This study examines surfactant dysfunction in rats with inflammatory lung injury from intratracheal instillation of hydrochloric acid (ACID, pH 1.25), small nonacidified gastric particles (SNAP), or combined acid and small gastric particles (CASP). Rats given CASP had the most severe lung injury at 6, 24, and 48 h based on decreases in arterial oxygenation and increases in erythrocytes, total leukocytes, neutrophils, total protein, and albumin in bronchoalveolar lavage (BAL). The content of large surfactant aggregates in BAL was reduced in all forms of aspiration injury, but decreases were greatest in rats given CASP. Large aggregates from aspiration-injured rats also had decreased levels of phosphatidylcholine (PC) and increased levels of lyso-PC and total protein compared with saline controls (abnormalities for CASP were greater than for SNAP or ACID alone). The surface tension-lowering ability of large surfactant aggregates on a bubble surfactometer was impaired in rats with aspiration injury at 6, 24, and 48 h, with the largest activity reductions found in animals given CASP. There were strong statistical correlations between surfactant dysfunction (increased minimum surface tension and reduced large aggregate content) and the severity of lung injury based on arterial oxygenation and levels of albumin, protein, and erythrocytes in BAL (P < 0.0001). Surfactant dysfunction also correlated strongly with reduced lung volumes during inflation and deflation (P = 0.0004–0.005). These results indicate that surfactant abnormalities are functionally important in gastric aspiration lung injury and contribute significantly to the increased severity of injury found in CASP compared with ACID or SNAP alone.
- lung surfactant
- surfactant dysfunction
- gastric aspiration
- acute lung injury
gastric aspiration is a frequent occurrence in unconscious surgical patients, with an incidence of one in every 2,000–3,000 anesthetics (22, 29, 38). Gastric aspiration is also a common complication in patients with blunt thoracic trauma (22), a widespread cause of hospital admission for intensive care. In addition, gastric aspiration is a known complication in patients with impaired glottic competency from alcohol use, pregnancy, or other causes (22). Although many episodes of gastric aspiration lead only to transient pneumonitis that resolves without incident, this condition can progress to severe clinical acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Aspiration-associated ALI/ARDS carries a 30% mortality and accounts for up to 20% of all deaths attributable to anesthesia (22, 38). Aspiration pneumonitis can also predispose patients to subsequent bacterial pneumonia (5, 22, 38).
The mechanistic pathophysiology of progressive gastric aspiration lung injury is not fully understood. One important factor in the severity of aspiration lung injury appears to involve the presence of concurrent (“two-hit”) insults. We have previously reported that the severity of lung injury is increased by the aspiration of combined hydrochloric acid and small gastric particles (CASP) compared with hydrochloric acid (ACID) or small nonacidified gastric particles (SNAP) alone (16, 19, 33). Lung injury is similarly worsened following the combined administration of acid and milk products in rats (28). Acid aspiration also exacerbates the injury-inducing effects of hyperoxia and nitric oxide (18, 23–25). Our prior studies of aspiration lung injury have emphasized pulmonary inflammation (7, 16, 17, 19, 23, 25, 33) and have not investigated associated surfactant dysfunction. The present study focuses on surfactant abnormalities and their functional importance in acute pulmonary injury following the aspiration of CASP, ACID, and SNAP.
Active pulmonary surfactant is required to normalize alveolar stability, compliance, and gas exchange (26, 27). Because of the essential physiological roles of lung surfactant, its dysfunction may be an important determinant in the severity and progression of aspiration lung injury in animals and patients. Aspiration injury was assessed here by measurements of arterial oxygenation [arterial partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) ratios], pressure-volume (P-V) mechanics, and levels of erythrocytes, neutrophils, total protein, albumin, and phospholipid in cell-free bronchoalveolar lavage (BAL) at 6, 24, and 48 h following intratracheal instillation of CASP, ACID, or SNAP. The relative content of large to small surfactant aggregates in BAL was also measured, along with the dynamic surface activity of resuspended large aggregates on a pulsating bubble surfactometer. Statistical analyses were carried out to examine the degree of correlation between these measures of surfactant dysfunction and the severity of lung injury based on PaO2/FiO2 ratios, P-V mechanics, and BAL concentrations of albumin, protein, and cells.
MATERIALS AND METHODS
Aspiration pneumonitis in rats.
