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The abnormal lung development in CDH results in a combination of pulmonary hypoplasia and pulmonary hypertension(1–3). Even with modern advances in neonatal and pediatric surgical care, the overall mortality is still 50% or greater(4). At birth the lungs in CDH are abnormally developed with a reduction in bronchial divisions, a total reduction in alveoli, and a corresponding decrease in the pulmonary vasculature(2, 3). Despite this, the pulmonary surfactant system in CDH has previously been considered normal(5). However, there are a number of clinical reports which show that the amount of DPPC, the most bioactive phospholid in surfactant, is decreased in full-term babies with CDH(6, 7). In addition, postmortem studies of CDH lungs show features of hyaline membrane disease, similar to that seen in the surfactant-deficient premature infant with respiratory distress syndrome(7). Laboratory work has recently confirmed these clinical reports, showing that the amounts of total phospholipid and PC are significantly decreased in both the alveolar space and in the lung parenchyma in CDH(8, 9). There was also an increase in plasma proteins in the alveoli, which are known to inhibit surfactant biophysical function(8, 10). These studies indicate that there is a qualitative and quantitative surfactant deficiency in CDH. Surfactant replacement studies in the lamb CDH model, and in small clinical series, suggest that this observed surfactant deficiency may play an important role in the pathophysiology of CDH(11, 13).

Histologic examination of the hypoplastic lungs in CDH reveal that, although both lungs are affected, the ipsilateral lung is more severely altered by the abnormal development(2). The purpose of this study was to measure the contribution of each lung to the surfactant pool. This was established by determining the surfactant status in the bronchoalveolar lavage and by assessing the alveolar type II cell function in each lung independently.

METHODS

Creation of the CDH model. Nineteen time-dated pregnant ewes were operated on. Diaphragmatic hernias were created surgically in the fetal lambs at 80 d of gestation (term 140-145 d). The ewe was anesthetized with 5% sodium thiamyl via the left external jugular vein, intubated, and maintained with 2.0% halothane in oxygen on a Harvard piston-type ventilator (Harvard Apparatus, Harvard, MA). The ewe received 500 mg of ampicillin and 40 mg of gentamicin intramuscularly. After a midline abdominal incision, a hysterotomy was performed, using GIA™ stapling device to open and a TA-90™ stapler (U.S. Surgical Corp., Norwalk, CT) to close the uterus. The upper torso of the fetus was carefully delivered, and a posterolateral left thoracotomy was made in the 10th intercostal space. The diaphragm was incised widely over the superior edge of the spleen and the fundus of the stomach; the stomach, omentum, and small intestine were gently pulled into the chest. The thoracotomy was closed in one layer using interrupted 4-0 silk pericostal sutures. Before closure of the hysterotomy, 500 mg of ampicillin, 40 mg of gentamicin, and warmed normal saline were instilled into the amniotic cavity. The maternal abdomen was closed with absorbable sutures. The ewes were housed in the animal care facility until term. Sham-operated twins acted as controls in some cases, wherein all procedures were identical but a daphragmatic hernia was not created. We found no differences between these animals and nonoperated controls.

Bronchoalveolar lavage. For all experimental animals, the left and right lungs were individually lavaged after they had been isolated from each other and weighed. This was achieved by ligating the right mainstem and right upper bronchus to lavage the left lung, and then ligating the left mainstem bronchus to lavage the right lung. For control and CDH animals both left and right lungs were lavaged using a total of 250 mL of normal saline in 50-mL aliquots. Each aliquot was instilled and withdrawn three times as previously described(8, 10). Earlier studies confirmed that this serial lavage protocol was equally effective at recovering total lipids and protein from both control and CDH lungs.

The phospholipids were extracted from the lavages by chloroform and methanol as described by Bligh and Dyer(14). The microphosphorus technique of Chen et al.(15) was then used to determine the total phospholipid content of lavage from each animal. Compositional analysis of phospholipid classes was also performed on extracted samples using one-dimensional thin layer chromatography as described by Touchstone et al.(16). SP-A and SP-B were measured in the bronchaoveolar lavage by the laboratory of Dr. Jeffrey Whitsett using a capture ELISA as described by Pruyhuber et al.(17). For each assay, polyclonal antibodies showing specificity for SP-A or SP-B via Western blot analysis were used. These ELISA have demonstrated good cross-reactivity between species due to the highly conserved nature of the surfactant proteins. Total protein content in the lavages was determined by a modification of the method originally described by Lowry et al.(18). In addition, the lavaged protein was characterized by standard SDS-PAGE(19). All data are expressed per g of lung tissue. This represents wet lung weight because dry lung weight measures would have precluded further cell isolations. However, additional experiments have shown that the same phenomena are observed when dry lung weights are used for normalization(25).

