Critical Care Medicine Issue: Volume 24(6), June 1996, pp 1025-1033 Copyright: (C) Williams & Wilkins 1996. All Rights Reserved. Publication Type: [Laboratory Investigation] ISSN: 0090-3493 Accession: 00003246-199606000-00024 [Laboratory Investigation] Surfactant replacement in the treatment of sepsis-induced adult respiratory distress syndrome in pigs Nieman, Gary F. BA; Gatto, Louis A. PhD; Paskanik, Andrew M.; Yang, Bennett MD; Fluck, Robert MS; Picone, Anthony MD PhD Author Information From the Departments of Surgery and Respiratory Care (Mr. Nieman, Drs. Yang and Picone, Mr. Fluck, and Mr. Paskanik), SUNY Health Science Center at Syracuse, Syracuse, NY, and the Department of Biological Sciences (Dr. Gatto), SUNY College at Cortland, Cortland, NY. Supported, in part, by a grant from The American Respiratory Care Foundation. Infasurf was donated by ONY Company. Address requests for reprints to: Gary F. Nieman, Department of Surgery, SUNY Health Science Center, 750 E. Adams Street, Syracuse, NY 13210. ---------------------------------------------- Outline Abstract MATERIALS AND METHODS Protocol. Calculations. Procedures at Necropsy: Wilhelmy Balance. Histometric Evaluation. Lung Water. Statistical Analysis. RESULTS DISCUSSION REFERENCES Abstract Objective: To evaluate the efficacy of treating sepsis-induced adult respiratory distress syndrome (ARDS) by instillation of exogenous surfactant in a porcine endotoxin model. Design: Prospective trial. Setting: Laboratory at a university medical center. Subjects: Fifteen hybrid pigs, weighing 15 to 20 kg. Interventions: Pigs were anesthetized and surgically prepared for hemodynamic and lung function measurements. Animals were randomized into three groups: a control group (group I; n equals 4) that received sham Escherichia coli lipopolysaccharide (endotoxin); an endotoxin group (group II; n equals 6) that received endotoxin (25 micro gram/kg); and an endotoxin plus surfactant (Infasurf, ONY, Amherst, NY) instillation group (group III; n equals 5) that received endotoxin (25 micro gram/kg) followed by surfactant (100 mg/kg) instillation; all groups were studied for 6 hrs after the start of endotoxin injection. At necropsy, lung water and surfactant function (Wilhelmy balance) were measured and the right middle lung lobe was fixed for histologic analysis. Surfactant function was expressed as the surface tension at the minimum trough area. Measurements and Main Results: Surfactant treatment (group III) significantly (p less than .05) decreased venous admixture (group III equals 41.5 plus minus 9.1%; group II equals 61.6 plus minus 4.7%), PaCO2 (group III equals 46.6 plus minus 1.3 torr [6.2 plus minus 0.2 kPa]; group II equals 54.4 plus minus 2.6 torr [7.25 plus minus 0.34 kPa]), and surface tension minimum (group III equals 8.8 plus minus 1.8 dyne/cm; group II equals 20.0 plus minus 2.0 dyne/cm), as compared with endotoxin without treatment (group II) 6 hrs after endotoxin infusion. However, surfactant instillation did not significantly improve PaO2 (group III equals 62.8 plus minus 6.8 torr [8.4 plus minus 0.9 kPa]; group II equals 50.3 plus minus 3.7 torr [6.7 plus minus 0.49 kPa]) or reduce the amount of pulmonary edema (group III equals 7.1 plus minus 0.39 ratio; group II equals 6.8 plus minus 0.24 ratio) seen 6 hrs following endotoxin injection. Histologic analysis showed that endotoxin caused edema accumulation around airways and pulmonary vessels, and a large increase in the number of marginated leukocytes with or without surfactant treatment. Surfactant treatment significantly increased the total number of leukocytes in the pulmonary parenchyma. Conclusions: We conclude that endotoxin caused lung injury typical of ARDS as demonstrated by pulmonary edema, an increase in PaCO2 and a decrease in PaO sub 2, a decrease in static lung compliance, and inhibition of surfactant function. Exogenous surfactant treatment effected only moderate improvements in lung function (i.e., reduced venous admixture and restored surfactant function) in this sepsis-induced ARDS model. (Crit Care Med 1996; 24:1025-1033) ---------------------------------------------- KEY WORDS: septic shock; endotoxemia; lipopolysaccharide; acute respiratory distress syndrome; porcine; pulmonary surfactant; lungs; critical illness; pulmonary function Numerous conditions (severe trauma, fat embolism, surface burns, etc.) are associated with the development of adult respiratory distress syndrome (ARDS), but the most common cause is sepsis [1]. The major mediator in sepsis-induced ARDS seems to be endotoxin or lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, which initiates a whole body inflammatory response resulting in ARDS as