Animal experiments utilized protocols approved by the Institutional Animal Care and Use Committee at the State University of New York (SUNY) at Buffalo. A total of 72 male Long-Evans rats (250–300 g; Harlan Sprague-Dawley, Indianapolis, IN) were assigned randomly to one of four injury groups and one of three time points (see below). Rats were anesthetized with halothane, the trachea was exposed by a 2-cm ventral incision, and one of four solutions (1.2 ml/kg) was tracheally instilled through a 14-gauge catheter: 1) 0.15 M NaCl (NS controls), pH 5.3; 2) NS adjusted to pH 1.25 with HCl (ACID); 3) gastric particles, 40 mg/ml in NS (SNAP); or 4) gastric particles 40 mg/ml in NS adjusted to pH 1.25 with HCl (combined acid and small particles, CASP) (7, 18, 19). Gastric particles were prepared from the stomach contents of healthy Long-Evans rats at necropsy (7, 19). Particles were washed three times in normal saline (pH 5.3) by filtration through sterile gauze sponges to remove debris, autoclaved for 25 min (20 psi, 121°C), pelleted by centrifugation at 2,000 g for 2 min, and resuspended in normal saline (pH 5.3) for aspiration studies. The distribution of particle diameters in equivalent preparations has previously been reported by Knight et al. (19) to be bimodal, with peaks at ∼4.5 and 13 μm and an average diameter of <10 μm. After instillation of ACID, SNAP, CASP, or NS, the tracheal incision was repaired with a 6-0 suture, and the ventral neck incision was closed with surgical staples. Rats were housed in the vivarium and examined for lung injury and surfactant parameters at 6, 24, and 48 h postaspiration (6 rats per injury per time point). One animal died before evaluation (NS at 48 h), and technical problems affected the lavage procedure for another (ACID at 24 h), resulting in a total of 70 rats analyzed postaspiration. Eleven additional male Long-Evans rats were studied for pulmonary histology at 48 h postaspiration (five rats) or to determine preinjury baseline levels (time 0 values) for physiological and BAL parameters (six rats).
Blood gas and P-V measurements.
At 6, 24, or 48 h postaspiration, rats were reanesthetized with 2% halothane in oxygen, and a sample of blood was collected with a heparinized syringe from the descending aorta for blood gas analysis (ABL5 blood gas analyzer; Radiometer American, West Lake, OH). The efficiency of gas exchange was determined from the ratio of the PaO2 to the FiO2 (fixed at 0.98 for 5 min before blood samples were obtained). A 14-gauge steel cannula was inserted into the trachea and secured with a silk suture, and the animals were exsanguinated through the vena cava. Pulmonary P-V mechanics were assessed by injecting air into the lungs at a rate of 25 ml/min by a syringe pump (31). Inflation pressures were monitored by an in-line pressure transducer connected to an Apple PowerBook G4 computer equipped with a signal conditioner and data acquisition board (National Instruments, Austin, TX), and custom software was written in LabVIEW 6 (National Instruments). When inflation pressure reached 40 cmH2O, the syringe pump was reversed, and deflation was begun. Pressure values during inflation and deflation were recorded every 0.2 s (every 83 μl of injected or withdrawn volume), and complete inflation/deflation curves were obtained in <1 min. Lung volumes at fixed pressure were calculated from the rate of air injection or withdrawal and were normalized to kilogram of body weight (31). Final P-V curves were plotted in pressure increments of 5 cmH2O.
BAL methods and cell counts.
After P-V measurements, the vasculature was flushed by injecting 20 ml (at 37°C) of Hanks' balanced salt solution (HBSS) with Ca2+ and Mg2+ (Life Technologies, Grand Island, NY) into the right ventricle. BAL was performed with 50 ml (5 × 10 ml at 37°C) of NS instilled through the tracheal cannula. Recovered BAL fluid was pooled on ice and immediately centrifuged at 1,500 g for 3 min at 4°C (GS-6R centrifuge with GH-3.8 rotor; Beckman Instruments, Palo Alto CA) to pellet cells, and the supernatant was stored at −80°C for biochemical and surface activity measurements.
Phospholipid and protein measurements on whole BAL and centrifuged large surfactant aggregates.
