INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The most common complication in survivors of prematurity and lung immaturity is the chronic lung disease called bronchopulmonary dysplasia (BPD) with an incidence in surviving infants with birth weights less than 1,000 g of about 50% (1). The disease is characterized by a prolonged need for supplemental oxygen, elevated PCO₂ values, and abnormal lung mechanics that include decreased compliance and increased airway resistance. Lungs from very preterm infants who have died from BPD have an arrest in alveolar septation, hyperplasia of type II cells, and thickening of the alveolar-capillary barrier (2, 3). This injury resulted in alveolar hypoplasia in preterm baboons that received oxygen and ventilatory support (4). A similar interference with alveolar development was reported after prolonged ventilation of preterm lambs (5), and ventilation of preterm infants can decrease alveolar numbers (6).

There has been very little attention given to the possibility that BPD may result in persistent abnormalities of the surfactant system. Infants who progress from respiratory distress syndrome to BPD had a delay in the appearance of phosphatidylglycerol in surfactant from airway samples (7), and the development of BPD was associated with low levels of surfactant protein A (SP-A) (8). Preterm ventilated and oxygen-exposed baboons with diffuse alveolar damage also had alveolar surfactant that had a low phosphatidylglycerol and high phosphatidylinositol composition typical of immaturity and lung injury as well as a decreased SP-A (9, 10). Most references to surfactant in BPD focus on the hypothesis that any abnormalities in surfactant likely result from inhibition of surfactant function by proteins and other substances in edema and inflammatory fluid in the air spaces. Pandit and colleagues (11) attributed improved oxygenation after intratracheal surfactant treatment of 10 infants with BPD to overcoming surfactant inhibition. We hypothesized that surfactant metabolism and function would be abnormal in BPD because of alveolar injury and type II cell hyperplasia. We also hypothesized that antenatal glucocorticoid therapy would mature the preterm lung and favorably alter the surfactant system (12). To test these hypotheses very preterm baboons randomized to receive antenatal glucocorticoids were surfactant-treated and mechanically ventilated for 6 d for studies of surfactant metabolism and function.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Fetal Treatment

The fetal treatments and delivery studies were performed at the Southwest Foundation for Biomedical Research (San Antonio, TX). All animal husbandry and animal handling and procedures were reviewed and approved to conform with AAALAC guidelines. Pregnancies were dated using cycle dates and growth parameters from prenatal ultrasounds at 70 and 100 d estimated fetal gestational age. The pregnant baboons were sedated intramuscularly with ketamine (10 mg/kg) for each of the prenatal ultrasounds. At 123 ± 2 d gestation (term = 185 d) the baboons were randomly assigned to receive 6 mg betamethasone (Celestone Soluspan; Schering Pharmaceuticals, Kenilworth, NJ) or saline by intramuscular injection 48 and 24 h before delivery.

Delivery

The pregnant baboons were sedated intramuscularly with ketamine (10 mg/kg) at 125 ± 2 d gestation, intubated, and anesthetized with 1.5% halothane. The preterm fetuses were delivered by cesarean section, weighed, and intubated using 2.0- or 2.5-mm endotracheal tubes. The newborns received 100 mg/kg surfactant (Survanta; donated by Ross Products, Columbus, OH) by tracheal instillation and ventilated with pressure-limited infant ventilators. After delivery of the fetus and repair of the maternal incisions, recovery of the female baboons was monitored daily for 2 wk, with all animals released to outside gang cages after 4 wk.

