Animal
Male C57BL/6 J mice (7 weeks old) were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed one per cage in polycarbonate ventilated cages, which were provided HEPA-filtered air, with fluorescent lighting from 0700 to 1900 hours. Autoclaved Alpha-Dri virgin cellulose chips and hardwood Beta-chips were used as bedding. Mice were monitored to be free of endogenous viral pathogens, parasites, mycoplasms, Helicobacter and CAR Bacillus. Mice were maintained on Harlan Teklad Rodent Diet 7913 (Indianapolis, IN), and tap water was provided ad libitum. Animals were allowed to acclimate for at least 5 days before use. All animals used in this study were housed in an AAALAC-accredited; specific pathogen-free, environmentally controlled facility. All animal procedures were studied under Protocol # 10-DP-M-008 approved by the NIOSH Institutional Animal Care and Use Committee.
Carbon nanotube source
MWCNTs used in this study were obtained from Hodogaya Chemical Company (MCWNT-7, lot #061220-31) and were manufactured using a floating reactant catalytic chemical vapour depositon method followed by high thermal treatment in argon at 2500°C furnace. This lot of MWCNT-7 was fully characterized in our prior report in which acute inhalation exposures were conducted with this lot of MWCNTs [3]. Briefly, MWCNT trace metal contamination was 1.32%, with iron (1.06%) being the major metal contaminants. Average MWCNT surface area measured by nitrogen absorption-desorption technique (Brunauer-Emmett-Teller method, BET) was 26 m2/g [11].
MWCNT aerosol generation and aerosol characterization
Mice were exposed to a MWCNT aerosol (5 mg/m3, 5 hours/day) for 12 days, using an acoustical-based computer controlled system designed and constructed by our laboratory [10] Details of the exposure system, aerosol control performance and aerosol characterization have previously been published in detail [3]. In brief, the inhalation exposure system combines air flow controllers, aerosol particle monitors, data acquisition devices, and custom software with automated feedback control to achieve constant and repeatable exposure chamber temperature, relative humidity, pressure, aerosol concentration, and particle size distributions. The generator produces airborne particles continuously for long periods of time, e.g. 35 hours of continuous operation, with minimal fluctuations during an exposure period. The uniformity of test atmosphere in the chamber was evaluated to have a total variation of < 5%. In this study, the MWCNT aerosol mass concentration was continuously monitored with a Data RAM (DR-40000 Thermo Electron Co, Franklin, MA), and gravimetric determinations (37 mm cassettes with 0.45 μm pore-size Teflon filters) were used to calibrate and verify the Data RAM readings. The mass mode aerodynamic diameter was 1.3 μm with a count mode aerodynamic diameter of 0.42 μm [3]. When characterized by lognormal statistics, the distribution was shown to have a mass median aerodynamic diameter (MMAD) of 1.5 μm and a geometric standard deviations (GSD) of 1.67 [3]. As described previously [3], the resultant inhaled lung burden in the mouse from this exposure is equal to the predicted human lung burden on an equivalent alveolar surface area basis for a person performing light work at 7 ug/m3 for 13 years.
Lung fixation and section preparation
At 1 day, 14 days, 84 days, 168 days and 336 days after the 12 day exposure period, mice were anesthetized, euthanized by an overdose of pentobarbital (>100 mg/kg body weight, i.p.) and lungs and systemic tissues were preserved by whole body vascular perfusion of paraformaldehyde while the lungs were inflated with air via a tracheal cannula. Separate, clean-air control groups were studied. Following fixation the diaphragm, heart, kidney, liver and brain were removed and sliced into 2–3 mm thick tissue blocks. The left lung and sliced tissue blocks were embedded in paraffin. The chest wall was decalcified in formic acid prior to embedding. Sections (5 micron thick) were collected on ultrasonically cleaned, laser cut slides (Schott North America, Inc, Elmsford, N.Y. 10523) to avoid nanoparticle contamination from the ground edges of traditional slides. To enhance the contrast between tissue and MWCNTs, sections were stained with Sirius Red. Sirius Red staining consisted of immersion of the slides in 0.1% Picrosirius solution (100 mg of Sirius Red F3BA in 100 ml of saturated aqueous picric acid, pH 2) for 1 hour followed by washing for 1 minute in 0.01 N HCl. Sections were then briefly counterstained in freshly filtered Mayer’s hematoxylin for 2 minutes, dehydrated, and coverslipped. For serial section analysis of lung clearance of MWCNT fibers and the number of MWCNT fibers per MWCNT structure in the lungs, three serial sections of the lungs were collect on one slide and stained as described above.