Total phospholipid in cell-free BAL was measured by the phosphorus assay of Ames (2), and total protein was determined by the method of Lowry et al. (21) modified by the addition of 15% SDS to allow accurate quantitation in the presence of lipid. Albumin concentrations in cell-free BAL were measured by ELISA with a polyclonal rabbit anti-mouse albumin antibody (a gift from Dr. Daniel Remick, University of Michigan, Ann Arbor, MI) and horseradish peroxidase-labeled goat anti-rabbit immunoglobulin (Pharmingen, San Diego, CA) (7). Rat albumin (Sigma, St. Louis, MO) was used as a standard. Additional studies examined the content and composition of large surfactant aggregates centrifuged from cell-free BAL at 12,000 g for 30 min. The content of large aggregates as a percentage of total BAL phospholipid was determined by phosphate assay (2), and levels of total protein in aggregates were measured by the modified assay of Lowry et al. (21) above. Hydrophobic protein levels in large aggregates were also determined following extraction into chloroform (4), and phospholipid classes in aggregates were defined by thin layer chromatography using a solvent system of chloroform-methanol-2-propanol-triethylamine-water (30:9:25:25:7 by volume) (36).
Pulsating bubble measurements of surface activity.
The surface activity of centrifuged large aggregates from rats given ACID, SNAP, CASP, or NS was assessed during cycling at a physiological rate of 20 cycles/min at 37 ± 0.5°C on a pulsating bubble surfactometer (General Transco, Largo, FL; formerly Electronetics, Buffalo, NY) (8). A small air bubble, communicating with ambient air, was formed in a 40-μl aliquot of surfactant in a plastic sample chamber mounted on the pulsator unit of the surfactometer. The bubble was oscillated between maximum and minimum radii of 0.55 and 0.4 mm while the pressure drop across the air-water interface was measured with a precision pressure transducer. Surface tension at minimum bubble radius (minimum surface tension) was calculated as a function of time of pulsation from the measured pressure drop at end-compression and the Laplace equation for a spherical interface (8, 10). Surfactant samples were examined at a uniform phospholipid concentration of 1 mg/ml in 10 mM HEPES, 2 mM CaCl2, and 150 mM NaCl, pH 7.0.
In five rats (ACID, SNAP, CASP, or NS at 48 h plus an uninjured control) not lavaged or studied for P-V mechanics, lungs were removed en bloc following tracheal cannulation and flushing of the vasculature with 20 ml of HBSS injected into the right ventricle. The lungs were fixed with 10% neutral buffered formalin at an inflation pressure of 20 cmH2O for 24 h, and tissue was stained with hematoxylin and eosin in 4-μm sections by standard methods (19). Slides were assessed for lung injury by an experienced pathologist (Dr. James Woytash, Department of Pathology, SUNY at Buffalo) blinded to animal group assignments.
Graphic displays and descriptive statistics for each outcome over time were expressed as means ± SE. Group means were compared using one-way analysis-of-variance (ANOVA) with Scheffé's post hoc analysis to adjust for multiple comparisons. Two-way ANOVA was performed to test for interaction between ACID and SNAP aspiration injury. Statistical evaluations for P-V mechanical data were done with Student's t-tests (corrected for multiple comparisons at a level of α = 0.05/6 = 0.0083) at selected pressures on the inflation and deflation limbs of the hysteresis curves. Pearson's product moment correlations with Fisher's z-transformation were also calculated to examine correlative relationships between specific parameters of surfactant dysfunction and the severity of aspiration lung injury.
Severity of aspiration lung injury.
Rats instilled intratracheally with ACID, SNAP, or CASP all had pulmonary injury, but its severity varied among the different forms of aspiration studied. At 6 h postaspiration, arterial oxygenation based on PaO2/FiO2 was reduced most substantially in rats with CASP aspiration. PaO2/FiO2 ratios in rats given CASP showed a synergistic decrease compared with the component injuries of ACID or SNAP alone at 6 h (Fig. 1A, P = 0.014 for interaction by two-way ANOVA). PaO2/FiO2 ratios in rats given CASP improved slightly at 24 h but were still significantly lower than NS controls at 48 h (Fig. 1A, P < 0.02). Arterial oxygenation in rats given CASP met the criteria for clinical ARDS at 6 h (PaO2/FiO2 = 158 ± 26 mmHg) and for clinical ALI at 24 and 48 h (PaO2/FiO2 = 271 ± 53 mmHg at 24 h and 250 ± 29 mmHg at 48 h) (Fig. 1A).
