Six-week old female pathogen-free CDF (F344)/CrlBR rats were purchased from Charles River Breeding Laboratories, Kingston, NY, and housed in an International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited humidity and temperature controlled facility. Rats were housed in microisolator cages on Alpha-dri cellulose bedding and supplied water and cereal-based diet NIH07 (Zeiger Brothers., Gardners, PA) ad libitum. The animal studies were approved by the CIIT Centers for Health Research Institutional Animal Care and Use Committee.
General experimental design
The objective of this study was to determine whether SWCNT are fibrogenic in the lungs of rats after exposure by oropharygneal aspiration. Moreover, we sought to determine whether SWCNT increase mRNA levels of growth factors that mediate a fibrogenic response. We hypothesized that SWCNT would increase the expression of pro-fibrotic growth factors (PDGF, CTGF, TGF-β1) and thereby induce interstitial fibrosis. Oropharyngeal aspiration is a standard method for delivering particle suspensions to the lung and results in better particle distribution throughout the lung as compared to intratracheal instillation. Quantitative real time RT-PCR is the optimal method for measuring changes in growth factor mRNA levels in the lungs of exposed animals and changes in mRNA can be normalized for several standard housekeeping genes (described below under Taqman quantitative real time RT-PCR). Animals were acclimated for two weeks and then randomly assigned into treatment groups according to body weight prior to particle exposure. Rats were exposed to 2 mg/kg of SWCNT by oropharyngeal aspiration. Negative control animals were treated with Ca2+ and Mg2+ -free phosphate-buffered saline (PBS) with 0.1% pluronic. Carbon black (CB) particles were used as a negative control particle that does not cause fibrosis. Vanadium pentoxide (V2O5) was used as a positive control for causing a fibrotic response in the lungs of rats. Animals were killed by pentobarbitol overdose at 1 and 21 days following particle exposure. The lungs were lavaged with PBS. The left lungs were used to assess lung cell proliferation and histopathology. The right lung lobes were snap-frozen in liquid nitrogen stored at -80°C until used for RNA isolation and assessment of gene expression by real time quantitative RT-PCR.
High-purity SWCNT were purchased from Helix Material Solutions, Richardson, TX . These SWCNT were produced by a chemical vapor deposition process yielding particle external diameters of less than 2 nm with lengths ranging from 0.5 to 40 microns and a purity > 90% as determined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Raman spectroscopy and thermogravimetric analysis (TGA). SWCNT contained less than 5% amorphous carbon. The specific surface area of SWCNT was 300–600 m2/g. The elemental composition of SWCNT as determined by energy dispersive X-ray diffraction was 89.6% carbon, 6.1% oxygen, 2.6% cobalt (Co), and 1.7% molybdenum (Mo). Co and Mo were metal catalysts used in the chemical vapor deposition process. Nanosized CB particles (Raven 5000 Ultra II) were obtained from Columbian Chemicals Company, Marietta, GA . The Raven 5000 Ultra II CB had a mean particle size of 8 nm and specific surface area between 350–583 m2/g. Vanadium pentoxide V2O5, (also referred to as vanadium oxide V) was obtained from Sigma-Aldrich, (St. Louis, MO). V2O5 was used as a positive profibrogenic agent and was purchased as an 8 micron mesh size (i.e., particles <8 micron). However, V2O5 is at least partly soluble in aqueous solutions and was therefore not compared on a size basis to CB and SWCNT. Dry CB and SWCNT were milled in a Retsch Mixer Mill (Retsch Inc., Newtown, PA) for 5 minutes at 30 cycles per second. The milled nanoparticles were then suspended in a biocompatible nonionic surfactant, 1% Pluronic F-68 (BASF Corp., Florham Park, NJ) in PBS and wet milled for an additional 5 min. The CB and SWCNT suspensions were further diluted with PBS to achieve the desired final dosing concentration of 0.1% Pluronic F-68. V2O5 particles were suspended in PBS with 1% Pluronic F-68, sonicated for 30 minutes, then further diluted 1:10 with PBS. Lastly, all particles were kept dispersed by placing suspensions in an ultrasonic waterbath until vortex mixed just prior to instillation. All particles were sterilized prior to instillation.
