Fourier transform infrared and differential scanning calorimetric studies of a surface-active material from rabbit lung.

A surface-active material with a chemical composition consistent with lung surfactant and with the ability to lower surface tension on a Wilhelmy balance to about 6 mN/m, has been isolated from rabbit pulmonary lavage. The thermotropic properties have been characterized with the techniques of Fourier transform infrared spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC). FT-IR melting curves were constructed from the temperature-dependence of the lipid CH2 symmetric stretching vibrational frequencies near 2850 cm-1. A broad gel-liquid crystal phase transition with an onset temperature of about 22 degrees C, and a completion temperature of about 38 degrees C was observed, with slight sample-to-sample variations in temperatures. A similar completion temperature was noted in DSC endotherms. Ca2+ (5-10 mM) increased the onset temperature of the lipid-melting event, and induced an ordering of surfactant and of its lipid extract at all temperatures studied. The effect on the lipids was suggestive of a Ca2+-induced phase separation caused by ion binding to phosphatidylglycerol and other acidic components. Evidence for a direct interaction between Ca2+ and the phosphate groups was suggested through small Ca2+-induced shifts in the 1090 cm-1 symmetric PO2 stretching frequency. Removal of most of the protein component from a 10:1 (lipid/protein, w/w) sample caused an ordering of the resultant lipid fractions. In contrast, removal of most of the protein component from a 20:1 sample resulted in no change in lipid order or thermotropic behavior. These observations are discussed in light of the roles played both by Ca2+ and protein in the spreading of surfactant. The power of FT-IR to acquire useful structural information from complex biological tissues is demonstrated.

Rapid in vitro tests of surfactant film formation: advantages of the Exerowa black film method.

Based on our research, the natural configuration of surfactant from birth through adulthood takes the form of intraalveolar bubbles. Thus, bubble film analysis would seem to be the specific in vitro testing method for lung surfactant. In the present study we report a battery of five in vitro tests for assessing structural and functional properties of surfactant bubbles and bubble films from hydrophobic extracts, namely, the therapeutic surfactants Survanta (SU) and Infasurf (IN) (full strength and diluted to 3 mg phospholipid/mL) and from aqueous extracts from rabbit lung lavage at 3 mg phospholipid/mL (SAM). Each substrate was assessed by: 1) Shake test: stable bubbles from SU, IN, and SAM (50/50, v/v in 95% ethanol) covered the peripheral surfaces, indicating positive response; bubble production by IN and SAM always exceeded SU; 2) Click test: bubble clicking began immediately in all preparations except for undiluted SU, in which the onset of clicking was delayed more than 40 sec; 3) Pattle's stability test: diameters of SU, IN, and SAM bubbles were unchanged for more than 20 min in aerated solution, indicating stable very low surface tension; 4) bubble generation by gas dispersion from a single capillary: full-strength concentration of SU and IN produced relatively large bubbles-bubble rate (number/min) and size were comparable; all SU bubbles rupture in < 25 min, whereas IN bubbles were stable for > 30 min; and 5) Exerowa black film method: in contrast with each of the preceding methods for studying intact bubbles, the Exerowa method focuses on the contact between bubble films and permits direct observation of film formation and determination of film structure. Stable black films were formed spontaneously by both IN (full strength and diluted) and SAM. Conversely, SU (full strength) formed no black films but stable rheological films. Diluted SU films ruptured in 50% of trials. Since methods 1, 2, and 3 were nondiscriminatory and method 4 produced unphysiologically large bubbles for most mammals, we concluded that the black film method of Exerowa is the most discriminating of the tests studied here. It provides a unique visual record of foam film formation and stability and clearly defines differences relative to both the nature and concentration of the preparations.

Temperature affects mechanics and stability during initial inflation-deflation of mature fetal lung.

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)

Fluid dynamics during initial aeration of mature fetal lung and effect of temperature.

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)

Intranasal surfactant aerosol therapy for otitis media with effusion.