Management of Newborns

The newborn baboons were maintained sedated intramuscularly with ketamine (10 mg/kg) and intravenously with diazepam (0.1 to 0.2 mg/ kg) if needed. An arterial line was placed either by percutaneous insertion into the radial artery or via an umbilical artery into the descending aorta for blood pressure monitoring and blood sampling. A venous line was placed percutaneously via the saphenous vein into the inferior vena cava for administration of fluids and medications. The animals were cared for on servo-controlled infrared warmers, given parenteral fluids containing glucose, amino acids, and multivitamins, and were not fed. Fluids were administered intravenously with appropriate electrolytes at 12.5 ml/kg/h initially, with infusion rates increased when heart rates were above 180 beats/min, for hematocrit increases of > 10%, or for an increasing metabolic acidosis. Sodium bicarbonate was administered (2 mEq/kg) when the base deficit (metabolic acidosis) exceeded 8 mEq/kg. Ampicillin (50 mg/kg/d in two divided doses) and gentamicin (5 mg/kg/d in two divided doses) were given intravenously. Local anesthesia with 2% lidocaine and additional ketamine were administered for any invasive procedures. In addition to routine arterial blood samples for assessments of pH, PO₂, PCO₂, and hematocrit, blood samples were collected at least daily for plasma electrolytes. Blood samples were replaced volumetrically with heparinized adult baboon blood. Arterial blood pressure, heart rate, oxygen saturation, and EKG were monitored continuously.

Pulmonary Management and Measurements

To standardize management, peak inspiratory pressures were adjusted to target a tidal volume of 5 ml/kg, and the positive end-expiratory pressure (PEEP) was set at 3 cm H₂O with an inspiratory time of 0.6 s. Arterial PCO₂ values within the target range of 35 to 50 mm Hg were maintained by adjusting ventilatory rates. Oxygenation was regulated by adjusting inspired oxygen content to achieve a target value of 60 to 80 mm Hg. Tidal volumes and dynamic compliances were measured with a VT1000 Vital Station Neonatal plethysmograph (Vitaltrends Technology, Inc., Wallingford, CT).

To evaluate the loss of dipalmitoylphosphatidylcholine (DPPC) from the treatment dose of 100 mg/kg Survanta, [¹⁴C]choline-labeled DPPC (New England Nuclear, Boston, MA) was associated with the surfactant before treatment (13), and each animal received 4.4 µCi [¹⁴C]DPPC/kg birth weight. The airways of the animals were suctioned every 12 h after instillation of 0.5 ml saline using a 5-Fr catheter, and the aspirated material was recovered. At 120 h of age (5 d) each animal also received an intravascular infusion of 200 µCi ³H-palmitic acid that was bound to albumin in saline in order to label de novo synthesized Sat PC (13).

At 144 h (6 d) after delivery, each animal was ventilated with 100% oxygen for 5 min, and deep anesthesia was achieved by the slow infusion of pentobarbital to decrease the blood pressure by 50%. The endotracheal tube was clamped to allow for adsorption atelectasis, and after 2 min the heart was stopped with additional pentobarbital. The chest was opened and a pressure-volume curve was measured by increasing the pressure on the lung to 35 cm H₂O in 5 cm H₂O pressure increments using a syringe and mammometer and then decreasing the pressure with measurement of volume after 30 s at each pressure (14). The volumes were corrected for the compression volumes of the measurement system.

Processing of Lungs

After the pressure-volume curve measurement, an alveolar wash was performed in situ by filling the lungs with 0.9% NaCl at 4° C until full distention was achieved visually and the fluid was recovered by syringe (13). The lavage procedure was repeated five times, the recovered saline was pooled, and the total volume was measured. The lungs were then weighed, and 1 to 2 g of lung tissue was cut from the left lower lobe and frozen with liquid nitrogen. The residual lung was again weighed and frozen. Subsequently, the lung was homogenized in saline and aliquots were used for measurements of Sat PC.

Aliquots of the alveolar wash were frozen for subsequent assay, and the residual fresh alveolar wash was used for the separation of large and small surfactant aggregate forms (15). Each alveolar wash was centrifuged at 140 × g for 10 min and the pellet of cellular debris was discarded. The supernatant was centrifuged at 40,000 × g for 15 min and the pellet containing the large aggregate surfactant was resuspended in 0.9% NaCl, layered over 0.8 M sucrose in 0.9% NaCl, and centrifuged again at 40,000 × g for 15 min (15). The interface was recovered, diluted with 0.9% NaCl, and recovered as a pellet after centrifugation at 40,000 × g for 15 min. The large aggregate surfactant was suspended in a small volume of 0.9% NaCl and frozen for subsequent testing.

