Background: Having confirmed (Scarpelli et al. 1996. Anat. Rec. 244:344-357 and 246:245-270) the discovery of intraalveolar bubbles and films as the normal anatomical infrastructure of aerated alveoli at all ages, we now address three questions. Why have these structures been so elusive? Visible in fresh lungs from the in vivo state, can they be preserved by known laboratory methods? Can they be preserved intact for study in tissue sections?
Methods: Lungs of adult rabbits and pups were examined in thorax directly from the in vivo state to confirm normal bubbles both at functional residual capacity and at maximal volume; other lungs were permitted to deflate naturally to minimal volume. The fate of bubbles in situ (either intact, transected, or diced lung tissue) and of isolated bubbles was assessed (1) during conventional histopreparative processing, (2) during inflation-deflation after degassing, (3) after drying in air, (4) during and after quick freezing in liquid N2, and (5) after preservation in fixed and stained tissue sections prepared by a new double-impregnation procedure in which glutaraldehyde-fixed tissue was preembedded in agar, dehydrated and clarified chemically, embedded in paraffin, sectioned, and stained. Control studies included both blocking of bubble formation by rinsing the air spaces with Tween 20 prior to double impregnation and preparation of normal tissue without preembedding in agar.
Results: (1) Each of the following procedures in conventional processing dislocated and disrupted bubbles and films: osmium tetroxide and glutaraldehyde:formaldehyde:tannic acid mixture fixation; chemical dehydration (70-100% ethanol) and clarification (xylene and acetone); and embedding in paraffin or epoxy resin. Transection and dicing of the tissue aggravated the untoward effects. In contrast, bubbles and films remained stable in either glutaraldehyde or formaldehyde, which, however, did not protect against the other agents. (2) Degassing destroyed all bubbles as expected; however, bubbles and films re-formed immediately with reinflation. (3) Topography of fixed bubbles and films was retained after air drying. The dry polygonal configuration reverted to spherical-oval either in saline solution or in 50% ethanol, whereas vulnerability to upgraded ethanol concentrations was unchanged. (4) Normal topography and shape appeared to be retained during quick freezing and after thawing. (5) Intraalveolar and intraductal bubbles and films were preserved and photographed in sections from tissue prepared by the double-impregnation procedure; they were not seen either when bubble formation had been blocked (double-impregnation procedure) or when preembedding in agar had been omitted.
Conclusions: (1) Whether or not fixed in glutaraldehyde or formaldehyde, preservation of intraalveolar and intraductal bubbles and films is not to be expected in tissue prepared by conventional histopreparative procedures, whereas product artifacts may be expected from bubble rupture in situ. (2) Degassing cannot be recommended for studies of alveolar structure-function interrelations because all natural bubbles are disrupted in the process, and bubble re-formation may not parallel their "natural history" in vivo. (3) Compared with glutaraldehyde or formaldehyde fixation, air drying offers no added protection against the untoward effects of conventional processing. (4) Quick-frozen tissue is equally at risk. (5) A new double-impregnation procedure does preserve bubbles and films during processing, sectioning, and staining.