OBJECTIVE: To determine the effect of surfactant alone and with other medications delivered intranasally as a metered dose inhaler (MDI) aerosol on the resolution of experimentally induced otitis media with effusion (OME). BACKGROUND: Eustachian tube dysfunction is a primary factor in the pathogenesis of OME. Intranasal surfactant via MDI has been shown in this laboratory to reduce passive opening pressure of the eustachian tube in normal gerbils and mice. STUDY DESIGN: OME was developed in 35 gerbils by transtympanic injection of 10 microg lipopolysaccharide from Klebsiella pneumoniae. Pretreatment otomicroscopy and tympanometry were performed to exclude pre-existing middle ear disease, and postinfection evaluations were performed on alternate days for a period of 30 days. Five animals received no treatment (control group); four were treated with propellant only (placebo); seven received surfactant alone; eight received surfactant and betamethasone; and six received surfactant with phenylephrine. All medications were sprayed intranasally as an aerosolized MDI and administered daily from postinfection day 2 onward. RESULTS: OME resolved after 16.0 +/- 0.44 days (mean + SD) in controls. There was no difference seen in the placebo or the surfactant with phenylephrine groups. Treatment with surfactant yielded resolution in 10.57 +/- 0.37 days; this was reduced to 8.57 +/- 0.37 days with surfactant plus betamethasone. These differences are statistically significant. There was no recurrence of OME in any group. CONCLUSION: This study demonstrates that using an aerosolized MDI surfactant with and without betamethasone decreases the duration of OME in this in vivo gerbil model.

Dosage regimens of intranasal aerosolized surfactant on otitis media with effusion in an animal model.

OBJECTIVE: To determine optimal dosage regimens of intranasal metered dose aerosolized surfactant with and without other medications in the treatment of otitis media with effusion (OME). STUDY DESIGN: Resolution of experimental OME in gerbils was determined based on otomicroscopy and tympanometry. Experimental intranasal drugs were: surfactant, surfactant with betamethasone, surfactant with phenylephrine, and a normal saline solution placebo. Medications were administered once or twice daily via a metered dose inhaler. RESULTS: For twice-daily dosing, mean days to OME resolution were 8.5 for the aerosolized surfactant, 6.3 for the surfactant with betamethasone, 18.7 for the surfactant with phenylephrine, and 16 each for control and placebo. Resolution with the once-daily dosage was longer for all conditions. Results were comparable using tympanometry. CONCLUSION: OME resolved faster than the natural course when treated with intranasal surfactant with and without steroids. Twice-daily dosing was statistically superior. SIGNIFICANCE: This study reiterates the effectiveness of OME treatment with an aerosolized synthetic surfactant with and without steroids and establishes a superior twice-daily dosage schedule.

Surface biophysics of the surface monolayer theory is incompatible with regional lung function.

The surface monolayer theory of Clements was tested on open surface films of calf lung surfactant extract in a leak-free vertical film surface balance in which alveolar area (A) changes in each lung zone were simulated in accordance with the theory. We found that: 1) physiologically necessary low surface tension (gamma), < 4 dyn/cm, was sustained only by continuous film compression ("expiration"); 2) compression from A equivalent to total lung capacity to functional residual capacity produced fleeting gamma reduction in all zones and quick reversal to high gamma with A changes that simulated tidal volume (VT) breathing at both 14 (adult) and 40 (neonatal) cpm; 3) phase differences between gamma and A axes of VT loops that indicate mixed surface film composition may be attributable to film inertia and viscoelasticity; 4) estimated alveolar retraction pressure due to gamma (P gamma) exceeds "net" transpulmonary pressure, i.e., favors alveolar collapse, under virtually all conditions of the theory in all zones; 5) return to transient, fleeting low gamma in successive VT cycles was determined by the inherent difference in compression and decompression rates, which results in exhaustion of available A in very few cycles; 6) the "sigh", which restores stable low gamma according to the theory, actually produced unstable high gamma during virtually all phases of the maneuver. In contrast, closed bubble films of the surfactant were structurally stable and produce stable near 0 gamma and P gamma.

Whole cell potassium currents in fetal rat alveolar type II cells cultured on Matrigel matrix.

