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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.

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BACKGROUND: Intraalveolar bubbles and bubble films have been shown to be part of the normal alveolar architecture in vivo from birth through the first 2 days of extrauterine life of rabbit pups (Scarpelli et al., 1996a. Anat. Rec. 244:344-357). The intraluminal boundary between air-way free gas and alveolar bubbles at the level of respiratory bronchioles is established within 1 hour after birth. We now examine the lung through the rest of development, namely, 2 weeks, 1, 2, and 3 months, and adulthood. METHODS: In quick succession in anesthetized spontaneously breathing rabbits, the abdominal aorta was transected and trachea was occluded either after an end-tidal exhalation at functional residual capacity (FRC) or after volume expansion in vivo by a single inflation from FRC to 20 or 25 cm H2O pressure (V20, V25). Immediately the thorax was opened and lungs were examined (anterior, anterolateral) through a dissecting stereomicroscope while still in the chest, unperturbed (pleural surface temperature 34 degrees C). Heart and lungs were then removed en bloc and re-examined (anterior, lateral, posterior) to confirm that architecture had not changed (22-27 degrees C). After these immediate examinations, lungs were entered into one of the protocols enumerated in Results. RESULTS: Immediate examination revealed bubbles in all aerated subpleural and deep ("central") alveoli from apex to base at all ages and temperatures. Bubbles were confirmed from two views (top and tangential) and from their individual mobility in response to gentle microprobe pressure. A "common bubble" (> 30 microns to approximately 120 microns inside diameter at FRC) appeared to occupy a single alveolus, sometimes arranged in clusters and collectively accounting for approximately 84% of the total bubble population. Few "large bubbles" appeared to be intraductal. We concluded that "small bubbles" (< or = 30 microns; approximately 16% of the total population) were contracted common bubbles. The free gas-bubble film boundary of the airways was at the level of respiratory bronchioles. Subsequent protocols: (1) Common bubbles moved out of adjoining tissue following subpleural incision. Adjacent bubbles either moved into vacated spaces or into the outside liquid medium. Large bubble(s) followed common bubbles out of the tissue. Small bubbles were less mobile and distal common bubbles did not move. The sequence of bubble movement at V25 was the same. Isolated bubbles had normal surfactant content and surface tension according to "Pattle's stability ratio." Transection revealed analogous conditions in central alveoli. (2) Bubble size increased during inflation from FRC to V25. Airless spaces were aerated with bubbles during inflation. (3) The bubble surface was compressed during deflation to 81% of maximal volume (Vmax) and below, including deflation to minimal volume (Vmin). (4) Bubble/alveolar shape changed from spherical-oval to polygonal when the pleural surface dried at FRC and V25. The original shape was restored when the surface was re-wet. Dry tissue showed but did not emit bubbles when cut; re-wet tissue did. (5) Lung liquid content and volume-pressure were normal at FRC. (6) As expected, conventionally fixed, dehydrated, and embedded sections showed no bubbles. CONCLUSIONS: Bubbles and bubble films are fundamental to normal architecture of aerated alveoli at all lung volumes from birth through adulthood. As infrastructure, they sustain aeration and resist deformation. With ductal films, they may be expected to form an alveolar surface liquid (foam film) network (Scarpelli, 1988. Surfactants and the Lining of the Lung) that modulates liquid balance principally at Plateau borders. They expand and contract respectively during inflation and deflation, maintaining their closed film integrity. Films are compressed to "film collapse" in situ during deflation from volumes well above FRC to Vmin. At these volumes, intact films sustain aeration; some may disperse into t

