INTRODUCTION

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Alterations in the surfactant system are proposed to contribute significantly to the pathophysiology of human acute respiratory distress syndrome (ARDS) (1). Changes in lung surfactant composition and function (2) are detectable in the lung lavage fluid of patients with ARDS. To date, four surfactant proteins (SP) have been identified (3): two high-molecular-weight proteins, surfactant protein-A (SP-A) (molecular weight: ~ 29 kD) and SP-D (molecular weight: ~ 43 kD), both of which are water soluble; and two low-molecular-weight proteins, SP-B (molecular weight: ~ 8 kD) and SP-C (molecular weight: ~ 5 kD), both of which are highly hydrophobic. Lung surfactant contains about 1% of these latter two hydrophobic proteins (4). SP-C is exclusively expressed in type II cells in the lung. Attempts to purify SP-C from extrapulmonary sources failed (5), and in transgenic mice SP-C could not be detected in any other organ than the lung (6). This is not the case for other surfactant proteins, since, for example, SP-B and SP-D have been detected in intestinal tissue (7), and a protein similar to SP-A has been found in the middle ear (8).

Recently, we showed that in an animal model of ARDS (9, 10), an SP-C-based surfactant was as effective as bovine-derived surfactants. The SP-C-based surfactant contained only dipalmitoylated SP-C, whereas the bovine-derived surfactant preparations contained both hydrophobic surfactant proteins, SP-B and SP-C. The animal model of surfactant depletion that was used was created by repetitive total lung lavage, and exhibited histopathologic changes similar to those seen in ARDS (9). The pathologic changes observed were atelectasis, protein leakage leading to formation of hyaline membranes and edema (9), and infiltration of polymorphonuclear neutrophils (PMN) (9, 11). These changes led to severe deterioration of gas exchange (9). According to results of several investigators (12), the efficacy of surfactant preparations can be tested under even more severe conditions when administration of surfactant preparations is delayed. This can be achieved by treatment 1 h after surfactant depletion.

In clinical trials, treatment with the synthetic surfactant Exosurf (13), which does not contain any surfactant protein, produced no improvements in survival rates in patients with ARDS. In contrast, pilot studies with the bovine-derived surfactants Alveofact (14), Survanta (15), and bLES (16) showed increased oxygenation and in some cases increased survival rates as compared with ventilation alone in patients with ARDS. All bovine-derived surfactants tested contained both SP-B and SP-C. In preterm babies suffering from respiratory distress syndrome (RDS) of the neonate, a randomized comparison was made of the bovine-derived surfactant Infasurf and the synthetic and protein-free surfactant Exosurf (17). It was shown that Infasurf led to significant alleviations in the severity of respiratory disease and in the incidence of air-leak-related complications as compared with Exosurf. Furthermore, in a clinical trial comparing Infasurf with Survanta (18), Infasurf produced a longer-lasting effect than Survanta in preterm babies with RDS and contains more surfactant proteins (19).

We were interested in testing these bovine-derived surfactants in an animal model under more stringent conditions, in comparison with a surfactant containing recombinant SP-C (rSP-C) and with the plain phospholipid (PL) surfactant (PL surfactant), without rSP-C. The rSP-C used in this comparison is an analogue of the human dipalmitoylated SP-C used in previous studies (9, 10), and is further defined in the METHODS section. Because of variations in the protein content and ratio of protein-containing surfactants in SP-B and SP-C, all surfactants were compared at doses related to their phospholipid content. The specific aims were: (1) to see whether the addition of rSP-C was sufficient to achieve oxygenation at least comparable to that with bovine-derived surfactants; (2) to test the surfactants with respect to ameliorating hyaline-membrane formation; (3) to investigate how the accumulation of polymorphonuclear neutrophils (PMN) in the lungs with injury acted on the activity of the different surfactants; and (4) to explore whether the different surfactants influenced PMN infiltration in this model.

