Assessment of morphometry of pulmonary acini in mouse lungs by nondestructive imaging using multiscale microcomputed tomography

Abstract

Establishing the 3D architecture and morphometry of the intact pulmonary acinus is an essential step toward a more complete understanding of the relationship of lung structure and function. We combined a special fixation method with a unique volumetric nondestructive imaging technique and image processing tools to separate individual acini in the mouse lung. Interior scans of the parenchyma at a resolution of 2 µm enabled the reconstruction and quantitative study of whole acini by image analysis and stereologic methods, yielding data characterizing the 3D morphometry of the pulmonary acinus. The 3D reconstructions compared well with the architecture of silicon rubber casts of mouse acini. The image-based segmentation of individual acini allowed the computation of acinar volume and surface area, as well as estimation of the number of alveoli per acinus using stereologic methods. The acinar morphometry of male C57BL/6 mice age 12 wk and 91 wk was compared. Significant increases in all parameters as a function of age suggest a continuous change of the lung morphometry, with an increase in alveoli beyond what has been previously viewed as the maturation phase of the animals. Our image analysis methods open up opportunities for defining and quantitatively assessing the acinar structure in healthy and diseased lungs. The methods applied here to mice can be adjusted for the study of similarly prepared human lungs.

Fig. 1.

Flowchart of acinar segmentation in HRES images. Sample images for the main steps of the acinar segmentation procedure. (A) Initial contrast enhancement using unsharp mask filtering. (B) Result of thresholding and connected component finding. (C) Inverted and smoothed mask. (D) Seed points inside and outside the acinus of interest. (E) Remaining acinar mask. (F) 3D surface rendering of the segmented acinus with original orthogonal image slice.

Fig. 2.

Means of verifying the correct separation into individual acini at a terminal bronchiole based on the definition of acini starting at the first occurrence of alveoli on a TB (arrowheads). (A) HRES CT section with partial 3D simulation of a surface scan in which two acini were identified to branch off the TB in a C57BL/6 mouse. (B) Scanning electron micrograph of a perfusion-fixed Balb/C lung with two acini splitting from a TB.

Fig. 3.

Adjacent acini, TB, and supplying vasculature. (A–C) Orthogonal views (transxial, sagittal, and coronal) of the images at 2 µm with overlays of the segmented structures. (D) The 3D rendering of all segmented components. (E) TB with artery (blue) and vein (red). (F) TB supplying the three individual acini. Dynamic representations of the reconstruction process are shown in Movies S1 and S2.

Fig. 4.

Comparison of 3D volumetric renderings of acinar segmentations in young (12 wk) mice (A and B) and old (91 wk) mice (C and D) (both C57BL/6) with two representative acinar casts (Balb/C young mature mouse) (E and F). The 3D renderings are displayed in a top view (A and C) as well as in a side view (B and D). In E and F, the casts were imaged with a scanning electron microscope and spread out on the supporter to allow visualization of their architecture. The 3D renderings show the acini in their natural shape and state at an inflation pressure of ∼60–70% of TLC. The acini of the C57BL/6 mice correspond in size to the average size of each age group (young, 0.15 mm³; old, 0.38 mm³). The silicon rubber casts correspond to an inflation of ∼60% of TLC, with volumes of 0.11 mm³ (E) and 0.24 mm³ (F).

Fig. 5.

Frequency distribution of acinar volume (in mm³) for three groups of mice. The bars show the volume distribution of silicon rubber casts of acini in the young Balb/C mice. The overlaid data points represent the distribution of acini in the young and old C57BL/6 mice. Acinar size matches the silicon rubber casts for the young mice and is larger in the old mice.

Fig. 6.

Example of counting alveoli in the disector slice pairs by the appearance of alveolar entrance rings in form of bridges (B), where gaps of septal walls are present in the other section, as indicated by arrows. Counting is limited to the area of the acinus marked by the red outline of the alveolar surface area, resulting in number of alveoli per acinus (Methods).