Alterations of the pulmonary vasculature are an important feature of human lung diseases such as chronic obstructive pulmonary disease, pulmonary hypertension, and bronchopulmonary dysplasia. Experimental studies to investigate the pathogenesis or a therapeutic intervention in animal models of these diseases often require robust, meaningful, and efficient morphometric data that allow for appropriate statistical testing. The gold standard for obtaining such data is design-based stereology. However, certain morphological characteristics of the pulmonary vasculature make the implementation of stereological methods challenging. For example, the alveolar capillary network functions according to the sheet flow principle, thus making unbiased length estimations impossible and requiring other strategies to obtain mechanistic morphometric data. Another example is the location of pathological changes along the branches of the vascular tree. For developmental defects like in bronchopulmonary dysplasia or for pulmonary hypertension, it is important to know whether certain segments of the vascular tree are preferentially altered. This cannot be overcome by traditional stereological methods but requires the combination of a three-dimensional data set and stereology. The present review aims at highlighting the great potential while discussing the major challenges (such as time consumption and data volume) of this combined approach. We hope to raise interest in the potential of this approach and thus stimulate solutions to overcome the existing challenges.

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.