Structure-function studies of blood and air capillaries in chicken lung using 3D electron microscopy

Abstract

Avian pulmonary capillaries differ from those of mammals in three important ways. The blood-gas barrier is much thinner, it is more uniform in thickness, and the capillaries are far more rigid when their transmural pressure is altered. The thinness of the barrier is surprising because it predisposes the capillaries to stress failure. A possible mechanism for these differences is that avian pulmonary capillaries, unlike mammalian, are supported from the outside by air capillaries, but the details of the support are poorly understood. To clarify this we studied the blood and air capillaries in chicken lung using transmission electron microscopy (EM) and two relatively new techniques that allow 3D visualization: electron tomography and serial block-face scanning EM. These studies show that the pulmonary capillaries are flanked by epithelial bridges composed of two extremely thin epithelial cells with large surface areas. The junctions of the bridges with the capillary walls show thickening of the epithelial cells and an accumulation of extracellular matrix. Collapse of the pulmonary capillaries when the pressure outside them is increased is apparently prevented by the guy wire-like action of the epithelial bridges. The enlarged junctions between the bridges and the walls could provide a mechanism that limits the hoop stress in the capillary walls when the pressure inside them is increased. The support of the pulmonary capillaries may also be explained by an interdependence mechanism whereby the capillaries are linked to a rigid assemblage of air capillaries. These EM studies show the supporting structures in greater detail than has previously been possible, particularly in 3D, and they allow a more complete analysis of the mechanical forces affecting avian pulmonary capillaries.

Fig. 1

A. Pulmonary capillary with three epithelial bridges linking it to surrounding blood capillaries. The expansions of the bridges at the junctions with the capillaries can be seen and also a nucleated red blood cell. ac, air capillary; bc, blood capillary; ep, epithelial bridge; er, erythrocyte. This is a screenshot from the video clip submitted as Supplementary Material SM1. B. High-power EM showing the detail of an epithelial bridge. The two extremely thin epithelial cells are clearly seen and both cell membranes of each can be identified. There is a small amount of matrix material between the cells. ep, epithelial cell. C. Another example of an epithelial bridge. Part of the bridge is very thin as in Fig. 1B but other parts show thickening of one of one of the epithelial cells. This may be because the plane of the section is not exactly at right angles to the plane of the cells. ep, epithelial cell.

Fig. 2

A. High-power EM of a junction of an epithelial bridge with a capillary wall. Marked enlargement of one of the epithelial cells is well shown. This section of the cell clearly shows extremely small circular inclusions that have a diameter of approximately 20 nm. These may be microtubules. The space between the two epithelial cells making up the bridge is contiguous with extracellular matrix of the capillary wall. ecm, extracellular matrix; en, endothelial cell; ep, epithelial cell; er, erythrocyte; mt, microtubule; pl, plasma. B. Another example of a junction between them an epithelial bridge and a capillary wall. Again there is some enlargement of the epithelial cells near the junction. The small circular inclusions referred to in relation to Fig. 2A are just visible. Also the space between the two epithelial cells is contiguous with extracellular matrix of the capillary wall. ecm, extracellular matrix; en, endothelial cell; ep, epithelial cell; pl, plasma. C. Another example of a junction showing the continuation of the extracellular matrix of the capillary wall into the space between the two epithelial cells of the bridge. This section was cut diagonally which explains the thickness of the extracellular matrix layer. ep, epithelial cell; mt, microtubule.

Fig. 3

Epithelial bridge connecting two pulmonary capillaries. The ribbon is 1 µm wide because this was the thickness of the tissue section but of course the bridge is much wider. The expansion of the epithelial cells at the junctions with the capillary walls is also shown. ep, epithelial bridge; bc, blood capillary. This is a screenshot from the video clip submitted as Supplementary Material SM2.

Fig. 4

A. Plane view of an epithelial plate which is one of the two cells making up the bridge. Note that it is connected to the surrounding pulmonary capillaries around its complete perimeter. The epithelial bridge is so thin that it is possible to see structures on the other side of the plate in the video clip. ep, epithelial plate; bc, blood capillary. B. Side view of an epithelial bridge showing the two epithelial plates. For this view the capillaries were removed by the operator using appropriate software. epi pl, epithelial plate. Both Figs. 4A and 4B are from the video clip submitted as Supplementary Material SM3.

Fig. 5

Screen short from the video clip submitted as Supplementary Material SM4. This shows the network of pulmonary capillaries with the clear spaces formed by the air capillaries between them.

Fig. 6

Screenshot from the video clip submitted as Supplementary Material SM5. The blood and air capillaries can be seen with the epithelial bridges. ac, air capillary; bc, blood capillary; ep, epithelial bridge.

Fig. 7

A. Diagram to show how the epithelial bridges could exert tension on the blood capillaries and prevent their collapse. The direction of the tension is indicated by the arrows. The bridges might also resist enlargement of the capillaries thus reducing the hoop stress in the wall. Modified from West (2009). B. Diagram showing how the junctions between the epithelial cells and the capillary wall could reduce the wall hoop stress just as the iron hoops do the same around a barrel of beer. In this diagram, the junction of the epithelial cell with the capillary wall runs around the circumference of a capillary. In Figure 7A, the junction is shown running along the direction of the axis of the capillary. These drawing show the two extremes of directions. In practice, the junctions presumably have a random orientation.