Analytic Techniques

Samples for Sat PC were extracted with chloroform-methanol (2:1, vol:vol) and treated with osmium tetroxide. Sat PC was isolated by column chromatography using alumina and quantified by phosphorus assay (16, 17). Total protein in alveolar washes was measured using the assay of Lowry and colleagues (18). Blood pH, PO₂, and PCO₂ values were determined with a Model 995 blood gas analyzer (AVL Scientific Corp., Roswell, GA). Blood sodium, potassium, and chloride concentrations were determined with a NOVA electrolyte analyzer (NOVA Biomedical, Waltham, MA). Plasma cortisol was measured with chemiluminescence kits (Nichols Diagnostic, San Juan Capistrano, CA).

Surface Tension Measurements

A Wilhelmy balance with a platinum dipping plate was used to measure minimum surface tensions after the fourth cycle from a maximum area of 64 cm² to a minimum area of 12.8 cm² at a temperature of 37° C and a rate of 3 min/cycle (19). Surface tensions for large aggregate surfactant fractions were measured at dilutions from 1 to 20 pmol Sat PC in 35 ml saline. To evaluate sensitivity of the surfactants to inhibition, increasing amounts of plasma were added to 20 pmol/ml surfactant and mixed with a glass rod, and surface tension measurements were repeated (19).

Preterm Rabbits

Preterm rabbits were used to evaluate the surfactant used to treat the preterm baboons, the large aggregate surfactants recovered from alveolar washes of preterm baboons, and alveolar washes of term and adult baboons (20). New Zealand white rabbits at 27 d ± 2 h gestation were lightly anesthetized with pentobarbital followed by spinal anesthesia using 1.5 ml of 2% lidocaine-0.5% bupivacaine (1:1, vol/vol). The does received oxygen and the preterm rabbits were sequentially delivered by cesarean section. The newborns were given acepromazine (0.1 mg/kg) and ketamine (10 mg/kg) by intraperitoneal injection. A tube made from an 18-gauge stainless steel needle was secured into the trachea of each rabbit. Controls for each litter received no surfactant, and the treated rabbits were randomized to receive 50 mg/kg of a surfactant by intratracheal instillation before lung inflation. The amount of each surfactant was estimated by measuring Sat PC content. Initial lung inflation was with five breaths of 100% oxygen using an anesthesia bag with just enough pressure to visibly move the chest to establish a gas volume in the lungs. The control and surfactant-treated rabbits were then transferred to a temperature-controlled (37° C) ventilator-plethysmography system on initial settings: peak inspiratory pressure (PIP), 35 cm H₂O; rate, 30 breaths/min; inspiratory time, 1 s; FI_O2, 1; PEEP, 3 cm H₂O. The PIP was individually adjusted for each rabbit to achieve tidal volumes of about 8 ml/kg as measured with a pneumotachometer (20). At the end of 15 min of ventilation, the endotracheal tube was obstructed for 5 min to allow complete atelectasis to occur by oxygen adsorption. Each rabbit was killed with intrathecal lidocaine. The rabbits were then transferred to temperature-controlled (37° C) plastic boxes. Quasi-static pressure-volume curve measurements were performed by inflating the lungs in 5 cm H₂O pressure increments to 35 cm H₂O and deflating in the same increments back to zero cm H₂O pressure. Lung volumes were corrected for the compression volumes of the system and expressed as ml/kg body weight.

Microscopy

Lung specimens from three additional 125-d baboons that did not receive radioisotopes but were treated with surfactant and ventilated comparably for 6 d, were intrabronchially fixed with phosphate-buffered 4% paraformaldehyde and 0.1% glutaraldehyde at 20 cm H₂O constant pressure for 24 h (21). A representative tissue section was processed for routine light microscopy. For transmission electron microscopy, 20 blocks were randomly collected and pooled from the fixed lung lobe, postfixed in isotonic osmium fixative, stained en bloc with uranyl acetate, dehydrated in graded ethanols, and embedded in resin. The blocks were thick-sectioned at 1 µm and stained with toluidine blue, from which three blocks were selected for thin-sectioning. The sections were stained with lead citrate and uranyl acetate and examined with a JEOL 100-CX electron microscope (JEOL, Peabody, MA).

