development of transgenic/knockout models as well as for safety and efficacy testing. B6.129X1 MPO-deficient mice were created by targeted disruption of the MPO gene in RW4 embryonic stem cells. The mutant allele was transferred to the C57BL/6J background using a marker assisted screening protocol. Polymor- phisms between parental strain 129/SvJ and recipient strain C57BL/6J were screened at approximately 20 cM intervals to select for the mice containing the most C57BL/6J composition. Heterozygous mice (approximately 91% C57BL/6J) were inter- crossed and all experiments used 2–6-month-old female knockout, heterozygote, and wild-type littermate animals. The absence of MPO in MPO k/o mice was proved by Northern blot or Western blot analysis of bone marrow. Neutrophils and monocytes from both peripheral blood and bone marrow of MPO k/o animals failed to exhibit endogenous peroxidase activity. In contrast, eosinophils, which possess eosinophil peroxidase, demonstrated peroxidase staining [30]. Animals were housed one mouse per cage receiving HEPA filtered air in AAALAC-approved NIOSH animal facilities. All animals were acclimated in the animal facility under controlled temperature and humidity for one week prior to use. Animals were supplied with water and certified chow 7913 (Harlan Tekland, Indianapolis, IN) ad libitum, in accordance with guidelines and policy set forth by the Institute of Laboratory Animals Resources, National Research Council. All animal studies were carried in compliance with the policies of the Institute of Laboratory Animal Resources (National Research Council) and the experimental protocol (#07-AS-M-010), approved by the National Institute for Occupational Safety and Health (NIOSH) Institutional Animal Care and Use Committee. Experimental Design Experimental protocols for the present study included pharyn- geal aspiration exposure of w/t and MPO k/o mice to 40 mg/ mouse SWCNT, while the corresponding control mice were administered sterile Ca2++Mg2+-free phosphate-buffered saline (PBS) vehicle. Particulate instillation was performed as described in Methods S1. Mice were sacrificed on days 1 and 28 following the exposure. Inflammation was evaluated by total cell counts, cell differentials, and accumulation of cytokines in the bronchoalveolar lavage (BAL) fluid. Fibrogenic responses to exposed materials were assessed by morphometric measurements and collagen deposition. Obtaining bronchoalveolar lavage (BAL) from mice Mice were sacrificed with intraperitoneal injection of sodium pentobarbital (.100 mg/kg) and exsanguinated. The trachea was cannulated with a blunted 22 gauge needle, and BAL was performed using cold sterile PBS at a volume of 0.9 ml for first lavage (kept separate) and 1.0 ml for subsequent lavages. Approximately 5 ml of BAL fluid per mouse was collected in sterile centrifuge tubes. Pooled BAL cells for each individual mouse were washed in PBS by alternate centrifugation (8006g, 10 min, 4uC) and resuspened. BAL aliquots were frozen at 280uC until processed. Procedures used for BAL cell counting and cytokine analysis is described in Methods S1. Sirius red staining The distributions of type I and type III collagen in the lung tissue were determined by morphometric evaluation of the Sirius red-stained sections [3]. To identify collagen fibers under the microscope, deparaffinized and dehydrated lung sections were stained with F3BA/picric acid for 1–2 h, washed with 0.01N HCL for 1 min, and counterstained with Mayer’s hematoxylin for 2 min [31]. Type I and III collagen stained by Sirus red was visualized, Myeloperoxidase Clearance of Carbon Nanotubes and 6 randomly selected areas were scored under polarized microscopy and average thickness of Sirius red positive connective tissues in the alveolar wall was quantitatively measured. Volume and surface density was measured using standard morphometric analyses of points and intercept counting [32]. Average thickness of the Sirius red positive connective tissues of the alveolar wall was computed from two times the ratio of volume density of points to the surface density of the alveolar wall. Lung collagen measurements Whole lungs from each mouse were homogenized in 0.7 ml of 0.5 M acetic acid containing pepsin (Accurate Chemical and Scientific Corporation, Westbury, NY) with 1:10 ratio of pepsin: tissue wet weight. Total lung collagen content was determined by quantifying total soluble collagen using the Sircol Collagen Assay kit (Accurate Chemical and Scientific Corporation, Westbury, NY). Solubilization of lungs Lungs were homogenized using Biospec Products Inc, OK, USA tissue homogenizer in 500 mL of deionized water. After homogenization, 1% SDS was added and samples were heated to 100uC for 10 min. This was followed by sonication using the ultrasonic benchtop cleaner (Branson 2510, output power of 70 W at 40 KHz) for 10 min. After cooling the contents to room temperature, Tris-HCl buffer (final concentration 30 mM) (pH 8.0) was added. Finally, an 18 hrs incubation of the material with 100 mg/mL of proteinase K at 50uC was undertaken to completely solubilize the lungs. Raman maps Raman maps of lung tissue sections were acquired using an NTEGRA Spectra microscopic system integrated with Raman spectrometer (NT-MDT, Russia) 473 nm cobalt laser operated at 10%, 1006 oil objective, 2 s exposure of static spectra centred at 2070 cm21. Ten maps (32632 points, 20 mm620 mm or 10 mm610 mm) were recorded for each tissue section. The Raman maps were then imported into Matlab 7 (The Mathworks, USA) for analysis, where all of the spectra were normalized, smoothed, baseline corrected and K-means clustering was carried out. Raman spectroscopy of solubilized lung tissue as well as transmission electron microscopy was performed as described in Methods S1. Image analysis Lung preparation for microscopic evaluation was performed as described in Methods S1. Two color images were collected from mounted sections of lung using a Hamamatsu Orca ER cooled CCD camera (Hamamatsu, Bridgewater, NJ) on a Nikon 90i upright microscope (Melville, NY), equipped with a high speed linear encoded xy stage (Prior Scientific, Rockland, MA), and fluorescence imaging and bright field imaging capabilities. The motorized stage coupled with Elements software (Nikon NIS, Melville, NY) allows collection of large area images which consist of multiple stitched and aligned frames. Each image was a 10610 image field at 106 magnification. To collect the image of the lung tissue the autofluorescent signal from the paraffin embedment was detected using 530 nm excitation and 560–600 nm emission. To visualize nanotubes sections were illuminated with white light and blocking filter (750–840 nm transmission) was placed between the specimen and the camera. In this conditions nanotubes selectively absorbing light in the range 750–800 nm were seen as black spots. For image processing, the area of the lung in the section profile PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e30923