well as failure of other organ systems [2]. Although the pathologic response to endotoxemia is complex, the primary event leading to pulmonary vascular injury and ARDS is activation of leukocytes by various inflammatory mediators [3]. Endotoxin has been shown to increase pulmonary arterial pressure and pulmonary vascular resistance [4], which are associated with marked leukopenia [5]. In addition, granulocytes accumulate in the pulmonary microcirculation and leukocytes migrate into the interstitium [5]. The accumulation of neutrophils in the lung is associated with increased vascular permeability to plasma proteins and alveolar flooding with a serous exudate [3]. Plasma proteins entering the alveolus are believed to be the major mechanism of surfactant deactivation [6]. In this study, we modeled sepsis-induced ARDS by injecting pigs with endotoxin and then tested the efficacy of the exogenous surfactant Infasurf (ONY, Amherst, NY), in ameliorating the hypoxemia, hypercapnia, surfactant inhibition, pulmonary edema, and histologic alterations associated with this ARDS model. We presumed that endotoxin infusion would best simulate the complex proinflammatory response elicited in humans with ARDS, and therefore, constitute an appropriate model to study surfactant replacement therapy. MATERIALS AND METHODS Fifteen hybrid pigs (15 to 20 kg) were pretreated with atropine (0.05 mg/kg im) 10 to 15 mins before intubation, and preanesthetized with ketamine (30 mg/kg im), and xylazine (2 mg/kg im). Anesthesia was induced and maintained with intravenous sodium pentobarbital, a tracheostomy was established, and the animals were ventilated with a mixture of 50% nitrous oxide and 50% oxygen delivered via an anesthesia ventilator (Narkomed Drager AV, Telford, PA). Sodium pentobarbital (6 mg/kg/hr) was delivered iv continually via a Harvard infusion pump (907, Harvard Apparatus, Millis, MA), and pancuronium bromide given in bolus (2 mg) to maintain anesthesia and eliminate respiratory muscle activity. A femoral artery cutdown was established with 2-mm inner diameter polyethylene tubing for blood gas (ABL2, Radiometer, Copenhagen, Denmark) and blood oxygen content (OSM3, Radiometer, Copenhagen, Denmark) sampling. Parameters measured on the ABL2 analyzer (Radiometer, Copenhagen, Denmark) included arterial and venous blood pH, PO2, PCO2, and base excess (mmol/L). Parameters measured on the OSM3 (Radiometer, Copenhagen, Denmark) analyzer included arterial and venous blood hemoglobin (Hgb [g/dL]), oxygen content (vol%), and mixed venous oxygen saturation (%). A 7-Fr flowdirected pulmonary artery thermodilution catheter was passed through the femoral vein into the pulmonary artery for mean pulmonary arterial (MPAP), pulmonary artery occlusion (PAOP), and central venous pressure measurements, mixed venous blood gas and oxygen content sampling, and cardiac output determinations (COM-1, Baxter Edwards Critical-Care, Irvine, CA). Cardiac output measurements were made in duplicate at end-expiration. A triple lumen catheter was inserted into the jugular vein for fluid, anesthesia, drug, and Escherichia coli lipopolysaccharide (endotoxin) infusion. A carotid artery cutdown was used for systemic arterial pressure measurements. Pressures were measured using transducers (Transpac MK4-04DTNVF, Sorensen Research, Triangle Park, UT) leveled at the heart and recorded on a recorder (7754A, Hewlett-Packard, Andover, MD). Peak airway pressure was measured from a side port located 2 cm from the end of the tracheal tube. Static pulmonary compliance was measured by disconnecting the ventilator and injecting twice the calculated tidal volume (VT equals wt [kg] times 12) into the lung with a Collins 1-L syringe (Collins Pulmonary Instruments, Braintree, MA). Plateau airway pressure was recorded and used to calculate static pulmonary compliance (injected volume/plateau pressure [mL/mm Hg]). A cystotomy was performed with a midline incision and a Foley catheter placed into the bladder. Base deficit below normal limits (minus 3 mmol/L) was corrected with intravenous sodium bicarbonate and adjustments made in tidal volume and ventilatory rate to bring PaO2 and PaCO2 values within normal range. After endotoxin infusion, respiratory rate was adjusted to maintain a PaCO2 of less than 50 torr (less than 6.7 kPa), when possible, and sodium bicarbonate was administered to maintain base deficit above minus 3 mmol/L. Heating pads and warmed intravenous fluids served to maintain core temperature between 37 degrees C to 39 degrees C. Protocol. All pigs were studied from t equals 0 to 360 mins while being infused with lactated Ringer's solution (25 mL/kg/hr). Cardiac output was measured every 15 mins