Efficient estimation of the total number of acini in adult rat lung

Abstract

Pulmonary airways are subdivided into conducting and gas-exchanging airways. An acinus is defined as the small tree of gas-exchanging airways, which is fed by the most distal purely conducting airway. Until now a dissector of five consecutive sections or airway casts were used to count acini. We developed a faster method to estimate the number of acini in young adult rats. Right middle lung lobes were critical point dried or paraffin embedded after heavy metal staining and imaged by X-ray micro-CT or synchrotron radiation-based X-rays tomographic microscopy. The entrances of the acini were counted in three-dimensional (3D) stacks of images by scrolling through them and using morphological criteria (airway wall thickness and appearance of alveoli). Segmentation stopper were placed at the acinar entrances for 3D visualizations of the conducting airways. We observed that acinar airways start at various generations and that one transitional bronchiole may serve more than one acinus. A mean of 5612 (±547) acini per lung and a mean airspace volume of 0.907 (±0.108) μL per acinus were estimated. In 60-day-old rats neither the number of acini nor the mean acinar volume did correlate with the body weight or the lung volume.

Keywords: Lung; X‐ray microcomputed tomography; X‐ray tomographic microscopy; pulmonary acinus; stereological analysis.

Figure 1.

Cross section of a right middle rat lung lobe. The arrows point to acinar entrances as they are recognized in one section. Morphological criteria (thickness of the wall of the airways and appearance of alveoli) were used to detect the transition from purely conducting to gas‐exchanging airways. This transition represents the entrance of an acinus.

Figure 2.

Structural alterations at the entrance of an acinus. Every acinus is ventilated by its transitional bronchiole (TrB, dotted line), which itself is ventilated by a purely conducting airway (CA). The conducting airway may be a terminal or larger bronchiole. Its inner surface is covered with cuboidal epithelium which looks like cobble stone pavement in this kind of reconstructions. The entrance of the acinus (dashed line) is located at the sudden transition of the thick wall typical for a conducting airway to the thin wall typical for the gas‐exchange area. The most proximal alveoli (arrow) appear directly distal of this transition. Dotted lines, wall of the transitional bronchiole.

Figure 3.

Schematic drawing comparing unbranched and branched transitional bronchioles. Typically the last branching point before the entrance of an acinus is located in the purely conducting part of the bronchial tree (A). In this case, the next branching point is located inside the acinus and represents a branching of alveolar ducts (B). However, relative frequently a branching point is located in the zone of transition from the conducting to the gas‐exchanging airways (C). Hence, the branching point itself shows still the thick wall of the conducting airways, but alveolated airway walls were observed proximal and distal of the branching point. Dark gray, terminal bronchiole (TeB); light gray, transitional bronchiole (TrB); white, alveolar duct (AD); dotted line, transition from the conducting to the gas‐exchanging airways.

Figure 4.

Dual acinar entrance in one transitional bronchiole. If the transitional bronchiole (TrB) contains a branching point (BP), two acini (A1, A2) share the same transitional bronchiole. The shown branching point is still inside the transitional bronchiole and not part of one larger acinus, because at the branching point itself the wall of the airway is still thick and does not contain alveoli. Dashed line, transition conducting airways–gas‐exchange area; dotted lines, wall of the transitional bronchiole.

Figure 5.

Tree of conducting airways shown inside a right middle lobe (young adult rat). The walls of the conducting airways are shown in light gray and the spheres represent the acini entrances. The gray shadows delimit the lobe tissue and the sample holder. This 3D reconstruction illustrates that the bronchial tree of rat does not follow the dichotomous branching pattern of the human lung.

Figure 6.

3D visualization of the location of acinar entrances. A small part of a secondary bronchus (in gray) and the acinar entrances are shown (colored balls). The skeletons of the conducting airways are drawn in gray, the ones of the transitional bronchioles in color. Arrow, single entrance to an acinus; double‐arrow, two acinar entrances very close together as shown in Figures 3C, 4.

Figure 7.

Correlation among lung volume, body weight, and number of acini at postnatal day 60. The coefficient of determination (R²) was calculated to analyze the correlation between different lung parameters. and . These coefficients indicate that the number of acini (mean: 5612) is independent of the lung volume and the body weight at postnatal day 60 of rat lung development.