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Published ahead of print on November 20, 2003, doi:10.1165/rcmb.2003-0279OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 641-650, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0279OC

Effect of Acute Lung Injury on Structure and Function of Pulmonary Surfactant Films

Amiya K. Panda*, Kaushik Nag{dagger}, Robert R. Harbottle, Karina Rodriguez-Capote, Ruud A. W. Veldhuizen, Nils O. Petersen and Fred Possmayer

Department of Chemistry, Behala College, Kolkata, India; Department of Chemistry, and Departments of Obstetrics and Gynecology and Biochemistry, University of Western Ontario, London, Ontario; Departments of Physiology and Pharmacology and Medicine, Lawson Health Research Institute, London, Ontario, Canada; and Victoria de Giron Medical School and Research Centre, Havana, Cuba

Address correspondence to: Dr. Fred Possmayer, Departments of Ob/Gyn and Biochemistry, The University of Western Ontario, 339 Windermere Road, London, ON, N6A 5A5 Canada. E-mail: fpossmay{at}uwo.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The structural and functional alterations in pulmonary surfactant that occur during acute lung injury were studied using rat lung surfactant large aggregates (LA) isolated from normal nonventilated lungs (N), and from standard ventilated (V) and injuriously ventilated (IV) excised lungs. N lungs inflated significantly better than IV lungs, with V lungs intermediate. Although IV LA phosphatidylcholine levels were unchanged, cholesterol and protein were elevated. V LA exhibited PC/cholesterol and PC/protein ratios intermediate between N and IV. In contrast to total cholesterol and protein levels, these ratios were not significantly different from IV LA. N and V LA, but not IV LA, adsorbed rapidly and were able to generate surface pressures ({pi}) near 70 mN/m during surface area reduction at 37°C on a captive bubble tensiometer. Langmuir-Wilhelmy surface balance studies at 23°C showed N LA films consistently attained {pi} approaching 70 mN/m during ten compression–expansion cycles. IV films were less effective and failed to achieve high {pi} consistently after the sixth cycle. V films were intermediate. Epifluorescence studies revealed compression of adsorbed N LA films formed well-defined liquid-condensed (LC) domains, but fewer, smaller domains were observed with IV films and, to a lesser extent, V films. Atomic force microscopy on Langmuir-Blodgett N films transferred at {pi} = 30 mN/m showed high, well-defined LC domains. IV films showed thinner, intermediate height, possibly fluid domains, which contain large numbers of small higher domains with heights corresponding to LC domains. V films were intermediate. We conclude that acute lung injury induced by hyperventilation, and to a lesser extent standard ventilation, of excised lungs alters surfactant surface activity and the ability of natural surfactant to form surface structures at the air–water interface.

Abbreviations: atomic force microscopy, AFM • acute lung injury, ALI • acute respiratory distress syndrome, ARDS • bovine lipid extract surfactant, BLES • dipalmitoylphosphatidylcholine, DPPC • injuriously ventilated, IV • large aggregates, LA • Langmuir-Blodgett, LB • liquid condensed, LC • liquid expanded, LE • normal, N • 1-palmitoyl-2-[12-{(7-nitro-1,3-benzooxadiazol-4-yl) amino}docecanoyl]-sn-glycero-3-phosphocholine, NBD-PC • positive end-expiratory pressure, PEEP • phospholipids, PL • phosphatidylcholine, PC • small aggregates, SA • noninjuriously ventilated, V


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pulmonary surfactant is essential for normal lung function. Through its ability to adsorb rapidly to the equilibrium spreading pressure of ~ 47 mN/m (surface tension ~ 23 mN/m), surfactant increases lung compliance, thereby reducing the work of breathing (1–6). The ability of surfactant films to attain surface pressures approaching 70 mN/m (surface tension 0 mN/m) during dynamic lateral compression stabilizes lung alveoli at end expiration by counteracting transudation of fluid from interstitial spaces and prevents alveolar collapse. The essential role of pulmonary surfactant in normal lung function is illustrated by the proven efficacy of exogenous replacement surfactant treatment for neonatal respiratory distress (3, 6, 7). It is also evident that surfactant dysfunction contributes to the pathophysiology associated with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (8–10). In a number of lung diseases, such as ARDS, surfactant composition is altered and surfactant subtype ultrastructure is modified (8–10). Although the initiating events leading to ALI and ARDS have been investigated in detail, the proximate causes of surfactant dysfunction in these syndromes have not been identified. Furthermore, the effect of surfactant inactivation on the structural organization of surfactant at the air–water interface has not been reported (3, 5, 9).

Previous investigations by our group and others have demonstrated that subjecting lungs to ventilation at high tidal volumes can lead to a significant decrease in compliance compared with nonventilated lungs (11, 12; see Refs. 13, 14 for review). Examination of bronchoalveolar lavage from excised rat lungs ventilated under injurious conditions revealed an increase in the surfactant small aggregates (SA) fraction but no significant changes in the large aggregates (LA) fraction (12). A number of studies have shown that surfactant LA are highly surface active, whereas SA have poor surface activity (3, 6, 8, 10). Consequently, the diminished compliance associated with injurious ventilation could not be attributed to the alterations in surfactant subtype levels. Examination of LA from injuriously ventilated (IV) excised rat lungs by electron microscopy revealed lower levels of highly organized lipid-protein structural elements such as lamellar bodies, tubular myelin, and multilamellar structures relative to LA from normal (N) lungs or excised lungs exposed to standard ventilation (V) (12). In addition, LA from IV lungs showed poor surface activity in captive bubble tensiometer studies. These studies demonstrated that mechanical ventilation under injurious conditions led to alterations in LA morphologic characteristics and an impairment in LA biophysical function. However, the bases of these alterations remain unidentified.