Alveolar type II cell isolation. Alveolar type II cells were isolated from lamb lungs by modification of the method previously described by Glick et al.(8). The chest was opened, and the lungs were perfused via the pulmonary artery with 0.15 M NaCl until blanched. The lungs were excised and lavaged as described above. After lavage both lungs were separated, so that ipsilateral and contralateral alveolar type II pneumocytes were isolated. To facilitate macrophage removal, lungs were instilled with 50 mL of normal saline containing 5 mg of barium sulfate, incubated for 10 min at 37 °C, and then lavaged with an additional 400 mL of normal saline. After the second lavage, the lungs were fully inflated with Joklik-modified MEM containing 10 μg/mL DNase I, 20 mg/mL trypsin, and 1.3 U/mL elastase, and incubated at 37 °C for 35 min. The digestion was stopped by the addition of cold (4 °C) Joklik MEM with 50 mg/mL DNase I, 2.5 mg/mL trypsin inhibitor, and 10% fetal bovine serum.

After enzymatic digestion, major bronchi were removed with microdissecting forceps, and the tissue was minced with dissecting scissors. The tissue mince was then stirred in the retained media for 15 min at 4 °C, filtered through three Nitex nylon gauze filters (160, 41, and 15 μm), and washed free of protease. Type II cells were isolated from the crude cell preparation by centrifugation on a discontinuous density gradient consisting of 1.08 g/mL and 1.035 g/mL density Percoll. Cells were added to the gradient in Joklik MEM and spun for 20 min at 1000 × g. The cell band above the 1.035 g/mL Percoll layer was removed and washed free of Percoll. Cell number, usually greater than 80 million, and viability (>90%) was determined by hemocytometer counts and trypan blue exclusion(8). Type II cell purity was 85-90% in all experimental groups, as determined by Papanicoulou staining.

PC biosynthesis by alveolar type II cells. The freshly isolated cells were resuspended in MEM. Two million cells were placed in test tubes containing 2 μCi/mL radiolabeled choline and 0.1 mM choline chloride. The cells were “preincubated” at 37 °C for 1 h to allow for substrate equilibration as previously described(10). After substrate equilibration, another 37 °C incubation for PC synthesis rate determination was performed, wherein reactions were stopped at 0, 30, and 60 min by the addition of cold saline. The cells were washed to remove unincorporated label and resuspended in 0.8 mL of normal saline. To this suspension, 3 mL of chloroform:methanol (1:2, v/v) were added, and phospholipids were extracted by the method of Bligh and Dyer(14). PC was isolated from the extract by thin layer chromatography using the solvent system of Touchstone et al.(16) and comparison to known standards after rhodamine visualization. The radioactivity of each spot was determined for each time point using a scintillation counter. Data points were then analyzed via linear regression analysis to determine choline incorporation into PC/million type II cells/h.

Assessment of in utero fluid mixing. The ability of surfactant to mix from one lung to the other was assessed in four CDH lambs at 140 d of gestation. This was performed using radiolabeled surfactant, which was prepared by adding [3H]DPPC to the chloroform phase of the calf lung surfactant extract mixture (Ony, Inc., Buffalo, NY). The radiolabeled extract was then resuspended into saline at a concentration of 30 mg of phospholipid/mL and 25 μCi of [3H]DPPC/mL. This stock preparation was diluted 50:50 with unlabeled calf lung surfactant extract. The lumbs were delivered as described above. While on placental bypass, a right thoracotomy was made through the 10th intercostal space. The lung was visualized, and 1.5 mL of radiolabeled surfactant were instilled into the base of the right lung using a 24-gauge needle; the surfactant was added in 200-μL aliquots slowly so as to minimize artifactual distribution. After surfactant administration the thoracotomy was closed, and the lamb was returned to the uterus. The ewe was kept anesthetized for 4 h, and lambs showed no signs of instability. The lamb was then delivered and sacrificed as described above. The lungs were individually lavaged using the same protocol, and the radioactivity in each lavage was then assessed.

Statistical analysis. Data are expressed as mean ± SEM. Groups were compared using the Mann Whitney U test. Comparisons between left and right lung were performed using the Wilcoxon signed rank test(20). Statistical significance was taken when p < 0.05.