Lung and Surfactant Samples from Other Baboons

Frozen lung tissues from fetal baboons at 125 d (n = 7), 140 d (n = 6), 160 d (n = 3), 175 d gestation (n = 3), and adult baboons (n = 6) were provided by the Southwest Research Foundation. These samples were used for Sat PC pool size measurements. Fresh alveolar washes from healthy term newborn and adult baboons were used for isolation of the large aggregate surfactants as described above for the alveolar washes of the preterms (15).

Data Analysis

Results are given as means ± SEM. Two-way analysis of variance was used for comparison of differences in physiologic measurements during the 6-d ventilation period. All other comparisons were by one-way analysis of variance, and the Student-Newman-Keuls test was used as the discriminating post-test. Comparisons of two groups were by two-tailed t tests. The level of significance accepted was p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Respiratory Status of Preterm Baboons

The six baboons exposed antenatally to betamethasone and the six control baboons had similar birth weights (Table 1). Cortisol levels in cord blood indicate suppression of cortisol with antenatal glucocorticoid exposure. The two groups of animals maintained similar PO₂ values of about 70 mm Hg while receiving similar inspired oxygen as indicated by similar AaPO₂ (a/A ratios) (Figure 1A). The animals achieved similar PCO₂ values with similar mean tidal volumes of about 5 ml/kg and similar peak inspiratory pressures (Figure 1B). Ventilatory rates were 20 to 40 breaths/min and were similar for both groups. The peak inspiratory pressures at 6 d of 23 cm H₂O for control animals and 21 cm H₂O for betamethasone-exposed animals indicated significant residual lung disease. The compliance values also were not different between groups to 3 d of age and at 6 d of age. The separation of the compliance curves from Days 3 to 6 may indicate a trend for improved compliance (Figure 1C). Pressure-volume curves were not different between control and betamethasone-exposed preterm baboons at 6 d of age (Figure 2). Therefore, there was no consistent improvement in the initial severity of lung disease or the progression of the lung disease over 6 d as a result of the antenatal betamethasone treatments.

View larger version (23K): [in this window] [in a new window]   Figure 1.   Arterial to alveolar Po2 ratio (a/A ratio) (A), peak inspiratory pressures (PIP) (B), and dynamic compliance values over 6 d (C ) for control and antenatally betamethasone-treated preterm baboons. There were no differences between groups.

View larger version (21K): [in this window] [in a new window]   Figure 2.   Pressure-volume curves measured after 6 d of ventilation for control on antenatally betamethasone-treated preterm baboons. There were no differences in the curves.

Sat PC Pool Sizes and Loss of ¹⁴C-Sat PC

The 100 mg/kg dose of surfactant given at the initiation of ventilation contained about 68 µmol Sat PC/kg. About 4 µmol/ kg Sat PC was removed from the lungs by the initial airway sampling at 12 h (Figure 3A). The recovery of Sat PC in the airway samples taken every 12 h decreased to very small amounts by Day 3 and subsequently increased by Day 6. The average total amount of Sat PC recovered over 6 d by airway sampling was 15.4 µmol/kg, an amount equivalent to 23% of the Sat PC in the surfactant given at delivery. There were no differences between control and betamethasone-treated animals in the amounts of Sat PC recovered by airway suctioning. Specific activity measurements (CPM/µmol Sat PC) for the airway samples demonstrated an exponential decrease in specific activities to 6 d after no change in specific activities initially for about 36 h (Figure 3B). The regression curves for samples from the control and antenatal betamethasone-treated groups were not different.

View larger version (15K): [in this window] [in a new window]   Figure 3.   Recoveries and specific activities of saturated phosphatidylcholine (Sat PC) from airway samples collected every 12 h for 6 d. (A) The recoveries of Sat PC for the control and betamethasone-treated animals were not different, and a composite curve for both groups is shown. (B) Specific activities as CPM/µmol Sat PC were normalized to the specific activity of the surfactant used to treat the preterm baboons. The specific activity values were close to 1.0 until 36 h. All subsequent values were used to calculate log-linear regression curves by the method of least-squares. The specific activity-time curves for the control and antenatally betamethasone-treated animals were not different, and the regression curves intercepted the specific activity of 1.0 at about 36 h. Although values for Sat PC in alveolar washes were higher than for the final airway samples, the differences were not significant.