Patch-clamp studies were performed on fetal rat alveolar type II cells isolated at 19 days of gestation and cultured on either plastic for 7 days or Matrigel matrix (40-50 microliters/cm2) for 10 days. Before study, cells cultured on Matrigel matrix were dissociated from alveolar-like structures with enzymes, replated, and washed with cold buffer at a constant flow rate to remove residual gel. This wash significantly improved obtaining of successful seals. Potassium current-voltage relationships and maximum whole cell K+ conductance (normalized to membrane capacitance) were significantly changed with time in cells cultured on plastic, but no significant change occurred in cells cultured on Matrigel matrix. Application of 20 mM tetraet hyl ammonium, 2mM 4-aminopyridine, and 5mM BaCl2 significantly inhibited K+ currents, showing differences in channel sensitivity to these agents and a voltage-dependent blockage between culture groups or with time in culture. To conclude, we have developed a new method by which epithelial cells cultured on Matrigel matrix can be successfully studied with the use of patch-clamp techniques. Furthermore, these studies show that fetal type II cells have voltage-activated K+ channels and that channel density and their sensitivity to channel blockers are modulated by the substratum on which the cells are cultured.

Surface chemistry of binary mixtures of phospholipids in monolayers. Infrared studies of surface composition at varying surface pressures in a pulmonary surfactant model system.

Phospholipid monomolecular films at the air/water interface were studied using Langmuir-Blodgett (L-B) surface chemistry, 31P NMR spectroscopy, and infrared (IR) spectroscopy. These monolayers were composed of binary mixtures of acyl chain perdeuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (i.e., DPPC-d62) with 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (i.e., DPPG). This particular PC-PG binary mixture was chosen for study since this lipid system has been used as a model for pulmonary surfactant, especially in conjunction with the so-called "squeezing-out" hypothesis of pulmonary mechanics. This theory predicts that upon successive compression-expansion cycles, a surfactant surface film will reorganize to exclude all components except DPPC, thus resulting in a stable, low surface tension film. Several general results were obtained from these experiments. First, we have developed a combined spectroscopic assay using high-resolution 31P NMR spectroscopy in combination with the C-H and C-D vibrational intensities obtained from the IR spectroscopy of binary mixtures in which one component is acyl chain perdeuterated. Using attenuated total reflectance IR spectroscopy of transferred L-B films, this combined spectroscopic approach allows us to quantitatively describe the fractional composition of each component in the binary monomolecular film. Second, when these methods are applied to transferred monolayer films of DPPC-d62 and DPPG (at an initial PC:PG mole ratio of 7:1), we find no evidence for a "squeezing-out" of the DPPG monolayer component at high surface pressure resulting in an enrichment of the DPPC component in the transferred monolayer film.(ABSTRACT TRUNCATED AT 250 WORDS)

Ventilation and pulmonary mechanics during high-frequency oscillation of immature lungs at birth.

Ventilation was initiated and maintained by high-frequency oscillation (HFO) in nine immature lambs not expected to survive for more than a few minutes out of the uterus. They were delivered to HFO (18 to 20 Hz) and ventilated only with air at the setting that yielded optimal blood gases and pH. Once removed from HFO, cardiorespiratory arrest occurred within minutes despite the return of previously inhibited natural inspiratory efforts. At autopsy, the lungs were liquid-filled (i.e., fetal). Thus, HFO had maintained viability without promoting either lung stability or fetal pulmonary fluid (FPF) absorption. Additional experiments on excised immature lungs showed that aeration is determined by distending pressure, which is regulated by the flow through the oscillator circuit and monitored most accurately as transpulmonary pressure (TPP). Once air-entry has begun, alveolar recruitment and negative compliance establish the limits of tolerable lung expansion via TPP. Within these limits, the immature lung can be ventilated effectively with air. However, since these lungs do not produce intrapulmonary foam, lung stability and FPF absorption are not effected. Complications of HFO (including pneumothorax, decreased arterial pressure and heart rate, and metabolic acidemia) can be related to overdistention of the lungs.

Fourier-transform infrared spectroscopy studies of lipid/protein interaction in pulmonary surfactant.