after endotoxin or sham endotoxin infusion and maintained at 1 SD from normal cardiac output (normal cardiac output for anesthetized pigs in our laboratory equals 297.9 plus minus 78.3 mL/kg/min) by bolus infusion of 6% dextran 70 in saline [7]. Pigs in the sepsis groups were infused with endotoxin (111:B4, 25 micro gram/kg; Sigma Chemical, St. Louis, MO) mixed in 500 mL of saline and delivered via a Flo-Guard 8000 volumetric infusion pump (Travenol, Deerfield, IL) from t equals 0 to 60 mins. The control group received 500 mL saline without endotoxin. The animals were grouped according to three treatment conditions: Control (no endotoxin [group I; n equals 4]) animals received 500 mL saline without endotoxin; endotoxin animals (group II; n equals 6) received endotoxin infusion with no treatment other than the previously described fluids; and endotoxin plus surfactant animals (group III; n equals 5) received endotoxin infusion with surfactant instilled into the airway 120 mins after the beginning of endotoxin infusion and the previously described fluids. The exogenous surfactant, Infasurf, was instilled into the lungs by the following procedure: Animals were placed on their right side, they were taken off the ventilator, and a suction catheter was advanced down the trachea to a point near the carina. The suction catheter was modified by occluding the main lumen and cutting multiple small holes in the catheter tip such that surfactant were delivered in numerous directions. Five milliliters of surfactant was injected, the animal was placed back on the ventilator, and following five normal tidal volumes, the animal was sighed to an airway pressure of 20 mm Hg to facilitate delivery. The above procedure was repeated with the animal in the prone position and on its left side until all of the surfactant (100 mg/kg in a 35 mg/mL concentration) was delivered. The entire process took approximate 15 mins. After instrumentation, a period of approximate 15 mins was allowed for the stabilization of hemodynamic and blood gas parameters, at which point, values were considered baseline. All vascular and airway pressures, cardiac output, arterial and mixed venous blood gas samples, oxygen content, oxygen saturation, hemoglobin, and urine output measurements were recorded during the baseline period and every 30 mins for 6 hrs following endotoxin injection. The pigs were killed by exsanguination and the right diaphramatic lung lobe was excised with the airway intact. In addition to these measurements made during the study, the following measurements were made after necropsy: surfactant function, lung water (wet/dry weight ratio), and histologic assessment (see Histometric Evaluation). Calculations. Based on the collected data, the following calculations were performed: Venous admixture (Qva/Qt) equals (CcO2 minus Ca/CcO2 minus CvO2), where CaO2 and CvO2 are arterial and venous blood oxygen content, Qva is venous admixture blood flow, and Qt is total blood flow. CaO2 and CvO2 were measured with the OSM3 (Radiometer). Capillary content values (CcO2) were calculated from the alveolar gas equation with the assumption that pulmonary capillary oxygen saturation (ScO2) is 100% [8]: Oxygen content (Vol%) equals ([Hb times ScO2 times 1.39] plus [0.003 times PO2]), where Hb is hemoglobin. Whole animal oxygen consumption (VO2) was calculated with the formula: VO2 (mL O2/min) equals CO times (CaO2 minus CvO2), where CO is cardiac output. Oxygen delivery (DO2) was calculated with the formula: DO2 (mL O2/min) equals CO times CaO2. Pulmonary (PVR) and systemic (SVR) vascular resistance (mm Hg/L/min) were calculated with the formula: PVR equals (MPAP minus PAOP)/CO, and SVR equals (Psys minus CVP)/CO, where Psys is mean systemic artery pressure, and CVP is central venous pressure. Procedures at Necropsy: Wilhelmy Balance. Dynamic surfactant function on surfactant extracted from minced lung tissue was measured on a modified Wilhelmy balance. Lung tissue extracts were prepared according to the technique of Lum and Mitzner [9]. The right diaphragmatic lobe was inflated to remove any atelectasis. Surfactant concentration was standardized by lung weight, assuming that the amount of surfactant/weight of lung tissue (1 g) was equal under baseline conditions in all groups [9]. Since endotoxin resulted in pulmonary edema, which increases lung weight/given lung volume, a 1.5-g sample of lung tissue was used in the groups exposed to endotoxin (groups II and III). It was presumed that increasing the weight of the lung tissue by 50% adequately accounted for the increased weight due to edema, since preliminary data demonstrated that endotoxin effects an increase in lung water of approximate 