Pulmonary surfactant in birds: coping with surface tension in a tubular lung

As birds have tubular lungs that do not contain alveoli, avian surfactant predominantly functions to maintain airflow in tubes rather than to prevent alveolar collapse. Consequently, we have evaluated structural, biochemical, and functional parameters of avian surfactant as a model for airway surfactant in the mammalian lung. Surfactant was isolated from duck, chicken, and pig lung lavage fluid by differential centrifugation. Electron microscopy revealed a uniform surfactant layer within the air capillaries of the bird lungs, and there was no tubular myelin in purified avian surfactants. Phosphatidylcholine molecular species of the various surfactants were measured by HPLC. Compared with pig surfactant, both bird surfactants were enriched in dipalmitoylphosphatidylcholine, the principle surface tension-lowering agent in surfactant, and depleted in palmitoylmyristoylphosphatidylcholine, the other disaturated phosphatidylcholine of mammalian surfactant. Surfactant protein (SP)-A was determined by immunoblot analysis, and SP-B and SP-C were determined by gel-filtration HPLC. Neither SP-A nor SP-C was detectable in either bird surfactant, but both preparations of surfactant contained SP-B. Surface tension function was determined using both the pulsating bubble surfactometer (PBS) and capillary surfactometer (CS). Under dynamic cycling conditions, where pig surfactant readily reached minimal surface tension values below 5 mN/m, neither avian surfactant reached values below 15 mN/m within 10 pulsations. However, maximal surface tension of avian surfactant was lower than that of porcine surfactant, and all surfactants were equally efficient in the CS. We conclude that a surfactant composed primarily of dipalmitoylphosphatidylcholine and SP-B is adequate to maintain patency of the air capillaries of the bird lung.     

Keeping lung surfactant where it belongs: protein regulation of two-dimensional viscosity

Lung surfactant causes the surface tension, gamma, in the alveoli to drop to nearly zero on exhalation; in the upper airways gamma is approximately 30 mN/m and constant. Hence, a surface tension gradient exists between alveoli and airways that should lead to surfactant flow out of the alveoli and elimination of the surface tension gradient. However, the lung surfactant specific protein SP-C enhances the resistance to surfactant flow by regulating the ratio of solid to fluid phase in the monolayer, leading to a jamming transition at which the monolayer transforms from fluidlike to solidlike. The accompanying three orders of magnitude increase in surface viscosity helps minimize surfactant flow to the airways and likely stabilizes the alveoli against collapse.     

A comparison of the molecular species compositions of mammalian lung surfactant phospholipids

While dipalmitoyl phosphatidylcholine (PC16:0/16:0) is essential for pulmonary surfactant function, roles for other individual molecular species of surfactant phospholipids have not been established. If any phospholipid species other than PC16:0/16:0 is important for surfactant function, then it may be conserved across animal species. Consequently, we have quantified, by electrospray ionisation mass spectrometry, molecular species compositions of phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylinositol (PI) in surfactants from human, rabbit, rat and guinea pig lungs. While PC compositions displayed only relatively minor variations across the animal species studied, there were wide variations of PG and PI concentrations and compositions. Human surfactant PG and PI were enriched in the same three monounsaturated species (PG16:0/18:1, PG18:1/18:1 and PG18:0/18:1) with minimal amounts of PG16:0/16:0 or polyunsaturated species, while all animal surfactant PG contained increased concentrations of PG16:0/16:0 and PG16:0/18:2. Animal surfactant PIs were essentially monounsaturated except for a high content of PI18:0/20:4 (29%) in the rat. As these four surfactants all maintain appropriate lung function of the respective animal species, then all their varied compositions of acidic phospholipids must be adequate at promoting the processes of adsorption, film refinement, respreading and collapse characteristic of surfactant. We conclude that this effectively monounsaturated composition of anionic phospholipid molecular species is a common characteristic of mammalian surfactants.     