Rats with CASP aspiration also had the most severe lung injury based on increased leakage of albumin into the alveolar spaces (Fig. 1B). Levels of albumin in cell-free BAL at 6 h following CASP (766 ± 153 μg/ml) were approximately seven to nine times higher than those for rats that received SNAP (104 ± 22 μg/ml) or ACID (81 ± 16 μg/ml), indicating a synergistic increase with respect to this parameter (Fig. 1B, P = 0.0009 for interaction by two-way ANOVA). Animals given CASP or SNAP had more severe intra-alveolar hemorrhage based on numbers of erythrocytes in BAL (Fig. 1C). At 6 h postaspiration, erythrocyte numbers were significantly higher in rats given CASP (1.3 ± 0.2 × 108, P < 0.01) or SNAP (1.1 ± 0.3 × 108, P < 0.05) compared with NS (1.0 ± 0.5 × 107). Erythrocyte numbers remained elevated in CASP aspiration at 48 h compared with NS or ACID (P < 0.002, Fig. 1C). Numbers of total leukocytes (Fig. 2A) and neutrophils (Fig. 2B) in BAL were significantly elevated at 6, 24, and 48 h for rats given CASP or SNAP compared with either NS controls (P < 0.01) or animals given ACID (P < 0.02) (Fig. 2).
The morphology of representative stained lung tissue sections from rats with ACID, SNAP, or CASP aspiration is shown in Fig. 3 at 48 h postinjury. Lung histology at earlier times (6 and 24 h) has been previously published by Knight et al. (19). Animals given ACID had minimal pulmonary histopathology at 48 h (Fig. 3A), with lung tissue generally similar in appearance to NS and uninjured controls (not shown). Animals given SNAP had well-formed noncaseating granulomas scattered throughout the lung parenchyma (Fig. 3B). In the alveolar epithelium, some type II pneumocytes were larger than normal, consistent with injury-induced activation. Lung tissue from animals given CASP had increased interstitial and alveolar septal edema compared with SNAP at 48 h (Fig. 3C). Granulomas were distributed throughout the parenchyma as in SNAP, with a more acute stage of formation indicated by decreased organization, increased neutrophils, and increased necrotic material. As in SNAP, large type II pneumocytes were apparent in the alveolar epithelium in CASP injury, sometimes in association with granulomas.
Measurements of quasistatic pulmonary P-V mechanics indicated significant decreases in inflation/deflation volumes at 24 and 48 h in rats injured with CASP (Fig. 4). Rats given SNAP had statistically equivalent decreases in inflation/deflation volumes, whereas rats given ACID were not significantly different from NS controls (P-V curves for ACID and SNAP are not shown explicitly on Fig. 4 for visual clarity). At 24 h, maximum inspired lung volumes at 40 cmH2O were significantly decreased in rats given CASP (31.5 ± 1.5 ml/kg) or SNAP (32.6 ± 1.0 ml/kg) compared with NS controls (38.5 ± 1.4 ml/kg, P < 0.0083). At 48 h, maximum inspired lung volumes at 40 cmH2O were decreased in rats given CASP (27.5 ± 1.1 ml/kg) or SNAP (30.3 ± 1.0 ml/kg) compared with either ACID (37.0 ± 0.6 ml/kg) or NS controls (38.3 ± 0.9 ml/kg) (Fig. 4C, P < 0.0083). At both 24 and 48 h, lung volumes at lower pressures (10–35 cmH2O) were also significantly reduced in rats given CASP or SNAP compared with NS controls. In addition, inflation/deflation volumes in rats that received CASP or SNAP were significantly reduced at 48 h compared with rats instilled with ACID (Fig. 4C).
Lung surfactant abnormalities in aspiration injury.
