Cournand's and Richards' Post-Nobel Challenge: "Do Anything on the Structure of the Lung That Is of Interest for Physiology".

In their Nobel Lectures of 1956, the two cardiopulmonary physiologists André Cournand and Dickinson Richards described the future projects that they considered important for rounding up their impressive scientific achievements. One of them called for work that brought together structure and function of the lung in a systematic way. They challenged a young anatomist to undertake this in their cardiopulmonary laboratory at Bellevue Hospital. The first steps established the need for quantitative information on the structures that form the pulmonary gas exchanger as well as the airway tree. This resulted in a new approach to lung anatomy: morphometry would provide the quantitative information for modeling structure-function relations in the lung, and eventually in the entire respiratory system, thus allowing a rational analysis of how structures affect or limit functions.

Lung morphometry: the link between structure and function.

The study of the structural basis of gas exchange function in the lung depends on the availability of quantitative information that concerns the structures establishing contact between the air in the alveoli and the blood in the alveolar capillaries, which can be entered into physiological equations for predicting oxygen uptake. This information is provided by morphometric studies involving stereological methods and allows estimates of the pulmonary diffusing capacity of the human lung that agree, in experimental studies, with the maximal oxygen consumption. The basis for this "machine lung" structure lies in the complex design of the cells building an extensive air-blood barrier with minimal cell mass.

Morphometric differences between central vs. surface acini in A/J mice using high-resolution micro-computed tomography.

Through interior tomography, high-resolution microcomputed tomography (µCT) systems provide the ability to nondestructively assess the pulmonary acinus at micron and submicron resolutions. With the application of systematic uniform random sampling (SURS) principles applied to in situ fixed, intact, ex vivo lungs, we have sought to characterize morphometric differences in central vs. surface acini to better understand how well surface acini reflect global acinar geometry. Lungs from six mice (A/J strain, 15-20 wk of age) were perfusion fixed in situ and imaged using a multiresolution µCT system (Micro XCT 400, Zeiss). With the use of lower-resolution whole lung images, SURS methods were used for identification of central and surface foci for high-resolution imaging. Acinar morphometric metrics included diameters, lengths, and branching angles for each alveolar duct and total path lengths from entrance of the acinus to the terminal alveolar sacs. In addition, acinar volume, alveolar surface area, and surface area/volume ratios were assessed. A generation-based analysis demonstrated that central acini have significantly smaller branch diameters at each generation with no significant increase in branch lengths. In addition to larger-diameter alveolar ducts, surface acini had significantly increased numbers of branches and terminal alveolar sacs. The total path lengths from the acinar entrance to the terminal nodes were found to be higher in the case of surface acini. Volumes and surface areas of surface acini are greater than central acini, but there were no differences in surface/volume ratios. In conclusion, there are significant structural differences between surface and central acini in the A/J mouse.

Lung Structure and the Intrinsic Challenges of Gas Exchange.

Structural and functional complexities of the mammalian lung evolved to meet a unique set of challenges, namely, the provision of efficient delivery of inspired air to all lung units within a confined thoracic space, to build a large gas exchange surface associated with minimal barrier thickness and a microvascular network to accommodate the entire right ventricular cardiac output while withstanding cyclic mechanical stresses that increase several folds from rest to exercise. Intricate regulatory mechanisms at every level ensure that the dynamic capacities of ventilation, perfusion, diffusion, and chemical binding to hemoglobin are commensurate with usual metabolic demands and periodic extreme needs for activity and survival. This article reviews the structural design of mammalian and human lung, its functional challenges, limitations, and potential for adaptation. We discuss (i) the evolutionary origin of alveolar lungs and its advantages and compromises, (ii) structural determinants of alveolar gas exchange, including architecture of conducting bronchovascular trees that converge in gas exchange units, (iii) the challenges of matching ventilation, perfusion, and diffusion and tissue-erythrocyte and thoracopulmonary interactions. The notion of erythrocytes as an integral component of the gas exchanger is emphasized. We further discuss the signals, sources, and limits of structural plasticity of the lung in alveolar hypoxia and following a loss of lung units, and the promise and caveats of interventions aimed at augmenting endogenous adaptive responses. Our objective is to understand how individual components are matched at multiple levels to optimize organ function in the face of physiological demands or pathological constraints.

Stereological assessment of mouse lung parenchyma via nondestructive, multiscale micro-CT imaging validated by light microscopic histology.