Surface properties of sulfur- and ether-linked phosphonolipids with and without purified hydrophobic lung surfactant proteins

Two novel C16:0 sulfur-linked phosphonolipids (S-lipid and SO(2)-lipid) and two ether-linked phosphonolipids (C16:0 DEPN-8 and C16:1 UnDEPN-8) were studied for surface behavior alone and in mixtures with purified bovine lung surfactant proteins (SP)-B and/or SP-C. Synthetic C16:0 phosphonolipids all had improved adsorption and film respreading compared to dipalmitoyl phosphatidylcholine, and SO(2)-lipid and DEPN-8 reached maximum surface pressures of 72mN/m (minimum surface tensions of <1mN/m) in compressed films on the Wilhelmy balance (23 degrees C). Dispersions of DEPN-8 (0.5mg/ml) and SO(2)-lipid (2.5mg/ml) also reached minimum surface tensions of <1mN/m on a pulsating bubble surfactometer (37 degrees C, 20cycles/min, 50% area compression). Synthetic lung surfactants containing DEPN-8 or SO(2)-lipid+0.75% SP-B+0.75% SP-C had dynamic surface activity on the bubble equal to that of calf lung surfactant extract (CLSE). Surfactants containing DEPN-8 or SO(2)-lipid plus 1.5% SP-B also had very high surface activity, but less than when both apoproteins were present together. Adding 10wt.% of UnDEPN-8 to synthetic lung surfactants did not improve dynamic surface activity. Surfactants containing DEPN-8 or SO(2)-lipid plus 0.75% SP-B/0.75% SP-C were chemically and biophysically resistant to phospholipase A(2) (PLA(2)), while CLSE was severely inhibited by PLA(2). The high activity and inhibition resistance of synthetic surfactants containing DEPN-8 or SO(2)-lipid plus SP-B/SP-C are promising for future applications in treating surfactant dysfunction in inflammatory lung injury.     

Increased palmitoyl-myristoyl-phosphatidylcholine in neonatal rat surfactant is lung specific and correlates with oral myristic acid supply

Surfactant predominantly comprises phosphatidylcholine (PC) species, together with phosphatidylglycerols, phosphatidylinositols, neutral lipids, and surfactant proteins-A to -D. Together, dipalmitoyl-PC (PC16:0/16:0), palmitoyl-myristoyl-PC (PC16:0/14:0), and palmitoyl-palmitoleoyl-PC (PC16:0/16:1) make up 75-80% of mammalian surfactant PC, the proportions of which vary during development and in chronic lung diseases. PC16:0/14:0, which exerts specific effects on macrophage differentiation in vitro, increases in surfactant during alveolarization (at the expense of PC16:0/16:0), a prenatal event in humans but postnatal in rats. The mechanisms responsible and the significance of this reversible increase are, however, not understood. We hypothesized that, in rats, myristic acid (C14:0) enriched milk is key to lung-specific PC16:0/14:0 increases in surfactant. We found that surfactant PC16:0/14:0 in suckling rats correlates with C14:0 concentration in plasma chylomicrons and lung tissue triglycerides, and that PC16:0/14:0 fractions reflect exogenous C14:0 supply. Significantly, C14:0 was increased neither in plasma PC, nor in liver triglycerides, free fatty acids, or PC. Lauric acid was also abundant in triglycerides, but was not incorporated into surfactant PC. Comparing a C14:0-rich milk diet with a C14:0-poor carbohydrate diet revealed increased C14:0 and decreased C16:0 in plasma and lung triglycerides, respectively. PC16:0/14:0 enrichment at the expense of PC16:0/16:0 did not impair surfactant surface tension function. However, the PC profile of the alveolar macrophages from the milk-fed animals changed from PC16:0/16:0 rich to PC16:0/14:0 rich. This was accompanied by reduced reactive oxygen species production. We propose that nutritional supply with C14:0 and its lung-specific enrichment may contribute to decreased reactive oxygen species production during alveolarization.     