The thermotropic behavior of intact bovine lung surfactant and its hydrophobic extract has been monitored via the temperature dependence of the 2850 cm-1 phospholipid acyl chain CH2 symmetric stretching frequencies in the IR spectrum. A broad, reversible, melting event was noted from about 15 to 40 degrees C in both the lipid extract and the native surfactant. Slight protein-induced disordering of the lipid acyl chains was evident. The melting event was confirmed by differential scanning calorimetry. The major surfactant protein, a 30-36-kDa class of glycoprotein (SP-A), has been isolated from bovine lung lavage and purified by affinity chromatography. SP-A was reconstituted into a binary lipid mixture of acyl chain perdeuterated dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC-d62/DPPG, 85:15 w/w), a ratio which approximates that in surfactant. Use of DPPC-d62 permitted the FT-IR determination of the effect of protein on the thermotropic behavior of individual phospholipids in the binary mixture. High levels of SP-A induced an ordering of the phospholipids, as shown by an increase in the transition temperature of DPPC-d62 compared to the lipid model. In contrast, a mixture of the other surfactant proteins induced a progressive disordering of the phospholipids and disruption of the cooperativity of the melting event. Transition widths of about 3 degrees, 9 degrees, and 27 degrees were noted for protein:lipid ratios of 0, 1:1, and 2:1 (w/w), respectively. Possible roles for the various proteins in surfactant function are discussed in light of these data.

Fourier transform infrared studies of secondary structure and orientation of pulmonary surfactant SP-C and its effect on the dynamic surface properties of phospholipids.

SP-C, a highly hydrophobic, 3.7-kDa protein constituent of lung surfactant, has been isolated from bovine lung lavage, purified, and reconstituted into binary lipid mixtures of 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylglycerol (DPPG). Fourier transform infrared (FT-IR) spectroscopy has been applied to examine SP-C secondary structure, the average orientation of alpha-helical segments relative to the bilayer normal in membrane films, and the effect of protein on the thermotropic properties of the phospholipid acyl chains. In addition, dynamic surface measurements were made on phospholipid films at the A/W interface in the presence and absence of SP-C. SP-C (0.5 mol %) was found to possess about 60% alpha-helical secondary structure in lipid vesicles. Higher levels (1.5 mol %) of SP-C resulted in a slight increase of beta-forms, possibly resulting from protein aggregation. The helical segments exhibited an average angle of orientation of about 24 degrees with respect to the bilayer normal, suggesting a trans-bilayer orientation of the peptide. The observation that 70% of the peptide bond hydrogens are hard to exchange in D2O further reflects the hydrophobic nature of the molecule. SP-C produced little effect on the thermotropic properties of the binary lipid mixture, as measured from acyl chain C-H and C-D stretching frequencies. However, the presence of 1 mol % protein markedly reduced the viscance and increased the elasticity of surface films suggesting a mechanism by which SP-C facilitates the spreading of phospholipids on an aqueous surface. The possible physiological consequences of these observations are discussed.

Intraalveolar bubbles and bubble films. I. formation and development during the first 48 hours of extrauterine life in rabbits.

BACKGROUND: Aeration of mature lungs at birth depends on formation of intraalveolar bubbles and bubble films (Scarpelli 1978. Pediatr. Res., 12:1070-1076). Bubbles establish immediately structural stability and pulmonary gas exchange. Given that air spaces are cleared in minutes of fetal liquid (the initial substrate for bubble formation), in formation possible beyond this period? If so, is this related to early development of pulmonary function and structure? METHODS: Mature, spontaneously breathing rabbit pups at 1-10 min and 1, 3, 8, 24, and 48 h after vaginal birth were anesthetized, trachea was occluded at "resting volume" (approximately functional residual capacity), and lungs were rapidly exposed to preserve in vivo intrapulmonary status. The entire lung was examined by stereomicroscopy. Other determinations included resting volume, lung wet weight, volume-pressure curves, histological sections, lung dry weight, tissue hydroxyproline (OH-Pro), and lavage phospholipids (PL). Bubble mobility in situ was tested. Bubbles were released into bathing liquid by incision of peripheral units and monitored over time. RESULTS: Pup activity and gross appearance of the lungs, together with septal thinning, secondary septal development, clearance of intraluminal liquid, increasing tissue OH-Pro, and PL distribution indicated normal postnatal development. Each aerated unit examined at resting volume (all lobes, all ages) contained intraalveolar bubbles. Transition to free gas exclusively in conducting airways and bubbles/bubble films in peripheral gas exchange units occurred within 1 h. Bubbles appeared to be exclusively within alveoli at 4 h and thereafter. Bubbles persisted and new bubbles were formed during subsequent inflation to maximal volume and deflation to atmospheric pressure (P0). Volume of intact lungs at P0 was maintained by the counterforce of rigid bubble films against tissue retraction. When bubbles were released either at resting volume or at P0, the bubble-free loci became airless. Constant size and stability of released bubbles support preferential incorporation of surfactants into bubble films and constant "near-zero surface tension" (Scarpelli 1978. Pediatr. Res., 12:1070-1076). CONCLUSIONS: We show the ubiquitous presence of intraalveolar bubbles and bubble films in vivo throughout the first 48 h of postnatal life. Bubble film rigidity sustains aeration and prevents collapse, while low surface tension of the films facilitate liquid removal from the air spaces. Bubbles in situ are stable and, within apparent limits, mobile; after birth they are quickly restricted to the alveolar spaces, leaving airways bubble free.

