1 Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, and 2 Division of Pulmonary and Critical Care Medicine, Beth Israel Deaconess Medical Center, Boston 02115; and 3 Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215
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ABSTRACT |
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Relationships between lung function and surfactant function and composition were examined during the evolution of acute lung injury in guinea pigs. Lung mechanics and gas exchange were assessed 12, 24, or 48 h after exposure to nebulized lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) fluid was processed for phospholipid and protein contents and surfactant protein (SP) A and SP-B levels; surfactant function was measured by pulsating bubble surfactometry. Lung elastance, tissue resistance, and arterial-alveolar gradient were moderately elevated by 12 h after LPS exposure and continued to increase over the first 24 h but began to recover between 24 and 48 h. Similarly, the absolute amount of 30,000 g pelleted SP-A and SP-B, the phospholipid content of BAL fluid, and surfactant function declined over the first 24 h after exposure, with recovery between 24 and 48 h. BAL fluid total protein content increased steadily over the first 48 h after LPS nebulization. In this model of acute lung injury, the intra-alveolar repletion of surfactant components in early recovery led to improved surfactant function despite the presence of potentially inhibitory plasma proteins.
acute respiratory distress syndrome; lipopolysaccharide; apoprotein; pulsating bubble surfactometry; optimal ventilator waveform
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INTRODUCTION |
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SURFACTANT, A COMPLEX MIXTURE of lipids and proteins, reduces surface tension at the air-liquid interface in the alveoli and small airways. In the absence of surfactant, surface tension forces exceed the counterbalancing tethering forces generated by adjacent lung tissue, leading to airway and alveolar instability and collapse. Prior studies (7, 16, 21) have demonstrated that the pathophysiology of acute respiratory distress syndrome (ARDS) is largely determined by abnormal surfactant function. Surfactant extracts from patients with ARDS demonstrate loss of surface activity, including an inability to achieve low (<5 dyn/cm) surface tensions during film compression, and loss of elastic behavior during surface area cycling (9, 16). Comparison of air- and fluid-filled pressure volume profiles in isolated lungs of animals with experimental ARDS indicates that increases in static elastic recoil after acute inflammation are due to abnormalities in surfactant function and not to changes in tissue elastic recoil (13). An in vivo animal study (1) has demonstrated surfactant dysfunction after capillary leakage due to chemical injury. To date, the biochemical and biophysical processes that relate inflammation to alterations in surfactant function are not well understood.
Several hypotheses have been proposed to account for the functional changes that occur in ARDS surfactant (9, 16, 23). Studies demonstrating decreased phospholipid and apoprotein contents in lung lavage samples from both patients with ARDS (9) and animal models (17) have suggested loss of surfactant through decreased production or accelerated destruction. Increased levels of oxidized lipids and serum proteins have been observed in lung lavage samples from animals with experimental ARDS (14) and from humans (22). Functional alterations in the affected surfactant included a decreased stability index, increases in minimum surface tension and compressibility, and delayed adsorption onto the air-water interface. These observations have led to efforts to restore normal surfactant function in ARDS by the addition of supplemental surfactant. However, the limited efficacy of surfactant replacement in restoring normal surfactant function (10) or improving clinical outcomes (2) among patients with ARDS remains puzzling.
This laboratory (15) has previously shown that endotoxin exposure with a combination of intravenous infusion and nebulization of lipopolysaccharide (LPS) in guinea pigs causes a reproducible pattern of acute lung injury (ALI) with many of the characteristics of ARDS. This was associated with surfactant dysfunction and a reduction in surfactant protein (SP) A levels in the surfactant of the injured animals (15). The studies presented here are aimed at a more extensive characterization of the relationship between alterations in lung mechanics, impairment of gas exchange, and alterations in lung surfactant in ALI produced by LPS exposure in guinea pigs. The results presented shed some light on the changes that occur in lung surfactant during early ALI and the early recovery phase.