	METHODS

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Surfactants

The rSP-C surfactant (Byk Gulden, Konstanz, Germany) used in the study contained 2% recombinant SP-C. The rSP-C is an analogue of human SP-C. It contains phenylalanine instead of two cysteines in positions 4 and 5 of the human SP-C sequence, and isoleucine instead of methionine in position 32. The rSP-C was associated in a 70:30 ratio with PL consisting of dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylglycerol plus 5% (wt/wt) palmitic acid. The rSP-C surfactant was resuspended with 0.9% NaCl solution to achieve a concentration of 25 mg PL per ml.

The protein-free, plain PL surfactant used in the study (Byk Gulden) contained the same lipids without rSP-C. The PL surfactant was processed similarly to the rSP-C surfactant. It was resuspended with 0.9% NaCl solution to a concentration of 25 mg PL per ml.

Alveofact (bovine-derived lung surfactant; Dr. K. Thomae, GmbH, Biberach, Germany) is a phospholipid fraction obtained by cow-lung lavage. Each vial contained a suspension of 50 mg PL per 1.2 ml. Seeger and associates (20) determined the PL profile of this suspension through high performance thin-layer chromatography. According to these published data, Alveofact contained about 82 ± 1.4% phosphatidylcholine, 4.0 ± 0.2% lysophosphatidylcholine, 9.0 ± 1.3% phosphatidylglycerol, 0.5 ± 0.2% phosphatidylinositol, 3.0 ± 0.4% phosphatidylethanolamine, and 1.4 ± 0.3% sphingomyelin. The SP-B content was determined to be 1.7% in relation to the total amount of PL. The SP-B content was determined with an enzyme-linked immunosorbent assay (ELISA). There are no specifications regarding the amount of SP-C in Alveofact.

bLES (bovine lipid extract surfactant; BLES Biochemicals Inc., London, ON, Canada) is a phospholipid fraction of cow lung obtained by lavage. Each vial holds a suspension containing 27 mg PL per ml. The PL profile was determined by Yu and colleagues (21), using gas- liquid chromatography. According to Yu and colleagues (21), the PL profile of bLES was: 79 ± 1.6% phosphatidylcholine, 1.5 ± 0.4% lyso-bis-phosphatidic acid, 11.3 ± 0.5% phosphatidylglycerol, 1.8 ± 0.3% phosphatidylinositol, 3.5 ± 0.5% phosphatidylethanolamine and 2.6 ± 0.5% sphingomyelin. The protein content was determined by the Lowry procedure, and therefore only the total protein content can be cited. It was 0.97 ± 0.07% in relation to the total amount of PL. Because of the method used, no specifications could be given about the amount of surfactant protein B or C in bLES.

Infasurf (a kind gift of G. Enhorning, Buffalo, NY) is a phospholipid fraction obtained from the lungs of freshly killed calves. The total PL content is 210 mg in 6 ml. Notter and coworkers (22) determined the phospholipid profile of Infasurf by thin-layer chromatography. According to these investigators (22), the phospholipid profile was 83% phosphatidylcholine, < 1% lysophosphatidylcholine, 6% phosphatidylglycerol, 5% phosphatidylinositol (including phosphatidylserine), 3% phosphatidylethanolamine, and 2% sphingomyelin. The protein content was determined by a modified Lowry procedure, and therefore only the total protein content can be cited. The protein content was approximately 1%. Again, because of the method used, Notter and colleagues could provide no specifications about the amount of surfactant protein B or C in Infrasurf (22). However, Seeger and colleagues using an ELISA (20), determined the SP-B content to be 1.7%.

All doses of surfactant were related to the total amount of PL. Because of the different techniques used for determining the protein content of the different surfactants, a comparison based on protein content seemed to be problematic. Also, relating the doses to total PL allowed comparison with the protein-free plain PL surfactant. All surfactants were instilled intratracheally as boluses at doses of 25, 50, and 100 mg PL per kg body weight, in a volume of 1.2 ml per animal. The surfactants were diluted with 0.9% saline solution to achieve the required concentrations of 6.25, 12.5, and 25 mg total PL per 1.2 ml.