The recoveries of ¹⁴C-Sat PC in alveolar washes were 4.8% for the control group and 3.6% for the betamethasone group, values that were not different (Figure 4A). Recoveries of ¹⁴C-Sat PC in the total lungs (alveolar wash plus lung tissue) were 15.4 and 17.7% for control and betamethasone-exposed baboons 6 d after treatment. Therefore, about 84% of the Sat PC in the initial dose of surfactant was lost from the lungs over 6 d, yielding an approximate half-life of 2 d. The amounts of Sat PC in alveolar washes were 22.7 µmol/kg for the control group and 24.2 µmol/kg for the betamethasone group (Figure 4B). The amounts of Sat PC in the total lungs were about 190 µmol/kg for both groups of preterm baboons at 6 d. The antenatal betamethasone treatment did not alter the amounts of Sat PC.

View larger version (16K): [in this window] [in a new window]   Figure 4.   Recovery of 14C-labeled saturated phosphatidylcholine (14C-Sat PC) (A) and total Sat PC (µmol/kg) (B) from alveolar washes, lung tissue, and total lung (the sum of alveolar wash plus lung tissue). The 14C-Sat PC came from 14C-dipalmitoylphosphatidylcholine in the treatment dose of surfactant given at birth. There were no differences for any of the measurements between the control and antenatally betamethasone-treated groups.

These Sat PC pool sizes in the total lungs were corrected for the residual Sat PC contributed by the initial surfactant treatment (based on ¹⁴C-Sat PC recoveries) and compared with the amounts of Sat PC found in fetal baboon lungs at different points in gestation and with the adult baboon (Figure 5). The 6-d ventilation period resulted in a 4.5-fold increase in the amount of Sat PC from about 38 µmol/kg (the average for 125 and 140 d gestation) to 170 µmol/kg. The amount of Sat PC was quantitatively higher than the 128 µmol/kg measured at 175 d gestation and 8.9 times higher than the amount of Sat PC in the lungs of the adult baboon.

View larger version (25K): [in this window] [in a new window]   Figure 5.   Amount of saturated phosphatidylcholine (Sat PC) in the total lungs of baboons at different gestational ages (hatched bars) and in the adult (open bar). The amount of Sat PC µmol/kg body weight increased from 140 d to 175 d gestation (term is 184 d gestation). The animals in the 125 + 6 group (solid bar) are the 12 animals delivered at 125 d gestation and ventilated for 6 d. The total lung Sat PC pool was corrected for Sat PC contributed to these lungs by the surfactant treatment at birth.

Secretion of Sat PC

These preterm baboons received ³H-palmitic acid 24 h before the alveolar wash procedure. About 1% of the ³H-palmitate given by intravascular injection became incorporated into Sat PC in the lungs. Of the labeled Sat PC, 8.7 ± 1.4% was recovered by alveolar wash (estimated percent secreted) for control animals and 6.5 ± 2.6% was in the alveolar washes of the animals exposed antenatally to betamethasone, values that were not different.

Large Aggregate Surfactant

The amounts of Sat PC in large aggregate surfactant as a percent of the total Sat PC in alveolar washes were 35 ± 10% for the control group and 37 ± 3% for the betamethasone group, values that were not different. The amount of protein in the alveolar washes also was similar: 82 ± 12 mg/kg for the control group and 77 ± 19 mg/kg for the betamethasone group.

Surfactant Function

Minimum surface tensions were measured at different concentrations for the surfactant used to treat the lambs, large aggregate surfactant from adult baboons, and large aggregate surfactant from the preterm baboons (Figure 6). At high dilution the surfactant recovered at 6 d of age from the preterm baboons was less effective than the other surfactants. Addition of plasma to suspensions of large aggregate surfactants containing 20 pmol/ml Sat PC resulted in inactivation curves that were not different for preterm and adult baboons.

View larger version (16K): [in this window] [in a new window]   Figure 6.   Minimum surface tensions and sensitivities to inhibition of the surfactant used to treat the preterm baboons (treatment surfactant), large aggregate surfactant recovered after 6 d ventilation from pooled samples from control and antenatally betamethasone-treated preterm baboons (preterm LA), and large aggregate surfactant from adult baboons (adult LA). The surfactant from the preterm baboons had higher minimal surface tensions at high dilution than did the other surfactants (p < 0.05). Addition of plasma to the surfactants resulted in a similar interference with minimal surface tensions.

