Specialized Histomorphic Techniques

Following euthanasia, lungs are inflation-fixed at 25 cm of water pressure with 4% paraformaldehyde in PBS for 1 min. The trachea is ligated, and the excised lungs and heart allowed to equilibrate in cold fixative. Lung and heart volumes are determined by fluid displacement. Each lobe is measured along its longest axis, bisected perpendicularly to the long axis, and processed into paraffin blocks. Five-micrometer sections are cut in series throughout the length of each lobe and stained with hematoxylin and eosin, Masson's trichrome stain for collagen, or orcein for elastin. Morphometric measurements are performed on mice at various ages. The overall proportion (% fractional area) of respiratory parenchyma and airspace is determined by using a point counting method, video images, and specialized software. Pressure-volume curves are generated by inflating lungs in 75-pL increments every 10 sec to a maximum pressure of 28 cm of water and then deflated. Pressure-volume curves are measured as lung volumes (mL/kg) at 10, 5, and 0 cm of water during the deflation curve.

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