Preparation of Rats

The study protocol was reviewed and approved by the Laboratory Animal Care Committee at the district presidency of Freiburg, Germany. The study was performed with a total of 251 male Sprague- Dawley rats (Harlan CBP, Zeist, The Netherlands), with a body weight (b.w.) of 230 to 270 g.

The anesthetic and surgical methods used in the study were the same as previously described (9, 10). Briefly, after introduction of inhalational anaesthesia, a catheter was placed into one carotid artery. Thereafter, the animals received an intraperitoneal injection of pentobarbitone (stock solution: 60 mg/ml; 1 ml/kg b.w.). After completion of the tracheotomy, a tube was secured into the trachea of each animal. The animals received an intramuscular injection of pancuronium bromide (1 ml/kg b.w.; solution concentration: 2 mg/ml) and ventilation was started, using a Servo Ventilator (900C; Siemens-Elema, Solna, Sweden). The tracheal tubes of six animals were connected to a distributor. The animals were ventilated simultaneously at a respiratory rate of 30 breaths/min, an FI_O2 of 1.0, an inspiration expiration ratio of 1:2, and a peak inspiratory pressure (PIP) of 15 cm H₂O, which included a positive end-expiratory pressure (PEEP) of 2 cm H₂O. Additional pentobarbitone (0.25 ml/kg intraperitoneally of the stock solution) and pancuronium bromide (1 ml/kg b.w.) intramuscularly were given when appropriate.

Protocols for Animal Experiments

The reported variable was arterial Pa_O2. Blood-gas analysis was performed with a blood-gas analyzer (Radiometer Copenhagen ABL 500; Radiometer Deutschland GmbH, Willich, Germany). After determination of pretreatment values, only animals with Pa_O2 values of more than 480 mm Hg were included in the experiments. PIP was raised to 28 cm H₂O and PEEP to 8 cm H₂O, and the animals were subjected to multiple lung lavage (six to eight times) with 1 ml/30 g b.w. of isotonic saline solution. Only those animals that had Pa_O2 values between 50 and 110 mm Hg were included in the study. Blood gases were determined at 5, 30, and 60 min after the last lavage. One hour after the last lavage, the surfactants were instilled as described earlier. Untreated controls received sham treatment with air. Subsequently, blood gases were determined at 30, 60, 90, 120, and 150 min after surfactant instillation (equivalent to 90, 120, 150, 180, and 210 min after the last lavage). The PIP and PEEP were kept constant throughout the experimental period at 28 cm H₂O and 8 cm H₂O, respectively. The experimental scheme is shown in Figure 1.

View larger version (22K): [in this window] [in a new window]   Figure 1.   Mean values of PaO2 (mm Hg) (A) and PaCO2 (mm Hg) (B) after ventilation only of lung-lavaged rats (control group, n = 36). "Lavage" marks the time at which repetitive lavage was performed. (C ) Corresponding time course of PIP (cm H2O) and PEEP (cm H2O) during the experiment.

Preparation of Lungs

Two control groups were used for histologic investigations. In Group 1 the animals were killed 1 h after the last lavage, and in Group 2, in which the animals did not receive any treatment, the animals were killed at 210 min after the last lavage. All surfactant-treated animals were killed after the last blood-gas determination (equivalent to about 210 min of experimental time). The histopathologic procedures used in the study were the same as previously described (9). From each rat lung, all lobes were evaluated, using a hematoxylin-and-eosin (H&E)-stained, 5 µm-thick tissue slice. The lobes were trimmed in a longitudinal manner, using the largest area of each lobe. Slides were coded so that the pathologist could evaluate and diagnose all lung lobes of the same animal at once. For each lobe, five consecutive fields were analyzed. After randomization and codification, each section was examined under light microscopy. Hyaline-membrane formation was assessed semiquantitatively according to the technique reported previously (9), with a modification regarding grading. The severity of hyaline-membrane formation was graded from 0 to 4+ (0 = no hyaline membrane formation; 1+ = occasional fields showing hyaline-membrane formation in a low number (from one to three) of membranes per viewed field [minimal]; 2+ = occasional fields showing hyaline-membrane formation in an increased number (more than three) of membranes per viewed field [mild]; 3+ = many but not all fields showing hyaline membrane formation [moderate]; 4+ = hyaline-membrane formation in all fields examined [severe]). The distribution and severity of intraalveolar accumulation of PMN were graded semiquantitatively from 0 to 4+, as with the grading of hyaline-membrane formation, but with respect to the number of inflammatory cells and the location of these cells.

