Pathophysiology of Congenital Diaphragmatic Hernia II: The Fetal Lamb CDH Model Is Surfactant Deficient By Philip L. Glick, Victoria A. Stannard, Corinne L. Leach, Jon Rossman, Yotaro Hosada, Frederick C. Morin, Donald R. Cooney, James E. Allen, and Bruce Holm
Buffalo, New York 0 The high mortality for congenital diaphragmatic hernia (CDH) has been attributed to a combination of pulmonary hypoplasia and pulmonary hypertension. We hypothesize that a surfactant deficiency may in part be contributing to the pathophysiology of CDH. This study documents the functional, quantitative, and qualitative aspects of the surfactant status of the alveolar air-liquid interface and the type II pneumocyte in the fetal lamb CDH model. Ten lamb fetuses (gestational age, 80 days) had a CDH created via a left thoracotomy and then were allowed to continue in utero development until term. Three litter mates and three nonoperated time-dated fetuses served as controls. At term, pressure-volume curves were performed to measure pulmonary compliance and total lung capacity. Alveolar lavage was then performed to measure the quantitative and the qualitative aspects of pulmonary surfactant. Finally, isolation of type II pneumocytes allowed quantification of phospholipid synthesis. When compared with controls (N = 8). the CDH lambs (N = 5) had significantly smaller lungs (P = .OOg), decreased total lung capacity (P c .OOl) and compliance (P < .OOl), reduced total lavaged phospholipids (P = .008), and decreased percent phosphatidylcholine (P = .02). CDH lambs also had increased total lavaged proteins (P = .05) and higher minimum dynamic surface tension (P -z .OOl). A surfactant deficiency may be contributing to the pathophysiology of CDH. Surfactant replacement therapy in premature infants has been shown to improve lung compliance, decrease morbidity, and improve survival. Exogenous surfactant may also benefit infants with CDH. Copyright o 1992 by W.B. Saunders Company INDEX WORDS: Congenital diaphragmatic diagnosis, fetal therapy; surfactant.
hernia, prenatal
T
HE MORTALITY for congenital diaphragmatic hernia (CDH) remains between 50% and 88% despite recent advances in prenatal diagnosis, mater-
nal transport, delivery room resuscitation, expeditious repair, and modern intensive care nursery care.“’ The high mortality in these infants has been attributed to the combination of pulmonary hypoplasia and pulmonary hypertension, both of which are consequences of herniation of the viscus into the chest during critical stages of pulmonary development.1’3,4In addition, studies in animal models and clinical reports have demonstrated that newborns with CDH have lung compliance, pressure-volume (P-V) curves, and hyaline membrane formation resembling the changes seen in surfactant-deficient premature newborns.5-7 Also similar to premature newborns, amniotic fluid (AF) analysis has shown that full-term fetuses with prenatally diagnosed CDH have immature AF lecithin-sphingomyelin (L/S) ratios and absent AF phosphatidylglycerol (PG).‘.lo Surfactant replacement therapy in premature infants with immature L/S ratios and absent PG has been shown to improve lung compliance, decrease morbidity, and improve survival.‘* However, controlled experimental studies have not been done to determine the surfactant status in CDH. This study documents the status of the pulmonary surfactant system in the fetal lamb CDH model. Specifically, pulmonary mechanics, alveolar surfactant levels, surfactant biophysical activity, and type II pneumocyte function were measured in newborn lambs with CDH. MATERIALS AND METHODS
Animal Model From the Buffalo Institute of Fetal Therapy; The Children’s Hospital of Buffalo Perinatal Center; and the Departments of Surgery, Pediatrics, and ObstetticslGynecology, School of Medicine and Biomedical Sciences, The University at Buffalo, State University of New York, The Children S Hospital of Buffalo, Buffalo, NY. Supported in part by The Women’s and Children’s Health Research Foundation of the Children’s Hospital of Buffalo, Buffalo, NY, BSRGIRDF Award, State University of New York at Buffalo, NIH Grant HL40896, and The United States Surgical Corporation, Norwalk, CT. Presented at the 22nd Annual Meeting of the American Pediatric SutgkalAssociation, Lake Buena Vuta, Florida, May 15-18, 1991. Address reprint requests to Philip L. Click, MD, The Buffalo Institute of Fetal Therapy, The Children’s Hospital of Buffalo, 219 Btyant St, Buffalo, NY 14222 Copyright o I992 by W B. Saunders Company 0022-3468/92/2703-0020$03.00/O 392