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MATERIALS AND METHODS |
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Animal model. Male Hartley guinea pigs (weight 500-600 g; Charles River Laboratories, Needham, MA) were studied under an institutionally approved animal protocol. All were housed in a virus-free facility, were cared for in a similar fashion, and were studied within 2 wk of arrival from the distributor. The guinea pigs were randomized to either endotoxin [lipopolysaccharide (LPS); n = 18 total] or no treatment (control; n = 6). For LPS nebulization, 2.5 mg of Pseudomonas serotype 10 LPS (Sigma, St. Louis, MO) were dissolved in 5 ml of normal saline and delivered via a nebulizer. The animals were then studied 12, 24, or 48 h after exposure. The LPS was suspended in saline by vortexing and then sonicated for 25 3-s cycles with a tip sonicator (Heat Systems-Ultrasonics, Farmingdale, NY) at maximal microprobe tip power. The solution was then administered at a continuous flow of 6 l/min via a DeVilbiss acorn nebulizer in a sealed chamber over a 15-min period. The nebulizer was thoroughly cleaned between administrations. Control animals, also referred to as time 0, were not subjected to nebulization.
The animals were anesthetized with intraperitoneal xylazine (5 mg/kg) and pentobarbital sodium (40 mg/kg), were tracheally cannulated, had a carotid arterial line placed for blood pressure and arterial blood gas measurements, and underwent a partial midsternal thoracotomy to allow for exposure of the pleural surfaces. The animals were mechanically ventilated at 60 breaths/min with a tidal volume of 5.1 ml/kg, a positive end-expiratory pressure of 3 cmH2O, and an inspired fraction of O2 of 21% with a Flexivent rodent ventilator (SCIREQ, Montreal, PQ).Lung mechanics. Lung mechanics were assessed by the optimal ventilator waveform (OVW) technique as described by Lutchen et al. (19). The OVW is a forced oscillatory volume signal consisting of six sine waves with frequencies between 0.5 and 14.25 Hz. These were selected according to a nonsum-nondifference criterion (26) that minimizes the nonlinear interactions between frequencies of the pressure output. The phase angles and amplitudes of the volume input are optimized so that the volume signal has a tidallike shape, which allows the measurement of lung impedance over a range of frequencies. The peak-to-peak volume was scaled to the same value as the tidal volume so that disruption to mechanical ventilation was minimal.
In these experiments, the airway opening pressure was low-pass filtered, and the flow was calculated from piston displacement. The Flexivent system delivered three complete OVW cycles (12 s) and discarded the first cycle to remove any transients in the calculation. Fast Fourier transforms of pressure and flow were calculated on overlapping segments of data. Lung input impedance (ZL) was determined at each of the six selected frequencies as the ratio of the cross-power spectrum of pressure and flow (GPF) and the autopower spectrum of flow (GFF): ZL = GPF/GFF. The calculated impedance was then fit to a mechanical impedance model to partition ZL into airway resistance (Raw), airway inertance, tissue damping (G), and tissue elastance (H) according to Hantos et al. (11).Lavage fluid collection and processing.
After characterization of in vivo lung physiology, the animals received
a lethal dose of intracardiac pentobarbital sodium (100 mg/kg) and
underwent two whole lung lavages with 10 ml each (total of 20 ml) of
warmed saline. The lavage return volume was measured, and the lavage
fluid was centrifuged at low speed (450 g for 10 min at
4°C) to remove cells. The supernatant was stored under nitrogen at
20°C for subsequent surfactant isolation. The cell pellet was
resuspended in 5 ml of red blood cell lysis buffer and brought up to
the initial lavage return volume for total cell count by hemocytometry
and determination of a differential count with rapid Wright's stain.
Surfactant isolation and surface tension studies.
Surfactant was pelleted by high-speed centrifugation (30,000 g for 45 min at 4°C). The crude pellet was resuspended in
0.5 ml of 0.15 M normal saline, and total phospholipid content was measured (25). The surfactant was dissolved in 5 mM
CaCl2 in 0.15 M NaCl to a final concentration of 1 mg/ml,
and interfacial properties were measured at 37°C with a pulsating
bubble surfactometer (Electronetics, Amherst, NY). For equilibrium
surface tension (equil) measurements, a bubble was
formed and observed without oscillations, and surface tension
measurements were recorded at 30 s, 5 min, and 15 min. The 5- and
15-min values were in close agreement, and the 15-min value is reported
as the
equil. Dynamic measurements were made at 20 cycles/min and a relative surface area change of 62% (standard
settings) until stable readings (unchanging over 5 min) were achieved.
