A comparison of airway pressures for inflation fixation of developing mouse lungs for stereological analyses

The morphometric analysis of lung structure using the principles of stereology has emerged as a powerful tool to describe the structural changes in lung architecture that accompany the development of lung disease that is experimentally modelled in adult mice. These stereological principles are now being applied to the study of the evolution of the lung architecture over the course of prenatal and postnatal lung development in mouse neonates and adolescents. The immature lung is structurally and functionally distinct from the adult lung, and has a smaller volume than does the adult lung. These differences have raised concerns about whether the inflation fixation of neonatal mouse lungs with the airway pressure (P(aw)) used for the inflation fixation of adult mouse lungs may cause distortion of the neonatal mouse lung structure, leading to the generation of artefacts in subsequent analyses. The objective of this study was to examine the impact of a P(aw) of 10, 20 and 30 cmH(2)O on the estimation of lung volumes and stereologically assessed parameters that describe the lung structure in developing mouse lungs. The data presented demonstrate that low P(aw) (10 cmH(2)O) leads to heterogeneity in the unfolding of alveolar structures within the lungs, and that high P(aw) (30 cmH(2)O) leads to an overestimation of the lung volume, and thus, affects the estimation of volume-dependent parameters, such as total alveoli number and gas-exchange surface area. Thus, these data support the use of a P(aw) of 20 cmH(2)O for inflation fixation in morphometric studies on neonatal mouse lungs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00418-020-01951-0.