20%. The Wilhelmy balance technique utilized in our laboratory has been described elsewhere [10]. Lung tissue from the dorsal portion of the right diaphragmatic lobe was excised and finely minced into 60 mL of saline and strained through cotton gauze. The surfactant extract was placed into the Teflon trough (351 cm2) of the Wilhelmy balance which had been primed with 500 mL of saline. The samples were allowed to ``cure'' for 30 mins before surface compression. The trough surface was compressed with a movable barrier from 100% (maximum) to 20% (minimum) of total trough area and then decompressed back to 100% of the area. The surface tension was continually measured with a platinum paddle connected to a Gould UC3 transducer with microscale accessory (Oxnard, CA), Hewlett-Packard HP8805B amplifier, and HP7754 strip-chart recorder. Each cycle time was 2 mins and all samples were run at room temperature. The alveolar pressure necessary to maintain alveolar patency is a function of the alveolar radius (r) and surface tension (ST), and is described by the LaPlace equation: Alveolar pressure equals 2.ST/r. Thus, for surfactant to be physiologically functional, it must decrease alveolar surface tension as alveolar radius decreases [10]. This parameter is assessed in vitro by compressing the surfactant in the trough of a Wilhelmy balance and recording the surface tension at minimum trough surface area (surface tension minimum [dyne/cm]). Surface tension minimum is used in this study as an index of functional surfactant. Histometric Evaluation. Tissues were processed for morphologic observations in the first four animals studied within each treatment condition. At the end of the experiment, the right cardiac lobe was excised and its hilar airway was cannulated and connected to a mercury manometer. Glutaraldehyde fixative (2.5%, phosphate-buffered) was instilled slowly through the cannula until air was no longer displaced from the airway; then, the lung was immersed in glutaraldehyde and additional fixative was infused with a syringe while pressure was monitored with the manometer. When the airway pressure of the fixative stabilized at 25 mm Hg, the cannula was clamped and the lung was placed in a chamber and exposed to a vacuum at minus 500 mm Hg for approximate 1 min, causing the lobe to sink in the fixative. Tissues were removed from vacuum and then stored at 25 mm Hg airway pressure in glutaraldehyde, at room temperature for at least 24 hrs. One tissue block from the fixed lobe of each animal was randomly chosen, processed for routine paraffin sectioning, and 100 serial sections made at 5 micro meter were stained with hematoxylin and eosin. Five equidistant sections from each animal were identified for histometric evaluation as follows: One section was chosen randomly from among the first 20 in the series, followed by another four consecutive sections 40 micro meter apart from each other. A sampling probe consisting of four equidistant sampling points was established for each section, as 4 points 4 mm apart were designated along the long axis of the section using the x/y vernier scales of the microscope stage. In this manner, 20 unbiased sampling areas were identified in each animal, amounting to n equals 80/treatment condition. Each sampling area was located blindly according to its x/y coordinates, and then observed at high magnification using a high-resolution video camera. Areas featuring bronchi, connective tissue septa, or blood vessels other than capillaries were discarded by advancing the stage 0.5 mm along the short axis of the section; thus, quantifications were limited to alveolar parenchyma. The sampling area amounted to 6400 micro meter2 and was overlaid with a grid consisting of 64 intersections at 10-micro meter intervals. Tissue density/sampling area was estimated as the ratio of intersection points falling on tissue vs. points corresponding to empty space. Alveolar macrophages and leukocytes were subsequently tallied in all focal planes of each sampling area following an unbiased counting procedure. Lung Water. Equal sized samples from both dorsal and ventral portions of lung lobes were dissected free of nonparenchymal tissue, placed in a dish, weighed, and placed in an oven (65 degrees C) until completely dry. Lung water was expressed as a wet/dry weight ratio. Statistical Analysis. Statistical significance between groups was determined using a one-way analysis of variance; significance between parameters within the same group was determined with a repeat analysis of variance. Whenever the F ratio indicated significance, a Newman-Keuls' test was used to identify the individual differences. A significant difference was assumed if the