Myeloperoxidase Clearance of Carbon Nanotubes Figure 2. Changes in the content of collagen and average thickness of alveolar connective tissue in the lungs of w/t and MPO k/o mice at days 1 and 28 after pharyngeal aspiration of SWCNT. a. Accumulation of collagen in the lung of w/t or MPO k/o mice. Mean 6 SEM (n = 6 mice/group). *p,0.05, vs control PBS-exposed mice, # p,0.05, vs w/t mice 28 days post exposure. b. Morphometric assessments of average thickness of alveolar connective tissue in the lung of w/t or MPO k/o mice. Mean 6 SEM (n = 6 mice/group). *p,0.05, vs control PBS-exposed mice, # p,0.05, vs w/t 28 days post exposure. doi:10.1371/journal.pone.0030923.g002 in the lung of MPO-deficient mice compared to w/t animals (Fig. 2b). Assessment of SWCNT aggregates in the lung of w/t and MPO k/o mice To evaluate the content of SWCNT and their aggregates we employed quantitative imaging by illuminating lung sections with the light in the spectral range of 750–840 nm selectively absorbed by SWCNT. In the images presented in Fig. 3a, the lung tissue is shown in red, the SWCNT image was inverted such that the nanotubes look bright and pseudocolored in green. The volume occupied by SWCNT at day 1 post-exposure (,07–1.0% of the total lung volume) markedly decreased by day 28 (to 0.02–0.03%). Elimination of SWCNT from the lung of w/t mice was almost 2 times more effective than in MPO k/o animals (Fig. 3b). Additionally, we employed another approach for collecting SWCNT images: we scanned unstained paraffin embedded lung sections using INCell Analyser 1000 high content screening; optically detectable SWCNT aggregates were counted in each field throughout the lung sections. The content of SWCNT aggregates in each field on day 1 post-exposure was approximately the same in both groups of mice but markedly decreased by day 28 after exposure (Fig. 3c). Assuming the content of aggregates on day 1 in both groups as 100%, the relative content of SWCNT aggregates dropped to ,20% in w/t samples but remained at the level of .60% in the MPO k/o lung sections – a more than 3-fold difference. To obtain further evidence for ‘‘cutting’’ of SWCNT by MPO, we performed TEM analysis of the lung samples after solubiliza- tion of the tissue and evaluated the size distribution of SWCNT and their aggregates (Fig. 4). On day 1 after exposure, ,35–40% of SWCNT were represented by large aggregates of .0.75 mm whereas 20–25% of aggregates were .0.5 mm and 35–40% had the size range from 0.5–0.75 mm in w/t and k/o animals. This distribution had changed markedly at day 28 after exposure in favour of small aggregates. The ‘‘cutting’’ pattern was different in w/t and k/o animals: a ,2-fold increase for aggregates ,0.5 mm, mostly at the expense of the decreased amount of large aggregates, was observed in the lungs of w/t mice whereas these changes in the SWCNT size distribution were much less pronounced in MPO k/o mice (Fig. 4b, c). This suggests that MPO was involved in the cutting of SWCNT into smaller fragments - in accord with our previous in vitro experiments [14]. Raman spectroscopic evaluation of defects in SWCNT in the lungs of w/t and MPO k/o mice MPO-driven biodegradation occurs via oxidative modification of SWCNT with the appearance of characteristic defects detectable by Raman spectroscopy [14]. Therefore, we utilized two different Raman spectroscopic techniques to determine whether these oxidation-associated defects could be detected in the samples of the lung from exposed mice. First, we performed Raman spectroscopy after solubilization of the lung tissue. To assess oxidative biodegradation of SWCNT, the D-band/G-band ratios were calculated, whereby an increase in the ratio indicates oxidative degradation [15,16]. Typical Raman spectra document- ing the signals from non-oxidized SWCNT with a pronounced G band and a very weak D-band were recorded on day 1 post- exposure resulting in low D-band/G-band ratios for both w/t and MPO k/o animals (Fig. 5a). On day 28 post-exposure, the D- band/G-band ratio remained almost unchanged for the samples from k/o animals whereas an ,2-fold increase was found in the lungs of w/t mice. Next, we obtained Raman maps of ten different areas that contained SWCNT within each lung tissue section. Fig. 5b shows examples of bright-field images of samples from w/t and MPO k/o mice, respectively, at day 1 post-exposure, with a red box indicating the area where the Raman maps were acquired; (5c, 1–4) shows typical Raman maps obtained based on the clusters (5c, 5–8) per pixel. These bright-field images and Raman maps were generated for every sample on days 1 and 28 after the SWCNT exposure. SWCNT degradation increased over time. Importantly, 28 days post exposure there was a significant difference (p = 0.0277) between the D-band/G-band ratios of the SWCNT indicative of a markedly higher SWCNT degradation in w/t mice than in MPO k/o animals (Fig. 5d). While the difference in clearance of SWCNT from the lungs of w/t and MPO k/o mice was significant, it is also obvious that additional, yet to be identified, factors contribute to biodegrada- tion and elimination of SWCNT. Indeed, SWCNT clearance occurred, although at a lower rate, in MPO k/o animals. PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e30923