A major emphasis of investigation here was on surfactant-related abnormalities in aspiration lung injury. Compositional analyses of whole cell-free BAL showed that both total protein and the ratio of total protein to phospholipid were increased in rats with aspiration injury compared with NS controls (Table 1). Increases in protein in whole BAL were greatest in rats with CASP aspiration at the three time points of injury studied (6, 24, and 48 h; Table 1). Total phospholipid was increased in BAL at 6 h for rats given CASP, but in most cases total phospholipid levels in BAL were similar for NS controls and animals with aspiration injury (Table 1). However, the percentages of total BAL phospholipid that sedimented in the large surfactant aggregate fraction during centrifugation at 12,000 g were consistently lower in all three forms of aspiration compared with NS controls at 6, 24, and 48 h (Fig. 5). Decreases in large aggregate content at a given time were greatest in rats given CASP. At 6 h postaspiration, the large aggregate fraction made up only 19.8 ± 1.9% of total BAL phospholipid in rats given CASP compared with 37.9 ± 2.9% for NS controls (Fig. 5). The percentage of large aggregates in BAL at 6 h was also reduced in rats given SNAP or ACID compared with NS (26.1 ± 1.6% of total phospholipid for SNAP and 25.3 ± 2.0% for ACID). A similar pattern of decrease in large surfactant aggregate content as a percentage of total BAL phospholipid was present at 24 and 48 h for rats with aspiration injury (CASP > SNAP ∼ ACID, Fig. 5).
Measurements of protein and phospholipid in centrifuged large surfactant aggregates from rats with different forms of aspiration lung injury are given in Tables 2 and 3. Large aggregates from rats with all three forms of aspiration had substantially greater levels of total protein (weight percentage relative to phospholipid) compared with NS controls (Table 2). However, this effect was most pronounced in large aggregates from rats injured with CASP, which had total protein contents that were 50–100% higher than those in aggregates from rats given ACID or SNAP (Table 2). In contrast to total protein, the levels of hydrophobic protein [indicative of surfactant proteins (SP)-B/C] in large aggregates were similar for aspiration-injured and control animals (Table 2). In addition to having abnormally high contents of total protein, large aggregates from rats with aspiration injury had decreased levels of phosphatidylcholine (PC) and increased levels of lyso-PC compared with NS controls (Table 3). As with total protein, the magnitudes of changes in phospholipid class composition were greater for rats given CASP compared with ACID or SNAP alone (Table 3).
In addition to having abnormal phospholipid and total protein contents, large aggregates were also impaired in surface activity during aspiration injury (Fig. 6). Centrifuged large aggregates were resuspended in buffered saline at a uniform phospholipid concentration of 1 mg/ml and studied for overall dynamic surface tension-lowering ability on a pulsating bubble surfactometer. Large aggregates obtained at 6 h following the aspiration of CASP reached minimum surface tensions of only 21.2 ± 0.4 mN/m after prolonged cycling (20 min) on the bubble surfactometer, compared with minimum surface tensions <1 mN/m for NS controls (Fig. 6A). Minimum surface tensions after 20 min of bubble pulsation were also elevated for resuspended large aggregates obtained at 6 h from rats given SNAP or ACID (13.4 ± 3.1 mN/m and 12.7 ± 1.2 mN/m, respectively) (Fig. 6A). Significant reductions in surface activity were also apparent in resuspended large aggregates lavaged from rats with aspiration lung injury at 24 h (Fig. 6B) and 48 h (Fig. 6C). Surface activity detriments were most pronounced in animals given CASP at all three time points of injury studied (Fig. 6).
Pearson's product moment analyses were used to define more specifically the degree of correlation between surfactant dysfunction and the severity of lung injury over the full time period studied (Table 4). In these analyses, surfactant dysfunction was based on percent large aggregate content in BAL and on minimum surface tension at 20 min of bubble pulsation for the various injury groups at 6, 24, and 48 h postaspiration. There were strong correlations between the extent of surfactant dysfunction based on these parameters and the severity of lung injury. Both measures of surfactant dysfunction were strongly correlated with PaO2/FiO2 ratios, BAL erythrocyte numbers, and BAL albumin and total protein content (P < 0.0001 for all variables, Table 4). Surfactant dysfunction based on minimum surface tension and large aggregate content also correlated strongly with the midpoint inflation lung volume at 20 cmH2O (Table 4, P = 0.0053 and P = 0.0031, respectively), which was chosen as a representative point on the inspiration limb of the P-V curve. In addition, surfactant dysfunction based on minimum surface tension and large aggregate content correlated strongly with maximum lung volume at 40 cmH2O (Table 4, P = 0.0005 and 0.0004, respectively). Similar strong levels of statistical correlation were also found between these measures of surfactant dysfunction and deflation lung volumes at pressures ≥20 cmH2O (data not shown).