probability of the null hypothesis was less than 5%. The experiments described in this study were performed in adherence with the National Institutes of Health guidelines for the use of experimental animals in research. The protocol was approved by the Committee for the Humane Use of Animals (CHUA) at the SUNY Health Science Center, Syracuse, NY. RESULTS In the control group, the respiratory rate was 8.8 plus minus 0.8 breaths/min at the conclusion of the experiment, and 5.7 plus minus 3.4 mL of bicarbonate had been given to maintain normal arterial pH. Endotoxin caused a significant increase in both the amount of bicarbonate (endotoxin equals 55.2 plus minus 8.1; endotoxin plus surfactant equals 76.6 plus minus 11.9 mL) required to correct for metabolic acidosis and in the respiratory rate (endotoxin equals 27.7 plus minus 4.2; endotoxin plus surfactant equals 26.2 plus minus 5.6 breaths/min) necessary to maintain a PaCO sub 2 of less than 50 torr (less than 6.67 kPa) with or without surfactant. There was no significant difference in the amount of dextran-70 given between groups. No significant difference was found between groups in systemic or pulmonary artery occlusion pressure, or systemic vascular resistance at any time period Table 1. Cardiac output was greater in the endotoxin group as compared with the endotoxin plus surfactant group at 360 mins postendotoxin infusion Table 1. Static compliance was decreased (p less than .05) in both groups II and III as compared with the control group from 120 mins to the end of the study Table 1. Pulmonary arterial pressure was greatly increased 30 mins after the start of endotoxin infusion in both groups II and III, decreased to near baseline values at the end of endotoxin administration (1 hr), and increased progressively over the subsequent 3 hrs Figure 1, A. PaO2 and mixed venous oxygen saturation were significantly reduced following endotoxin administration (groups II and III) as compared with baseline and the control group Table 2. Although both PaO2 and mixed venous oxygen saturation tended to be higher in the surfactant treated group, as compared with endotoxin without surfactant, they were not significantly different. There was a significant decrease in DO2 with time in all groups, but VO2 remained unchanged Table 2. There was a large decrease in urine output in both groups II and III from 120 mins to the end of the experiment (p less than .05 vs. control) Table 2. Arterial pH decreased below the control group at 3 hrs post endotoxin infusion in both groups II and III Figure 1, B. The pH in the surfactant-treated group increased above that in group II from 4 to 6 hrs Figure 1B. The increase in arterial pH was associated with a decrease in PaCO2 with surfactant treatment Figure 1C. Endotoxin significantly increased venous admixture over baseline values and those values of the control group, both with (group III) and without (group II) surfactant treatment Figure 1D. However, surfactant (group III) halted the continual increase in venous admixture seen in the endotoxin group without surfactant (group II) and effected a significant decrease in venous admixture, as compared with group II 6 hrs after the beginning of endotoxin infusion. Pulmonary surfactant function was inhibited 6 hrs following endotoxin (group II), as demonstrated by a significant increase in surface tension minimum Figure 2. Surfactant treatment (group III) reduced surface tension minimum to near control values Figure 2. Endotoxin significantly (p less than .05) increased lung water (group II: 6.2 plus minus 0.5; group III: 6.3 plus minus 0.6 wet/dry lung weight ratio) as compared with group I (5.5 plus minus 0.1 wet/dry lung weight ratio). The qualitative evaluation of histologic preparations revealed substantial differences between control (group I) and treated animals (groups II and III). The control group exhibited slender and well-defined alveolar septa Figure 3a with occasional areas of marked cellularity and, in some specimens, discrete accumulations of diffuse lymphoid tissue highlighted the adventitia of large airways. In contrast, the animals receiving endotoxin featured more prominent alveolar walls Figure 3b, frequent areas of marked cellularity, and enlarged connective tissue cuffs around airways and pulmonary vessels, regardless of surfactant treatment. Pulmonary vessels in the treated animals showed large numbers of marginated leukocytes without obvious evidence of clotting. Leukocyte infiltration was manifest in the interstitium and extended into the lumen of the alveoli, where the sporadic presence of erythrocytes suggested the occurence of minor pulmonary hemorrhages. Endotoxin was associated with patchy