Surface tension and hysteresis results represent those measured 15 min
after pulsations were initiated (all samples had achieved steady state
at this time). Static and dynamic calibrations were performed before
and after each run with distilled H2O.
Phospholipid assay. The method of Stewart (25) was used. Fifty microliters of sample were added to glass tubes containing 2 ml of spectroscopic-grade chloroform. Two milliliters of 3.04% (wt/vol) ammonium ferrocyanate and 2.7% (wt/vol) ferric chloride hexahydrate in distilled H2O were added, and the mixture was vortexed for 1 min. Standards (0-100 µg/ml) were prepared with phosphatidylcholine in chloroform. The lower chloroform phase was withdrawn, and absorption was measured at 488 nm with a quartz cuvette.
Measurement of thiobarbituric acid-reactive substances. Lipid peroxidation products were measured following the procedure of Gillard et al. (8). Briefly, samples containing 1 mg/ml of phospholipid were added to a solution of 15% TCA, 0.375% thiobarbituric acid, 0.25 N HCl, and 0.01% butylated hydroxytoluene, heated to 95°C, and centrifuged, and the absorbance in the supernatant was measured at 535 nm. Malonaldehyde was used for standard calibration.
Total protein assay. The bicinchoninic acid spectrophotometric assay (Pierce, Rockford, IL) was used for protein determinations according to manufacturer's specifications. Protein concentration is proportional to absorbance at 562 nm, with bovine serum albumin as a standard. Detection of protein content in surfactant pellets (at 1 mg/ml phospholipid concentration) was determined by solubilizing samples in 0.5% SDS and preparing standards in a solution of 1 mg/ml aqueous phosphatidylcholine to account for bicinchoninic acid binding by lipid.
Anti-apoprotein antibody development. SP-A was purified from calf lung surfactant with a modification of the method of McCormack et al. (20). Whole calf lung surfactant (10 mg in 1 ml) was injected into 45 ml of butanol on ice to precipitate the aqueous proteins. After 15 min, the samples were centrifuged at 10,000 g for 30 min, and the pellet containing SP-A and serum proteins was dried under nitrogen. The dried pellet was resuspended in 20 mM octylglucopyranoside by repeated injection through a 25-gauge needle. The sample was ultracentrifuged for 60 min at 100,000 g at 4°C, and the pellet containing SP-A was resuspended in 5 mM Tris · HCl. Residual detergent was removed by microdialysis against a 5 mM Tris · HCl bath followed by passage over a Bio-Bead SM-2 (Bio-Rad) low-pressure chromatography column. Sample purity was verified by gel electrophoresis. Rabbit antiserum against SP-A was developed for our laboratory commercially (Tana Laboratories, Houston, TX).
SP-B was purified with a modification of the method of Beers et al. (4). SP-B purification from calf lung surfactant by the published protocol did yield a highly pure preparation of SP-B, which demonstrated a single band with a relative molecular weight of 6,000 with SDS-PAGE and Coomassie staining. However, when the preparation was tested for the presence of SP-A with Western blotting with anti-SP-A antibody, trace amounts of SP-A were found in several preparations. Because of concern about possible cross-reactivity of an antibody raised by this preparation, the SP-B purified by selective extraction was subjected to preparative electrophoresis, with recovery by electroelution. The preparation yielded a single band with Coomassie staining and no detectable SP-A with anti-SP-A Western blotting. Rabbit anti-serum against SP-B was developed for our laboratory commercially (Charles River Laboratories).Western blotting. Ten to fifty micrograms of purified whole surfactant from each sample (equal volumes from all pellets for comparative purposes) were applied to 12 or 15% SDS-polyacrylamide gels. Proteins were separated under reducing conditions, blotted onto polyvinylidene difluoride membrane, and analyzed with Western blot with primary antibodies raised to purified calf lung surfactant SP-A and SP-B. Cross-reactivity of these antibodies between the calf surfactant and guinea pig surfactant apoproteins was demonstrated during antibody development. Blots were developed with a streptavidin-biotin-peroxidase amplification system (Sigma) and subjected to quantitative densitometry with the Gel-Pro analyzer software package (Media Cybernetics, Silver Spring, MD).