Statistics

The experiment was started with 12 rats for each dose level of all surfactant preparations (with the exception the low dose of the PL surfactant), and with 12 animals in both untreated control groups. Because of the time interval from the first to the last experiment, the controls were repeated three times. The time interval over which this was done was 11 mo. The influence of surfactant instillation on Pa_O2 was demonstrated by time-effect curves, using mean ± SD values. Dose-response curves were plotted using means ± SD of the Pa_O2 values at 30 min (Pa_O2[30']) as well as the values of Pa_O2 at 120 min (Pa_O2[120']) after treatment (equivalent to 90 and 180 min of experimental time, respectively). The results for formation of hyaline membranes were presented in tabular form using means ± SD and median and range. The primary variables (Pa_O2[30'], Pa_O2[120'], Pa_CO2[30'], and Pa_CO2[120']) were analyzed for monotone dose dependence with the nonparametric Jonckheere-Terpstra test (23). Subsequently, the effects of rSP-C surfactant on these variables for each dose level were compared with the effects of the other surfactants, through one-sided Wilcoxon's tests. An adjustment for multiple Type I error was made for each dose level according to the method of Bonferroni and Holm (24). The effects of the surfactants on the histopathologic variables (hyaline-membrane formation and infiltration of PMN) were compared with the findings in controls killed at 60 min and at 210 min after lavage for monotone dose dependence, using the Jonckheere- Terpstra test. Furthermore, at the highest dose, the comparison between rSP-C surfactant and the other surfactants was made in an exploratory manner, using Wilcoxon's test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Time Course of Pa_O2 and Pa_CO2

After lavage, only two of the 36 animals in the control group that were ventilated for the entire experimental period (210 min) showed a spontaneous improvement in Pa_O2 or Pa_CO2 values. During the 1 h period after the last lavage, no groups (treated and untreated controls) showed any major changes in Pa_O2 or Pa_CO2 values. The Pa_O2 values showed only a small change (Figure 1) and as time passed, the SD values increased. This was due to the spontaneous improvements in the two animals.

Figure 2 summarizes the effects of treatment with the various doses of the different surfactants. All protein-containing surfactants improved oxygenation at 30 min after administration of each of the three doses used. The 25-mg dose of Infasurf produced minimal improvement in oxygenation (Figure 2A), and the dose of 50 mg produced an intermediate response (Figure 2B). Only the protein-containing rSP-C surfactant, bLES, and Infasurf showed stable activity for the entire observation period (Figures 2A, 2B, and 2C) when oxygenation was improved at 30 min after treatment. In contrast, Alveofact produced initially good oxygenation, after which the Pa_O2 values decreased gradually toward the end of the observation period after administration of all three doses (Figures 2A, 2B, and 2C). After administration of all doses of PL surfactant (Figures 2A, 2B, and 2C), the values of Pa_O2 remained clearly below the corresponding values with the protein-containing surfactants.

View larger version (34K): [in this window] [in a new window]   Figure 2.   Time-response curves of PaO2 (mm Hg) after administration of rSP-C surfactant (solid squares), PL surfactant (open squares), bLES (solid diamonds), Alveofact (solid circles), and Infasurf (solid triangles). All values are given as means ± SD. (A) Values after administration of 25 mg, (B) after administration of 50 mg, and (C ) after administration of 100 mg surfactant per kg body weight; n = 11 or 12 for each dose. "Lavage" marks the time point at which repetitive lavage was performed. The arrow marks the time when surfactant was administered. (D) Corresponding time course of PIP (cm H2O) and PEEP (cm H2O) during the experiment.

