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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.

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