The present studies sought to further characterize the effects of ALI, induced by mechanical hyperventilation of excised rat lungs, on surfactant levels, composition, surface activity, and, in particular, on interfacial structural organization. This injury model was used not only because it is well controlled and highly consistent, but particularly because the ex vivo model limits the injury to lung specific influences (11, 12; see Refs. 13, 14 for review). Recent studies on LA from children suffering mild infections show differences in LA phospholipid molecular species composition, apparently arising from the presence of membrane components from neutrophils and monocyte/macrophage influx from the circulation (15). LA from infected children had poor surface activity on the pulsating bubble surfactometer. For the studies described here, surfactant LA obtained from excised rat lungs subject to IV for 1 h ex vivo were compared with V and N LA. Surfactant was recovered through bronchoalveolar lavage and phosphatidylcholine (PC), cholesterol, and protein levels determined by chemical and enzymatic assays of individual components. Surface activity was estimated by monitoring adsorption and the ability to achieve high surface pressures during film compression at 37°C using a captive bubble tensiometer. These determinants were correlated with the ability of the three surfactants to generate liquid condensed (LC) surface domains during compression on a Langmuir-Wilhelmy surface balance at 23°C using direct epifluorescence microscopic examination of the films at the air–water interface. To further investigate differences in interfacial structure between these surfactants, Langmuir-Blodgett films were examined by atomic force microscopy (AFM). The results show that mechanical ventilation using injurious conditions generates alterations in lavage composition, a significant deterioration of surface activity and a marked alteration in the number and the size of LC domain structures at the air–liquid interface.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Fluorescently labeled phospholipid, 1-palmitoyl-2-[12-{(7-nitro-1,3-benzooxadiazol-4-yl) amino}docecanoyl]-sn-glycero-3-phosphocholine (NBD-PC) was purchased from Avanti Polar Lipids (Pelham, AL). Unless indicated, biochemicals were obtained from Sigma Chemicals (St. Louis, MO). Standard reagent chemicals and solvents (high-performance liquid chromatography grade) were purchased from Canlab (Mississauga, ON, Canada) and were used as received. Doubly distilled water (resistivity >18 M {Omega}/cm) was used for the film subphase.

Animal Procedures
Male Sprague-Dawley rats (Charles River Labs, St. Constant, PQ, Canada), weighing 350–400 g, were used in this study. Surfactant was isolated as previously described (12). Briefly, all animals were anesthetized with an intraperitoneal injection of ketamine hydrochloride (75 mg/kg) and zylazine (5 mg/kg) and intubated under lidocaine local analgesia. After the animal was exsanguinated, the lungs were removed and randomized to one of the three groups. The first was a nonventilated group (N), the second was a 1-h mechanically ventilated group (V), and the third was a 1-h injuriously ventilated group (IV). The second group, V, was subjected to the noninjurious mechanical ventilation in a humidified chamber at 37°C using a tidal volume (VT) of 10 ml/kg, a positive end-expiratory pressure (PEEP) of 3.0 cm H2O, and a respiratory rate of 60 breaths/min. The third group, IV, was subjected to an injurious mechanical ventilation strategy using a VT of 20 ml/kg, a PEEP of 0 cm H2O, and a respiratory rate of 30 breaths/min (12). At the end of the 1-h period, lung compliance was determined by constructing static pressure–volume (P–V) curves through the stepwise inflation of the lungs in 2-cm-H2O increments to a pressure of 25 cm H2O and subsequent deflation in 2-cm-H2O increments to a pressure of 0 cm H2O. All animal procedures were approved by the University of Western Ontario Animal Care and Use Committee.

Surfactant Isolation
After the P–V maneuvers, the isolated rat lungs were lavaged five times with saline as previously described (12). The combined lavages were spun at 150 x g for 10 min to remove cells. After an aliquot was removed, the remaining supernatant was further centrifuged at 40,000 x g for 15 min to obtain the LA fraction, which was resuspended with 2.0 ml saline. All samples were kept frozen at –20°C before use.

Compositional Analysis
Surfactant concentration was estimated as PC content using an enzymatic, colorimetric phospholipid (PL) assay kit supplied by Roche Diagnostics GmbH (Mannheim, Germany) as previously described (16). In this assay, total PC (i.e., both saturated and unsaturated molecular species) is hydrolyzed by phospholipase C to yield phosphorylcholine, from which choline is released by alkaline phosphatase. Choline is then measured through oxidation with choline oxidase to generate hydrogen peroxide, which reacts with a colorimetric dye. Bovine lipid extract surfactant (BLES Biochemicals, London, ON, Canada) and choline were used as standards. This assay measures both PC, which accounts for ~ 80%, and sphingomyelin, which accounts for 1–2%, of surfactant PL. These values will be referred to as PC (3, 6). Total cholesterol (free and cholesterol esters) present in the LA of the three groups was determined using an enzymatic colorimetric method using a kit supplied by Wako Chemicals USA, Inc. (Richmond, VA). In this assay, free cholesterol is released by cholesterol esterase and total cholesterol is estimated by cholesterol oxidase, which yields hydrogen peroxide to react with a colorimetric dye. For both assays, absorbances were measured at 490 nm in an enzyme-linked immunosorbent assay reader after 30 min incubation at room temperature (16). Total protein in LA was measured spectrophotometrically using the method of Lowry and coworkers (17), using bovine serum albumin as a standard with 2 mM sodium dodecylsulphate to dissolve lipids (16).