Alveolar surface forces and lung architecture

The entire alveolar surface is lined by a thin fluid continuum. As a consequence, surface forces at the air-liquid interface are operative, which in part are transmitted to the delicate lung tissue. Morphologic and morphometric analyses of lungs show that the alveolar surface forces exert a moulding effect on alveolar tissue elements. In particular, in lungs at low degrees of inflation, equivalent to the volume range of normal breathing, there is a derecruitment of alveolar surface area with increasing surface tensions which reflects equilibrium configurations of peripheral air spaces where the sum of tissue energy and surface energy is minimum. Thus, changes in surface tension alter the recoil pressure of the lung directly and indirectly by deforming lung tissue and hence changing tissue tensions. However, the interplay between tissue and surface forces is rather complex, and there is a marked volume dependence of the shaping influence of surface forces. With increasing lung volumes the tissue forces transmitted by the fiber scaffold of the lung become the predominant factor of alveolar micromechanics: at lung volumes of 80% total lung capacity or more, the alveolar surface area-volume relation is largely independent of surface tension. Most important, within the range of normal breathing, the surface tension, its variations and the associated variations in surface area are small. The moulding power of surface forces also affects the configuration of capillaries, and hence the microcirculation, of free cellular elements such as the alveolar macrophages beneath the surface lining layer, and of the surfaces of the peripheral airways. Still enigmatic is the coupling mechanism between the fluid continua of alveoli and airways which might also be of importance for alveolar clearance. As to the surface active lining layer of peripheral air spaces, which determines alveolar surface tension, its structure and structure-function relationship are still ill-defined owing to persisting problems of film preservation and fixation. Electron micrographs of alveolar tissue, of lining layers of captive bubbles, and scanning force micrographs of surfactant films transferred on mica plates reveal a complex structural pattern which precludes so far the formulation of an unequivocal hypothesis.     

Cell deformation at the air-liquid interface induces Ca2+-dependent ATP release from lung epithelial cells

Extracellular nucleotides regulate mucociliary clearance in the airways and surfactant secretion in alveoli. Their release is exquisitely mechanosensitive and may be induced by stretch as well as airflow shear stress acting on lung epithelia. We hypothesized that, in addition, tension forces at the air-liquid interface (ALI) may contribute to mechanosensitive ATP release in the lungs. Local depletion of airway surface liquid, mucins, and surfactants, which normally protect epithelial surfaces, facilitate such release and trigger compensatory mucin and fluid secretion processes. In this study, human bronchial epithelial 16HBE14o(-) and alveolar A549 cells were subjected to tension forces at the ALI by passing an air bubble over the cell monolayer in a flow-through chamber, or by air exposure while tilting the cell culture dish. Such stimulation induced significant ATP release not involving cell lysis, as verified by ethidium bromide staining. Confocal fluorescence microscopy disclosed reversible cell deformation in the monolayer part in contact with the ALI. Fura 2 fluorescence imaging revealed transient intracellular Ca(2+) elevation evoked by the ALI, which did not entail nonspecific Ca(2+) influx from the extracellular space. ATP release was reduced by ～40 to ～90% from cells loaded with the Ca(2+) chelator BAPTA-AM and was completely abolished by N-ethylmalemide (1 mM). These experiments demonstrate that in close proximity to the ALI, surface tension forces are transmitted directly on cells, causing their mechanical deformation and Ca(2+)-dependent exocytotic ATP release. Such a signaling mechanism may contribute to the detection of local deficiency of airway surface liquid and surfactants on the lung surface.     

