Quantification of Lung Fibrosis and Emphysema in Mice Using Automated Micro-Computed Tomography

<div><h3>Background</h3><p><em>In vivo</em> high-resolution micro-computed tomography allows for longitudinal image-based measurements in animal models of lung disease. The combination of repetitive high resolution imaging with fully automated quantitative image analysis in mouse models of lung fibrosis lung benefits preclinical research. This study aimed to develop and validate such an automated micro-computed tomography analysis algorithm for quantification of aerated lung volume in mice; an indicator of pulmonary fibrosis and emphysema severity.</p> <h3>Methodology</h3><p>Mice received an intratracheal instillation of bleomycin (n = 8), elastase (0.25U elastase n = 9, 0.5U elastase n = 8) or saline control (n = 6 for fibrosis, n = 5 for emphysema). A subset of mice was scanned without intervention, to evaluate potential radiation-induced toxicity (n = 4). Some bleomycin-instilled mice were treated with imatinib for proof of concept (n = 8). Mice were scanned weekly, until four weeks after induction, when they underwent pulmonary function testing, lung histology and collagen quantification. Aerated lung volumes were calculated with our automated algorithm.</p> <h3>Principal Findings</h3><p>Our automated image-based aerated lung volume quantification method is reproducible with low intra-subject variability. Bleomycin-treated mice had significantly lower scan-derived aerated lung volumes, compared to controls. Aerated lung volume correlated with the histopathological fibrosis score and total lung collagen content. Inversely, a dose-dependent increase in lung volume was observed in elastase-treated mice. Serial scanning of individual mice is feasible and visualized dynamic disease progression. No radiation-induced toxicity was observed. Three-dimensional images provided critical topographical information.</p> <h3>Conclusions</h3><p>We report on a high resolution <em>in vivo</em> micro-computed tomography image analysis algorithm that runs fully automated and allows quantification of aerated lung volume in mice. This method is reproducible with low inherent measurement variability. We show that it is a reliable quantitative tool to investigate experimental lung fibrosis and emphysema in mice. Its non-invasive nature has the unique benefit to allow dynamic 4D evaluation of disease processes and therapeutic interventions.</p> </div

Qualitative and quantitative assessment of lung volume by <i>in vivo</i> µCT imaging.

<p>(A) The crucial steps of the automated analysis protocol are illustrated in a normal and a fibrotic lung. On each tomogram, pixels with grayscale indices below 50 Houndsfield Units (HU) are selected, segmenting air-containing pixels. Further despeckling and thresholding steps eliminate contamination by extrapulmonary air and result in stack tomogram-based 3D parameter calculation and reconstruction of a 3D model that visualizes aerated lung volumes. Note that the mouse has four lobes in the right lung with one retrocardiac lobe. (B) Retrospective gating allowing surface rendering of inspiratory (gray) and expiratory (red) air volumes. (C) Calculated aerated lung volumes of a representative normal lung, scanned 4 consecutive times in 1 day, expressed in total voxel number, plotted versus respiratory cycle phase divided into four phases and extending from the end of inspiration (EIV) to the end of expiration (EEV). (Data are mean +/- SD, additional thin lines above and below the main line are mean + and – repeatability coefficients, respectively (n = 5 mice)).</p

Pressure-Volume (PV) relationships.

<p>(A) PV-curves of bleomycin-induced pulmonary fibrosis, based on µCT data. (B) PV-loops in bleomycin-induced pulmonary fibrosis, based on flexiVent measurement, with inflating and deflating (<) curve. (C) Area under the curve (AUC) of µCT based PV-curves in pulmonary fibrosis (data are mean & 95% CI, *p = 0.0065 versus PBS). (D) Compliance in bleomycin-induced lung fibrosis, measured by flexiVent (data are mean & 95% CI, *p = 0.0102 versus PBS).</p

Number of mice used in the experiments.