Ten time-dated pregnant ewes at 78 days of gestation were fasted for 48 hours, induced with ketamine (2 to 4 mg/kg), intubated, and maintained with halothane-oxygen (1.0% to 2.0%) on a Harvard ventilator (Billerica, MA). This gestational age was specifically chosen to create the CDHs during the crucial glandular stage of lung development.‘~” Similar to human newborns with CDH, this model has documented that there is a decrease in the total size of the pulmonary vascular bed, a decrease in the number of vessels per unit lung, and an increase in the muscularization of the pulmonary arterial tree when compared with the controls.) One gram of ampicillin and 40 mg of gentamicin were given to the ewe intramuscularly, and 500 mg of ampicillin and 20 mg of gentamicin were instilled into the AF prior to closing the uterus. We used the GIA stapling device to open the uterus and the TA-90 stapler (US Surgical Corp, Norwalk, CT) to close the hysterotomy.13 The diaphragmatic hernias were created as originally described by deLorimier et alI4 and later modified by Adzick et al,’ Pringle et Journalof Pediatric Surgery, Vol27, No 3 (March), 1992: pp 382-388
THE FETALLAMBMODELIS SURFACTANTDEFICIENT al,” and Harrison et al.” A hysterotomy was performed over the fetal left hemithorax. The upper torso of the fetus was carefully brought out of the uterus and a posterolateral left thoracotomy was made in the 11th intercostal space. The lung was retracted superiorly and the posterior-lateral diaphragm was incised widely; the stomach, omentum, and small intestine were then gently pulled up into the chest. The thoracotomy was closed in one layer, followed by closure of the hysterotomy. At 136 to 141 days gestation, the pregnant ewes were fasted for 48 hours and underwent cesarean section as described previously. Via a hysterotomy, the fetal head and neck were exposed with the umbilical cord intact. An endotracheal tube was placed proximal to the right upper lobe bronchus and secured with an circumferential umbilical tape placed via a transverse neck incision, before any spontaneous breaths occurred. The lambs were killed by intravenous potassium chloride injection as the cord was divided. The lambs were delivered, dried, and weighed. The sternal portion of the thoracic wall of each lamb was removed to expose the lungs, mediastinum and diaphragmatic hernia. Three sham-operated (ie, uterus opened and closed) litter mates and three additional time-bred ewes underwent cesarean section at 139 to 145 days gestation and the lambs were prepared for study as described previously. These animals served as gestational agematched controls.
Lung Mechanics To measure the functional status of the alveolar air-liquid interface, pulmonary compliance of this system was measured. Pulmonary compliance in the absence of chest wall compliance was assessed by removing the anterior thoracic cage prior to performing quasistatic P-V measurements. The P-V measurements were made by the method described by Bermel et alI6 and modified by Holm et al.” Transpulmonary pressure (P,,) was measured between the trachea and the atmosphere with a P23AC Statham transducer connected to a Gould 2800s g-channel recorder (Cleveland, OH). The airless lungs were slowly inflated with air introduced by a Harvard syringe pump from 0 to 30 cm H,O P,, at a rate of 7.64 mlimin. Total lung capacity (TLC) was defined when P,, of 30 cm H,O was achieved and maintained for 1 minute. At this point stress adaptation of the lungs was performed by intermittently delivering air to maintain P,, at the plateau value. Absence of air leakage was verified by observing a constant P,, for at least 1 minute. Deflation was at a rate of 4.0 mL/min and continued until P,, of zero to define a quasistatic P-V deflation curve.
Bronchoalveolar Lavage Analysis To measure the quantitative and qualitative aspects of the pulmonary surfactant status the lungs were carefully removed from the lamb and weighed, after completion of the P-V measurements. The intact lungs were gently lavaged via the endotracheal tube with 30-mL volumes of normal saline (0.15 mol/L NaCl). This was repeated for a total lavage volume of 1 L. The lavage fluid was immediately centrifuged at 250 g for 30 minutes to remove ceils. Aliquots of the supernatant were saved for protein and phospholipid analysis. The remaining cell free supernatant was centrifuged at 40,000 g for 30 minutes to isolate surfactant phospholipids for surface activity measurements. The phospholipids were extracted from lavages with chloroform and methanol as described by Bligh and Dyer. ‘” The microphosphorus technique of Chen et al” 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.” Protein content in the reserved aliquots was determined by a
modification of the method addition, dynamic surface surfactant was measured at phospholipid per milliliter, described by Enhorning.”