This study demonstrates that the pathophysiology of severe aspiration lung injury includes functionally important reductions in the surface activity and large aggregate content of lavaged pulmonary surfactant. Rats instilled intratracheally with ACID, SNAP, or a combination of the two (CASP) had significant reductions in the percentage of total lavage phospholipid in the large surfactant aggregate fraction (Fig. 5). In addition, large aggregates from animals with aspiration injury had abnormally high levels of total protein and altered phospholipid class distributions (Tables 2 and 3), and their surface activity was significantly impaired at a fixed phospholipid concentration in pulsating bubble studies (Fig. 6). Abnormalities in surfactant aggregate composition and activity were consistently greatest in rats injured with CASP compared with SNAP or ACID alone. Rats with CASP aspiration also had the most severe lung injury based on PaO2/FiO2 ratios, BAL albumin and total protein, and BAL erythrocyte numbers (Fig. 1, Table 1). Rats with CASP and SNAP aspiration had the highest levels of pulmonary inflammation in terms of numbers of leukocytes and neutrophils in BAL (Fig. 2) and also exhibited abnormal P-V mechanics (Fig. 4). Decreases in the content and surface activity of lavaged large aggregates were strongly correlated statistically with the severity of lung injury and P-V mechanical detriments across all forms of aspiration and time points of injury studied (Table 4).
There are multiple pathways by which lung surfactant abnormalities can enter the pathophysiology of acute aspiration lung injury. One mechanism that almost certainly contributes to the surfactant dysfunction observed here involves biophysical interactions of alveolar surfactant with blood-derived proteins such as albumin (e.g., Refs. 12–14). In the present study, albumin concentrations and levels of total protein were elevated to the greatest extent in BAL from rats receiving CASP (Table 1, Fig. 1). Previously published results have also reported that CASP aspiration induces elevated levels of albumin in BAL that are synergistically greater than for ACID or SNAP alone (16, 19). Consistent with higher levels of albumin and total protein in BAL, rats injured with CASP had the greatest reductions in large aggregate surface activity (Fig. 6). Albumin and other blood proteins including hemoglobin and fibrinogen are known to compete with lung surfactant components during adsorption to the air-water interface (12, 14, 26). Active lung surfactant components are not able to penetrate preformed interfacial films of albumin or related blood proteins, resulting in increased equilibrium (adsorption) surface tensions and elevated minimum surface tensions during dynamic compression (12, 14, 26).
A second mechanism of surfactant dysfunction in our experiments involved decreases in the content of large surfactant aggregates obtained from BAL by centrifugation at 12,000 g for 30 min (Fig. 5). Alveolar surfactant is known to contain a size-distributed microstructure of phospholipid-rich aggregates, the largest of which normally have the highest surface activity and apoprotein content (see Refs. 26, 39 for review). Several studies have shown that large surfactant aggregates can be depleted in content or impaired in activity during acute pulmonary injury (11, 20, 30). Reductions in the content of large surfactant aggregates during lung injury can occur from increased conversion to small aggregates in the alveolar hypophase, as well as from alterations in surfactant recycling or metabolism in alveolar type II pneumocytes. Substantial alterations in type II cell surfactant metabolism appear unlikely in our studies, since total BAL phospholipid was not depleted in aspiration-injured animals (Table 1), and hydrophobic protein consistent with SP-B/C was unchanged in the large aggregate fraction (Table 2). However, large aggregates in our study did have several aspiration-induced compositional abnormalities that are mechanistically relevant for their decreased intrinsic surface activity as discussed below.