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Air volume-pressure (VP) curves were recorded simultaneously on pairs of mature rabbit fetuses from the same litter with one member of the pair at 37 degrees C and the other at 22 degrees C. Intrasaccular bubbles, formed primarily during inflation, were assessed for stability and surface tension (gamma). Average air flow rates (dV/dt) were calculated from the VP data. In separate experiments, liquid VP curves were recorded at 37 degrees and 22 degrees C: maximal liquid V was matched to maximal air V at 37 degrees and 22 degrees C, respectively. Fetal pulmonary liquid (FPL) viscosity (eta) and density (rho) were determined by standard methods. Both the effect of temperature on lung mechanics as reported previously, and the reliability of the rabbit model were confirmed in the paired fetuses. Analysis of fluid dynamics revealed that of the six parameters relevant to initial inflation-deflation of FPL-filled lungs, liquid rho, distensibility (recoil), and gamma were not altered significantly by temperature increase from 22 degrees to 37 degrees C. Enhanced lung mechanics at 37 degrees C (including enhanced inflation at lower P, higher maximal V, increased production of intrasaccular bubbles, and higher V at end-deflation) was primarily due to lowering of FPL eta at the higher temperature which appears to have an effect by augmenting bulk liquid flow and liquid drainage. Lower eta increases bulk flow through airways directly. Consequent recruitment and distention of these conducting units effectively increases radius (r) and further enhances flow. (The ultimate "brake" to airways flow at both temperatures is counter P from gamma at air/liquid menisci.).(ABSTRACT TRUNCATED AT 250 WORDS)

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Volume-pressure (VP) curves of initial aeration of mature (0.94-0.97 term) rabbit fetuses were compared in three groups, respectively, at 37 degrees C, with maximal inflation pressure of 25 cm H2O (P25); 22 degrees C, P25; and 22 degrees C, P30. Anesthetized fetuses were delivered through uterotomy; chest was opened; trachea of fetal pulmonary liquid (FPL)-filled lungs cannulated; and lungs inflated-deflated in 5 cm H2O, 2 min steps under continuous microscopic observation. As distending pressure was increased, FPL moved peripherally with airways inflation by free gas and with saccular recruitment by free gas and bubbles. Saccular aeration continued during initial reduction of P from Pmax. At end-deflation, air was retained in saccules virtually exclusively as bubbles. Airways inflation required less P at 37 degrees C, though airways volume (V) was the same at both temperatures. Opening P was lower, and saccular aeration was larger and more rapid at 37 degrees C. The apparently higher distensibility at 37 degrees C was most likely due to temperature effects on fluid dynamics rather than on tissue elasticity. Maximal V attained during early P reduction in all groups, was total lung capacity (TLC) at 37 degrees C and less than TLC at 22 degrees C. Air retention at end-deflation, with films of near-zero surface tension, was greatest at 37 degrees C and least at 22 degrees C, P25. Lung stability, greater at 37 degrees C than at 22 degrees C, was best discriminated when V at P0 was taken into account.(ABSTRACT TRUNCATED AT 250 WORDS)

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Previous studies have suggested that passive smoking (involuntary inhalation of tobacco smoke by nonsmokers) reduces small airways function. We evaluated the exposure to passive smoking and its effects on pulmonary function and symptoms in a group of 12- to 17-year-old high school athletes (N = 209; 119 boys and 90 girls) at their annual presport participation physical examinations. A structured interview was used to assess pulmonary symptoms, personal smoking habits, and passive cigarette smoke exposure. All athletes performed forced expiratory maneuvers on a portable spirometer. We measured forced vital capacity, forced expiratory volume in 1 second, and forced expiratory flow 25% to 75% (FEF25-75). The best of three FEF25-75 measured was used. Less than 70% of predicted FEF25-75 was considered abnormal. Of the 209 athletes, 7.7% were active smokers and were excluded. Of the remaining 193 athletes, 68.4% were currently exposed to passive smoking. We found a fourfold increase in incidence of low FEF25-75 and/or cough in athletes exposed to passive smoking compared with athletes not exposed: 18 of 132 exposed athletes (13.6%) had low FEF25-75 and/or cough compared with two of 61 unexposed athletes (3.3%) who had low FEF25-75 and cough (P = .02). Boys were more frequently exposed to passive smoking than girls (74% of boys [80/108] v 61% of girls [52/85] ), but the effects were more pronounced in girls. These data show a relationship between exposure to passive smoking and early pulmonary dysfunction in young athletes. The frequent exposure to passive smoke and the high prevalence of dysfunction in this population, generally considered to be healthy, is of particular concern.