Dose-Response Curves of the Different Surfactant Preparations

Dose-response curves based on the Pa_O2 values at 30 min after treatment are given in Figure 3A for each surfactant. A dose-dependent improvement in Pa_O2 values was observed at 30 min after treatment for all protein-containing surfactants, based on all three doses used. In contrast, the PL surfactant produced a minimal increase in Pa_O2 with increasing doses. Comparisons at each dose level were made for the 30 min point after treatment. At a dose of 25 mg PL per kg b.w. rSP-C surfactant produced significantly higher Pa_O2 values than did Alveofact (p  0.05), Infasurf (p  0.001), or PL surfactant (p  0.001). rSP-C surfactant produced lower Pa_O2 values than did bLES (p  0.05). At a dose of 50 mg PL, the Pa_O2 values after treatment with rSP-C surfactant differed only from those for Alveofact (p  0.05) and the PL surfactant (p 0.001). At a dose of 100 mg PL there were no statistically significant differences between the rSP-C surfactant and Alveofact and Infasurf. As observed for the two lower doses, the rSP-C surfactant produced significantly higher Pa_O2 values than did the PL surfactant (p  0.001). The values at this dose were lower than after treatment with bLES (p  0.05).

View larger version (23K): [in this window] [in a new window]   Figure 3.   Dose-response curves based on values for PaO2 at 30 min (A) and for PaO2 at 120 min (B) after administration of different doses of rSP-C surfactant (solid squares), PL surfactant (open squares), bLES (solid diamonds), Alveofact (solid circles), and Infasurf (solid triangles). The values represent means ± SDs. Dose-response calculations based on all three doses resulted in a monotone increase in dose dependence for the PaO2 values at 30 min after treatment, which was significant at p < 0.001 for rSP-C surfactant, Alveofact, bLES, and Infasurf, and at p  0.05 for PL surfactant. Comparisons of all three doses of rSP-C surfactant with the plain PL surfactant resulted in significance with p  0.001 for each dose. The calculations based on PaO2 values at 120 min after treatment (B) resulted in significance, with p < 0.001 for rSP-C surfactant and Infasurf, p < 0.01 for Alveofact, and p  0.05 for bLES, and no significance for plain PL surfactant. Comparisons of all three doses of rSP-C surfactant with the PL surfactant resulted in significance with p  0.001 for each dose.

The dose-response curves based on the Pa_O2 values at 120 min after administration of the different surfactants are presented in Figure 3B. All protein-containing surfactants caused a dose-dependent increase in Pa_O2 at 120 min after treatment. The PL surfactant did not induce a statistically significant increase in dose dependence at 120 min after treatment. All doses of rSP-C surfactant led to significantly higher Pa_O2 values at 120 min than did the respective doses of PL surfactant. The statistically significant differences between these two surfactants were similar to those in the comparisons of Pa_O2 at 30 min (Figures 3A and 3B). Furthermore, there were no statistically significant differences between rSP-C surfactant and bLES at any dose. However, at 120 min after treatment, the differences between rSP-C surfactant and the other protein-containing surfactants became even more prominent. Based on a dose of 25 mg PL per kg b.w., significantly higher Pa_O2 values were found after rSP-C surfactant than after Alveofact (p  0.01) or Infasurf (p  0.01). The comparison based on a dose of 50 mg showed statistically significant differences between rSP-C surfactant and Alveofact (p  0.001). Although the mean Pa_O2 after administration of Infasurf was lower than after rSP-C surfactant (Figure 3B), the differences were not significant. At a dose of 100 mg, the rSP-C surfactant did not show statistically significant differences from bLES or Infasurf. However, the rSP-C surfactant did produce statistically significantly higher Pa_O2 values than did Alveofact (p  0.01) at 120 min after treatment.

