Three-dimensional measurement of alveolar airspace volumes in normal and emphysematous lungs using micro-CT

Abstract

In pulmonary emphysema, the alveolar structure progressively breaks down via a three-dimensional (3D) process that leads to airspace enlargement. The characterization of such structural changes has, however, been based on measurements from two-dimensional (2D) tissue sections or estimates of 3D structure from 2D measurements. In this study, we developed a novel silver staining method for visualizing tissue structure in 3D using micro-computed tomographic (CT) imaging, which showed that at 30 cmH20 fixing pressure, the mean alveolar airspace volume increased from 0.12 nl in normal mice to 0.44 nl and 2.14 nl in emphysematous mice, respectively, at 7 and 14 days following elastase-induced injury. We also assessed tissue structure in 2D using laser scanning confocal microscopy. The mean of the equivalent diameters of the alveolar airspaces was lower in 2D compared with 3D, while its variance was higher in 2D than in 3D in all groups. However, statistical comparisons of alveolar airspace size from normal and emphysematous mice yielded similar results in 2D and 3D: compared with control, both the mean and variance of the equivalent diameters increased by 7 days after treatment. These indexes further increased from day 7 to day 14 following treatment. During the first 7 days following treatment, the relative change in SD increased at a much faster rate compared with the relative change in mean equivalent diameter. We conclude that quantifying heterogeneity in structure can provide new insight into the pathogenesis or progression of emphysema that is enhanced by improved sensitivity using 3D measurements.

Fig. 1.

A: 3 orthogonal planes intersecting the 3-dimensional (3D) structure reconstructed from micro-computed tomographic (μ-CT) slices of an emphysematous mouse lung tissue (day 14 group). The dark regions represent airspaces and the lighter regions represent silver-stained tissue. We focus our attention on the airspace that is marked by the red arrow. The 3 planes are moved to intersect this airspace and the 3 orthogonal views of this airspace are shown in axial (A; horizontal), coronal (C; vertical), and sagittal (D; vertical) view. The 2 perpendicular lines indicate the other 2 orthogonal planes. The mouth of the airspace where it opens to the duct was manually identified using these 3 views. E and F: a 3D reconstruction, showing the branching from the top of the largest airway to an airspace (in blue and arrow). All other branches have been eliminated for ease of visualization.

Fig. 2.

μ-CT images of silver-stained mouse lung tissue fixed at 30 cmH₂O. A: normal lung tissue. B: lung tissue 7 days after elastase treatment. C: lung tissue 14 days after elastase treatment.

Fig. 3.

Laser scanning confocal microscopic (LSCM) images of mouse lung tissue fixed at 30 cmH₂O. A: normal lung tissue. B: lung tissue 7 days after elastase treatment. C: lung tissue 14 days after elastase treatment.

Fig. 4.

The probability distribution of 2D equivalent diameters of normal mouse lungs measured using μ-CT and LSCM. Kolmogrov-Smirnoff test shows no statistical differences between the 2 distributions.

Fig. 5.

Comparison of 3D and 2D mean equivalent diameters of alveolar airspaces calculated from alveolar volumes measured from μ-CT and cross-sectional areas measured from LSCM. The bar and the error indicate the mean and SD of diameters in each treatment group. *3D mean diameter was significantly higher than the corresponding 2D mean diameter. § and +Significant increases in mean and SD of equivalent diameters between treatment groups (statistical significance of the difference in mean and SD was the same in both 2D and 3D).

Fig. 6.

LSCM was used to image the same region of lung tissue at different depths and these images were used to reconstruct the tissue structure in 3D. A: one typical slice from such a stack of images obtained from normal lung tissue. B: the block of tissue that was reconstructed from the slices. Note the increased noise at the bottom of the block. This is due to the multiple scattering events that restrict the detection and recovery of photons beyond ∼100 μm. C: a view from inside the lung tissue showing opening of an alveolus. The opening is ∼40 μm wide.