Immunofluorescence study and morphometric analysis of collagen-IV in the exchange tissue of the quail (Coturnix coturnix) lung by confocal laser scanning microscopy

Collagen type-IV (Col-IV) is the principal basement membrane structural component of the blood-gas barrier (BGB) to which the strength, i.e., the structural integrity, of the profoundly thin tissue barrier of the lung has been attributed. In this study, Col-IV was immunolocalized and the spatial organization, and relative volume density in the exchange tissues (ETs) of the lung of an adult common quail (Coturnix coturnix) was determined. Formalin-fixed paraffin-embedded lung sections were immunolabeled with anti-collagen IV monoclonal primary antibody and detected using Alexa Fluor 488-conjugated secondary antibody, prior to confocal laser scanning microscopy. Three-dimensional reconstructions were prepared from the acquired image datasets to allow visualization of the spatial arrangement of Col-IV in the ETs. The volume densities of Col-IV in the BGB, the epithelial-epithelial cell connections, and the blood capillary-blood capillary connections were determined by the stereological method of point-counting. Three-dimensional reconstructions showed heterogeneous, vast distribution of Col-IV within the BGB. In the parabronchial ETs, Col-IV continues centripetally, i.e., inwards, to connect to the atrial smooth muscles. The structural feature shows existence of a functional continuum that may strengthen the parabronchus, contributing to the rigidity of the air- and the blood capillaries. The high-volume density of Col-IV in the ETs (∼20%) of the lungs of the investigated adult common quail indicates its significance as a structural component. The abundance and the organization of Col-IV in the ETs underpins its importance in stabilizing the lung histo-architecture, particularly the exceptionally thin BGB. The findings provide insights that may inform on optimal biomimetic designs of artificial gas-exchange membranes.