Captive Bubble Tensiometer Measurements
Surfactant functionality was monitored at 37°C using a captive bubble tensiometer (18). The area, volume, and surface tension of a surfactant film were monitored at the bubble air–aqueous interface by monitoring the bubble shape with a video system (19; see also Refs. 20, 21). Measurements were on surfactant samples at 300 µg/ml (0.4 mM) PC as determined with the microassay described above in saline-1.5 mM CaCl2. Lipid adsorption was followed as a function of time. After equilibrium was attained, surface activity was examined during cycles of compression and expansion in quasi-static and dynamic modes as previously described (21). Briefly, quasi-static cycles were conducted by decreasing bubble volume ~ 10% per step and waiting 10 s between steps. Compression cycles ceased when {pi} was ~ 69.0 mN/m or the bubble "clicked". Bubble clicking refers to a sudden decrease in bubble volume accompanied by a decrease in {pi}. This response is thought to arise through the shedding of surface lipid (18). After {pi}max was attained, expansion curves were generated by stepwise increases in volume, as above, until the original volume was attained. Bubbles were allowed to equilibrate for 1.5 min between cycles and five quasi-static compression:expansion curves were examined for each bubble. For dynamic cycling, a quasi-static compression-expansion curve was generated as above, after which the bubbles were dynamically cycled between the required surface area limits at 30 cycles/min.

Langmuir-Wilhelmy Surface Balance
In addition to the captive bubble tensiometry, surface activity was also determined with the Langmuir-Wilhelmy balance. Surface pressure ({pi})-area (A) isotherms were obtained at 23 ± 1°C on a Wilhelmy surface balance ({pi}-Trough; Kibron Inc., Helsinki, Finland) with an operational area of 125 cm2. Surfactant, dispersed in saline, was mixed with a solution of NBD-PC at 1% PC (wt/wt) and kept overnight at 4°C after thorough mixing on a vortex mixer. To prepare films for imaging, 100 µl of surfactant emulsions containing 300 µg/ml of PC were applied to the surface of the air–aqueous interface and allowed to adsorb to an initial surface pressure of ~ 1.5 mN/m. After a 1-h equilibration, the film was slowly compressed at a rate of 0.5 Å2/molecule/min, starting from a nominal film area of 125 cm2 (~ 120 Å2/molecule). This value was based on the molecular area of dipalmitoyl-phosphatidylcholine (DPPC) at these particular {pi}s, determined during preliminary studies using the same balance.

To measure the {pi}-A isotherms on the Wilhelmy balance, 100-µl samples of emulsions containing 300 µg/ml PC were injected at multiple points on a clean air–water interface and allowed to adsorb. One hour was allowed for equilibration. The surface pressure ({pi}) of the film was monitored continuously until sufficient amounts of surfactant were adsorbed to attain a constant level of {pi} = ~12 mN/m. The film was compressed and expanded at a rate of 85 Å2/molecule/min.

Fluorescence imaging of the adsorbed surface monolayers containing 1% NBD-PC was achieved by exciting the probe at 470 nm (blue) and observing the fluorescence emission at 530 nm (green). The Langmuir trough rests on a commercial epifluorescence microscope (Axiovert 10; Carl Zeiss, Jena, Germany) equipped with a Sony charge coupled device (CCD) camera (Empix Imaging, Inc., Mississauga, ON, Canada), attached to a Stardan II intensifier (Empix Imaging, Inc.). Fluorescence images were recorded at four different {pi}s, 15, 20, 25, and 30 mN/m, while the pressure was kept constant for ~ 5 min. Fluorescence image analysis was conducted using Northern Eclipse (Version 6.0 software; Empix Imaging Inc.). The percentage of dark probe-excluding area, presumed to be LC phase, was estimated by measuring the total amount of nonfluorescent area of each image divided by the total area of the image. An average of ten images was analyzed at each surface pressure and the data presented as percentage of area covered by the dark phase. The average diameters of the condensed lipid domains were obtained as previously reported (20, 22).

The {pi}-A isotherms were processed by defining characteristic parameters as follows. The surface pressure at the plateau region is denoted as the squeeze-out pressure {pi}s because it is generally assumed that the plateau occurs because fluid non-DPPC components are forced out of the monolayer (4, 18, 23). The maximum pressure that could be attained was defined as {pi}max. The isothermal compressibility of the film at {pi} = 30 mN m-1 (kA) was determined by using Equation 1:

(1)
where A30 is the surface area of the film and (dA/d {pi})30 is the inverse of the slope of the isotherm at {pi} = 30 mN/m.

AFM
Langmuir-Blodgett films were prepared for AFM on freshly cleaved mica. The substrate was submerged into the ring well of the surface balance before film adsorption and compression to {pi} = 30 mN/m. Following a slow compression (0.5 Å2/molecule/min) and a 5-min wait, the surface film was deposited onto the mica by raising it vertically at a rate of 10 mm/min while maintaining the surface pressure at a constant value of 30 mN/m. This {pi} was chosen because it allows for good transfer of surface films with minimal distortion, even though it is lower than the {pi}max achieved in surface balances and in the lung.

The Langmuir-Blodgett deposit on mica was mounted on the magnetic stainless steel disk in the AFM scanner where the samples were imaged within 2 h of deposition with a Nanoscope III SPM (Digital Instruments, Santa Barbara, CA). Images were measured on 60 µm x 60 µm and 10 µm x 10 µm regions using a J scanner. The measurements were in contact mode using a silicon nitride tip on a cantilever having a force constant of 0.12 N/m. The 10-µm images were flattened and analyzed using the AFM software to determine the height differences between the observed domains.