MWCNT lung burden
At 1, 14, 28, 84, 168 and 336 days post-exposure, mice were euthanized with an i.p. injection of sodium pentobarbital (>100 mg/kg body weight) followed by exsanguination. After euthanasia, lungs were removed and stored at −80°C. MWCNT burden determinations were conducted using a method previously described by our laboratory [3, 32] with minor modification. The lung tissue was digested in 25% KOH/methanol (w/v) at 60°C overnight, followed by centrifugation at 16,000 × g for 10 minutes. The supernatant was removed, the remaining pellet was mixed with 50% HNO3/methanol (v/v), and incubated at 60°C overnight, followed by centrifugation (16,000 × g, 10 minutes). The supernatant was removed, and the pellet resuspended in 10% NP-40 (v/v) in dH2O, followed by 60 second sonication (≈3,100 Joules) using water-cooled cup horn sonicator. MWCNT standards were processed in parallel with the lung samples. The optical densities of the solutions were measured at 700 nm using a UV/visible spectrophotometer. Lung MWCNT content was determined from a standard curve.
Whole lung lavage
At 1, 14, 28, 84, 168 and 336 days post-exposure, mice were euthanized with an i.p. injection of sodium pentobarbital (>100 mg/kg body weight) followed by exsanguination. A tracheal cannula was inserted and bronchoalveolar lavage (BAL) was performed through the cannula using ice cold Ca2+ and Mg2+-free phosphate buffered saline, pH 7.4, supplemented with 5.5 mM D-glucose (PBS). The first lavage (0.6 ml) was kept separate from the rest of the lavage fluid. Subsequent lavages, each with 1 ml of PBS, were performed until a total of 4 ml of lavage fluid was collected. BAL cells were isolated by centrifugation (650 × g, 5 minutes, 4°C). An aliquot of the acellular supernatant from the first BAL (BAL fluid) was decanted and transferred to tubes for analysis of lactate dehydrogenase (LDH) and albumin. The acellular supernatants from the remaining lavage samples were decanted and discarded. BAL cells isolated from the first and subsequent lavages for the same mouse were pooled after resuspension in PBS, centrifuged a second time (650 × g, 5 min, 4°C), and the supernatant decanted and discarded. The BAL cell pellet was then resuspended in PBS and placed on ice. Total BAL cell counts were obtained using a Coulter Multisizer 3 (Coulter Electronics, Hialeah, FL) and cytospin preparations of the BAL cells were made using a cytocentrifuge (Shandon Elliot Cytocentrifuge, London). The cytospin preparations were stained with modified Wright-Giemsa stain, and cell differentials were determined by light microscopy.
BAL fluid LDH activity and albumin
BAL fluid LDH activities were evaluated as a marker of cytotoxicity. BAL fluid LDH activities were determined by monitoring the LDH catalyzed oxidation of lactate to pyruvate coupled with the reduction of NAD+ at 340 nm using a commercial assay kit (Roche Diagnostics Systems, Montclair, NJ). Both the BAL fluid albumin and LDH assays were conducted using a COBAS MIRA Plus (Roche Diagnostic Systems, Montclair, NJ).
