INTRAALVEOLAR BUBBLES AND BUBBLE-FILMS .3. VULNERABILITY AND PRESERVATION IN THE LABORATORY

Background: Having confirmed (Scarpelli et al, 1996, Anat. Rec. 244,-3 44-357 and 246:245-270) the discovery of intraalveolar bubbles and fil ms as the normal anatomical infrastructure of aerated alveoli at all a ges, we now address three questions, Why have these structures been so elusive? Visible in fresh lungs from the in vivo state, can they be p reserved by known laboratory methods? Can they be preserved intact for study in tissue sections? Methods: Lungs of adult rabbits and pups we re examined in thorax directly from the in vivo state to confirm norma l 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 tis sue) and of isolated bubbles was assessed (1) during conventional hist oprepapative processing; (2) during inflation-deflation after degassin g (3) after drying in air, (4) during and after quick freezing in liqu id N-2, and (5) after preservation in fixed and stained tissue section s prepared by a new double impregnation procedure in which glutaraldeh yde-fixed tissue was preembedded in agar, dehydrated and clarified che mically, 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 followin g procedures ill conventional processing dislocated and disrupted bubb les and films: osmium tetroxide and glutaraldehyde:formaldehyde:tannic acid mixture fixation; chemical dehydration (70-100% ethanol) and cla rification (xylene and acetone); and embedding in paraffin or epoxy re sin, Transection and dicing of the tissue aggravated the untoward effe cts, In contrast, bubbles and films remained stable in either glutaral dehyde or formaldehyde, which, however, did not protect against the ot her agents, (2) Degassing destroyed all bubbles as expected; however, bubbles and films re-formed immediately with reinflation. (3) Topograp hy of fixed bubbles and films was retained after air drying, The dry p olygonal configuration reverted to spherical-oval either in saline sol ution or in 50% ethanol, whereas vulnerability to upgraded ethanol con centrations was unchanged, (4) Normal topography and shape appeared to be retained during quick freezing and after thawing. (5) Intraalveola r and intraductal bubbles and films were preserved and photographed in sections from tissue prepared by the double-impregnation procedure; t hey were not seen either when bubble formation had been blocked (doubl e-impregnation procedure) or when preembedding in agar had been omitte d. Conclusions: (1) Whether or not fixed in glutaraldehyde or formalde hyde, preservation of intraalveolar and intraductal bubbles and films is not to be expected in tissue prepared by conventional histopreparat ive procedures, whereas product artifacts may be expected from bubble rupture in situ. (2) Degassing cannot be recommended for studies of al veolar structure-function interrelations because all natural bubbles a re 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 tiss ue is equally at risk. (5) A new double-impregnation procedure does pr eserve bubbles and films during processing, sectioning, and staining. (C) 1997 Wiley-Liss, Inc.