interstitial edema and focal areas of consolidation interspersed with areas that appeared less congested. This regional inhomogeneity occurred throughout the specimens and was not affected by surfactant treatment. Occasional atelectasis was confined to much larger areas delimited by connective tissue and commensurate with lung lobules. Interlobular connective tissue septa in all treated animals were thickened by enlarged lymph vessels containing numerous leukocytes. At the cellular level, endotoxin (groups II and III) was followed by a marked enlargement of alveolar macrophages Figure 3b and pneumocytes type II caused by the extensive presence of clear cytoplasmic vesicles Figure 3c. Cell enlargement was more marked in alveolar macrophages, especially after surfactant administration (group III). Pneumocytes type I exhibited no obvious differences between treatment conditions. The systematic sampling and histometric analysis showed statistically significant differences in the frequency of wandering cells (leukocytes and alveolar macrophages), which were always consistent with endotoxin treatment Figure 3 b, c. Average numbers of wandering cells/sampling area Table 3 doubled after endotoxin administration and almost tripled in the group that also received surfactant. This change was caused to a large extent by increases in the numbers of leukocytes, particularly those leukocytes found in open spaces (as opposed to wandering within cellular areas). Leukocytes in open spaces showed a five-fold increase with endotoxin and a nine-fold increase with endotoxin plus surfactant. Alveolar macrophages exhibited a relatively moderate response to endotoxin treatment, regardless of surfactant. Estimates of tissue density, expressed as percentages of points falling over tissue (as opposed to open spaces) averaged 18.5 plus minus 8.1 in the control, 32.3 plus minus 12.7 in the endotoxin, and 32.7 plus minus 9.0 in the endotoxin plus surfactant group. DISCUSSION In this study, we tested the efficacy of treating respiratory failure with exogenous surfactant instillation in an established model of endotoxin-induced ARDS. Surfactant treatment improved venous admixture, PaCO2, and surfactant function but failed to prevent hypoxemia or pulmonary edema. The failure of exogenous surfactant to completely correct the ventilation/perfusion abnormalities in this study may be due to atelectasis, which limited access to and subsequent adsorption of instilled surfactant onto the alveolar surface. Histologic changes were consistent with ARDS and were not altered by exogenous surfactant. Surfactant instillation increased the number of sequestered leukocytes in the pulmonary parenchyma. The mechanism of increased leukocyte sequestration and its pathologic significance are unknown. The pathophysiology that occurs in pigs following endotoxin injection is very similar to that of ARDS. The clinical course of late phase human ARDS comprises pulmonary hypertension, massive bilateral consolidation, hypoxemia, white blood cell sequestration in the pulmonary parenchyma, high permeability pulmonary edema, surfactant deactivation, and increased venous admixture and PaCO2 [11-14]. We observed pathophysiologic and histologic changes similar to those changes described in human patients [11-14] and animal models of ARDS [7,15-22]. Endotoxin infusion caused acute pulmonary hypertension similar to other studies utilizing the pig [7,20], dog [21], and sheep [22]. We also demonstrated a sequestration of leukocytes in the pulmonary parenchyma, a decrease in PaO2, and an increase in venous admixture and wet/dry ratio consistent with the data from other laboratories [7,15,16] utilizing the porcine lipopolysaccharide endotoxicosis model. Endotoxin inhibited surfactant function as shown by the increased surface tension minimum. Surfactant dysfunction has been demonstrated in animal models of acute lung injury [17-19] and in patients with ARDS [13]. Inhibition of surfactant occurs very early in the course of human ARDS and correlates well with the severity of lung injury [13]. However, only one other study [19] has measured surfactant function in an animal model of sepsis-induced ARDS, where sepsis was modeled in sheep by cecal ligation and perforation. It was found that sepsis altered both surfactant aggregate form (ratio of small/large aggregate forms increased), and decreased surfactant protein (SP-A, B, C) concentrations, but these changes were not correlated with a significant decrease in PaO2. Surfactant replacement has been studied in only one other animal model [23] of endotoxin-induced ARDS; the model differed from ours, however, in that it exposed rats to endotoxin by intratracheal instillation. Unlike