Instillation of nanoparticles
Rats were administered particles by oropharyngeal aspiration. In brief, animals were anesthetized with isofluorane and ~100 μl volume of either CB, SWCNT or V2O5 (dose concentration equivalent of 2 mg of particles per kg of bodyweight) was placed at the back of the throat while holding the rat's tongue until the suspension was aspirated into the lungs. Control rats were administered an equivalent volume of PBS with 0.1% Pluronic F-68 surfactant.
Rats were euthanized by pentobartital overdose and lungs were lavaged five times with 5-ml volumes of PBS. Two bronchoalveolar lavage (BAL) samples representing the first two and subsequent three recovered lavages were pooled and placed on ice. We established that this gentle lavage procedure does not compromise lung architecture as compared to animals that were not lavaged nor does it induce mRNA levels encoding pro-inflammatory cytokines or pro-fibrotic growth factors. BAL cells collected by centrifugation were resuspended in culture medium and enumerated using an automated cell counter (Model ZM, Coulter, Marietta, GA). Cytospins were prepared with approximately 105 cells per slide. Cell differential counts were performed on HEMA-3 (Fisher Scientific, Pittsburgh, PA) stained cytocentrifuge slide preparations. Total protein and LDH in cell-free BALF from the first two pooled lavages were analyzed spectrophotometrically using a COBAS FARA II (Roche Diagnostic Systems Inc., Montclair, NJ).
Lung histopathology and cell proliferation
One hour prior to euthanasia, rats received a single intraperitoneal injection of 50 mg/kg body weight of bromodeoxyuridine (BrdU; Sigma-Aldrich). At necropsy, left lungs were pressure-infused intratracheally (30 cm H2O) with 10% neutral-buffered formalin. Lungs were fixed for approximately 48 h and then changed to 70% ethanol. Subsequently, the lungs were embedded in paraffin, sectioned at 5 μm, and stained with Masson's trichrome or immunostained for bromodeoxyuridine (BrdU) by established methods . Cell labeling indices were determined in the bronchiolar/alveolar region and in the bronchus-associated lymphoid tissue for each animal, and the mean labeling index was calculated for each group of five animals.
Cytology and carbon bridge assessment
In order to validate our observations in a quantitative, unbiased manner, BAL cell cytospin slides were scored in a blinded fashion; i.e., without knowing which cytospins were from CB or SWCNT exposure groups. Cell numbers were quantified by light microscopy using the 40× objective and a squared grid eyepiece graticule (field area = 0.173 mm2). Ten microscopic fields were counted per animal and at least 500 cells per animal were counted. Four parameters were measured; A) Total cells/field, B) Numbers of cells per field containing carbon inclusions, C) Carbon bridges between macrophages per field, and D) Mitotic figures per field.
Taqman quantitative real time RT-PCR
Total RNA from right anterior lungs of particle-treated rats was isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA), followed by RNA cleanup performed using RNeasy Midi spin columns (Qiagen, Valencia, CA). One or two micrograms of total RNA was reverse transcribed at 48°C for 30 minutes using Moloney murine leukemia virus reverse transcriptase (Eurogentec, San Diego, CA) in 1 × RT buffer, 5 mM MgCl2, 500 μM of each dNTP, 2.5 μM of random nonamers, and 0.4 U/μL RNase inhibitor in a volume of 100 μl. Twenty nanograms of the RT product was amplified using Taqman Gene Expression Assays specific for platelet-derived growth factor receptor alpha (PDGFRα), PDGF-A, PDGF-B, PDGF-C, transforming growth factor beta-1 (TGF-β1), osteopontin (OPN), connective tissue growth factor (CTGF), Type I procollagen (COL1A2), and 18S on the Applied Biosystems 7900 Prism® Sequence Detection System (Applied Biosytems, Foster City, CA). The PCR conditions and data analysis were performed according to the manufacturer's protocol described in User bulletin no.2, Applied Biosystems Prism 7700 Sequence Detection System. Gene expression was measured by the quantitation of cDNA converted from mRNA corresponding to the target genes relative to the vehicle-treated control groups and normalized to eukaryotic 18S reference endogenous control. Relative quantitation values (2-ΔΔCT) were expressed as fold-change over controls.
All data were tested for normality and homogeneity of variance. Gene expression data were log transformed and comparisons to controls were made using Dunett's test (P < 0.05). The software package JMP (SAS Institute, Cary, NC) was used for the statistical analysis.