Pa_CO2 values showed a pattern of response almost inverse to that of the Pa_O2 values after administration of the different surfactant preparations (data not shown). For all groups (untreated and treated), the mean Pa_CO2 before lavage was between 34 and 43 mm Hg. During lavage the mean Pa_CO2 increased to values between 53 and 76 mm Hg. The values remained constant until 60 min after the last lavage. After treatment with the different surfactant preparations, only minor decreases in Pa_CO2 were seen. The mean Pa_CO2 ranged from 44 to 66 mm Hg at 30 min after treatment. The Pa_CO2 of untreated controls was 53 mm Hg at this time. At 120 min after treatment the mean Pa_CO2 ranged from 44 to 61 mm Hg. The respective value for untreated controls was 51 mm Hg. No dose-dependent or dose-independent effects were detectable after administration of the different surfactants. Although there were detectable decreases in Pa_CO2 values after the administration of all surfactants, there were no statistically significant differences between the different surfactants with respect to Pa_CO2.

Histopathological Evaluation

In this investigation, two control groups were used for histologic examination. One group of animals was killed 1 h after the last lavage, and the other group of animals was killed after the experimental period (210 min after the last lavage). The histopathologic time course of ARDS development is shown by the comparison of unlavaged healthy rats (Figure 4A) with lavage control rats killed at 60 min (Figure 4B) and lavaged and untreated control rats killed at 210 min (Figure 4C). At 1 h after lavage, hyaline membranes and infiltration of PMN were present in the alveolar spaces of the lavaged rats (compare Figures 4A and 4B). Further histopathologic deterioration was obvious at 210 min after lavage in the untreated control group (Figures 4B and 4C). A statistically significant increase was found in the severity of hyaline-membrane formation, from 2.2 (mean) for the 60-min value to 3.3 (mean) for the 210-min value (Table 1 and Figure 5A). The increase in hyaline-membrane formation was accompanied by a statistically significant increase (p  0.01) in the grading for intraalveolar accumulation of PMN, from 1.7 (mean) at 60 min to 2.1 (mean) at 210 min (Figure 5B). This increase in grading was not as prominent as that seen for hyaline-membrane formation.

View larger version (77K): [in this window] [in a new window]   View larger version (114K): [in this window] [in a new window]   View larger version (113K): [in this window] [in a new window]   Figure 4.   (A) Lung of an unlavaged control rat. Focal, minimal (1+) agonal margination of PMN is seen in the capillary lumen (arrow). No hyaline membrane formation is visible. H&E: original magnification, ×380. (B) Lung of a rat killed at 60 min after the last lavage. Moderate (3+) margination of PMN is seen in the capillary lumen (arrowheads). Mild, 2+ formation of hyaline membranes (arrow) is detectable. H&E: original magnification, ×380. (C ) Lung of a rat killed at 210 minutes after the last lavage. Moderate (3+) margination of PMN is seen in the capillary lumen. Severe (4+) hyaline membrane formation (arrow) is detectable. H&E: original magnification, ×380.

View larger version (46K): [in this window] [in a new window]   Figure 5.   Survey of the grading for hyaline membranes (A) and the grading for intraalveolar accumulation of PMN (B) after treatment with the different doses of the tested surfactants. The values are given as means ± SDs in comparison with the control groups killed at 60 min and 210 min after the last lavage. All surfactant-treated animals were killed after the last blood-gas determination (equivalent to about 210 min of experimental time).

Treatment with the protein-containing surfactants led to dose-dependent reductions in the formation of hyaline membranes as compared with that in the control group killed at 210 min (Figure 5A). The PL surfactant did not show dose-dependent effects in preventing hyaline-membrane formation, and the grading was higher than after rSP-C surfactant, bLES, or Infasurf. At the dose of 100 mg PL per kg b.w. (Table 1 and Figure 5A), the PL surfactant had the highest value for hyaline-membrane formation (2.8; mean) and the rSP-C surfactant had the lowest value (0.9; mean). At this dose the rSP-C surfactant showed a statistically significant lower grading than did Alveofact (p  0.001).

