E (Agilent Technologies) on snap-frozen tissue. After PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28513872 freeze drying, tissues were digested by microwave and ICPMS performed in no-gas mode using mass 107 as the quantifier and mass 71 as the internal standard. The ICP-MS was calibrated using silver standards diluted from a 1 ppm stock.Measurement of malondialdehydeCytokines and chemokines including KC, CCL11 (eotaxin), Interferon (IFN)-, IL-1, IL-4, IL-6, IL-13, IL17A, CCL2 (MCP-1) and CCL3 (MIP-1) were measured in BAL supernatants using a Milliplex MAP rat cytokine panel (Millipore Analayte Kit Finder, Millipore Ltd, Watford, UK) according to the manufacturer’s specifications.Data analysisBAL malondialdehyde (MDA) was measured using a HPLC system with fluorescent detection (Waters, Milford, MA, USA) set at 532 nm for the excitation wavelength and 553 nm for the emission wavelength. A Nova-Pak C18 column (Waters, Milford, MA, USA) was used with a mobile phase that was composed of 40 methanol and 60 water containing 50 mM KH2PO4 (pH = 6.8). The detection limit, extraction recovery and analytical precision were 1.8 nM, 75.9, and 2.2 , respectively.Total phospholipid and surfactant proteins in BALData analysis was performed using Prism 5 software. Data were treated non-parametrically as data was generally not normally distributed when tested using the Shapiro-Wilk normality test. A non-parametric ANOVA (Kruskal-Wallis test) was performed at each time point and comparison of the means of the multiple groups was assessed by Dunn’s post-hoc test. P PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26655304 values <0.05 were considered significant.ResultsAgNP dose in the lungsBAL supernatant was separated into two fractions using differential centrifugation. 1 mL of BAL from the firstThe AgNPs had an average count median diameter (CMD) spanning between 13.4 ?1.0 nm and 15.9 ?0.8 nm on the different days of exposure with particle number concentrations between 3.68 ?0.48 ?107 and 4.55 ?0.70 ?107/cm3 (Table 1; Fig. 1). Mass concentrations measured gravimetrically ranged from 617 ?25 to 801 ?33 g/m3. Figure 1 shows representative images of separate Ag nanospheres collected during exposures, illustrating their spherical form. In SD rats, low and high lung doses were estimated using deposition fractionsSeiffert et al. Respiratory Research (2016) 17:Page 5 ofFig. 1 Panel a: Density of particles as a function of the diameter of silver nanoparticles measured during each of the experimental conditions for each rat strain. BN-LD: Brown Norway rats exposed to low Odanacatib dose; BN-HD: Brown Norway rats exposed to high dose; SD-LD: Sprague awley rats exposed to low dose; SD-HD: Sprague awley rats exposed to high-dose. Panel b: Representative high resolution transmission electron microscopy (TEM) images of aerosol particles delivered to the exposure manifold at 12,000X magnification (scale bar 200 nm) (Panel B a) and 800,000X magnification (scale bar 2 nm) (Panel B b)from the MPPD model as 8 and 28 g respectively, while in BN rats, these were 8 and 26 g. The equivalent values for alveolar deposition were 6 and 19 g for the SD rats and 6 and 18 g for the BN rat.Quantification and localisation of silver in lung tissueSilver levels in the lungs of SD rats exposed to an estimated deposited dose of 28 g of AgNP particles were 9.97 ?2.79 g/g (wet weight) at 24 h. Assuming a typical rat lung weight of 1 g this suggests significant clearance within the first 24 h, and lower levels of 4.99 ?2.21 g/g wet weight at day 7 (P < 0.05), indicate further clearance had occurred from the.