Field emission scanning electron microscopy
For scanning electron microscopy, sections of the lung were cut at 8 microns, placed on carbon planchets, deparaffinized and sputter coated. After coating, the specimens were examined with a Hitachi Model S-4800 Field Emission Scanning Electron Microscope (FESEM) at 5 to 10 kV and working distances of 4.5 mm to 6 mm for magnifications of 100,000× to 1000×, respectively. Photographs were taken in slow scanning mode at 1280 × 1024 pixels. Use of thin sections from paraffin embedded tissue was found to be preferable to large, unevenly cut blocks because it provided a uniform thickness of organic material on the carbon planchet. The 8 micron sections were thick enough to convey three-dimensional information but still thin enough to avoid effects due to charging and/or undergoing physical shifts when examined at the high magnifications necessary to study nanomaterials.
Enhanced-darkfield light microscopy imaging of MWCNTs
Carbon nanotubes in sections from exposed lungs were assessed using an enhanced-darkfield optical system. Nanomaterials, such as carbon nanotubes, have dimensions less than the wavelength of light, have closely packed atoms, and typically have a refractive index significantly different from that of biologic tissues and/or mounting medium. These characteristics produce significantly greater scattering of light by nanoparticles than by the surrounding tissues. The enhanced-darkfield optical system images light scattered in the section and, thus, nanomaterials in the section stand-out from the surrounding tissues with high contrast. Using this method of imaging, it is practical to scan whole lung sections at relatively low magnification (40-60× objectives) to identify CNTs that would not be detected by other means.
The optical system consisted of high signal-to-noise, darkfield-based illumination optics adapted to an Olympus BX-41 microscope (CytoViva, Auburn, AL 36830). Sections for dark-field examination were specifically cut from paraffin blocks and collected on ultrasonically cleaned, laser cut slides (Schott North America Inc, Elmsford, N.Y. 10523) to avoid nanoparticle contamination from the ground edges of traditional slides. After staining with Sirius Red-Hematoxylin, sections were coverslipped with Permount. After alignment of the substage oil immersion optics with a 10× objective, sections were examined with 60× air or 100× oil immersion objectives. Enhanced darkfield images were taken at 2400×4800 pixels with a Olympus DP-73 digital camera (Olympus America Inc., Center Valley, PA 18034).
Lung distribution of MWCNTs
The distribution of MWCNTs in the lungs was determined by counting the occurrence of MWCNTs under an eyepiece point counting overlay using standard morphometric point counting methods [33] as previously described for study of the distribution of CNTs [11, 34]. Point counting categories were subdivided into points over MWCNTs in airway region, points over MWCNTs in alveolar regions, and points over MWCNTs in the subpleural tissue region. Airway regions were defined as those containing airway tissue (airway epithelial cells-basement membrane and tissues of the broncho-vascular cuff), airway lumen, and associated blood vessels greater than 25 microns. Alveolar regions were those containing alveolar tissue and alveolar air space. The subpleura tissue region included MWCNTs in the subpleural tissue and MWCNTs in the visceral pleural surface. The subpleural tissue regions included the immediately subpleural alveolar interstitial-epithelium layer and subpleural lymphatics but did not include any portion of alveolar walls attaching to the pleura. Points in airway and alveolar regions were further subdivided into points over MWCNTs that were in the airspace, points over MWCNTs that were in tissue of the region, and points over MWCNTs that were partially or completely within macrophages.
To accomplish the counting, an eyepiece counting overlay consisting of 11 by 11 lines (121 total points for each throw of the overlay) was used with a 100× oil immersion objective. A grid pattern for throws of the counting overlay was used in order to ensure a uniform sampling of the section which did not overweight interior points. The counting overlay throws of the eyepiece were positioned over the section at 12 uniformly spaced grid points in both X and Y co-ordinates. These 12 grid points were determined using the stage micrometer scale to measure the X and Y bounds of the section. Using the bounding rectangle of these co-ordinates, a 3 by 4 grid was selected and the 12 intersections were used as the center point for each of the eyepiece counting overlay throws.
