity measurement
P-glycoprotein activity was quantified by measuring the passage of Vinblastine (0.1 μM), a P-glycoprotein substrate, across cell-based mouse BBB model. Serum (1/30 dilution in media) from Apo E−/− mice exposed to either MVE or FA was applied to the apical compartment of the BBB co-culture. At 4 and 24 hr post-application of the serum, [3H]-Vinblastine was measured in both endothelial and glial well supernatants by scintillation counting at 1 hr (37°C) and resulting ratio was calculated as reported previously [63, 64]. Experiments were performed in replicates of 3, two times.
Analyses were performed using the Prism 3.0 program (GraphPad Software, Inc, San Diego, CA) for in vitro experiments or Sigma Stat v10 program (Systat Software, Inc, San Jose, CA) for in vivo experiments. Data expressed as mean ± SEM, in vitro data expressed as mean ± SD. Statistical comparisons conducted herein were accomplished using the two-tailed Student’s t-test or variance analysis (one-way ANOVA) for both in vitro and in vivo experiments. A p < 0.050 was considered statistically significant.