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Mature rabbit fetuses produce intrapulmonary foam at the onset of breathing at birth. Bubbles establish a minimal volume immediately and require relatively little distending pressure for their formation. Stability of bubbles that are formed during both rapid spontaneous breathing in vivo and slow inflation-deflation of excised lungs is determined by the surfactant content of fetal pulmonary fluid (FPF). Mature bubbles can be delivered at atmospheric pressure from all aerated saccules by microdissection. When observed in air-equilibrated normal saline solution (NSS), their stability with time indicates that film surface tension (gamma) is very low, i.e., near-zero. When mature FPF is replaced with NSS, stable bubble production is absent. Conversely, supplementation of immature FPF with a surfactant dispersion prior to aeration induces bubbles that are as stable (near-zero gamma) as those from mature lungs. Proper mixing of the supplement, e.g., by repeated inflation-deflation, is required for proper distribution of foam in the immature fetal saccules. From these findings, it may be concluded that bubbles establish the condition for production of near-zero gamma in situ. The latter stabilizes the lung by sustaining normal liquid transfers (Pattle theory). In addition, bubble films promote mechanical stability by providing a saccular infrastructure that resists collapse and retards surface spreading.

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Immature rabbit lungs were inflated, then deflated from the fetal pulmonary fluid (FPF)-filled state. Stereomicroscopic observation and measurement of volume change (delta V) during each pressure step and after 15 and 120 sec at each pressure revealed the following: (1) Only airways inflate from atmospheric pressure (P0) to P25. Significant time-dependency here is due to FPF flow through the narrowest airways, airways dilation and recruitment as functions of tissue and surface forces, and, perhaps, interfacial adsorption of surfactants. (2) Saccular recruitment and distention are the principal transformations from P30 to P35. Time-dependency here is the result of FPF flow and labile bubble production. (3) Time-dependency during deflation from P25 to P10 is due to diminishing influence of inflation processes and to decreasing radii of curvature at air/liquid interfaces as FPF refills the saccular air-spaces. Redistribution of air and hypophase liquid probably also play a role. (4) Deflation from P10 to P0 is determined by FPF flow through the smallest airways, interfacial forces, and recoil of previously distended airways as liquid locks are formed. Some implications are that FPF flow through the smallest airways is a gate to saccular ventilation; time-dependent processes place airspaces at risk to rupture; and different time constants of saccules and airways renders 120 sec pressure steps adequate for evaluation of the latter but not the former.

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The mechanical behavior of immature rabbit fetal lungs in situ was assessed by air and saline volume-pressure diagrams. All lungs were in their natural fetal state, i.e., filled with fetal pulmonary fluid, prior to inflation. Anatomic correlates were determined by continuous stereomicroscopic monitoring of the lungs. We found the following to be characteristic of immature lungs: (1) Tissue retractive forces are similar to adults. (2) Fetal lungs are not 'plastic' above functional residual capacity. (3) Initial aeration is by 'axial filling' in which airways are distended several times their resting size. (4) Invariably, peripheral rather than central saccules are the first to be aerated and saccules are recruited by both pressure- and time-dependent processes. (5) Pressure-dependence is related to surface forces and terminal orifice size, while time-dependent processes include orifice enlargement, liquid flow through terminal conduits, and the formation of very short-lived, labile bubbles. (6) 'Opening pressure' inflection in the VP diagram is not coincidental with, but follows the onset of saccular aeration. (7) Negative compliance at the onset of deflation is due to saccular enlargement and recruitment. (8) Hysteresis is due to tissue conformational characteristics at high pressures and air entrapment at low pressures. (9) Surface tension cannot be measured reliably from the saline and air VP diagrams.

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