Brown-Norway rat dams and pups were procured from Harlan (IN, USA). Animals were maintained in ventilated cages housed in a specific, pathogen-free animal facility. Animal protocols were prepared in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at Louisiana State University Health Sciences Center.
Combustion Generated Ultrafine Particles (CGUFP)
Particles were synthesized essentially as previously described and had a mean diameter of 0.2 μm . In brief, EPFR containing CGUFP (DCB230) was formed as follows: 5% CuO supported on silica was dosed with adsorbate vapors (i.e. 1,2 dichlorobenzene) in a custom made vacuum exposure system for 5 min at 10 torr at 230°C. Any physisorbed dosant was removed by evacuation at 10-2 torr, and the particles were allowed to cool to room temperature under vacuum. The presence of free radicals was confirmed by electron paramagnetic resonance. Based on previous studies  it is known, that 1,2-dichlorobenzene adsorbed on CuO/Silica particles at 230°C forms primarily o-semiquinone EPFRs. Non-EPFR containing CGUFP (DCB50) was formed by dosing silica with adsorbate vapors (i.e. 1,2 dichlorobenzene) in a custom made vacuum exposure system for 5 min at 10 torr at 50°C. The particles were allowed to cool to room temperature and evacuated at 10-2 torr to remove remaining gas phase reactant, which allowed for the formation of particles containing silica matrix and adsorbed molecular 1,2-dichlorobenzene as reference material to 1,2-dichlorobenzene originating EPFRs. The absence of free radicals was confirmed by electron paramagnetic resonance. The size of the particles was confirmed prior to experiments using flow cytometry also as previously described .
Neonates (7 days of age) were divided into three exposure groups: Air, DCB50, and DCB230. Neonates were subjected to nose-only exposures of DCB230 or DCB50 at 200 μg/m3 or air using the In-expose inhalation system (SciReq) for 20 min/day for seven consecutive days. All assessments were performed 24 hours following the final exposure. Cytokines were additionally assessed at 72 hours following the final inhalation exposure. Each group consisted of 4-6 rats, and all experiments were performed in triplicate.
Respiratory mechanics were measured using the forced oscillation technique (FlexiVent; Scireq) as previously described . Exposed rats were anesthetized, intubated, and mechanically ventilated by a computer controlled piston ventilator. Rats were then challenged with an aerosolized bronchoconstrictor, methacholine (MeCh), at increasing doses (MeCh: 0, 6.25, 12.5, 25 mg/ml). At each dose, lung resistance, compliance, and elastance were calculated using the single compartment model and pressure-volume data were captured using a step-wise quasi-static inflation/deflation maneuver to total lung capacity/functional residual capacity, respectively. For comparison among the groups and across measurement days, all data were normalized to their individual baseline resistance values ((value-baseline)/baseline) and plotted as normalized resistance. Baseline values ranged from 0.701 to 0.805 cm H2O·s/ml and were not statistically different among the groups.
Briefly, tissue was homogenized in 5% sulfosalicylic acid (in ice-cold 5% metaphosphoric acid mixture) and centrifuged at 2500 g for 10 min, 4°C. An aliquot of the supernatant was used for the assay. Oxidation of GSH by 5,5'-dithio-bis (2-nitrobenzoic acid) to form nitrobenzoic acid and the enzymatic recycling of glutathione (GSH) from glutathione disulfide (GSSG) by glutathione reductase (GR) in the presence of NADPH were spectrocphotometrically measured at 412 nm .
Lipid peroxidation was measured by a competitive enzyme-linked immunosorbent assay (ELISA) for 8-iso-PGF with a commercial kit (Cayman Chemical, Ann Arbor, MI). The assay is based on the competition between 8-iso-PGF and 8-isoprostane-acetylcholinestase (AChE) conjugate for a limited number of binding sites in each ELISA plate well. The concentration of 8-iso-PGF is inversely proportional to the number of binding sites available, whereas AChE is held constant. For lung homogenates, samples were weighed and homogenized in 0.1 M PBS (1 mM EDTA, 0.005% butylated hydroxytoluene). Samples (BALF and lung homogenates) were transferred to the ELISA plate and incubated with the antibody for 18 hr. The absorbance of the colorimetric enzymatic reaction was read at 405 nm using the SpetraMax-M2 (Molecular Devices, Sunnyvale, CA) and compared with an 8-iso-PGF standard curve to calculate concentration.
Bronchoalveolar lavage fluid (BALF) was harvested in 1 ml of PBS containing 2% BSA. Isolated BALF was used to determine total number of leukocytes. Cytospin slide preparations were made, stained with Diff-Quik (Fisher Scientific), and used for differential cell counts. All counts were performed by two unbiased observers using standard morphological criteria to classify individual leukocyte populations. Six rats from each group were used for these analyses, and 200 cells were counted per animal.