Acute responses to phosgene inhalation and selected corrective measures (including surfactant).

We exposed 22 mongrel dogs to 94 ppm phosgene for 20 min from a non-rebreathing system. We expressed exposure to phosgene as ppm X VI X min X kg 1, i.e., the amount of gas containing a known phosgene concentration that was actually inhaled per min standardized to body weight, the "exposure index" (EI). In contrast, the conventional expression of exposure, i.e., ppm X min, fails to take volume inhaled (VI) and body weight into account. Five dogs received no intervention and served as controls. Fourteen dogs received basic therapy of oral cortisone (40 mg/kg) and NaHCO3 (3 mEq/kg) plus 100% O2 (FiO2 = 1.0) for 30 min after the exposure period. These animals were further divided according to the following selected additional therapies, which were started 30 min after exposure: Theophylline, 5 mg/kg iv for 20 min followed by 1 mg/kg/hr for 70 min (n = 5). Three dogs of this group were given a trial of 5 cm H2O expiratory resistance during the period of basic therapy. Because of the untoward response, expiratory resistance was discontinued and not used in other experiments. PGE2-hi, [1 microgram/kg/min] iv for 90 min (n = 3). PGE1-lo, [0.04 microgram/kg/min] iv for 90 min (n = 3). Atropine, 0.5 mg/kg iv at 30 and 50 min after exposure (n = 3). Three dogs [group 5] received oral cortisone and NaHCO3 plus inhaled supplementary surfactant, 2.7 mg/min ultrasonically nebulized (FiO2 = 0.5; phosphate buffer), for 30 min after exposure. All treated dogs, groups [1] through [4] and the surfactant group [5], received cortisone (40 mg/kg/hr iv), NaHCO3 to correct base deficit, and O2 to correct hypoxemia from 30 min to 120 min after exposure. Because of its clearly beneficial effect in group [1], theophylline was also given to all other treatment groups during this period. At the end of the study, all lungs were excised, examined and prepared for light microscopy. We found that EI, which varied among subjects because of spontaneous variations of VI during exposure, correlated significantly with the changes in base deficit induced by phosgene inhalation. We also found that the change in minute ventilation, delta VI X kg-1, correlated significantly with changes in lung compliance, peak flow and base deficit. Evaluation of the various therapeutic modalities revealed the following: Immediate therapy with O2 is vital and and FiO2 of 0.4 to 0.5 is sufficient.(ABSTRACT TRUNCATED AT 400 WORDS)

Surface tension of therapeutic surfactants (exosurf neonatal, infasurf, and survanta) as evaluated by standard methods and criteria.