Quantitative assessment of the lung microstructure using standard stereological methods such as volume fractions of tissue, alveolar surface area, or number of alveoli, are essential for understanding the state of normal and diseased lung. These measures are traditionally obtained from histological sections of the lung tissue, a process that ultimately destroys the three-dimensional (3-D) anatomy of the tissue. In comparison, a novel X-ray-based imaging method that allows nondestructive sectioning and imaging of fixed lungs at multiple resolutions can overcome this limitation. Scanning of the whole lung at high resolution and subsequent regional sampling at ultrahigh resolution without physically dissecting the organ allows the application of design-based stereology for assessment of the whole lung structure. Here we validate multiple stereological estimates performed on micro-computed tomography (µCT) images by comparing them with those obtained via conventional histology on the same mouse lungs. We explore and discuss the potentials and limitations of the two approaches. Histological examination offers higher resolution and the qualitative differentiation of tissues by staining, but ultimately loses 3-D tissue relationships, whereas µCT allows for the integration of morphometric data with the spatial complexity of lung structure. However, µCT has limited resolution satisfactory for the sterological estimates presented in this study but not for differentiation of tissues. We conclude that introducing stereological methods in µCT studies adds value by providing quantitative information on internal structures while not curtailing more complex approaches to the study of lung architecture in the context of physiological or pathological studies.

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.

Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography.

Utilizing micro-X-ray CT (µCT) imaging, we sought to generate an atlas of in vivo and intact/ex vivo lungs from normal murine strains. In vivo imaging allows visualization of parenchymal density and small airways (15-28 µm/voxel). Ex vivo imaging of the intact lung via µCT allows for improved understanding of the three-dimensional lung architecture at the alveolar level with voxel dimensions of 1-2 µm. µCT requires that air spaces remain air-filled to detect alveolar architecture while in vivo structural geometry of the lungs is maintained. To achieve these requirements, a fixation and imaging methodology that permits nondestructive whole lung ex vivo µCT imaging has been implemented and tested. After in vivo imaging, lungs from supine anesthetized C57Bl/6 mice, at 15, 20, and 25 cmH(2)O airway pressure, were fixed in situ via vascular perfusion using a two-stage flushing system while held at 20 cmH(2)O airway pressure. Extracted fixed lungs were air-dried. Whole lung volume was acquired at 1, 7, 21, and >70 days after the lungs were dried and served as validation for fixation stability. No significant shrinkage was observed: +8.95% change from in vivo to fixed lung (P = 0.12), -1.47% change from day 1 to day 7 (P = 0.07), -2.51% change from day 1 to day 21 (P = 0.05), and -4.90% change from day 1 to day 70 and thereafter (P = 0.04). µCT evaluation showed well-fixed alveoli and capillary beds correlating with histological analysis. A fixation and imaging method has been established for µCT imaging of the murine lung that allows for ex vivo morphometric analysis, representative of the in vivo lung.

The challenge of measuring lung structure. On the "Standards for the Quantitative Assessment of Lung Structure".

The purpose of this review is to call attention of respiratory scientists to an Official Policy Statement jointly issued by the American Thoracic Society and the European Respiratory Society on "Standards for the Quantitative Assessment of Lung Structure", based on an extended report of a joint task force of 20 experts, and recently published in the Am. J. Respir. Crit. Care Med. This document provides investigators of normal and diseased lung structure with a review of the stereological methods that allow measurements to be done on sections. It critically discusses the preparation procedures, the conditions for unbiased sampling of the lung for microscopic study, and the potential applications of such methods. Here we present some case studies that underpin the importance of using accurate methods of structure quantification and outline paths into the future for structure-function studies on lung diseases.

Is length an appropriate estimator to characterize pulmonary alveolar capillaries? A critical evaluation in the human lung.

Stereological estimations of total capillary length have been used to characterize changes in the alveolar capillary network (ACN) during developmental processes or pathophysiological conditions. Here, we analyzed whether length estimations are appropriate to describe the 3D nature of the ACN. Semi-thin sections of five human lungs, previously investigated by Gehr et al. (Respir Physiol 1978; 32:121-140), were used to estimate alveolar capillary length using a "design-based" or a "model-based" stereological approach. The design-based approach involves counting of capillary profiles related to a defined area of the reference space. The model-based approach bases on the assumption that capillaries are round tubes and length was calculated from capillary volume and surface area. The model-based approach provided a mean of 6,950 km (SD: 3,108 km) for total capillary length, the design-based approach resulted in a mean of 2,746 km (SD: 722 km). Because of the geometry of the ACN both approaches carry an unpredictable bias. The bias incurred by the design-based approach is proportional to the ratio between radius and length of the capillary segments in the ACN, the number of branching points and the winding of the capillaries. The model-based approach is biased because of the real noncylindrical shape of capillaries and the network structure. In conclusion, the estimation of the total length of capillaries in the ACN cannot be recommended as the geometry of the ACN does not fulfill the requirements for stereological length estimation. Until new methods are being developed, the unbiased estimates of capillary volume, and surface area should be preferred.