Fibrous Network of the Lung and its Change with Age

Abstract

The remarkable elastic behaviour of the lung entails both continuous tissue stress and a cyclic stress produced by the fluctuating difference between intrathoracic and intra-alveolar pressures throughout the entire span of life. In spite of these potentially disruptive forces, lung structure is preserved by the fibrous connective-tissue proteins-collagen, elastin, and reticulin. These extracellular proteins are characterized by their insolubility, resistance to destruction, and high tensile strength. Collagen and elastin both exhibit elastic behaviour, but great extensibility is a property only of elastin. The most reasonable assumption is that the elastic fibres, therefore, are principally responsible for the elastic behaviour of the lung tissue, but sure proof is lacking. Recently, Carton, Dainauskas, Tews, and Hass (1960) and Wright (1961) have described the nature of the network of elastic tissue in the lung. We sought to describe more fully the roles of collagenous and elastic fibres in the terminal air spaces of the lung, and thus their respective contributions to tissue elasticity and the general elastic performance of the lung. As the study developed, it seemed important to measure the amounts of collagen and elastin in the lung parenchyma and pleura. These findings have been correlated with the age of the subjects and interpreted according to knowledge about tissue elasticity. METHODS The study was based on 36 human lungs taken from patients who had died of a variety of diseases. No lungs were taken from patients with a history of cough, sputum production, dyspnoea, or chronic pulmonary disease, or from patients whose clinical history was incomplete. Of these 36, 27 lungs were dried in an inflated state by the extraction process using dilute (0-1 N) sodium hydroxide solution (Pierce, Hocott, and Ebert, 1959). Nine more lungs were also dried in the inflated state but were fixed with formalin vapour. The advantage of the alkaline extraction method is that it permits the morphology of the lung 1 Supported in part by grants HTS5333 and HE04031 from the U.S. Public Health Service to be examined and also the quantities of collagen and elastin to be measured. The lungs were cut into thin slices and examined grossly and with a stereomicroscope. Slices of tissue were variously stained (Lillie, 1954) but not embedded. Elastic tissue was stained by the Taenzer-Unna acid-orcein method (in absolute ethanol); Van Gieson's picric acid-acid fuchsin mixture was used to stain collagen; and Fraenkel's method of using orcein and indigo-carmine was employed to stain both collagen and elastin in the same slice. All slices were rapidly dehydrated in alcohol and then in ether, and dried in vacuo to prevent collapse. This procedure enabled one to study the structure of the alveoli, alveolar ducts, and respiratory bronchioles as well as to visualize the relationship of the fibres in three dimensions. Preparations extracted with sodium hydroxide provided a more clearly defined fibrous network than did the formalin-vapour preparations. Thin slices of the alkali-prepared lung tissue were dissected under the stereomicroscope, and the large blood vessels, bronchi, septa, and pleura were removed from the walls of the alveoli, alveolar ducts, and respiratory bronchioles. It was impossible to remove all vessels less than 50 microns in diameter completely, but all non-respiratory bronchioles and all vessels greater than 50 microns in diameter were entirely removed. The final preparations consisted of only the walls of the parenchymal air spaces together with very small vessels. Approximately 200 mg. of this light fluffy material was used for analysis. Collagen and elastin were measured by the method of Lowry, Gilligan, and Katersky (1941). Since the entire lung had been exposed to sodium hydroxide solution for several days, the samples were immersed initially in 0-1 N sodium hydroxide solution for only two hours at room temperature. The samples were then neutralized with hydrochloric acid, washed free of chloride, and put in an autoclave in water for six hours at 30 p.s.i. to hydrolyse collagen to gelatin. The insoluble residue was washed with water, and gelatin was recovered from the solution by evaporation. Crude elastin was prepared by treating the residue from the autoclaved sample with 0-1 N sodium hydroxide for 30 min. at 100° C. After washing, the material was dried in a vacuum desiccator. The crude elastin was corrected for impurity on the basis of nitrogen analyses (Dumas) performed in duplicate. The mean difference between the duplicate results was 0-8%. 469 2N