In comparing the effects of the different surfactants with respect to the controls killed at 60 min (in order to judge the possible reversal of hyaline-membrane formation), the differences between rSP-C surfactant and the PL surfactant became even more prominent. The rSP-C surfactant was able to dose-dependently reverse hyaline-membrane formation. Figures 6A, 6B, and 6C show this dose-dependent decrease in hyaline membrane formation in comparison with the untreated controls (Figure 4B) at 60 min. The protein-containing surfactants bLES and Infasurf behaved similarly, but their dose-dependent effects were less pronounced than those seen after rSP-C surfactant (Table 1). The PL surfactant showed no effect when compared with the 60-min controls. The same lack of effect was observed for Alveofact.

View larger version (118K): [in this window] [in a new window]   View larger version (99K): [in this window] [in a new window]   View larger version (90K): [in this window] [in a new window]   Figure 6.   (A) Lung of a rat treated with 25 mg/kg r-SPC surfactant and killed at 210 min after the last lavage. Moderate (3+) margination of PMN is seen in the capillary lumen (arrowheads). A representative hyaline membrane, graded 3+ (moderate), is indicated by the arrow. H&E: original magnification, ×380. (B) Lung of a rat treated with 50 mg/kg r-SPC surfactant and killed at 210 min after the last lavage. Moderate (3+) margination of PMN is seen in the capillary lumen. A representative hyaline membrane, graded 1+ (minimal), is indicated by the arrows. H&E: original magnification, ×380. (C ) Lung of a rat treated with 100 mg/kg r-SPC surfactant and killed at 210 min after the last lavage. Moderate (3+) margination of PMN is seen in the capillary lumen (arrowheads). A representative hyaline membrane graded 1+ (minimal) is indicated by the arrow. Compare with Figures 4A, 4B, and 4C. H&E: original magnification, ×380.

The effects of all surfactants on intraalveolar accumulation of PMN are presented in Figure 5B. None of the tested surfactants had any statistically significant effects on this parameter. However, the different doses of bLES produced higher grading (mean values) than was found in the controls killed at 210 min. The highest dose of Alveofact produced the highest grading value. At the highest dose, of 100 mg, only rSP-C surfactant, PL surfactant, and Infasurf produced values comparable to the value of the untreated control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

With the rat-lung-lavage model used in this study, it is possible to characterize new surfactant preparations in a standardized and systematic manner (1) in comparison with commercially available surfactants. In contrast to previous reports (9, 10), the late treatment used in this study can provide information about the ability of surfactant preparations to improve the severe injury that follows repeated lavage. This injury results in physiologic and histologic abnormalities, which can be observed in the model (Figures 4A, 4B, and 4C). It therefore better reflects the situation in the lungs of patients with fully established ARDS (25, 26) than does the early treatment model (9, 10). In ARDS patients, an accumulation of PMN is present in the lungs. A similar influx of PMN is observed in the rat model described here when treatment is withheld for one hour after the last lavage (Figure 4B). In addition, an increased formation of hyaline membranes (compare Figures 4A and 4B) is present and is accompanied by an influx of factors that can inhibit surfactant function, such as albumin or fibrinogen (20). The influence of surfactants on hyaline-membrane formation can be investigated by comparing the 210-min control group in the present study with treated groups that were killed at the same time. Furthermore, in comparing the effects of surfactant treatment with the findings in the 60-min controls, the ability of surfactants to decrease existing hyaline membranes can be examined. The rSP-C surfactant was not only able to prevent further exacerbation hyaline-membrane formation, but was even able to reverse the formation of hyaline membranes (Table 1). This effect is shown by comparing Figures 6A, 6B, and 6C with Figure 4B (from a control rat killed at 60 min after lavage).