Data Analyses
Values are reported as means ± SE. Statistical significance between the three groups was determined by one-way ANOVA followed by Tukey's post hoc test. Statistical analyses were performed employing SPSS 9.0 statistical software. P values of < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P–V Relationships
Figure 1 depicts the changes in ex vivo lung volumes with applied air pressure in the different experimental groups. The N group demonstrated superior compliance to the IV group, in that throughout the inflation:deflation cycle, the maximum volume was significantly (P < 0.05) greater than the IV group lungs. Comparison between the two ventilated groups, V and IV showed that V reached higher volumes than IV at all pressures. Although these latter differences were not significant, the intermediate behavior of V suggests that even noninjurious ventilation ex vivo alters the P–V characteristics of the lung. P–V curves reflect lung compliance and therefore the effectiveness of the endogenous surfactant in promoting expansion in response to applied air pressure (12). These alterations, therefore, are consistent with surfactant dysfunction arising with ex vivo ventilation.



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  		Figure 1. Static P–V curves for three different groups of excised rat lungs: open circles, normal (n = 4); filled squares, noninjuriously ventilated (n = 4); and triangles, injuriously ventilated (n = 4) (mean ± SE are depicted). Lower parts of the curves represent the inflation limbs and upper parts of the curves represent deflation. Injuriously ventilated lungs exhibited lower compliance than the normal group (P < 0.05). The difference between the noninjuriously ventilated and the normal group was not significant.

 
Compositional Analyses
Table 1 summarizes the results of total PC, protein, and total cholesterol assays for the three groups of LA. Similar levels of surfactant PC were recovered per set of lungs from the three different groups, although the value for V LA PC was slightly lower than the other two groups. Because the enzymatic assay used measures both PC and sphingomyelin, the possibility exists that sphingomyelin levels were increased and PC levels decreased by the experimental procedures. Injurious ventilation led to an approximate doubling of cholesterol content and a 3-fold increase in the amounts of protein associated with LA. Interestingly, although the absolute amounts of cholesterol and protein recovered in IV LA were significantly lower than in V LA, neither the PC/cholesterol nor the PC/protein ratio showed statistical differences, although the PC/cholesterol ratios showed a trend (P < 0.07) toward being lower than the injurious group.


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  		TABLE 1. PC, total cholesterol, and protein levels in surfactant LA from N and from V and IV excised lungs

 
Captive Bubble Tensiometry
Adsorption isotherms were determined for LA from N, V, and IV at 300 µg PC/ml (0.4 mM) in a captive bubble tensiometer. N reached the equilibrium spreading pressure {pi}eq (~ 47 mN/m) within a minute while it took two minutes to reach {pi}eq in the case of V. IV did not reach {pi}eq during the first hour (data not shown). The rapid adsorption behavior of N and V was consistent with earlier results obtained with natural and lipid extract surfactants derived from other sources (18, 21, 23, 24).

Figure 2A depicts the initial and the fifth quasi-static compression–expansion curves on the captive bubble tensiometer for adsorbed N, V, and IV LA surfactants at 37°C. In each case, compression was initiated from the {pi}eq of ~ 47 mN/m. With N and V LA samples, surface area reduction resulted in a short rapid increase in {pi} followed by a plateau initiating at ~ 52 mN/m. Such behavior is usually referred to as "squeeze-out" because it has been interpreted as the elimination of the more fluid phospholipid species from the surface monolayer (18). Further compression resulted in a continued increase to high {pi} near 70 mN/m, although with both N and V a second minor break was observed around 65 mN/m. With an increase in bubble surface area, surface pressure fell rapidly until {pi}eq was attained at ~ 47 mN/m where surfactant respreading and/or adsorption tended to maintain relatively constant {pi} to the initial surface area. Due to the differences in compression and expansion characteristics, both films showed considerable hysteresis. In contrast to the above, quasi-static compression of IV LA films produced prolonged plateaus which only reach {pi}s of ~55 mN/m at the maximal compression (~ 80%) attainable. During expansion, the {pi} of such films decreased almost linearly to {pi} > 30 mN/m.




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  		Figure 2. Surface properties of N, V, and IV rat lung surfactant LAs in a captive bubble tensiometer at 37°C. (A) N, left panels. First and fifth (bottom) quasi-static (QS) compression–relaxation cycles for N (left panels), V (middle panels), and IV (right panels) (mean ± SEM, n = 4 for each group). Filled symbols indicate compression and open symbols indicate expansion. (B) First (top) and tenth (bottom) dynamic (Dyn) compression–relaxation cycles for N (left panels), V (middle panels), and IV (right panels) (means ± SEM, n = 4 for each group).

 
By the fifth cycle, N and V exhibited improved surface activity where the lengths of the putative squeeze-out plateaus were decreased. As a result, lower compression ratios were required to achieve {pi} {cong} 70 mN/m compared with the initial cycle. In addition, the expansion curves followed the compression curves more closely than in the first cycle, resulting in reduced hysteresis. V surfactant required slightly greater compression ratios to achieve high pressures and exhibited greater variability than N surfactant. Surface activity with IV LA remained poor by these criteria.

Figure 2B shows the dynamic compression–expansion cycles obtained at 30 cycles/min, a regimen which mimics regular breathing more closely. Dynamic compression of N and IV adsorbed films produced sharp, almost linear increases in {pi} so that {cong} 70 mN/m was attained with 20% or less surface area reduction during the first dynamic compression. During the subsequent film expansion {pi} fell almost linearly to {pi}eq and then less rapidly until the initial surface area was reached. Slightly more compression was required to attain high pressures with V films and hysteresis was more evident. IV LA films acted poorly in dynamic as well as in quasi-static cycling. These films were only able to achieve a {pi}max of ~ 55 mN/m and fell to ~ 25 mN/m during expansion. By the 10th dynamic cycle, N film profiles were almost linear and exhibited essentially no hysteresis. V films still required slightly more compression, and hysteresis remained evident. IV LA films showed no improvement over dynamic cycle 1.