Pulmonary Lymphocyte Characterization
A single cell suspension of lung cells was prepared using a standardized protocol . Briefly, lungs were perfused, excised, cut into small pieces and incubated at 37°C for 1 hour in RPMI-1640 media supplemented by 2% heat inactivated FBS, 1 mg/ml collagenase I (Invitrogen), and 150 μg/ml DNase I (Sigma). After incubation, single cells were obtained by mashing the lung pieces through a 40 μm cell strainer (BD Biosciences). Red blood cells were lysed using 1× RBC lysis buffer (eBioscience) and cells were stained with the following antibodies purchased from BD Pharmingen: APC-CD4, FITC-CD8, and Biotin-CD3, and from Santa Cruz: PE-OX62. Cell staining was determined with an LSRII (BD Biosciences) flow cytometer after gating on the lymphocyte population as determined by forward and side scatter properties. Flow data were analyzed and plotted using FlowJo software (Version 7.2.2 for windows, Tree Star, Inc).
Cytokine Levels in BALF
Cytokine levels were measured from 50 μl of cell-free BALF (rat cytokine assay; Millipore, MO, USA) using a high-throughput multiplex cytokine assay system according to the manufacturer's instructions. Each sample was analyzed in duplicate on the Bio-Plex 200 system (BioRad). A broad range of standards (4.88 to 20,000 pg/ml; depending on the analyte) was used to quantitate a dynamic range of cytokine concentrations. The concentrations of analytes in these assays were quantified using a standard curve and a 5-parameter logistic regression was performed to derive an equation that was then used to predict the concentration of the unknown samples. The following cytokines were assayed: IL-1β, IL-10, IL-18, IFN-γ, TNF-α, GRO/KC, VEGF, MCP-1, and MIP-1α. The data presented excluded any value outside the range of sensitivity for the particular analyte.
Lungs were perfused with PBS and heparin, inflated by gentle infusion of HistoChoice Tissue Fixative (Amresco, Inc.) to total lung capacity, and isolated. The fixed lungs were then dehydrated, embedded in paraffin, and sectioned at 4 μm sections. Each lung section was stained with hematoxylin and eosin (H&E). Lung sections were probed for E-cadherin and α-smooth muscle actin (SMA) using the following antibodies (Abcam) and dilutions: anti-SMA (1:1000), E-cadherin (1:400). Primary antibodies were respectively detected with goat anti-mouse Alexa Fluor 568 (1:500) and with Goat anti-Rabbit Alexa Fluor 488 (1:500) both from Invitrogen. Cell nuclei were stained with DAPI.
Alveolar septal lesioning was quantified from H&E stained lung sections of animals exposed to air, DCB50, and DCB230. The destruction of alveolar walls was quantified using the destructive index (DI) method . A grid with 42 points that were at the center of hairline crosses was superimposed on the lung field. Structures lying under these points were classified as normal (N) or destroyed (D) alveolar and/or duct spaces. Points falling over other structures, such as duct walls, alveolar walls, etc. did not enter into the calculations. The DI was calculated using the formula: DI = D/(D + N) × 100.
To quantify smooth muscle thickness, images of major airways were taken at 40× magnification from lung sections of rats exposed to DCB230, DCB50, or air. Using AxioVision software, a line perpendicular to the basal lamina was drawn from the basement membrane to the edge of the smooth muscle layer. Smooth muscle cells were identified according to morphology and stain. After this was repeated 4-5× per airway, an average was taken from the software-generated thickness values (μm). Averages from 3 different animals per exposure group were compared.
2D-Gel Electrophoresis and Mass Spectroscopy to Identify Proteins
Tissue Preparation and Protein quantification
Lungs from exposed animals (4 animals/group) were sonicated following the addition of 400 μl of Lysis Buffer (7 M Urea, 2 M ThioUrea, 4% CHAPS, 30 mM Tris, pH8.5, and 20% glycerol). Sonication was performed 4 times at 25% amplitude for 15 seconds each; returning to ice in between. Protein concentrations were determined by Bradford protein assay using an 8 point BSA standard concentration curve. For CyDye labeling, samples were aliquotted into 50 μg fractions.
The mixed internal standard methodology of  was used in these studies. Briefly, aliquots of 50 μg protein from each sample were labeled with 400 pmol of either Cy3 or Cy5 (vehicle and/or treated randomized). In a similar fashion, 50 μg of each of the samples (vehicle and treated) was pooled and labeled with 400 pmol Cy2 per 50 μg standard. Equal protein loading of replicate #1 versus replicate #2, and the standard-sample mixture was resolved between pH 3-10NL per gel. Samples were dissolved in rehydration buffer (7 M Urea, 2 M ThioUrea, 4% CHAPS, 20% glycerol) supplemented with IPG buffer (GE Healthcare).