The in vivo function of the large aggregate surfactants were tested by the surfactant treatment responses of preterm rabbits. The large aggregate surfactants from the control and betamethasone-exposed animals had been recovered after 6 d of ventilation. The preterm rabbits were of similar size (about 32 g) and were ventilated with similar tidal volumes of 8 ml/ kg. Compliances after 15 min of ventilation were higher for the surfactant from term and adult baboons, and the surfactant from the preterms was similar to the surfactant used to treat the animals (Figure 7). The same patterns of response were found for maximal lung volumes measured at 35 cm H₂O pressure and deflation volumes at 5 cm H₂O pressure measured from the pressure-volume curves. There was no effect of antenatal glucocorticoids on the function of the surfactants recovered from the preterm baboons.

View larger version (19K): [in this window] [in a new window]   Figure 7.   Compliance and lung volumes of preterm rabbits after treatment with the surfactant used to treat the preterm baboons (treatment surfactant), large aggregate (LA) surfactants recovered from term newborn and adult baboons, and LA surfactants recovered from control and antenatally betamethasone-treated (Beta) preterm baboons after 6 d of ventilation. (A) The surfactant used to treat the preterm baboons and the surfactant recovered after 6 d of ventilation increased compliance relative to control volumes (p < 0.05). The LA surfactants from term and adult baboons resulted in compliance values higher than in all other groups (p < 0.05). (B and C ) This pattern of responses also was noted for the maximal lung volumes measured at 35 cm H2O pressure and at 5 cm H2O on deflation. Lung volumes at 5 cm H2O for the surfactant recovered from the Beta-treated preterm baboons were less than for the control surfactant-treated (p < 0.5), but not different from untreated rabbits.

Anatomy

Specimens from 125-d animals ventilated for 6 d had thickened saccular walls, an absence of alveoli, and abundant cuboidal epithelial cells. Ultrastructurally, the type II epithelial cells showed considerable variation in lamellar body number; some cells contained only glycogen pools and no lamellar bodies, whereas others had a few to abundant lamellar bodies and minimal glycogen (Figure 8). The lamellar bodies varied in their osmiophilic staining intensity. The interstitium contained increased mesenchymal undifferentiated cells, scattered myofibroblasts, and occasional inflammatory cells. Capillaries were evident in the saccular walls. Hyaline membranes and edema were not present in the alveolar spaces.

View larger version (131K): [in this window] [in a new window]   Figure 8.   The variability in epithelial type II cell morphology includes a cell with a more vesicular nucleus, a cytoplasmic glycogen pool (glycogen not retained during fixation) and a poorly osmicated lamellar body (*). The cells to each side show more mature chromatin patterns, and one of the cells contains several well- osmicated lamellar bodies (arrow), whereas the other cell contains a poorly osmicated lamellar body (double arrows). AS = alveolar space, I = interstitium. Magnification: ×3,800. Uranyl acetate and lead citrate stains.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We found that surfactant-treated very preterm baboons had large amounts of Sat PC in lung tissue after 6 d of ventilation. The animals lost most of the Sat PC that they received in the treatment dose of surfactant from the lungs over 6 d and, despite the large tissue pools, only about 7% of de novo synthesized Sat PC was secreted to the air spaces in 24 h. The large aggregate surfactant present in the alveolar washes contained 36% of the Sat PC, yielding a pool size of functional surfactant of 8 µmol Sat PC/kg. This surfactant was less effective at lowering minimum surface tension at high dilution and was not as effective a surfactant for the treatment of surfactant deficiency as surfactant recovered from term and adult baboons. Therefore, ventilated preterm baboons have metabolic and functional abnormalities of the surfactant system. We found no effect of antenatal glucocorticoids on lung function or the surfactant system.

