Anisotropic Nature of Mouse Lung Parenchyma

Abstract

Lung parenchyma is normally considered to be isotropic, that is, its properties do not depend upon specific preferential directions. The assumption of isotropy is important for both modeling of lung mechanical properties and quantitative histologic measurements. This assumption, however, has not been previously examined at the microscopic level, in part because of the difficulty in large lungs of obtaining sufficient numbers of small samples of tissue while maintaining the spatial orientation. In the mouse, however, this difficulty is minimized. We evaluated the parenchymal isotropy in mouse lungs by quantifying the mean airspace chord lengths (Lm) from high-resolution histology of complete sections surrounded by an intact continuous visceral pleural membrane. We partitioned this lung into 5 isolated regions, defined by the distance from the visceral pleura. To further evaluate the isotropy, we also measured Lm in two orthogonal spatial directions with respect to the section orientation, and varied the sample line spacing from 3 to 280 μm. Results show a striking degree of parenchymal anisotropy in normal mouse lungs. The Lm was significantly greater when grid lines were parallel to the ventral–dorsal axis of the tissue. In addition the Lm was significantly smaller within 300 μm of the visceral pleura. Whether this anisotropy results from intrinsic structural factors or from nonuniform shrinkage during conventional tissue processing is uncertain, but it should be considered when interpreting quantitative morphometric measurements made in the mouse lung.

Appendix: Measurement of Lm with ImageJ

This is the plugin macro for ImageJ that will logically combine an image called “Section 2U.tif” on the desktop, with an image called “Lm analysis grid.tif” consisting of a grid of parallel lines located in the Documents folder. The data window will list the length of all the chords that run through open space. Chords that hit the edge of the camera rectangle are not counted.

• saveAs(“Tiff”, “/Users/physiologydivision/Desktop/ Section 2u.tif “);

• run(“Set Scale...”, “distance=0.680 known=1 pixel=1 unit=μm global”);

• open(“/Documents/Lm analysis grid.tif “);

• run(“Threshold”);

• run(“Invert”);

• imageCalculator(“AND create”, “Section 2u.tif”,” Lm analysis grid.tif “);

• //run(“Image Calculator...”, “image1= Section 2U.tif operation=AND image2=[ Lm analysis grid.tif.tif] create”);

• run(“Threshold”, “stack”);

• run(“Set Measurements...”, “area standard perimeter feret’s redirect=None decimal=2”);

• run(“Analyze Particles...”, “minimum=1 maximum=999 bins=20 show=Outlines display exclude size clear record stack”);