1<p>Number of mice at end-stage represents survivors in the experiment² Mice in this analysis were scanned 4 times without any other intervention. ³ Only 4 mice had successful scans at all time points and were included in the longitudinal analysis.</p

Cytotoxicity of Cadmium-Free Quantum Dots and Their Use in Cell Bioimaging

The use of quantum dots (QDots) as bright and photostable probes for long-term fluorescence imaging is gaining more interest. Thus far, (pre)­clinical use of QDots remains limited, which is primarily caused by the potential toxicity of QDots. Most QDots consist of Cd²⁺ ions, which are known to cause high levels of toxicity. In order to overcome this problem, several strategies have been tested, such as the generation of cadmium-free QDots. In the present study, two types of cadmium-free QDots, composed of ZnSe/ZnS (QDot_ZnSe) and InP/ZnS (QDot_InP), were studied with respect to their cytotoxicity and cellular uptake in a variety of cell types. A multiparametric cytotoxicity approach is used, where the QDots are studied with respect to cell viability, oxidative stress, cell morphology, stem cell differentiation, and neurite outgrowth. The data reveal slight differences in uptake levels for both types of QDots (maximal for QDot_ZnSe), but clear differences in cytotoxicity and cell functionality effects exist, with highest toxicity for QDot_ZnSe. Differences between cell types and between both types of QDots can be explained by the intrinsic sensitivity of certain cell types and chemical composition of the QDots. At concentrations at which no toxic effects can be observed, the functionality of the QDots for fluorescence cell visualization is evaluated, revealing that the higher brightness of QDot_ZnSe overcomes most of the toxicity issues compared to that of QDot_InP. Comparing the results obtained with common Cd²⁺-containing QDots tested under identical conditions, the importance of particle functionality is demonstrated, revealing that cadmium-free QDots tested in this study are not significantly better than Cd²⁺-containing QDots for long-term cell imaging and that more work needs to be performed in optimizing the brightness and surface chemistry of cadmium-free QDots for them to replace currently used Cd²⁺-containing QDots

Validation of µCT-based quantification of aerated lung volumes in fibrosis.

<p>(A) End-expiratory aerated volumes (EEV), calculated by µCT, in bleomycin-induced pulmonary fibrosis (data are mean and 95% CI, * p = 0.024). (B) Representative histology of a saline (PBS) - (left) and a bleomycin- (right) treated mouse (hematoxylin & eosin staining). (C) Ashcroft score (data are mean and 95% CI, * p = 0.0005). (D) Total collagen content (data are mean and 95% CI, * p = 0.0007). (E) Agreement between EEV and histopathological fibrosis (F) and hydroxyproline content (G) based on linear regression (R²  = 0.523; p = 0.0002 and R²  = 0.598; p = 0.0004 respectively). Plots show the linear regression line and 95% prediction intervals.</p

Serial µCT imaging during bleomycin-induced lung fibrosis.

<p>(A) 3D micro-CT imaging of progressive pulmonary fibrosis in a representative bleomycin-treated mouse over time. (B) Mean EEV over time after induction of fibrosis. (data are mean +/− SEM, differences were significant over time (p = 0.0061) and per group (p = 0.0291) when comparing treated vs non-treated mice exposed to bleomycin (n = 4 vs 5). Adding the non-bleomycin treated control group (n = 7) also showed significant interaction between time and treatment group (p = 0.007).</p

Specific topographic information conveyed by µCT imaging.

<p>(A) 3D image of an individual mouse identified as an outlier in the correlation between Ashcroft score and µCT derived end-inspiratory volume. The image shows an almost normal right lung, but selective damage to the upper part of the left lung, which was sampled for histology. (B) Corresponding histological image (hematoxylin and eosin staining) confirming disease in the upper part of the left lung.</p

post hoc and a priori power analyses of the different experiments.

1<p>analyzed for unpaired t-test, ² Two way ANOVA for repeated measurements, ³ One way ANOVA (3 groups).</p

Comparison of the mean Dice-scores and their standard deviation between different initialization methods for the UZ Leuven dataset.

<p>The highest Dice-score per NMF method and per tissue class is marked in bold. * indicates statistically significantly higher Dice-scores with SPA initialization compared to direct SPA endmember extraction (right column), using a one-tailed Wilcoxon signed rank test (<i>p</i> < 0.05).</p