Figure 1.
Arrangement of multiple mice for high resolution CT of lungs using a clinical CT-scanner.
(A) Animal wood box holder with multiple anesthetized mice placed in prone position inside polypropylene tubes (anterior view). (B) Posterior view of the same structure. (C) Scout view or scanogram to plot the chest area where slice images are obtained (area labeled in red). (D) Panoramic view of multiple lungs in the same axial tomographic section.
Figure 2.
Quantification of lungs density in healthy BALB/c mice.
(A) Mouse pulmonary figure in coronal view from a healthy BALB/c mice showing four right lobes and a unique left lobe. Dotted lines correspond to the places where selected tomographic slices were taken: upper or hilar (B), central or middle (C) and lower or lung bases (D) The upper left symbol in figure B, gives the spatial position of posterior or dorsal (P), anterior or ventral (A), left (L) and right (R) regions. (E) Box plot showing the upper, central and lower pulmonary density in healthy BALB/c mice. Density was expressed by Hounsfield units (HU) scale and was measured in ten animals at 7, 11, 15, 19 and 23 weeks of age. One-way ANOVA was used to compare the groups. Upper lung density showed a statistically significant difference in relation to central and lower lung zones, p≤0.001. ns = non significant.
Figure 3.
Topographic location of the main pulmonary lesions and sequential changes of the lungs density in the experimental PCM model.
Representative HRCT images of a lung control (A, E, I) and P. brasiliensis infected mice (B, F, J) at 12 weeks of follow-up. The upper left symbol in figure A, gives the spatial position of posterior or dorsal (P), anterior or ventral (A), left (L) and right (R) regions. Tomographic slices, selected as described in figure 1, represent upper (A, B, C, D) central (E, F, G, H) and lower (I, J, K, L) pulmonary regions. Peri-bronchial and bilateral consolidations around pulmonary hilum in upper (B) and central lung regions (F) of a P. brasiliensis infected mice. Quantification of lung density in infected animals (C, D, G, H, K, L) was assessed selecting pulmonary parenchyma, excluding main vessels and bronchi (C, G, K), or including the hilum (D, H, L). Measures are expressed as Hounsfield units (HU) ± SEM and were acquired at 0, 4, 8, 12 and 16 weeks pos-inoculation. Gray squares represent control group and black circles, P. brasiliensis infected group. Two-way ANOVA was used to compare groups. Scale bar in tomographic images = 1 cm.
Figure 4.
Frequency of increased lung density according to different lung regions and time of Pb-infection.
The infected mice with increased lung density were selected using the upper limit value of density established in normal animals. Note upper left lung predominance of lesions (black bar) in the majority of mice during all evaluation times.
Figure 5.
Comparison of the main lung lesion patterns accessed by HRCT and histopathology in the experimental model of PCM.
Figures A, C, E, G correspond to HRCT images and B, D, F, H show the corresponding histopathological lesions taking in coronal plane. The upper left symbol in (A) indicates posterior or dorsal (P), anterior or ventral (A), left (L) and right (R) regions. Dotted lines show an approximated position of the tomographic section. (A) Large nodular lesion represented by a peri-bronquial consolidation (arrow) is located at right hilar region and other left small nodules are indicated by arrowhead. (B) Several nodules with varied sizes. Only the larger nodules are easily seen by tomography. (C) Confluent lesion expressed by left central peri-bronchial consolidation (arrow) extended from the hilum to large area of the parenchyma. (D) Consolidated areas of perivascular and justabronchial granulomatous lesions. (E) Pseudotumoral lesion defining a left central pulmonary mass (arrow). (F) Left central pseudotumoral mass obstructing the bronchus. (G) Left lung with accentuated atelectasis (arrow). (H) Lung section corresponding to figure G showing inflammatory periarterial nodules tending to confluence. Scale bar for HRCT images = 1 cm. Scale bar for histopathological images = 2 mm.
Figure 6.
Tomographic reconstruction of pulmonary air space and its quantification in the experimental model of PCM.
Three-dimensional aspect of lungs from control (A) and P.b infected mice (B), showing a none insufflated area in the upper left lung (arrow in B) and compensated hyper-insufflation in the opposite lung. Scatter dot-plot of air space volume of lung (cm3) (C) measured at 0, 4, 8, 12 or 16 weeks post inoculation with PBS (gray squares) or P. brasiliensis conidia (black circle). There was no significant difference between the groups at anytime during the experimental process.
Figure 7.
Percentage of the lung area with inflammatory reaction in P. brasiliensis infected mice at different times of infection.
The lung area with inflammation was assessed by histomorphometry as described in M&M. Five control (gray) and Pb-infected (black) mice were evaluated at 2h, 4, 8, 12 and 16 weeks after inoculation.
Figure 8.
Correlation between percentage of pulmonary area with inflammatory reaction and lung density.
Pulmonary inflammation measured by histomorphometry was correlated with HUs obtained in the upper (A), central (B) and lower (C) regions. Both parameters were directly correlated when the upper and the central lung density were used (r2 = 0.5095, p<0.0001 and r2 = 0.2578, p<0.0002, respectively); and inversely correlated with the lower lung density (r2 = 0.2788, p<0.0001).