Statistics. All data are presented as means ± SD for each treatment group unless otherwise specified. Data were analyzed by one-way analysis of variance and Leven's test for homogeneity of variance. Comparisons between individual treatment groups were carried out by the Bonferroni method when the variances were homogeneous and by Dunnett's T3 method when the variances were not homogeneous. Differences between groups were considered significant if P < 0.05.
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RESULTS |
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Pathophysiological profiles of LPS-exposed animals. Guinea pigs exposed to LPS and studied at various time points after exposure demonstrated no obvious behavioral changes between the time of exposure and surgery. The animals moved freely about, with no clear alteration in their intake of liquid or food. Initial mean arterial blood pressures as assessed with the carotid arterial line were in the range of 50-60 mmHg, with no difference between the untreated and various LPS-treated groups.
Lung mechanics, however, were markedly altered by nebulized LPS exposure in these animals. Figure 1 plots the dynamic elastance and resistance components of total lung impedance versus input frequency for control animals and for animals 12, 24, and 48 h after LPS exposure. Tissue G, tissue H, and Raw were calculated for each group as described in MATERIALS AND METHODS, and the results are summarized in Table 1. Tissue G and tissue H increased 10- and 6-fold, respectively, over the first 24 h after LPS exposure. Between 24 and 48 h, there was evidence of early recovery beginning to take place, with 10- and 4-fold falls from peak values in tissue G and tissue H, respectively. Raw was increased 12 and 48 h after exposure yet appears to be unchanged from baseline in the 24-h treatment group.
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Indexes of inflammation in LPS exposure.
As shown in Table 3, administration of
nebulized LPS produces a marked neutrophilic inflammatory response in
the alveolar space. Neutrophil counts in the bronchoalveolar lavage
(BAL) fluid peaked 12 h after exposure followed by a gradual
decrease between 12 and 48 h. The numbers of other cells remained
relatively stable over this time. As previously reported
(15), lung histopathology revealed an interstitial and
alveolar neutrophilic influx accompanied by exudative-appearing
material and mild hemorrhage in the alveolar space.
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BAL fluid biochemistry after LPS exposure.
Figure 2 displays the phospholipid and
protein concentrations in the surfactant pellets obtained from the BAL
fluid by ultracentrifugation as a function of time after LPS exposure.
In comparison with control samples, the phospholipid content of the
surfactant pellets was diminished by ~50% by 12 h after LPS
exposure and by ~75% at 24 h, the time point that corresponds
to the most profound gas-exchange and ventilatory impairment. There is
a modest but significant increase in pellet phospholipid between 24 and
48 h after LPS exposure, corresponding to the early recovery phase
as defined by ventilatory mechanics and oxygenation.
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Surfactant function in LPS-exposed animals.
A previous tissue mechanics study (15) of subpleural
parenchymal strips harvested from guinea pigs similarly exposed to LPS
revealed no significant difference in isolated tissue properties between control and LPS-exposed animals (15), pointing to
an interfacial- or surfactant-mediated mechanism for physiological dysfunction. Table 4 lists means ± SD of both static and dynamic parameters for the various treatment
groups.
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Alterations in surfactant apoproteins.
The levels of SP-A and SP-B in the surfactant pellets of animals in the
various treatment groups were assessed by Western blotting and
quantitative densitometry as described in MATERIALS AND
METHODS. Figure 3 shows
changes in the relative level of SP-A and SP-B as a function of time
after LPS exposure. Adjusted for the phospholipid level, SP-A levels in
the surfactant pellets remained relatively constant over the first
24 h after LPS administration, whereas SP-B levels decreased by
55% over the first 24 h and increased to 220% of baseline values
in the recovery period between 24 and 48 h. As shown in Fig.
3B, there was a substantial diminution in the levels of both
SP-A and SP-B in the surfactant pellet over the first 24 h after
LPS exposure. By 24 h after exposure, the levels of SP-A and SP-B
had decreased by 75 and 85%, respectively, relative to the control
values.
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Alteration in surfactant phospholipid profile.
To measure the extent of lipid peroxidation occurring in the surfactant
samples as a consequence of LPS exposure, thiobarbituric acid assays
were performed on all surfactant samples. The results are shown in
Table 5. There was no increase in
detectable aldehyde products of lipid peroxidation in samples from
LPS-exposed animals as opposed to those from control animals. The
positive control value shown in Table 5 represents control surfactant
samples that were exposed to oxidizing conditions in vitro, with
subsequent thiobarbituric acid measurements. Clearly, there is a
substantial population of readily oxidizable lipid in these samples,
and the end products are readily detectable with the assay system
employed.