This is unique information in very preterm animals. There is no information on surfactant in preterm humans developing BPD other than the persistence of a high phosphatidylinositol to phosphatidylglycerol ratio (7) and a delay in the appearance of SP-A (8). The modest improvement in oxygenation after surfactant treatment of infants with BPD is also consistent with abnormalities of surfactant function (11). The previously described model of BPD required 100% oxygen and conventional ventilation of 140-d preterm baboons to achieve a progressive lung injury characterized by the combination of focal overexpansion and alveolar atelectasis that resulted in decreased alveolar development at 8 mo of age (3, 4). After 16 d of ventilation with high oxygen, the 140-d preterm baboons had decreased SP-A, a persistence of a high phosphatidylinositol to phosphatidylglycerol ratio in surfactant, abnormal surface activity, and hyperplasia of type II cells (21). The same hyperplasia of type II cells was noted for human infants with BPD (22), and infants with BPD may not develop the normal number of alveoli with growth (6).

The present study utilized preterm baboons at 0.67 of gestation to better mimic the lung injury that develops in very preterm infants with respiratory distress syndrome. At birth these preterm baboons have immature type II cells that contain large glycogen pools and lack lamellar bodies, and there is essentially no surfactant in alveolar washes at delivery. The animals cannot be ventilated successfully without surfactant treatment. However, with surfactant they survive without the need for the amounts of supplemental oxygen generally needed to cause oxygen toxicity (3). Unlike the 140-d gestation baboon that has abundant lamellar bodies in the type II epithelial cells after ventilation with appropriate oxygen for 6 d (21), the comparably treated 125-d animals have type II cells with glycogen and variable numbers of lamellar bodies that show differences in osmiophilia. A delay in epithelial differentiation seems to exist in these more gestationally immature baboons. The significance of the variable osmiophilia of lamellar bodies is not known.

These are the first measurements of surfactant metabolism in very preterm surfactant-treated and ventilated animals. We measured the change in specific activity of the [¹⁴C]DPPC from the treatment surfactant in airway samples over 6 d because a similar measurement of the change in ratios of surfactant components was used to estimate biologic half-life values for those components in humans (23). In a result similar to the human data, we found an exponential decrease in the specific activity of Sat PC. The alveolar pool size estimate at 6 d of 23 µmol/kg Sat PC is 34% of the treatment dose that contained 68 µmol/kg Sat PC, and the normalized specific activity of the surfactant recovered by alveolar wash was 0.13 of that of the treatment surfactant. Therefore, very little of the Sat PC used for treatment remained in the air spaces after 6 d, the measured recovery being about 4%. A half-life for the alveolar [¹⁴C]DPPC estimated from the airway samples is about 30 h. However, this value is difficult to interpret because of the decrease in the alveolar pool size of Sat PC. An interesting aspect to the curves are the intercepts at the initial specific activity of the treatment surfactant at about 36 h. The lack of change of specific activity indicates that the animals were unable to contribute a significant amount of surfactant to the alveolar pool for about 1.5 d. This long delay probably results from the immaturity of the type II cells, and is consistent with the delay in secretion of surfactant phospholipids in infants with RDS (24). A caution about airway sampling is apparent from the quantities of Sat PC recovered. Airway suctioning every 12 h that was similar to clinical practice removed about 15 µmol/kg of Sat PC and about 8% of the [¹⁴C]DPPC from the lungs.

We gave the [¹⁴C]DPPC with the treatment dose of surfactant primarily to evaluate catabolic activity. The adult rabbit and mouse lung rapidly degrades DPPC associated with treatment doses of surfactant with a half-life of about 8 h (25, 26). Term sheep and rabbits degrade surfactant much more slowly with half-life values of about 6 and 3 d, respectively (27, 28). The preterm ventilated lamb does not degrade a significant amount of the Sat PC or the surfactant proteins associated with a surfactant treatment over 24 h (13, 28). The concept has developed from these measurements that the preterm or term newborn benefits from surfactant treatments in part because the surfactant is retained in the lungs and is reprocessed by the preterm lungs to preserve or enhance function (14). These preterm baboons degraded about 84% of the [¹⁴C]DPPC over 6 d. Nevertheless, the net accumulation of Sat PC in the lungs was large (about 22 µmol/kg/d) relative to the average pool size of Sat PC in the fetal lung over the interval of 125 to 140 d gestation of 37 µmol/kg. Assuming the same catabolic rate of 27% each day for 6 d and mixing of the alveolar and tissue Sat PC pools, the total synthetic rate for Sat PC would be about 28 µmol/kg/d, a value that is between estimates of 11 µmol/kg/d for newborn and 36 µmol/kg/d for adult rabbits (26). Such large accumulations of Sat PC were not noted by Jackson and colleagues (9) for more mature Macaca nemistrina monkeys that were recovering from respiratory distress syndrome after delivery at 83% of gestation. In those animals lung Sat PC increased from 46 µmol/kg at delivery to about 110 µmol/kg at 4 to 6 d of age.