Inhibition of surfactant activity by Pneumocystis carinii organisms and components in vitro

This study examines the direct inhibitory effects of Pneumocystis carinii (Pc) organisms and chemical components on the surface activity and composition of whole calf lung surfactant (WLS) and calf lung surfactant extract (CLSE) in vitro. Incubation of WLS suspensions with intact Pc organisms (10(7) per milligram of surfactant phospholipid) did not significantly alter total phospholipid levels or surfactant protein A content. Incubation with intact Pc organisms also did not impair dynamic surface tension lowering in suspensions of WLS or centrifuged large surfactant aggregates on a bubble surfactometer (37 degrees C, 20 cycles/min, 0.5 and 2.5 mg phospholipid/ml). However, exposure of WLS or CLSE to disrupted (sonicated) Pc organisms led to severe detriments in activity, with minimum surface tensions of 17-19 mN/m vs. <1 mN/m for surfactants alone. Extracted hydrophobic chemical components from Pc (98.8% lipids, 0.1 mM) reduced the surface activity of WLS and CLSE similarly to sonicated Pc organisms, whereas extracted hydrophilic chemical components from Pc (primarily proteins) had only minor effects on surface tension lowering. These results indicate that in addition to surfactant dysfunction induced by inflammatory lung injury and edema-derived inhibitors in Pc pneumonia, disrupted Pc organisms in the alveolar lumen also have the potential to directly inhibit endogenous and exogenous lung surfactants in affected patients.     

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.     

Relations among alveolar surface tension, surface area, volume, and recoil pressure

For pulmonary structure-function analysis excised rabbit lungs were fixed by vascular perfusion at six points on the pressure-volume (P-V) curve, i.e. at 40, 80, and 100% of total lung capacity (TLC) on inflation, at 80 and 40% TLC on deflation, and at 80% TLC on reinflation. Before fixation alveolar surface tensions (gamma) were measured in individual alveoli over the entire P-V loop, using an improved microdroplet method. A maximal gamma of approximately 30 mN/m was measured at TLC, which decreased during lung deflation to about 1 mN/m at 40% TLC. Surface tensions were considerably higher on the inflation limb starting from zero pressure than on the deflation limb (gamma-V hysteresis). In contrast, the corresponding alveolar surface area-volume (SA-V) relationship did not form a complete hysteresis over the entire volume range. There was a considerable difference in SA between lungs inflated to 40% TLC (1.49 +/- 0.11 m2) and lungs deflated to 40% TLC (2.19 +/- 0.21 m2), but at 80% TLC the values of SA were essentially the same regardless of the volume history. The data indicate that the gamma-SA hysteresis is only in part accountable for the P-V hysteresis and that the determinative factors of alveolar geometry change with lung volume. At low lung volumes airspace dimensions appear to be governed by an interplay between surface and tissue forces. At higher lung volumes the tissue forces become predominant.     

Arguments against and alternatives for an extracellular surfactant layer in the alveoli of mammalian lung

It is generally believed that lung alveoli contain an extracellular aqueous layer of surfactant material, which is allegedly required to prevent alveolar collapse at small lung volume; the surfactant's major constituent is a fully saturated phospholipid, referred to as dipalmitoyl lecithin or DPL. I herein demonstrate that the surfactant hypothesis of alveolar stability is fundamentally wrong. Although DPL is synthesized inside type II epithelial cells and stored in the typical inclusion bodies therein and lowers surface tension to zero in the surface balance, there is no evidence to the effect that type II cells secrete the DPL surfactant into the aqueous intra-alveolar layer which is shown by electron microscopy in support of the surfactant theory. To the contrary, all the evidence indicates that, when seen, such an extracellular layer is an artifact. This is probably upon the damage glutaraldehyde inflicts onto alveolar structures during fixation of air-inflated lung tissue. Furthermore, several cogent arguments invalidate the belief that an extracellular layer of DPL and serum proteins is present in the alveoli of normal lung. In light of these arguments, a surface tension role of DPL in alveolar stability is excluded. Three hypotheses for an alternative role of DPL in respiration mechanics are proposed. They are: (a) alveolar clearance by viscolytic and surfactant action (bubble or foam formation) on the aqueous systems which are present in lung alveoli during edema and in prenatal life and which would otherwise be impervious to air; (b) homeostasis of blood palmitate in normal lung; (c) modulation of the elasticity of terminal lung tissue by the intact inclusion bodies and parts thereof inside type II cells in normal lung.     