Langmuir-Wilhelmy Surface Balance Studies
When the surface areas of adsorbed surface films were decreased in the Langmuir-Wilhelmy balance, there was a comparable increase in {pi} for all samples (Figure 3). However, the behavior was distinctly different when the films were compressed and expanded rapidly for ten consecutive cycles. Following a gradual increase in pressure, a squeeze-out plateau was observed at {pi} ~ 42 mN/m. These observations agree with similar measurements using bovine and porcine lipid extract surfactants where the squeeze-out pressure is around 40 mN/m (20, 22, 25). The pressure at the onset of apparent squeeze-out ({pi}s) increases during the first few successive compression-expansion cycles but then levels off (Figure 4A). Values for {pi}s are lower with N compared with V and markedly lower than IV films during all cycles. After the sixth cycle, IV LA film did not show a squeeze-out plateau. This could suggest that with each cycle, there is progressive loss of surface active material from the surface resulting in the need for compression to a smaller surface area to reach comparable surface pressures and that such losses are greater for IV.



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  		Figure 3. Representative dynamic compression and expansion surface pressure: relative area isotherms for the three different rat surfactants on the Langmuir-Wilhelmy surface balance at 23 ± 1°C. Ten consecutive compression (C1–C10) and relaxation (R1–R10) cycles are depicted.

 


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  		Figure 4. Comparison of (A) {pi}s, (B) compressibility, (C) {pi}max, and (D) A0/A30 for different rat surfactants on the Langmuir-Wilhelmy balance at 23 ± 1°C (mean ± SEM). Circles, N (n = 5); triangles, V (n = 5); and squares, IV (n = 7).

 
After the first compression cycle, the shape of the compression curves changed very little, with the exception of IV films, where a dramatic change occurs after about the sixth cycle. This is illustrated quantitatively by the compressibility values measured at {pi} = 30 mN/m before the initial break, as shown in Figure 4B. An effective pulmonary surfactant should have a low compressibility value. N films maintained compressibilities of ~ 0.020 for all 10 cycles, whereas V films show a progressive increase to ~ 0.025. The IV LA films had compressibilities at {pi} = 30 mN/m of 0.026 with the initial cycle, but by the seventh cycle no longer reached {pi} = 30 mN/m. These studies show that N possesses good surface activity, whereas IV has poor surface activity, with V being intermediate.

The maximum pressure that can be attained on compressing the surface film ({pi}max), although limited by the minimum area attainable with the particular trough, is nevertheless an excellent indicator of surfactant film behavior. For N and V, {pi}max decreased slightly as the number of cycles proceeded (Figure 4C). This indicates that the characteristics of the film at high pressures did not change appreciably even though some material may be lost from the surface. For both groups, readsorption and/or respreading were relatively rapid. In contrast the {pi}max for IV fell dramatically once the number of cycles exceeded six.

Figure 4D shows film compressibility calculated as the ratio of the area at the onset of squeeze-out (A0) to the area at {gamma} = 30 mN/m (A30). With this index, higher ratios of (A0/A30) are indicative of superior surface activity and for an effective surfactant the ratio should be relatively independent of the number of compression cycles. Figure 4D shows intermediate behavior for V, which indicates that even normal ventilation of isolated lungs caused an alteration of surface activity.

Fluorescence Microscopy Studies
Fluorescence microscopy can be used to monitor the phase transition of lipid surface films (4–6, 26, 27). This technique has been applied to a number of pulmonary surfactant extracts (20, 22). A phase transition can occur during a decrease in the surface area of phospholipid monolayers as part of the liquid expanded (LE) phase is converted to the gel-like liquid condensed (LC) phase in response to the increased surface pressure. The phase-separated LC domain structures formed exclude the fluorescent probe used here and appear dark.

Typical fluorescence images for the three different groups of surfactants are shown in Figures 5A–5F at {pi}= 20 mN/m (Figures 5A–5C) and at {pi} = 30 mN/m (Figures 5D–5F). Figure 5G shows the variation in the % area of black domains as a function of {pi} and Figure 5H shows the average diameter of the domains as a function of {pi}. Consistent differences were observed between N and IV LA, with V demonstrating intermediate behavior. For N, there was a significant increase in both the % of area and the average diameters of the domains with increasing {pi}, whereas no increases were observed for IV. The fraction and sizes of the domains were always much larger for N than for IV.



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  		Figure 5. Top: fluorescence images of different rat surfactants at {pi} = 20 mN/m on the Langmuir-Wilhelmy balance at 23 ± 1°C (A, N; B, V; C, IV) and at {pi} = 30 mN/m (D, N; E, V; F, IV). Bottom: comparison of the percentage of area of coverage (G) and average diameter (H) for the three categories of surfactants (mean ± SEM). Circles, N (n = 5); triangles, V (n = 5); and squares, IV (n = 7).

 
AFM
LB films were transferred onto mica surfaces and examined by AFM, which has greater resolution than fluorescence and avoids measurements relying on probe partitioning between phases (5, 27). Representative AFM images are shown in Figure 6 for films compressed to {pi} = 30 mN/m before deposition. In these images, LC domains, seen as the brighter regions, are higher. The darker lower regions surrounding the LC domains correspond to the LE regions.