For each animal, three sections were counted, and the counts for the airways, alveolar and subpleural tissue regions were summed. Each counting category was divided by this total and multiplied by 100 to express the results as a percentage of total lung burden. To express the results in terms of absolute lung burden (ug), each percentage was then multiplied by 28 ug/lung based on measurements of initial total lung burden of MWCNT in the results Eight animals were analyzed per group. Clean-air controls were also cut and scanned for MWCNTs but none were found. Additional, negative controls to test for contamination by MWCNTs from the treated lungs included changes of the embedding processor solutions between treatment groups, changes of sectioning bath between blocks and use of a new sectioning knife edge for each block.
MWCNT fiber number per MWCNT structure
Serial section analysis at 1 day, 14 days and 168 days was used to measure the clearance of MWCNT fibers from the lungs and to assess how the composition of MWCNT structures was altered by redistribution and clearance. For each series of sections a series of 6 overlapping photographs were taken at 100× (oil immersion) using the enhanced darkfield optical system. Each MWCNT structure was identified in prints of the middle section. The prints from all three sections of the series were then used to determine if the MWCNTs identified in the middle section contained one, two, three, four or more than four fibers. Point counting of the middle section was then used to determine the volume density of MWCNTs for each fiber class (one, two, three, four and more than four fibers), total volume density of all MWCNT fibers, and the relative distribution in terms of fibers per MWCNT structure.
Morphometric analysis of collagen distribution
Morphometric analysis was conducted to determine the fibrillar collagen response to inhalation of MWCNTs at 1, 14, 84, 168 and 336 post-exposure and in clean-air controls. Collagen fibers in the lungs were detected with Sirius Red staining [35], which has been demonstrated to be a quantitative morphometric method for collagen fiber determination in the lungs [36, 37]. Quantitative morphometric methods were used to measure the average thickness of Sirius Red positive connective tissue fibers in the alveolar regions. Volume and surface density were measured using standard morphometric analyses [38, 39]. This consisted of basic point and intercept counting. Volume density was determined from counting the number of points over all tissues in the alveolar regions and points over Sirius Red positive connective tissue. Surface density of the alveolar wall was determined from intercepts between a line overlay and the alveolar wall. These point and intercept counts were made using a 121-point /11-line overlay graticule (12.5 mm square with 100 divisions), at 100× magnification, taken at six locations equally spaced across each section (one section per animal). This process was repeated twice for each animal. In order to limit the measurements to alveolar parenchyma, areas containing airways or blood vessels greater than 25 mm in diameter were excluded from the analysis. Average thickness of the Sirius Red positive connective tissue fibers of the alveolar wall was computed from two times the ratio of volume density of point to the surface density of the alveolar wall. Mean linear intercept length, a measure of the average size of the alveolar/alveolar duct airspaces dimensions in the alveolar region, was computed from the ratio of volume density to surface density [38].
Statistical analyses
Data were analyzed using analysis of variance (STATGRAF). Bartlett’s test was used to test for homogeneity of variances between groups. Statistical differences were determined using one-way analysis of variance with significance set at p ≤ 0.05. When significant F values were obtained, individual means were compared to clean-air controls using Duncan’s multiple range test [40], and P < 0.05 was considered to be significant. Data are given as Means ± S.E.
For the BAL data statistical comparisons between MWCNT and air controls were performed separately for each post-exposure time using analysis of variance (ANOVA). Since variance estimates were different across treatment groups, the ANOVA models were estimated using an unequal variance method available from SAS PROC MIXED [41]. Similarly, comparisons across post-exposure times were performed using ANOVA and post-hoc comparisons using the Tukey method to account for multiple comparison. All statistical tests were two tailed with significance level equal to 0.05. All statistical tests were two-tailed and performed at the 0.05 significance level.