RESULTS

Nineteen CDH lambs were operated upon, and 13 survived to be studied. Sham-operated twins acted as controls. A fetal wastage of less than 50%, using this CDH model, is in agreement with other published series(8, 21). There was no significant difference in the lamb body weights between the two groups (control, 3.6 ± 0.8 kg; CDH, 3.8 ± 0.6 kg). The total lung weights were significantly reduced in the CDH lambs and, as expected, combined left and right CDH lungs weighed less than controls (control, 129 ± 8.4 g; CDH, 60.6 ± 5.4 g,p < 0.01). In addition, the left CDH lung was smaller than the right lung (left CDH, 22 ± 2.4 g; right CDH, 37 ± 3.1 g,p < 0.01).

Bronchoalveolar lavage. Nine CDH and nine control animals were analyzed for BAL and type II cell characteristics in the ipsilateral and contralateral lungs. Figure 1 shows that the amount of total alveolar phospholipid per g of lung tissue was similar in both left lung and right lung of control animals (0.42 ± 0.16 versus 0.33±.04 mg/g, respectively). Similarly, total phospholipid in both CDH lung was also the same (0.11 ± 0.05 versus 0.10 ± 0.02 mg/g, respectively). However, both left and right CDH BAL phospholipid levels were significantly less than control (p < 0.01)(Fig. 1). Compositional analyses of the phospholipids are shown in Figure 2. There was no statistical difference between left and right lungs. However, there was a significant reduction in the percentage of PC in the CDH lavages compared with the control group(p < 0.01).

Figure 1
figure 1

Total phospholipid in bronchoalveolar lavage from left and right lungs from both CDH and control animals, expressed per g of lung weight. * p < 0.01.

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Figure 2
figure 2

Individual phospholipid composition of the bronchoalveolar lavage from the left and right lungs of both CDH and control animals, expressed as percentage of the total phospholipid. *p < 0.01.

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The levels of SP-A and SP-B were not different between the ipsilateral and contralateral lungs for both control and CDH animals. Both SPs were significantly reduced in the CDH lungs (p < 0.01). The mean SP-A levels in the control group were 1.75 ± 0.31 μg/g and 2.68 ± 1.33 μg/g for the left and right lungs, respectively. This was compared with 0.20 ± 0.51 μg/g and 0.29 ± 0.12 μg/g, for the left and right CDH lungs, respectively (Fig. 3A). Results for SP-B were 3.12 ± 0.59 μg/g, versus 2.21 ± 0.52μg/g, in left and right control lungs respectively, and 0.35 ± 0.11μg/g, and 0.87 ± 0.32 μg/g for the left and right CDH lungs (Fig. 3B).

Figure 3
figure 3

(A) SP-A concentration in the bronchoalveolar lavage from the left and right lungs from both CDH and control animals, expressed per g of lung weight. * p < 0.01. B) SP-B concentration in the bronchoalveolar lavage from the left and right lungs from both CDH and control animals, expressed per g of lung weight. *p < 0.01.

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Total protein recovered in the BAL was the same between ipsilateral and contralateral lungs of both the control and CDH animals. However, the amount of protein in the CDH lambs was significantly increased compared with controls(p < 0.01) (Fig. 4).

Figure 4
figure 4

Total protein in bronchoalveolar lavage from the left and right lungs from CDH and control animals, expressed per g of lung weight.* p < 0.01.

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Phospholipid synthesis. The rates of choline incorporation into PC by isolated type II cells are shown in Figure 5. PC synthesis by type II cells isolated from left and right control lungs were not different from each other. However, these PC synthesis rates were significantly greater than PC synthesis rates from cells from both CDH lungs. In addition, cells from the ipsilateral CDH lung demonstrated lower PC synthesis rates compared to the contralateral CDH lung.

Figure 5
figure 5

Choline incorporation rate in alveolar type II cells, isolated from the left and right lungs from both CDH and control animals.* p < 0.01 (CDH vs control). # p < 0.025 (left CDH vs right CDH).

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In utero surfactant distribution. Within 4 h the distribution of radiolabeled surfactant originally instilled into the lower right lung of CDH animals was found to be 57% ± 9 on the left side and 43% ± 9 on the right lung. These results demonstrate significant movement of surfactant within the alveolar fluid between the two lungs.

DISCUSSION

Surfactant is a complex mixture of phospholipids and SPs. The physiologic properties of surfactant are reliant on these two basic bioactive ingredients. The phospholipids, particularly DPPC, are important in lowering surface tension at the air liquid interface(22). SP-A, -B, and-C enable the phospholipids to adsorb at the air-liquid interface and allow rapid surface tension lowering during inspiration(23). Conversely, plasma proteins present at the air liquid interface are known to reduce surfactant biophysical activity, probably by interfering with the adsorptive process(23, 24). The data presented here show that all components contributing to surfactant biophysical function are abnormal in the CDH lamb model. There is a reduction in total phospholipids, in PC, and in SPs. In addition, the amount of inhibitory alveolar plasma proteins present is increased in the BAL of CDH animals at birth.