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  		Figure 6. Atomic force microscopic images of three different rat surfactants deposited on mica. Image field sizes were: (A) 20 x 20 µm, (B) 10 x 10 µm, and (C) 5 x 5 µm. Samples are Langmuir-Blodgett films collected at 30 mN/m for normal rat surfactant LA (N; A1, B1, C1), standard ventilated lung LA (V; A2, B2, C2), and injuriously ventilated lung LA (IV; A3, B3, C3). Approximate height differences are indicated by the scale to the right of the images. The lines and triangles in the B images represent lines used for height step analysis.

 
Normal rat surfactant LA (N), showed numerous bright (high), distinct, well-defined circular shapes (Figure 6A1). Domain sizes were slightly smaller than observed by fluorescence, but the overall pattern was similar. Further magnification showed the domains extended above the surrounding LE phase and possessed a very uniform texture (Figures 6B1 and 6C1). The surrounding LE phase was less uniform and appeared somewhat granular.

AFM of V LA showed some distinct rounded domains, but in keeping with the fluorescent images, the overall pattern was less uniform (Figure 6A2). The V domains showed thickness and AFM tip-interacting characteristics resembling those observed with N LA. However, these films also had regions which appeared disrupted and contained domains of intermediate height, which in turn contained smaller domains with height differences (Figures 6B2 and 6C2) similar to the large high domains in N films (Figures 6A1, 6B1, and 6C1). Examination of IV LA films by AFM revealed many disrupted areas with smaller domains relative to N and V LA (Figure 6A3). The higher resolution images showed numerous intermediate height domains of 2–10 µm that contained numerous smaller, more elevated domains (Figures 6B3 and 6C3). In addition, many very small intermediate domains containing higher regions could be observed in the LE phase. These rather small domains contributed to the rough appearance of the background regions.

The N, V, and IV LA domains were further characterized using height difference analyses. Height step differences for the N domains were 1.22 ± 0.13 nm (n = 12), similar to the LE-LC height difference we observe for bovine lipid extract surfactant (BLES) under the same conditions. Height differences between the LA phase and the outer intermediate domains were 0.99 ± 0.14 nm (n = 9). The smaller inner domains were 0.30 ± 0.09 nm (n = 9), higher than the inner domains (~ 1.3 nm higher than the LE phase). Height differences for the large IV intermediate domains were 0.78 ± 0.09 nm (n = 6) with a further 0.25 ± 0.09 nm (n = 6) height step between the intermediate and smaller, higher domains. This results in an ~ 1.1 nm height difference between the abundant surrounding LE phase and the inner domains. The height difference analyses are consistent with the large distinct domains observed with N LA surfactant being similar in composition to the smaller high domains located within the intermediate domains. Although IV and V AFM LB films both possessed intermediate domains containing the smaller, higher domains, the smaller domains were considerably more common with IV surfactant. With these latter films, the inner, higher domains were smaller and more numerous than with V surfactant. This observation is consistent with the intermediate behavior of V in all of the surface tests conducted.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The objective of the present study was to examine further the composition (i.e., protein and cholesterol content) and the biophysical properties (surface tension lowering ability) of functional and dysfunctional surfactants. Furthermore, the observations were extended to include the structural organization of surface films using epifluorescence and atomic force microscopy. Surfactant preparations in the current study were obtained using a ventilated isolated lung model, which provides a very consistent system for obtaining functional and dysfunctional surfactants. Unfortunately, in terms of experimental studies, there is no single animal model that represents all the features of ARDS and/or ventilation-induced lung injury. Therefore, the current study must be interpreted in the context of this particular experimental model, which is limited by ischemia and the absence of circulation. Future studies, with other models of lung injury, and possibly with samples from patients with ARDS, are required to further investigate the clinical relevance of the current observations.

Mechanical ventilation of the excised lung resulted in reduced compliance of the lung. Lungs from the IV group were significantly less compliant than the noninjuriously ventilated group, which in turn were less compliant (not significant) than the normal lungs. Total LA PC levels showed no difference between groups. As in our previous studies, electrospray ionization mass spectroscopy analysis showed no differences in PC molecular species composition (data not shown). In addition, PG molecular species were not affected. Cholesterol and protein (most likely serum protein) levels were elevated in IV LA over the other two groups. However, the PC/protein and PC/cholesterol ratios (P > 0.05 < 0.1) were not significantly lower than those observed for V LA. The overall alterations in lung compliance and surfactant LA composition reported here are in excellent agreement with previous experiments employing this model (12). The observed changes are consistent with injurious and, to a lesser degree, normal ventilation causing damage to alveolar walls allowing leakage of serum components even with the isolated lung model (14).

Captive bubble studies demonstrated that although V LA were not as effective as N LA, both groups adsorbed to equilibrium rapidly and achieved high {pi}s during quasi-static and dynamic surface cycling, as previously observed with rat and rabbit natural surfactants and with bovine and porcine lipid extract surfactants (4, 6, 7, 23, 24, 28). In contrast, LA isolated from IV lungs lack the capacity to adsorb rapidly and the ability to generate high surface pressures during compression as observed in previous studies employing this model (12).

The inferior surface properties of LA from IV were confirmed by compression–expansion cycling experiments with the Langmuir-Wilhelmy surface balance, which can be used to characterize the squeeze-out plateau, the maximal pressure attainable, and the compressibility of surface films (26). These studies showed that IV lung LA surfactant samples require higher pressures to reach the squeeze-out plateau, the compressibility is higher and the maximum pressure attained during the first compression is lower than for the N and V surfactant samples. After about six cycles, IV LA surfactant failed to attain high surface pressures, possibly because this surfactant does not respread at a rate high enough to keep up with the compression–expansion cycle rate. In these trough studies, the V surfactant displayed properties intermediate between the N and the IV surfactant, as was observed with the P–V curves, surfactant composition, and the captive bubble studies.