2-D gel electrophoresis and imaging
Cy-dye labeled samples for each subject (410 μl final volume) were actively rehydrated into 24 cm 3-10NL immobilized pH gradient (IPG) strips (GE Healthcare) for 15 hours, followed by isoelectric focusing using an IPGphor (GE Healthcare) step 1: Step 300 V for 2 hours, step 2: Gradient 1000 V for 6 hours, step3: Gradient 8000 V for 6 hours, step 4: Step 8000 V for 8 hours, and step 5: step 200 V for HOLD. The cysteines were reduced and carbamidomethylated, while the proteins were equilibrated into the second-dimensional loading buffer by incubating the focused strips in equilibration buffer (6 M Urea, 20% glycerol, 2% SDS, 375 mM Tris, pH8.8) supplemented with 20 mg/ml DTT for 15 min at room temperature with shaking, followed by 25 mg/ml iodoacetamide in equilibration buffer for an additional 15 min room temperature incubation. IPG strips were then cemented onto 2nd dimension gels using an overlay consisting of 0.5% agarose in SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, trace of bromophenol blue). Homogeneous polyacrylamide gels (12%) were used for the second-dimensional SDS-PAGE which was then carried out on all gels simultaneously using a DALT6 (GE Healthcare) at 5 W/gel for 30 min followed by 17 W/gel for 4 h. The Cy2 (standard), Cy3 and Cy5 (vehicle or treated) for each gel were individually imaged using mutually exclusive excitation/emission wavelengths of 488 nm (ex) and 520 nm (em) BP40 (bandpass) for Cy2, 532 nm (ex) and 580 nm (em) BP 30 for Cy3, and 633 nm (ex) and 670 nm (em) BP30 for Cy5 using a Typhoon 9400(GE Healthcare). After imaging for Cy-Dye components, the non-silanized glass plate was removed, and the gels were fixed in 10% methanol, 7% acetic acid for 1 h, rinsed in water three times and then incubated in SYPRO Ruby in the dark overnight. The SYPRO Ruby post-stain allows for the correction of unlabeled protein's migration in relation to the 1-3% CyDye labeled migration, and ensures accurate protein excision. Sypro Ruby images were acquired on the same imager using 450 nm (ex) and 610 nm BP40 filter, as well as re-imaged post-excision to ensure accurate protein excision.
DeCyder software (GE Healthcare) was used for simultaneous comparison of abundance changes across all samples, and for comparisons of individual Cy3 and Cy5 samples for each subject. Difference ratios or abundance changes and paired Student's t-test p-values for the variance of these ratios for each protein pair across all samples were calculated. Fold abundance changes are reported, whereby a fold increase is calculated directly from the volume ratio and a fold decrease by the inverse of volume ratio.
Proteins of interest were excised using the Ettan Spot Picker (GE Healthcare) based on a 'hit list' generated in DeCyder. Spots were de-stained by successive changes of 20 mM ammonium bicarbonate and 50% acetonitrile, followed by dehydration with a 20 minute incubation with 100% acetonitrile. Dehydrated gel plugs were automatically digested in-gel with 8 μL 20 μg/ml porcine modified trypsin protease (Promega) in 20 mM ammonium bicarbonate for 6 hours at 37°C. Tryptic peptides were then extracted from the gel plugs in two cycles of 50% acetonitrile, 0.1% trifluoroacetic acid and dried by evaporation. Peptides were reconstituted in 1 μl 50% Acetonitrile and 0.1% trifluoroacetic acid and mixed with an equal volume of 10 mg/ml α-Cyano-4-hydroxy-cinnamic acid for spotting onto a MALDI plate.
Mass Spectrometry and Identification of Proteins
Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS): The parent polypeptides were identified by comparing the profile of tryptic peptide masses generated by the mass spectrometer with predicted tryptic peptides from all known polypeptides using the MASCOT program. Since a covalent modification such as thiolation or nitration changes a peptide mass by a known amount, it is possible through MASCOT and other programs to identify both a protein and its posttranslational modifications.
All data were plotted as mean ± SEM and analyzed using GraphPad Prism (GraphPad Software Inc., Version 5.0.0). Two-way ANOVA (Bonferroni post-test) was used to evaluate the differences of airway responsiveness, airway resistance, elastance, compliance, and BALF cellularity. One-way ANOVA was used to evaluate the differences of PV loop, cytokine levels between groups, alveolar septal destruction index, and smooth muscle proliferation. Tukey's one-way analysis of variance was performed to test for significance between the groups. Differences between means were considered significant when p < 0.05.