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DISCUSSION |
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Exposure of guinea pigs to nebulized LPS at the concentrations employed produces a substantial influx of neutrophils into the alveolar space by 12 h after LPS exposure. This is followed by ALI, which is histologically similar to the exudative phase of ARDS (5), 24 h after LPS exposure. Finally, there is evidence of early recovery between 24 and 48 h after LPS exposure. The acute inflammatory process is marked by capillary leakage, resulting in plasma protein extravasation into the alveolar space, a severalfold increase in lung tissue H, and profound gas-exchange abnormalities. The most striking change in lung mechanics produced by LPS exposure is an increase in tissue H, most likely due to increased surface tension with concomitant airway closure and alveolar collapse. This increase in tissue H in LPS-treated animals corresponds to a dramatic decrease in static compliance as assessed by conventional quasi-static pressure-volume curves (data not shown). Tissue G shows a dramatic 15-fold increase at 24 h vs. the control value, whereas Raw appears to undergo a complex series of changes over time.
Compared with the control value, Raw first increases in the 12-h group by ~50%, then decreases by ~50% at 24 h, and finally increases again by ~100% at 48 h. The apparent decrease in Raw in the 24-h group is most likely a consequence of the development of mechanical time-constant inequalities, or heterogeneities, in the lung. The model used to fit ZL data for the purposes of this paper is a single-compartment model. Yet whereas the lung behaves as a homogeneous one-compartment system in control or healthy animals, it tends to behave as a multicompartment system in the setting of injury or disease. Heterogeneity will be most marked in the lungs of the most injured animals in the 24-h group. When the single-compartment model is fitted to the data from injured animals, time-constant inequalities are folded into an artificial increase in G and a decrease in Raw (18) to compensate for the increased frequency dependence of lung resistance at low frequencies. Thus we estimate that the true increase in tissue G is probably similar to that in H. Lung heterogeneity induced by injury not only results in an increase in average Raw but also leads to ventilation-perfusion mismatch.
The combination of ventilation-perfusion mismatch and atelectasis produced the highest arterial-alveolar gradient in the 24-h group. At the time that blood gas measurements were made, all animals were sedated and mechanically ventilated to the same weight-adjusted minute ventilation. Therefore, an increase in PCO2 in the 24 and 48 h post-LPS animals is consistent with an increase in the alveolar dead space quotient as a consequence of injury. Whether nebulized LPS produces an increase in CO2 production that contributes to this change cannot be determined based on the available data. The mild respiratory acidosis seen only in the 24-h animals under anesthesia and mechanical ventilation suggests that these animals had a higher spontaneous minute ventilation when awake. The effects of such relative hyperpnea would have been to normalize pH, reduce PCO2, and produce some improvement in PO2. The ability of guinea pigs to survive with such appallingly low PO2 values may be due in part to the relative high avidity of their hemoglobin for oxygen: even with PO2 values in the 42- to 46-mmHg range, oxygen saturation remained in the 80-85% range.
Alteration in lung mechanical properties in LPS-treated guinea pigs can be accounted for by an alteration in interfacial properties, which, in turn, result from dysfunction of lung surfactant. The degree of physiological dysfunction and surfactant dysfunction observed closely paralleled each other both during the development of lung injury over the first 24 h after LPS exposure and during early resolution between 24 and 48 h after LPS exposure. This animal model serves as a useful system for examining the mechanisms leading to and the consequences stemming from surfactant dysfunction during early ALI secondary to inflammatory processes such as sepsis.
The inflammation produced by nebulized LPS appears to alter surfactant metabolism in a variety of ways. The most obvious is a reduction in the size of the surfactant pellets recovered from BAL fluid, which corresponds to a reduction in the most active large-aggregate surfactant fraction. At the same time, there is a significant extravasation of plasma proteins into the alveolar space secondary to capillary leak. The reduction in surfactant SP-A level mirrors the overall decrease in surfactant phospholipid in the large-aggregate fraction. SP-B behaves differently, however, in that large-aggregate surfactant becomes relatively deficient in SP-B as a consequence of LPS exposure. SP-B is not only critical to surfactant function under normal conditions (28) but also appears to confer on surfactant a degree of resistance to the deleterious effects of plasma proteins (6, 24, 28).