When comparing the infiltration of PMN in unlavaged control rats (Figure 4A) with that of controls killed at 60 min (Figure 4B) or 210 min (Figure 4C) after the last lavage, the massive influx of PMN becomes obvious. As Ryan and colleagues (27) have shown, activated PMN are capable of impairing surfactant function in vitro. This situation can be tested in vivo in the rat-lung-lavage model. The intense infiltration of PMN seemed to have greater influence on surfactants without any surfactant proteins. This was obvious when looking at the influence of plain PL surfactant on improving oxygenation. We assume that the improvement in this case was poor because of the immense infiltration of PMN. In addition, such infiltration of PMN may contribute to the formation of hyaline membranes (28), which then hampers gas exchange, leading to reduced oxygenation. Further evidence of such an effect may be derived from results of our previous investigation (10), in which the surfactants were given shortly after the last lavage. At this time infiltration of PMNL into the airways of rats was not as intense as it was 60 min later, and the therapeutic activity of the plain PL surfactant was better than its activity in the present investigation. It is also likely that in the case of Infasurf, the influence of the intense infiltration of PMN on hyaline-membrane formation, leading to a reduced oxygenation response after the low dose of Infasurf, could have been surmounted if the dose had been high enough. The same can be concluded in the case of Alveofact. Even higher doses may be necessary to achieve a sustained response with regard to oxygenation and a better response with regard to inhibiting the formation of hyaline membranes. However, once it was established, the inflammatory response observed as an intraalveolar accumulation of PMN was not affected (Figure 5B) by any of the surfactants tested. Some surfactants even showed a tendency to increase the inflammatory response. The histologic results in this regard are consistent with the idea that treatment with surfactant should be initiated as early as possible in order to prevent inflammatory responses, whereas the formation of hyaline membranes may be reversed even by late treatment.

Comparison of the rSP-C surfactant with the bovine-derived surfactants containing both surfactant proteins SP-B and SP-C and with the plain PL surfactant shows that the addition of rSP-C to a PL surfactant is sufficient to produce the same activity with respect to oxygenation and hyaline-membrane formation as that with the bovine-derived surfactant bLES. Even between bovine-derived surfactants there are detectable differences. The rSP-C surfactant and bLES are both superior to the two other protein-containing surfactants, Alveofact and Infasurf, with respect to improving oxygenation. On the basis of cited literature (20), the bovine-derived surfactants show slightly differing PL profiles. The differences between these surfactants may be explained by the different detection methods used. Furthermore, this can also be said with respect to surfactant protein content. The protein content of surfactants can be compared only when using the same assay method. Seeger and colleagues (20) determined the SP-B content of Alveofact and Infasurf with an ELISA. Both surfactants contained about 1.7% SP-B according to Seeger and colleagues' results. However, because no specific ELISA for SP-C was available, the content of SP-C could not be determined. The content and ratio of SP-B and SP-C may be important for the activity of a surfactant preparation. The differences between bLES and the other bovine-derived surfactants are probably due to their different contents and/or ratios of SP-B and SP-C. Measurements of SP-C content done at Byk Gulden, using high-pressure liquid chromatography (HPLC), show that bLES contains three times more SP-C than do Alveofact and Infasurf. The higher SP-C content can probably explain the functional superiority of bLES and rSP-C surfactant to that of Alveofact and Infasurf in the present study.

In conclusion, in the more stringent rat-lung-lavage model of late treatment, used in the present study, an intense inflammatory response (Figure 4B and C) was present in the airways. This response was accompanied by formation of hyaline membranes. It was shown that a surfactant that contains rSP-C is sufficient to achieve oxygenation comparable or even superior to that achieved with bovine-derived surfactants that contain SP-B and SP-C. With respect to effects on ameliorating hyaline-membrane formation, the rSP-C surfactant showed a clear empirical difference from the other protein-containing surfactants. However, there was a statistically significant difference only between rSP-C surfactant and Alveofact. We assume that the intense infiltration of PMN and the presence of hyaline membranes had different influences on the activity of the surfactants tested. The protein-free surfactant was more sensitive than the surfactants that contained surfactant protein. None of the surfactants was able to affect the intense infiltration of PMN in this model. The present comparison of the effects of rSP-C surfactant with those of protein-containing bovine-derived surfactants that were shown to be effective in clinical trials of ARDS (14), suggests that the rSP-C surfactant could be an effective treatment in patients with ARDS.