Large surfactant aggregates from aspiration-injured rats were found to have substantial changes in both protein content (increased total protein, Table 2) and phospholipid content (decreased PC and increased lyso-PC, Table 3). Increased total protein levels in large aggregates are consistent with inhibitory blood-derived proteins being incorporated into or associated with these microstructures in the alveolar hypophase. This process would be expected to decrease the intrinsic surface activity of aggregates based on the known inhibitory effects of plasma proteins on surface active function noted earlier (12, 14, 26). Increases in total protein in large aggregates also potentially include hydrophilic surfactant proteins (SP-A/D), which were not specifically assessed in our experiments. However, it is very unlikely that increases in SP-A/D were responsible for the large elevations in total protein observed in aggregates from aspiration-injured animals in Table 2. Levels of hydrophobic surfactant proteins (SP-B/C) in large aggregates were unchanged based on measurements on chloroform-extracted aggregates (Table 2). In addition to effects from plasma proteins, reductions in PC and increases in lyso-PC in large aggregates could also contribute to surface activity detriments. These changes in phospholipid content are consistent with the action of phospholipases, which are known to be present in the lungs along with other lytic enzymes during inflammatory injury. Lyso-PC is a known inhibitor of lung surfactant activity (14, 26, 37) and has the ability to penetrate directly into interfacial films to impair surface tension lowering during dynamic compression (14, 26). Lyso-PC can also generate additive surfactant inhibition when present with albumin (37).
The results found here demonstrate that surfactant dysfunction is functionally important in the pathophysiology of aspiration injury and contributes to the increased severity of lung injury in rats given CASP compared with ACID or SNAP alone. However, our experiments did not address specific inflammatory or gene-based pulmonary responses and their significance for surfactant dysfunction. The observed synergy of acid and gastric particulate aspiration implies that diverse but complementary pathological inflammatory responses may be generated by ACID and SNAP. Exposure of pulmonary tissue to low pH causes direct injury that releases cellular constituents like proteinases and early cytokines and chemokines that activate local and systemic inflammatory cascades (17, 18, 25). SNAP, on the other hand, is unlikely to be directly injurious to tissue and more probably serves as a nidus for the progression and persistence of inflammation (19). When these two injury inducers are present together in CASP, they interact to cause a more severe and longer lasting inflammatory lung injury. The mechanistic coupling between specific inflammatory pathways and surfactant dysfunction in CASP aspiration is an important area for future investigation.
Our prior work has extensively examined inflammatory mediators in BAL from rats following the aspiration of CASP, SNAP, and ACID (e.g., 7, 16, 33), and measurements of this kind were thus not included in the present study. Among other findings, this prior work has demonstrated significant elevations in the CXC chemokines macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) in rats over a 24-h period following the tracheal instillation of CASP or SNAP (16), consistent with the increased levels of BAL neutrophils found in these two forms of aspiration injury in the present experiments (Fig. 2). Similarly, levels of the monotactic chemokine monocyte chemoattractant protein-1 (MCP-1) were increased over the 24-h period following instillation of CASP or SNAP (16), also consistent with the elevated levels of leukocytes in BAL measured in Fig. 2.
The strong statistical correlations found between multiple parameters of lung injury severity and decreases in large aggregate content and surface activity in BAL (Table 4) are consistent with the known essential physiological roles of pulmonary surfactant in reducing the work of breathing, normalizing pulmonary mechanics and gas exchange, and protecting against edema (26, 27). The presence of surfactant dysfunction in aspiration lung injury has potentially important implications for clinical therapies for severe gastric aspiration. Meconium aspiration lung injury in term infants is known to have a prominent component of surfactant inactivation, and this condition has been improved by the airway delivery of exogenous surfactants in multiple animal studies (1, 6, 34, 35) and in clinical trials in human infants (3, 9, 15). Although there are obvious differences between meconium and gastric contents, both have acidic (caustic) components as well as particulate components. In addition to meconium aspiration, surfactant abnormalities are well documented in clinical ALI/ARDS from multiple etiologies, and laboratory studies indicate that active exogenous surfactants can mitigate related acute inflammatory lung injury in a variety of animal models (see Refs. 26, 27, 32 for review). These findings provide a rationale for further studies investigating the use of exogenous surfactants, alone or in combination with agents or interventions targeting other aspects of inflammatory lung injury, to treat severe forms of gastric aspiration.
This study was supported by National Institutes of Health Grants AI-46534 (P. R Knight, B. A. Davidson, T. A. Russo), HL-48889 (P. R Knight, B. A. Davidson, T. A. Russo), HL-69763 (T. A. Russo, P. R Knight, B. A. Davidson), HL-03910 (P. R. Chess), and HL-56176 (R. H. Notter, B. A. Holm) and also by the John R. Oishei Foundation (T. A. Russo).
The authors gratefully acknowledge the assistance of Dr. James A. Woytash in examining histological sections for lung injury.
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. Section 1734 solely to indicate this fact.
- Copyright © 2005 the American Physiological Society