Table 1.
Number of mice used in the experiments.
Table 2.
post hoc and a priori power analyses of the different experiments.
Figure 1.
Qualitative and quantitative assessment of lung volume by in vivo µCT imaging.
(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)).
Figure 2.
Validation of µCT-based quantification of aerated lung volumes in fibrosis.
(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.
Figure 3.
Serial µCT imaging during bleomycin-induced lung fibrosis.
(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).
Figure 4.
Pressure-Volume (PV) relationships.
(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).
Figure 5.
µCT imaging can also quantify lung emphysema in mice.
(A) Representative histological image of an elastase-treated mouse (hematoxylin and eosin staining). (B) End-inspiratory aerated volumes (EEV), calculated by µCT, in elastase (PPE)-induced pulmonary emphysema (ANOVA p<0.0001; PBS versus both 0.25U and 0.5U PPE: * p<0.05, 0.25U versus 0.5U PPE: * p<0.05). (C) Pressure-Volume (PV)-curves of elastase-induced pulmonary emphysema, based on flexiVent measurement (ANOVA p<0.0001; PBS versus both 0.25U and 0.5U PPE: * p<0.05, 0.25U versus 0.5U PPE: * p<0.05).
Figure 6.
Specific topographic information conveyed by µCT imaging.
(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.