The cumulative effect of these processes accounts for the majority of surfactant dysfunction and physiological dysfunction observed in these animals over the first 24 h. Between 24 and 48 h after LPS exposure, there was a recovery in the levels of surfactant phospholipids, SP-A, and particularly SP-B. Surfactant harvested 48 h after exposure revealed an improvement in function observed in vitro despite the persistence of significant plasma protein contamination in the surfactant pellet. Lipid peroxidation per se did not appear to occur to any measurable degree in these samples (see Table 5).
The small-aggregate fraction of alveolar surfactant was not studied in these experiments, and, therefore, we cannot comment on potential changes in its function or composition. The nature of the larger molecular weight form of SP-B seen by Western blot in the LPS-exposed animals remains to be determined. Possible candidates include covalently formed aggregates of SP-B caused by free radical mechanisms or the presence of biosynthetic precursors of SP-B being released into the alveolar space in the setting of alveolar injury. Finally, the extent to which residual dysfunction in the sucrose-purified surfactant pellet can be corrected for by apoprotein replacement or other reconstitution approaches in vitro remains to be determined.
It is likely that a variety of mechanisms are contributing to surfactant dysfunction within the alveolar space in this model of ALI. The ability of both whole serum and albumin to inhibit surfactant function has been shown to be dependent on surfactant phospholipid concentration (12). The presence of surfactant apoproteins has been observed to protect surfactant from inhibitory effects of proteins (6, 24, 28). In clinical observations, decreased BAL fluid phospholipid content and SP-B have been reported among patients with ARDS (9).
The reasons for the limited efficacy of surfactant replacement strategies that have thus far been employed in ALI and ARDS are not well understood. A better understanding of this process will be needed to devise more effective approaches to the restoration of surfactant function in critically ill adults. The findings of this study have several potential implications for surfactant replacement strategies. Adequate surfactant replacement in this model of ALI will require that surfactant containing adequate amounts of both phospholipid and apoprotein components be delivered to the most affected lung segments in sufficiently high concentrations to overcome the inhibitory effects of plasma proteins and other unidentified factors. The application of a recruitment maneuver or positive end-expiratory pressure to open up collapsed areas of the lung before surfactant administration may therefore enhance the efficacy of surfactant replacement.
To the extent that there may be ongoing degradation of surfactant apoproteins in the areas of most intense inflammation, supranormal quantities of apoproteins may need to be included in the surfactant replacement mixture to act as a reserve and to supplement existing surfactant in those alveoli. On the basis of the results observed in this animal model, one would predict that a non-apoprotein-containing surfactant delivered by aerosolization would have limited clinical efficacy in ARDS as has been reported (2). On the other hand, a hydrophobic apoprotein-containing surfactant delivered by instillation in supraphysiological quantities might well have a positive impact in ARDS as has also been reported (10).
The presence of inhibitors to surfactant function in the alveolar space in ALI or ARDS points to the potential utility in performing alveolar lavage before surfactant replacement. Such a maneuver might remove those inhibitors, the effects of which are not readily overcome by increasing apoprotein and phospholipid concentrations at the interface, from the alveolar space. The potential utility of a lavage step before surfactant administration has been demonstrated in a piglet model of saline lavage-induced ALI (3) and in a limited clinical study in patients with ARDS (27).
The issues surrounding ALI, physiological dysfunction, surfactant dysfunction, and surfactant replacement are highly complex, and the clinical importance of understanding them is quite clear. It is our hope that the establishment of a small-animal model of ALI and surfactant dysfunction secondary to LPS administration, coupled with a detailed multipronged examination of the underlying processes, will make a contribution to our understanding of ARDS.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the assistance of the Respiratory Therapy Department of the Brigham and Women's Hospital Neonatal Intensive Care Unit (Boston, MA) in the performance of arterial blood gas analyses. Dr. Guo-Ping Shi provided technical assistance with development of the anti-surfactant protein B polyclonal antibodies.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-33009.
Address for reprint requests and other correspondence: R. Mora, Pulmonary and Critical Care Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (E-mail: rmora{at}caregroup.harvard.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 6 July 1999; accepted in final form 3 March 2000.
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