Lung protease/anti-protease network and modulation of mucus production and surfactant activity

Lung epithelium guarantees gas-exchange (performed in the alveoli) and protects from external insults (pathogens, pollutants…) present within inhaled air. Both functions are facilitated by secretions lining airway surface liquid, mucus (in the upper airways) and pulmonary surfactant (in the alveoli). Mucins, the main glycoproteins present within the mucus, are responsible for its rheologic properties and participate in lung defense mechanisms. In parallel, lung collectins are pattern recognition molecules present in pulmonary surfactant that also modulate lung defense. During chronic airways diseases, excessive protease activity can promote mucus hypersecretion and degradation of lung collectins and therefore contribute to the pathophysiology of these diseases. Importantly, secretion of local and systemic anti-proteases might be crucial to equilibrate the protease/anti-protease unbalance and therefore preserve the function of lung host defense compounds and airway surface liquid homeostasis. In this review we will present information relative to proteases able to modulate mucin production and lung collectin integrity, two important compounds of innate immune defense. One strategy to preserve physiological mucus production and collectin integrity during chronic airways diseases might be the over-expression of local ‘alarm’ anti-proteases such as SLPI and elafin. Interestingly, a cross-talk between lung collectins and anti-protease activity has recently been described, implicating the presence within the lung of a complex network between proteases, anti-proteases and pattern recognition molecules, which aims to keep or restore homeostasis in resting or inflamed lungs.     

Protein-lipid interactions and surface activity in the pulmonary surfactant system

Pulmonary surfactant is a lipid-protein complex, synthesized and secreted by the respiratory epithelium of lungs to the alveolar spaces, whose main function is to reduce the surface tension at the air-liquid interface to minimize the work of breathing. The activity of surfactant at the alveoli involves three main processes: (i) transfer of surface active molecules from the aqueous hypophase into the interface, (ii) surface tension reduction to values close to 0 mN/m during compression at expiration and (iii) re-extension of the surface active film upon expansion at inspiration. Phospholipids are the main surface active components of pulmonary surfactant, but the dynamic behaviour of phospholipids along the breathing cycle requires the necessary participation of some specific surfactant associated proteins. The present review summarizes the current knowledge on the structure, disposition and lipid-protein interactions of the hydrophobic surfactant proteins SP-B and SP-C, the two main actors participating in the surface properties of pulmonary surfactant. Some of the methodologies currently used to evaluate the surface activity of the proteins in lipid-protein surfactant preparations are also revised. Working models for the potential molecular mechanism of SP-B and SP-C are finally discussed. SP-B might act in surfactant as a sort of amphipathic tag, directing the lipid-protein complexes to insert and re-insert very efficiently into the air-liquid interface along successive breathing cycles. SP-C could be essential to maintain association of lipid-protein complexes with the interface at the highest compressed states, at the end of exhalation. The understanding of the mechanisms of action of these proteins is critical to approach the design and development of new clinical surfactant preparations for therapeutical applications.     

Theoretical modeling of the interaction between alveoli during inflation and deflation in normal and diseased lungs

Alveolar recruitment is a central strategy in the ventilation of patients with acute lung injury and other lung diseases associated with alveolar collapse and atelectasis. However, biomechanical insights into the opening and collapse of individual alveoli are still limited. A better understanding of alveolar recruitment and the interaction between alveoli in intact and injured lungs is of crucial relevance for the evaluation of the potential efficacy of ventilation strategies. We simulated human alveolar biomechanics in normal and injured lungs. We used a basic simulation model for the biomechanical behavior of virtual single alveoli to compute parameterized pressure–volume curves. Based on these curves, we analyzed the interaction and stability in a system composed of two alveoli. We introduced different values for surface tension and tissue properties to simulate different forms of lung injury. The data obtained predict that alveoli with identical properties can coexist with both different volumes and with equal volumes depending on the pressure. Alveoli in injured lungs with increased surface tension will collapse at normal breathing pressures. However, recruitment maneuvers and positive endexpiratory pressure can stabilize those alveoli, but coexisting unaffected alveoli might be overdistended. In injured alveoli with reduced compliance collapse is less likely, alveoli are expected to remain open, but with a smaller volume. Expanding them to normal size would overdistend coexisting unaffected alveoli. The present simulation model yields novel insights into the interaction between alveoli and may thus increase our understanding of the prospects of recruitment maneuvers in different forms of lung injury.     