The nature of the dark, probe-excluding domains observed by epifluorescence can be interpreted in terms of studies conducted with pure DPPC and simple model surfactant systems (29–34; see also Refs. 3, 5, 26, 27 for review). Previous studies on the organization of spread surfactant films during compression show that bovine and porcine lipid extract surfactants, form well-defined, fluorescent probe-excluding LC domains upon compression (20, 22, 25, 29, 35; see also Refs. 4, 5, 27 for review). The present studies demonstrate nonfluorescent domains are also formed with adsorbed natural surfactant. The normal group exhibited characteristics attributed to effective surfactants with well-defined domain structures that exclude the fluorescent probe and increased sizes and area coverage upon compression of the film. In contrast, the IV group surfactant contained only a few probe-excluding domains even at high pressures, and those that were detected were considerably smaller and are more poorly defined in terms of contrast. Again, the V group showed intermediate behavior in this system. Previous investigations using several approaches with bovine and porcine surfactant lipid extracts and the phospholipid fraction isolated from lipid extracts have concluded the nonfluorescent domains are highly enriched in DPPC, whereas the bright areas retain lipids which are fluid at room temperature (20, 22, 35). Assuming this is true for rat surfactant, the fluorescence data imply that fewer and smaller DPPC-rich LC structures are formed with IV surfactant and they cover less of the total surface area. This occurs despite the presence of a similar DPPC content (12).

The decreased ability to form large distinct nonfluorescent domains observed with V LA correlated with the intermediate behavior of this surfactant on the Langmuir-Wilhelmy balance used to prepare the Blodgett films, but less so with surface activity assayed, particularly during the first compression, on the captive bubble tensiometer. This apparent difference could be related to the higher concentration (300 µg/ml) assayed on the captive bubble tensiometer, because the ability of surfactant to attain high pressures during compression is highly concentration-dependent (36–38).

At present, the relation between the ability to form probe-excluding LC domains and the ability to achieve the high surface pressures required to stabilize the lung at low lung volumes is unknown (3, 5, 27). The current results show that this property correlates with the P–V characteristics and the inferior functionality of IV and, to a lesser extent, V LA with the captive bubble and Langmuir-Wilhelmy balances.

The high-resolution investigations of domain structure by AFM provide further insight into structural organization of the films. N LA formed large numbers of circular domains characteristic of LC phase domains observed with pure DPPC monolayers (4, 33, 35, 39). Domains observed by AFM were somewhat smaller than those observed using fluorescence microscopy but generated similar overall patterns. The domain heights of ~ 1.2 nm observed here were noticeably higher than the ~ 0.8 nm observed with pure DPPC domains using the same instruments and conditions (not shown). Step heights of 0.7 nm for pure DPPC domains have been reported by others (33, 39, 40). These height changes arise from increased tilt of the palmitates in DPPC, and potentially other fatty acids, in the LC phase relative to the plane of the monolayer compared with those in the LE phase (26, 27, 31).

With V LA, domains were observed approximating the size and height characteristics of the N domains, but occasionally "mixed" domains were apparent. These latter domains exhibited domains of intermediate heights of ~ 0.8 nm that contained a few smaller, circular domains that extended a further ~ 0.3 nm above the intermediate domains. IV LA showed almost none of the large high domains observed with N surfactant, but intermediate height domains, usually with a large number of small higher domains, were observed. To our knowledge, neither fluorescent nor AFM domains have been reported previously for natural surfactant from any species. The intermediate (~ 0.8 nm) domains do not exclude the fluorescent probe well, showing that they possess some LE characteristics. This indicates they are different from the ~ 0.8 nm LC domains observed with pure DPPC monolayers, despite the similar height differences. Given the lack of evidence to the contrary, it is assumed that the smaller, approximately circular domains within the intermediate domains represent residual LC. This is consistent with their combined height differences of ~ 1.2 nm relative to the abundant surrounding LE phase. A height step of ~ 1.2 nm has been observed between LE and LC domains with dipalmitoylphosphatidylglycerol:1-palmitoyl, 2-oleoylphosphatidylglycerol monolayers (30). The small higher domains are too small for detection by fluorescent microscopy, but could account, at least in part, for the mottled appearance with V and IV surfactants.

The basis for the formation of the intermediate domains with a height step of ~ 0.8 nm with the V and, to a greater extent, IV LA surfactants is not understood. The similarity of domain sizes and the intermediate height profiles are consistent with a partial fluidization of the LC domains observed with N LA. Ring- or halo-like regions which surround the LC domains generated by compression of BLES monolayers have been detected in time-of-flight secondary ion mass spectroscopic studies (35). Although these halo-like regions admit fluorescent probe, as do the intermediate domains reported here, they do not show intermediate height characteristics, indicating that they are structurally distinct from the DPPC-rich LC phase. Clearly, further studies are required to investigate the factors involved in the formation of the intermediate phase observed by AFM in V and IV LA surfactants.