The total phospholipids were significantly decreased in the CDH lambs, even when normalized per g of lung weight in the hypoplastic lung. In these experiments wet weight was used, because the lungs were further processed for type II cell isolation, thereby preventing dry weight determination. However, additional experiments have shown that the same phenomena are observed when lung dry weights are used for normalization(25). The percentage of phospholipids that were PC, the primary surfactant phospholipid, was also reduced in the CDH lambs. Therefore there was both a quantitative and qualitative reduction in the phospholipid component of surfactant in the lamb congenital diaphragmatic hernia model. This is in agreement both with previous animal experiments and with clinical reports(6–9).

SP-A and SP-B were both significantly reduced in the CDH lambs. Moreover, the quantity of SP-A and SP-B in proportion to total phospholipid present in the BAL was also decreased in the CDH lambs. This further discrepancy may explain why previous studies show that the BAL from CDH lambs had a reduced ability to lower surface tension even when phospholipid quantity was standardized compared with controls, and the inhibitory plasma proteins were removed(8).

Although surfactant-associated protein levels were decreased in CDH lungs, the quantity of total alveolar protein present in the BAL was significantly increased in the CDH lambs. Previous experiments using other animal lung injury models have shown that the presence of plasma proteins impairs surfactant function in vitro and in vivo(24). These results may be explained by an “in utero” increase of alveolar capillary permeability, as has been reported in the surfactant-deficient premature lamb model(26).

The surfactant deficiency observed in CDH has recently been shown to play a significant role in the pathophysiology of CDH. In studies using the lamb model, calf lung surfactant extract significantly increased gas exchange and improved pulmonary mechanics(13). This supported the two small clinical series which have been reported(11, 12). These results suggest a potential clinical role for surfactant replacement therapy in the treatment of newborns with CDH.

One possible mechanism for the surfactant biochemical abnormalities in lavages of CDH lambs is dysfunction of the type II pneumocyte. The ability of the lungs to synthesize surfactant phospholipid was assessed using the incorporation of radiolabeled choline into PC in freshly isolated type II cells. As was expected from the lavage data, there was a significant reduction in the choline incorporation rate in the CDH lungs when compared with the control lungs. These data agree with those of Zimmerman et al.(27) who showed decreased PC synthesis in type II cells from rats with CDH induced by nitrofen exposure. It was also interesting to observe that the rate of choline incorporation between the two lungs in CDH was significantly different. The ipsilateral lung had a significantly lower rate of synthesis than the contralateral lung, whereas in control lungs, the rate of synthesis was the same. These results are consistent with autopsy studies, which show that both lungs are affected in CDH, but that the ipsilateral lung is more severely altered by the embryologic maldevelopment(2).

The PC synthesis results showing a difference between the two lungs are interesting considering that the BAL parameters per g of lung tissue were similar between the CDH lungs. One possible explanation is that surfactant released in utero was able to mix freely in the lung fluid so giving an even distribution. To study this possibility radiolabeled surfactant was surgically injected into the peripheral alveolar portion of the right lung of four CDH lambs still in utero. After 4 h the animals were delivered, sacrificed, and then lavaged using the same protocol described above. Even after as little as 4 h, the radiolabeled surfactant had evenly distributed between the two sides. Although one must be appropriately cautious about the potential artifacts of in utero fetal manipulations, we did not see major alterations in the status of the ewe or fetus after the radiolabeled surfactant injection. Nevertheless, we cannot rule out possible subtle changes in fetal breathing movements after surgical manipulation, nor can we predict whether such an occurrence might impact fluid mixing.

Our results suggest that fluid mixing in utero is the most plausible explanation for the relatively even alveolar distribution of surfactant seen in the CDH lambs. If this is the case, one might expect to see the type II cell differences result in alveolar surfactant levels differ between the ipsilateral and contralateral lungs after several hours of air ventilation. However, further studies of surfactant secretion and uptake by type II cells in CDH are still needed to fully understand the surfactant dysfunction in CDH.

In conclusion, the lamb CDH model is surfactant-deficient. The ability of type II pneumocytes in both lungs to synthesize surfactant is decreased in CDH, but the ipsilateral lung is more severely altered by the maldevelopment. However, in utero fluid mixing between the two lungs may equalize the quantity and quality of surfactant recovered from alveoli of the two sides. Nevertheless, alveolar levels of both surfactant phospholipids and proteins are significantly lower in both CDH lungs compared with control lungs.