Surpellic films, lung surfactant, and their cellular origin in newt, caecilian, and frog

The lung lining and its surfactant have been studied in one species from each of the amphibian sub-classes Anura, Urodela, and Apoda. The surfactant was studied by observation of bubbles derived from the lungs, the morphology by electron microscopy. Other details of the apodan are also given.
The surfactant layers of Rana temporaria and Ichthyophis (species unknown) resemble one another, being somewhat less stable than that of mammals. That of Triturus vulgaris is even less stable, and shows qualitative differences from the other two. Nevertheless, it can reduce the surface tension to 2 mN/m.
The epithelial cells of amphibian lungs cannot be divided into two clearly defined types, as can those of mammals. Microvilli are present on the alveolar surface of all these cells. In the lungs of Rana and Ichthyophis the lining cells contain numerous lamellated osmiophilic bodies (which are intracellular depots of surfactant); in the former their appearance is mainly cross-barred, in the latter mainly concentric. Similar bodies exist in the lung epithelial cells of Triturus but are relatively rare.     

Dexamethasone and epinephrine stimulate surfactant secretion in type II cells of embryonic chickens

Pulmonary surfactant (PS), a mixture of phospholipids and proteins secreted by alveolar type II cells, functions to reduce the surface tension in the lungs of all air-breathing vertebrates. Here we examine the control of PS during lung development in a homeothermic egg-laying vertebrate. In mammals, glucocorticoids and autonomic neurotransmitters contribute to the maturation of the surfactant system. We examined whether dexamethasone, epinephrine, and carbamylcholine hydrochloride (agonist for acetylcholine) increased the amount of PS secreted from cultured type II cells of the developing chicken lung. In particular, we wanted to establish whether dexamethasone would increase PS secretion through a process involving lung fibroblasts. We isolated and cocultured type II cells and lung fibroblasts from chickens after 16, 18, and 20 days of incubation and from hatchlings (day 21). Epinephrine stimulated phosphatidylcholine (PC) secretion at all stages, whereas dexamethasone stimulated secretion of PC at days 16 and 18. Carbamylcholine hydrochloride had no effect at any stage. This is the first study to establish the existence of similar cellular pathways regulating the development of surfactant in chickens and eutherian mammals, despite the vastly different birthing strategies and lung structure and function.     

Analysis of elastic and surface tension effects in the lung alveolus using finite element methods

Displacement method finite element theory is used to examine the structural and elastic properties of the constituent network of elastin and collagen of the alveoli that form the mammalian lung. The role of the surface tension of pulmonary surfactant of the lung is also examined using an area-dependent relationship inferred from experimental studies. The pressure-volume (PV) curves of the resulting model are found to compare favourably with measured pressure-volume curves for whole lungs filled with air (surface tension included) and saline (no surface tension effects).     