Three commercial preparations for the treatment of neonatal respiratory distress syndrome (NRDS), Exosurf Neonatal (EX), Infasurf (IN), and Survanta (SU), were studied at 37 degrees C in both a pulsating bubble surfactometer and a vertical film surface balance. "Static" characteristics of adsorbed films were assessed with "bubble" at maximum radius (Rmax) and at minimum radius (Rmin). Adsorption time was time to stable film formation (gamma equilibrium). In dynamic experiments, the bubble was cycled (compressed-decompressed) between Rmax and Rmin at 20, 40, and 80 cpm. Spread films were cycled in the surface balance between maximum area (Amax) and minimum area (A(min)) at 1.5 cpm. In all experiments, maximum surface tension (gamma max) coincided with Rmax or Amax and gamma min with Rmin or A(min). All trials were continued until gamma max and gamma min were reproducible. Data were evaluated according to standard criteria for normal function both in vivo and in vitro of lipid mixtures (EX) or natural surfactants (IN, SU) prepared for treatment of NRDS. All preparations failed two of the four criteria, adsorption in the time of a deep breath and maintenance of stable, low gamma. Adsorption required 10 to 20 seconds and stable gamma was achieved only at equilibrium gamma, > 17 mN/m. IN and SU conformed in general to two criteria, gamma approaching zero on compression and low surface compressibility (< 0.09 m/mN), whereas spontaneously formed films of EX did not. EX was idiosyncratic in that these two criteria were met only after an apparent change of film conformation had been effected, possibly related to elimination of preparation additives from the surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraalveolar bubbles and bubble films: II. Formation in vivo through adulthood.

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

A direct test of the "squeeze-out" hypothesis of lung surfactant function. External reflection FT-IR at the air/water interface.

The current theory of pulmonary surfactant function requires that very low surface tension be achieved and maintained in the alveolar surface film during compression (expiration). To effect this condition, it has been hypothesized that the unsaturated and/or fluid components of surfactant are selectively excluded or "squeezed out" from mixed monolayers containing both saturated and unsaturated phospholipids, leaving a surface film of essentially pure 1,2-dipalmitoylphosphatidylcholine (DPPC). External reflection Fourier transform infrared (FT-IR) spectroscopy has been employed to quantitatively test this hypothesis. Mixed monolayer films of acyl chain-perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC-d62) with 1,2-dioleoylphosphatidylglycerol (DOPG), 1-palmitoyl, 2-oleoylPG (POPG), 1,2-dipalmitoylPG (DPPG) were examined in situ at the air/water interface as a function of surface pressure. The relative intensities of CD2 (CH2) stretching vibrations of the deuterated (proteated) components permitted quantitative determination of the relative concentrations of each in the film. For 7:1 (mol:mol) mixtures of DPPC-d62/DOPG, progressive, selective squeeze out of up to about 90% of the PG component is observed over a range of surface pressures from about 51 to 68 mN/m. The extent of maximal PG squeeze out was reduced to 61% for a 7:1 (mol:mol) mixture of DPPC-d62/POPG. This phenomenon, which is at least partially reversible, appears to require relatively high rates of film compression. Squeeze out was reduced (< 20%) for 7:1 (mol:mol) mixtures of DPPC-d62/DPPG or for 7:3 mixtures of DPPC-d62/POPG. Squeeze out requires that the lipid mixture achieve surface pressures greater than about 50-60 mN/m along with unsaturation (or at least conformational disorder) in the acyl chains of the non-DPPC component.(ABSTRACT TRUNCATED AT 250 WORDS)

External reflection absorption infrared spectroscopy study of lung surfactant proteins SP-B and SP-C in phospholipid monolayers at the air/water interface.

The interactions of the hydrophobic pulmonary surfactant proteins SP-B and SP-C with 1,2-dipalmitoylphosphatidylcholine in mixed, spread monolayer films have been studied in situ at the air/water interface with the technique of external reflection absorption infrared spectroscopy (IRRAS). SP-C has a mostly alpha-helical secondary structure both in the pure state and in the presence of lipids, whereas SP-B secondary structure is a mixture of alpha-helical and disordered forms. When films of SP-B/1,2-dipalmitoylphosphatidylcholine are compressed to surface pressures (pi) greater than approximately 40-43 mN/m, the protein is partially (15-35%) excluded from the surface, as measured by intensity ratios of the peptide bond amide l/lipid C==O stretching vibrations. The extent of exclusion increases as the protein/lipid ratio in the film increases. In contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids. The amide l peak of SP-C becomes asymmetric as a result of the formation of intermolecular sheet structures (1615-1630 cm-1) suggestive of peptide aggregation. The power of the IRRAS experiment for determination of film composition and molecular structure, i.e., as a direct test of the squeeze-out hypothesis of pulmonary surfactant function, is evident from this work.

Intrasaccular bubbles of near-zero surface tension stabilize neonatal lungs.

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.