Comprehensive stereological assessment of the human lung using multiresolution computed tomography.

The application of stereology to lung casts and two-dimensional microscopy images is the gold standard for quantification of the human lung anatomy. However, these techniques are labor intensive, involving fixation, embedding, and histological sectioning of samples and thus have prevented comprehensive studies. Our objective was to demonstrate the application of stereology to volumetric multiresolution computed tomography (CT) to efficiently and extensively quantify the human lung anatomy. Nontransplantable donor lungs from individuals with no evidence of respiratory disease (n = 13) were air inflated, frozen at 10 cmH2O, and scanned using CT. Systematic uniform random samples were taken, scanned using micro-CT, and assessed using stereology. The application of stereology to volumetric CT imaging enabled comprehensive quantification of total lung volume, volume fractions of alveolar, alveolar duct, and tissue, mean linear intercept, alveolar surface area, alveolar surface area density, septal wall thickness, alveolar number, number-weighted mean alveolar volume, and the number and morphometry of terminal and transitional bronchioles. With the use of this data set, we found that women and men have the same number of terminal bronchioles (last generation of conducting airways), but men have longer terminal bronchioles, a smaller wall area percentage, and larger lungs due to a greater number of alveoli per acinus. The application of stereology to multiresolution CT imaging enables comprehensive analysis of the human lung parenchyma that identifies differences between men and women. The reported data set of normal donor lungs aged 25-77 yr provides reference data for future studies of chronic lung disease to determine exact changes in tissue pathology.NEW & NOTEWORTHY Stereology has been the gold standard to quantify the three-dimensional lung anatomy using two-dimensional microscopy images. However, such techniques are labor intensive. This study provides a method that applies stereology to volumetric computed tomography images of frozen whole human lungs and systematic uniform random samples. The method yielded a comprehensive data set on the small airways and parenchymal lung structures, highlighting morphometric sex differences and providing a reference data set for future pathological studies.

Assessment of Small Airways with Computed Tomography: Mosaic Attenuation or Lung Density?

BACKGROUND: Computed tomography (CT) assessment of air trapping has been considered useful as a measure of small airway disease. Mean lung density (MLD) and the percentage of the lung field occupied by low attenuation area (LAA%) can be evaluated automatically, and their expiratory/inspiratory (E/I) ratios correlate with asthma severity and spirometry parameters. However, mosaic attenuation, another indicator of air trapping, has been assessed visually, and its functional relevance remains controversial. OBJECTIVES: This retrospective study was conducted to correlate mosaic attenuation, which was assessed visually and automatically, and the E/I ratios of MLD and LAA% (defined as areas <-960 Hounsfield units) with clinical and physiological variables, including impulse oscillometry (IOS) indices. MATERIAL AND METHODS: In 36 nonsmoking patients with stable asthma, the lungs were scanned at full inspiration and full expiration. Mosaic attenuation was measured visually and automatically, by counting areas with CT values higher than the surrounding areas. MLD and LAA% were measured using our validated method. Spirometry, IOS, exhaled NO and the sputum eosinophil count were evaluated. RESULTS: The automatic results and visual scores of mosaic attenuation correlated well on expiratory scans (r = 0.894) and to a lesser degree on inspiratory scans (r = 0.629; p < 0.0001 for both). However, only the E/I ratios of MLD and LAA% correlated with forced expiratory volume in 1 s/forced vital capacity of spirometry and the IOS indices of resistance from 5 to 20 Hz and the integrated area of low-frequency reactance. CONCLUSIONS: Our automatic method for analysis of mosaic attenuation is likely useful, but the results themselves may not be reflecting small airway involvement of asthma, unlike the E/I ratios of MLD and LAA%.

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

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.

Assessment of imaging techniques for evaluating small-airway disease in asthma.

The imaging techniques used to investigate patients with asthma and to assess the effects of asthma treatments include computed tomography (CT), helium magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron-emission tomography (PET). Only MRI does not involve radiation exposure. Technical improvements in CT, together with the imaging advantages inherent in the presence of air in the lung, have diminished the radiation exposure required for lung CT. High-resolution low-dose lung CT protocols deliver a dose roughly equal to 1 year of natural radiation exposure and can be used even in paediatric patients. To date, CT is the most extensively studied lung imaging method, the simplest to perform, and the least expensive. In patients with asthma, CT may show several structural changes related to small-airway disease including cylindrical bronchiolectasis, bronchial wall thickening, and air trapping; an indirect marker for bronchiolar obstruction. A robust body of evidence indicates that valid CT markers for small-airway disease can be derived from quantitative lung density measurements and that these markers correlate with clinical severity and lung function test results. In addition, these CT markers are sufficiently sensitive to demonstrate therapeutic effects.

Quantitative assessment of lung microstructure in healthy mice using an MR-based 3He lung morphometry technique.

The recently developed technique of lung morphometry using hyperpolarized (3)He diffusion magnetic resonance (MR) (Yablonskiy DA, Sukstanskii AL, Woods JC, Gierada DS, Quirk JD, Hogg JC, Cooper JD, Conradi MS. J Appl Physiol 107: 1258-1265, 2009) permits in vivo study of lung microstructure at the alveolar level. Originally proposed for human lungs, it also has the potential to study small animals. The technique relies on theoretical developments in the area of gas diffusion in lungs linking the diffusion attenuated MR signal to the lung microstructure. To adapt this technique to small animals, certain modifications in MR protocol and data analysis are required, reflecting the smaller size of mouse alveoli and acinar airways. This is the subject of the present paper. Herein, we established empirical relationships relating diffusion measurements to geometrical parameters of lung acinar airways with dimensions typical for mice and rats by using simulations of diffusion in the airways. We have also adjusted the MR protocol to acquire data with much shorter diffusion times compared with humans to accommodate the substantially smaller acinar airway length. We apply this technique to study mouse lungs ex vivo. Our MR-based measurements yield mean values of lung surface-to-volume ratio of 670 cm(-1), alveolar density of 3,200 per mm(3), alveolar depth of 55 µm, and mean chord length of 62 µm, all consistent with published data obtained histologically in mice by unbiased methods. The proposed technique can be used for in vivo experiments, opening a door for longitudinal studies of lung morphometry in mice and other small animals.