the results in the current study, Tashiro et al. [23] demonstrated that surfactant replacement reversed the respiratory failure caused by endotoxin. This difference in results could be caused by one or more of the following: a) a species-related response to surfactant treatment; b) intratracheal instillation of endotoxin may not elicit a whole body inflammatory response as would intravenous injection of endotoxin and, thus, the injury may be less severe; c) a pressure-controlled ventilator was used in the study by Tashiro et al. [23], such that the tidal volume may have been larger in the surfactant-treated group, which may have contributed to the improved oxygenation. There are a number of mechanisms by which exogenous surfactant may be rendered ineffective in the ARDS lung. Endogenous surfactant exists in a highly active large aggregate form and a poorly functional small aggregate form [24]. With lung injury, the small aggregates can increase, which may have an inhibitory effect on exogenous surfactant [25]. Alveolar type II cells can become injured, decreasing endogenous surfactant metabolism [26]. Disruption of the alveolar-capillary membrane causes exudation into the alveolus of plasma proteins, which are known to deactivate surfactant [6,27]. Also, the severity of the lung lesion may render surfactant replacement ineffective [28-30]. Surfactant treatment in most nonsepsis animal models of ARDS is very effective in ameliorating respiratory failure [17,31-35]. The saline lavage ARDS model [32-34] more closely duplicates infant respiratory distress syndrome than ARDS, since the primary injury is loss of surfactant [11] without the myriad of pathological components inherent in ARDS [3-14,35]. The clinical response to exogenous surfactant treatment may be less dramatic in ARDS than that documented in infant respiratory distress syndrome [11] or adult animals subjected to saline lavage [32-34]. Other studies utilizing acid aspiration [28], oleic acid [29], or N-nitroso-N-methylurethane [30] models of ARDS demonstrated only marginal improvement in oxygenation with surfactant replacement. Lewis et al. [30] demonstrated only modest physiologic improvements with either surfactant instillation or aerosolization following N-nitroso-N-methylurethane administration. Lamm and Albert [28] showed that surfactant instillation following acid-aspiration improved lung recoil, although it did not improve alveolar to arterial PO2 gradient. No benefit was measured with surfactant aerosol treatment in a sheep oleic acid ARDS model [29]. The latter models, like endotoxemia, are more complex pathologically and likely cause many of the same proinfammatory responses as does endotoxemia. Thus, surfactant replacement appears less successful in studies that closely model the complex pathophysiology of ARDS. The increased sequestration of leukocytes in the pulmonary parenchyma caused by exogenous surfactant was unexpected. The mechanism of exogenous surfactant-mediated leukocyte sequestration is unknown. It could be due to the mechanical effects of the instillation process, as well as to properties specific to the surfactant preparation. Surfactant instillation may stimulate either release of tumor necrosis factor, interleukin-1, C5a, or platelet activating factor, causing adhesion of leukocytes to the vascular endothelium [36]. However, the exogenous surfactants Exosurf Registered Trademark and Survanta Registered Trademark reduce inflammatory cytokine release from alveolar macrophages [37], suggesting that the physical process of instillation may play a significant role in this model. Instilled surfactant may have stimulated alveolar macrophages to release leukotriene B4, causing diapedesis of leukocytes from the pulmonary vasculature into the airspaces. Fink et al. [7] demonstrated, in a sepsis model similar to our own, that leukocyte sequestration in the pulmonary vasculature is probably mediated by cytokines (tumor necrosis factor, interleukin-1, etc.), whereas the diapedesis of leukocytes into the airspaces is mediated by leukotriene B4 released from alveolar macrophages. These authors [7] also showed that without diapedesis, endothelial permeability is not increased even though leukocytes are sequestered in the vasculature [7]. We demonstrated a nine-fold increase in the number of wandering cells in the airspaces as compared with a five-fold increase with endotoxin alone. Surfactant instillation, by some unknown mechanism, must have triggered a chemotactic signal responsible for the accumulation of wandering cells in the airspaces. This increase may have been associated with increased vascular damage. 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