We gave ³H-palmitic acid 24 h before recovery of the lungs to evaluate the amount of Sat PC derived from de novo synthesis that was secreted into the air spaces (13). In adult animals the interval between incorporation of palmitate until maximal accumulation of radiolabeled Sat PC in the air spaces is 8 to 12 h. However, in term newborn rabbits and preterm lambs, the interval is 24 to 48 h (26, 29). This interval may be as long as 3 d in the preterm infant with RDS (24). We selected a precursor administration to recovery period of 24 h, although there is no information to predict an optimal time for study in preterm primates. The net percent of the de novo synthesized Sat PC that was secreted over 24 h minus any clearance of that Sat PC was about 7%. We made a similar estimate in another group of preterm baboons ventilated for 24 h after delivery and recovered only 2.5% (n = 5). For comparison, adult rabbits and mice accumulate 25 to 40% of de novo synthesized Sat PC in the air spaces (25, 26), and values for newborn lambs are about 30% (30). Although Sat PC pool sizes in the lung tissue of term animals are large, the alveolar pool sizes also are large, on the order of 100 µmol Sat PC/kg. Therefore, these preterm baboons have the anomalous metabolic characteristics of a normal to high synthetic rate, very large tissue pools, small alveolar pools, and a low efficiency of secretion of de novo synthesized Sat PC.

The explanation(s) as to why the lung tissue accumulates so much Sat PC are not known. Several factors that may contribute are the hyperplasia of type II cell that are immature and the responses of the type II cells to cytokines. The type II cells of ventilated 140 d gestation baboons are large and have numerous lamellar bodies (21). Each cell may contain more Sat PC. Airway samples from infants with BPD contain more polymorphonuclear white blood cells, more macrophages, high levels of proinflammatory cytokines such as TGF, IL-1, IL-4, and IL-8, and no detectable anti-inflammatory IL-10 (31, 32). Overexpression or the lack of selective cytokines and growth factors cause metabolic abnormalities in transgenic mice, resulting in alveolar proteinosis (25). Mediators present in the air spaces of these preterm baboons with lung injury could be interfering with normal surfactant metabolism.

An initial hypothesis for this study was that antenatal betamethasone would favorably alter lung function and surfactant metabolism. There were no consistent differences for the measurements between the glucocorticoid-exposed and the control animals, except for the suppression of cord cortisol values. The control fetuses had high cortisol levels, much higher than cord values for preterm humans and similar to the stress responses generated by preterm humans with respiratory distress syndrome (33). Pepe and Albrecht (34) found that pregnant baboons contribute about 75% to the fetal cortisol levels at midgestation because the placenta leaks cortisol. There is very little research on antenatal maturation strategies with the baboon. Although other primate fetuses do seem to respond to antenatal glucocorticoids with an increase in surfactant pools (12), high dose antenatal glucocorticoids primarily influenced lung structure and not surfactant in other reports (35). We suspect that both the control and the betamethasone-treated fetuses were exposed to high levels of endogenous glucocorticoids as a result of the maternal stress responses to fetal ultrasound and to the administration of the betamethasone or saline 24 and 48 h before delivery.

The interpretations of these measurements of surfactant kinetics are limited by the inability to study multiple animals at multiple time points. However, the measurements are sufficient to demonstrate abnormalities of surfactant pools, secretion, and function. Surfactant abnormalities may contribute more than previously thought to the abnormalities of lung function reported in BPD. The mechanisms underlying the type II cell immaturity and the defective mobilization of surfactant to the alveolar space need to be explored.