Whether the alterations in surface organization of IV and, to a lesser extent, V LA surfactant contribute to the slow adsorption and, more importantly, the inability to attain low surface tension during compression is not known. It has long been thought that the ability of surfactant films to achieve low surface tension depends on monolayers highly enriched in DPPC (1–6, 18, 27). However, recent attempts to demonstrate DPPC enrichment of surface films either by selective adsorption (41) or the squeeze-out of fluid phospholipids (3, 5) have proven unsuccessful. Further, captive bubble tensiometer studies show that high surface pressures/low surface tensions can be attained at 37° by rapid compression of spread monolayers of dimyristoylphosphatidylcholine or palmitoyl-oleoyl phosphatidylcholine (3, 5, 42). These observations show that the ability of surfactant films to attain high surface pressures does not require alterations in chemical composition (i.e., high DPPC). It may be that the ability to attain such high surface pressures depends on surface organization. This would indicate that the alterations in LC domain formation could contribute to the dysfunctional nature of the IV surfactant.

Injurious ventilation of excised lungs led to a significant alteration in LA cholesterol content. As indicated above, the increase in cholesterol could conceivably contribute to the alterations in surfactant domain formation. Although its presence in surfactant has long been recognized, the role of this sterol in surfactant function is still unclear (6, 43). It has previously been reported that surfactant cholesterol increases rapidly with oleic acid–induced acute lung injury and is elevated in alveolar proteinosis (44). However, alveolar cholesterol also increases rapidly with exercise and these increases are more evident in fit individuals (45). It is known that cholesterol can interfere with the ability of DPPC-containing films to attain high surface pressures on the Langmuir balance, but at low concentrations this sterol has no effect on tensiometric behavior of surfactant on the captive bubble (46). Because cholesterol acts as a "fluidizing" agent, the increase in IV LA cholesterol could contribute to the formation of the intermediate height domains. Cholesterol can be involved with sphingolipids and/or disaturated PC in the formation of a liquid-ordered (L0) phase with characteristics intermediate between LE and LC phase (47, 48). L0 phase is considered the structural basis for the formation of lipid rafts and caveolae in cellular membranes. The ~ 20% cholesterol observed with IV LA could possibly contribute to the replacement of LC with intermediate height domains. Although the cholesterol content of V LA was not significantly increased over control values, the PC/cholesterol ratio was lower and only showed a trend (P < 0.1 > 0.5) toward differing from the injuriously ventilated group. Consequently, the possibility that the cholesterol in the V LA samples could contribute to formation of the intermediate domains could not be eliminated. However, whether the changes in sterol content observed here contribute to the overall dysfunction, possibly in combination with the increased protein and the differences in surface structure, is not known and must be investigated further.

In addition to the elevated cholesterol, increases in protein are detected in LA samples from the injuriously ventilated animals compared with the other groups (Table 1) (12). Numerous studies have documented increased levels of protein with experimental and clinical ARDS (8, 10). Further in vitro studies demonstrate distinct inhibitory effects of serum proteins on surfactant function (3, 10, 37, 49). It is thought likely that the rough, disorganized appearance of IV films represents protein incorporated into surface monolayers. This disorganization could contribute to the instability of these films at higher {pi} and the inability to reincorporate surfactant material during expansion. These considerations lead us to conclude that the 2- to 3-fold increase in serum proteins associated with IV LA could be the major factor responsible for the impaired surfactant activity even though the ex vivo isolated lung model was chosen to minimize the effects of nonpulmonary influences. It is interesting to note that the V LA which possessed PC/protein ratios intermediate and statistically indistinguishable from the other two groups also had diminished surface activity particularly on the Langmuir-Wilhelmy balance and altered surface structure. In addition, as indicated above, it is possible that combined alterations in cholesterol as well as protein may contribute to the overall effects. Moreover, other as yet nondefined changes, for example, in surfactant apoprotein content, could be involved in the observed dysfunction. Further studies involving more detailed analysis of surfactants from the experimental groups employed here are presently in progress.

In summary, standard ventilation of excised rat lungs led to a small, and injurious ventilation to a significant, decrease in lung compliance. Levels of total PC and PC molecular species profiles in IV LA were not altered, but cholesterol and protein levels were increased. Functional assessment of surfactant activity with the captive bubble tensiometer and the Langmuir-Wilhelmy surface balance demonstrated an impairment with IV and, to a lesser extent, V LA. Fluorescent imaging analysis of surface films revealed a decreased capacity for V films to generate probe-excluding LC domains, and LC domain formation was almost absent with IV films. AFM imaging studies revealed well-formed, large LC domains with a height step of ~ 1.2 nm with N and, to a lesser extent, V surfactant. V, and, to a greater extent, IV LA surfactant showed large, intermediate domains ~ 0.8 nm higher than the surrounding LE phase, which contained a number of smaller LC domains ~ 0.3 nm higher than the intermediate domains. V and IV LA surfactant also contained highly disordered surface areas. These results show that surfactant recovered from noninjuriously ventilated lungs show effects between normal and injurious samples in functional and imaging analyses despite having no obvious alteration in phospholipid composition. It is suggested that the alterations in surface structural organization may be related to dysfunctional surface activity, but direct cause–effect relationships must still be established.


   Acknowledgments
 
This work has been supported by grants received from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR). K.N. and K.R.-C. gratefully acknowledge financial support through CIHR-Canadian Lung Association (CLA) Fellowships. A.K.P. gratefully acknowledges the receipt of a BOYSCAST Fellowship from the Department of Science and Technology, Ministry of Science and Technology, New Delhi, India, to pursue the research at the University of Western Ontario. The technical assistance of Mr. Blayne Welk and the help of Mr. Marc Possmayer with some of the figures are appreciated.


   Footnotes
 
* On leave as a BOYSCAST fellow sponsored by Department of Science and Technology, Ministry of Science and Technology, India. Back

{dagger} Present affiliation: Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada. Back

Received in original form July 25, 2003

Received in final form October 27, 2003


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 Introduction
 Materials and Methods
 Results
 Discussion
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