Lung structure parameters estimated from modeling aerosol deposition in isolated dog lungs

A three-compartment model predicting the recovery of aerosol boli (i.e., the ratio of the number of particles expired to the number inspired) as a function of breath-holding time and bolus penetration was fitted to experimental data measured in nine isolated dog lungs. For each lung, the diameters of alveoli and alveolar ducts, as well as the volume fractions of alveoli, alveolar ducts, and airways, were determined as parameters providing the best fit. Parameter values were alveolar diameter = 0.116 +/- 0.007 (SE) mm, alveolar duct diameter = 0.284 +/- 0.015 mm, total alveolar volume/total lung capacity (TLC) = 0.68 +/- 0.02, total alveolar duct volume/TLC = 0.24 +/- 0.02, and total airway volume/TLC = 0.09 +/- 0.01. These values agreed with published values for linear dimensions and volumetric fractions in the canine lung. The mean alveolar diameter determined by the model in the nine lungs agreed closely with a mean value of 0.115 +/- 0.002 mm determined by morphometric analysis of photographs of the subpleural alveoli in the same lungs. The procedure of fitting the model to experimental data appears to have promise as a noninvasive probe of the lung periphery. However, aerosol-derived dimensions were more variable than morphometric ones, possibly because of interlung differences in aerosol distribution not accounted for in the model.     

A simple method for the differential characterization of alveoli and alveolar ducts in injured lungs

RATIONALE AND HYPOTHESIS: Previous studies evaluating the histoarchitecture of distal airspaces have been shown to be limited by the difficulty in adequately differentiating alveoli and alveolar ducts. This limitation has been specially noticed in studies addressing lung recruitment and in situations of diffuse alveolar damage (DAD), where generic nominations for distal airspaces had to be created, such as "peripheral airspaces" (PAS) and "large-volume gas-exchanging airspaces" (LVGEA). Elastic stains have been largely used to describe normal lung structures. Weigert's resorcin-fuchsin staining (WRF) demarcates the thickened free portions of the ductal septum facilitating its recognition. We hypothesized that this staining could help in differentiating alveoli from alveolar ducts in distorted lung parenchyma. MATERIAL AND METHODS: Samples of control lungs and of DAD lungs induced by mechanical ventilation (VILI) were stained with hematoxylin-eosin (HE) and with WRF. Using morphometry we assessed the volume proportion of alveoli, alveolar ducts and LVGEA in control and VILI lungs. RESULTS: WRF stained VILI lungs showed a significant decrease in the volume proportion of LVGEA and alveoli and a significant increase in the volume proportion of alveolar ducts when compared to HE stained samples. CONCLUSION: We conclude that WRF staining is useful to distinguish alveolar ducts from alveoli in a DAD model, and suggest that it should be routinely used when morphometric studies of lung parenchyma are performed.     

Biophysical inhibition of synthetic lung surfactants

The biophysical activity and inhibition of a series of synthetic surfactant mixtures was studied and correlated with physiological effectiveness in restoring pressure-volume (P-V) mechanics of excised lungs. Results showed that several simple mixtures of dipalmitoyl phosphatidylcholine (DPPC) with fatty acids or diacylglycerols could be formulated to give good adsorption facility and dynamic surface tension lowering to less than 1 mN/m in pulsating bubble measurements at 37 degrees C. However, although biophysical activity approached that of natural lung surfactant (LS) and a related surfactant extract (CLSE) under normal conditions, surface properties were sharply inhibited by relatively small amounts of the plasma protein albumin (2 mg/ml) with minimum surface tensions greater than 30 nM/m even at high surfactant concentrations (5-20 mg lipids/ml). This sensitivity to biophysical inhibition was markedly increased compared to LS and CLSE, and had direct consequences for physiological efficacy: in spite of initially high activity, synthetic surfactants did not exert beneficial effects on P-V mechanics when instilled into surfactant-deficient excised rat lungs. Endogenous protein material was shown to be present upon surfactant recovery by lavage, and bubble measurements confirmed surface activity well below pre-instillation levels. Moreover, full biophysical activity was restored when lavage fluid was extracted to separate the synthetic surfactants from endogenous inhibitors. These results show that it is important to define relative sensitivity to biophysical inhibition in the development of effective lung surfactant substitutes. In addition, the existence of inhibition effects can generate an apparent lack of correspondence between initial biophysical activity and ultimate physiological actions of exogenous surfactant mixtures.