Digital 3D reconstructions using histological serial sections of lung tissue including the alveolar capillary network.

Grothausmann R, Knudsen L, Ochs M, Mühlfeld C. Digital 3D reconstructions using histological serial sections of lung tissue including the alveolar capillary network. Am J Physiol Lung Cell Mol Physiol 312: L243-L257, 2017. First published December 2, 2016; doi:10.1152/ajplung.00326.2016-The alveolar capillary network (ACN) provides an enormously large surface area that is necessary for pulmonary gas exchange. Changes of the ACN during normal or pathological development or in pulmonary diseases are of great functional impact and warrant further analysis. Due to the complexity of the three-dimensional (3D) architecture of the ACN, 2D approaches are limited in providing a comprehensive impression of the characteristics of the normal ACN or the nature of its alterations. Stereological methods offer a quantitative way to assess the ACN in 3D in terms of capillary volume, surface area, or number but lack a 3D visualization to interpret the data. Hence, the necessity to visualize the ACN in 3D and to correlate this with data from the same set of data arises. Such an approach requires a large sample volume combined with a high resolution. Here, we present a technically simple and cost-efficient approach to create 3D representations of lung tissue ranging from bronchioles over alveolar ducts and alveoli up to the ACN from more than 1 mm sample extent to a resolution of less than 1 μm. The method is based on automated image acquisition of serially sectioned epoxy resin-embedded lung tissue fixed by vascular perfusion and subsequent automated digital reconstruction and analysis of the 3D data. This efficient method may help to better understand mechanisms of vascular development and pathology of the lung.

Grothausmann R ，Knudsen L ，Ochs M ，Mühlfeld C ... -  《-》

Estimation of the number of alveolar capillaries by the Euler number (Euler-Poincaré characteristic).

The lung parenchyma provides a maximal surface area of blood-containing capillaries that are in close contact with a large surface area of the air-containing alveoli. Volume and surface area of capillaries are the classic stereological parameters to characterize the alveolar capillary network (ACN) and have provided essential structure-function information of the lung. When loss (rarefaction) or gain (angiogenesis) of capillaries occurs, these parameters may not be sufficient to provide mechanistic insight. Therefore, it would be desirable to estimate the number of capillaries, as it contains more distinct and mechanistically oriented information. Here, we present a new stereological method to estimate the number of capillary loops in the ACN. One advantage of this method is that it is independent of the shape, size, or distribution of the capillaries. We used consecutive, 1 μm-thick sections from epoxy resin-embedded material as a physical disector. The Euler-Poincaré characteristic of capillary networks can be estimated by counting the easily recognizable topological constellations of "islands," "bridges," and "holes." The total number of capillary loops in the ACN can then be calculated from the Euler-Poincaré characteristic. With the use of the established estimator of alveolar number, it is possible to obtain the mean number of capillary loops per alveolus. In conclusion, estimation of alveolar capillaries by design-based stereology is an efficient and unbiased method to characterize the ACN and may be particularly useful for studies on emphysema, pulmonary hypertension, or lung development.

Willführ A ，Brandenberger C ，Piatkowski T ，Grothausmann R ，Nyengaard JR ，Ochs M ，Mühlfeld C ... -  《-》

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

Mühlfeld C ，Weibel ER ，Hahn U ，Kummer W ，Nyengaard JR ，Ochs M ... -  《-》

Assessment of the Alveolar Capillary Network in the Postnatal Mouse Lung in 3D Using Serial Block-Face Scanning Electron Microscopy.

The alveolar capillary network (ACN) has a large surface area that provides the basis for an optimized gas exchange in the lung. It needs to adapt to morphological changes during early lung development and alveolarization. Structural alterations of the pulmonary vasculature can lead to pathological functional conditions such as in bronchopulmonary dysplasia and various other lung diseases. To understand the development of the ACN and its impact on the pathogenesis of lung diseases, methods are needed that enable comparative analyses of the complex three-dimensional structure of the ACN at different developmental stages and under pathological conditions. In this study a newborn mouse lung was imaged with serial block-face scanning electron microscopy (SBF-SEM) to investigate the ACN and its surrounding structures before the alveolarization process begins. Most parts but not all of the examined ACN contain two layers of capillaries, which were repeatedly connected with each other. A path from an arteriole to a venule was extracted and straightened to allow cross-sectional visualization of the data along the path within a plane. This allows a qualitative characterization of the structures that erythrocytes pass on their way through the ACN. One way to define regions of the ACN supplied by specific arterioles is presented and used for analyses. Pillars, possibly intussusceptive, were found in the vasculature but no specific pattern was observed in regard to parts of the saccular septa. This study provides 3D information with a resolution of about 150 nm on the microscopic structure of a newborn mouse lung and outlines some of the potentials and challenges of SBF-SEM for 3D analyses of the ACN.

Buchacker T ，Mühlfeld C ，Wrede C ，Wagner WL ，Beare R ，McCormick M ，Grothausmann R ... -  《Frontiers in Physiology》

Stereological estimates of alveolar number and size and capillary length and surface area in mice lungs.

Knust J ，Ochs M ，Gundersen HJ ，Nyengaard JR ... -  《-》