originally described by Lowry et al.” In activity of the isolated protein-free a standardized concentration of 2 mg of using the oscillating bubble apparatus
Type II Pneumocyte Isolation After completion of bronchoalveolar lavage, the lungs were further processed to isolate the type 11 alveolar cell using the enzymatic dissociation technique of Finklestein and Shapiro.” Briefly, the lungs were inflated with Ca2+free balanced salt solution (BSS) containing 5 mg of BaSO, particles and incubated for 10 minutes at 37°C. After a second lavage to remove bariumcontaining macrophages, the lungs were reinflated with warmed Joklik modified minimum essential medium (JMEM) containing 10 ug/mL DNase I, 17.5 ug/mL trypsin, and 1.0 U/mL elastase and incubated at 37°C for 35 minutes. The digestion was stopped by the addition of cold JMEM with 50 u.g/mL DNase I, 2.5 mg/mL trypsin inhibitor, and 10% fetal bovine serum. At this point, the major airways were dissected from the lung tissue, which was minced with sharp scissors. All minced tissue was stirred at 4°C in retained media for 10 minutes. The resulting cell suspension was filtered through three consecutive Nitex nylon gauze filters (160, 41, and 15 urn; Tetko, Lancaster, NY) and washed free of protease. Type II cells were isolated from the crude cell preparation by centrifugation on a discontinuous density gradient consisting of 2 mL of Percoll (density, 1.08) and 10 mL of Percoll (density, 1.04). Five milliliters of crude cell suspension at 1 x IO’ were added to each gradient and the tubes centrifuged at 2,SOOgfor 20 minutes at 4°C. The cell band between the JMEM and Percoll (density, 1.04) was collected and washed. Cell counts were determined with a hemocytometer and viability assessed by trypan blue exclusion. This methodology yielded type II cells of 80% purity and >90% viability. There were no apparent differences in cell yield or viability between control and experimental animals.
Surfactant Synthesis Phospholipid synthesis in the purified type II cells was evaluated by measuring the rate of incorporation of [methyl-‘H] choline (75 Ciimmol; Amersham, Arlington Heights, IL) into phosphatidylcholine (PC) according to the method of Finkelstein and Mavi? as modified by Holm et al.” Isolated type II cells in suspension medium were incubated at 37°C in 12 x 75 mm polypropylene tubes. The suspension medium consisted of minimal essential media (MEM) ph 7.4,0.1 mmol/L choline chloride, and 2 p,Ci/mL [methyl-ZH] choline. The tubes were preincubated for 60 minutes to allow equilibration and the reactions subsequently stopped at 0, 30, and 60 minutes by the addition of 3 mL of cold 0.15 mol/L NaCl. The cells were washed to remove unincorporated label and resuspended in 0.8 mL of normal saline. Three milliliters of chloroform:methanol(1:2vol/vol) were added and the phospholipids extracted by the method of Bligh and Dyer.‘” The solvent system of Touchstone et al’” was used to separate the extracted phospholipids, which were visualized with rhodamine and compared with PC, phosphoglyceroi, sphingomyelin, and phosphatidylethanolamine standards (Sigma chemicals, St Louis, MO). The PC spot was isolated and its associated radioactivity measured in a scintillation counter.
Statistical Analysis Results are expressed as the mean + SEM. All animals were equally weighted statistically; significant differences compared with controls were determined using an unpaired Student’s f test for
GUCK ET
384
equal or unequal variances. Compliance was calculated by taking the slope of the regression line from 0 cm H,O to 5 cm H,O. RESULTS One ewe aborted her fetus shortly after the initial operation. The remainder apparently continued to term and the lambs were delivered by cesarean section. Five of the operated lambs had large diaphragmatic hernias evident on opening the thoracic cavity. In each the lungs were markedly hypoplastic, the left lung being completely obscured by herniated viscera that consisted of dilated stomach, small bowel, and liver. It was also noted that each of these animals had a paucity of lung fluid at the time of intubation. In the remaining four ewes the operated lamb was found to be dead or resorbed at cesarean section for an overall fetal wastage per in utero surgery of 50%. In three cases a viable litter mate was present and studied as a control animal. The three unoperated lambs were also delivered near term and were used as control animals. Therefore, a total of 11 lambs were evaluated, consisting of 5 CDH lambs and 6 normal lambs.
Lung Mechanics
The deflation limbs of the quasistatic P-V curves for the control and CDH lambs are shown in Figs 1A and 1B. It is apparent that there is a significant difference between the two curves, which still remains after correction for the differences in total lung capacity (Fig 1B). The CDH lambs had a marked decrease in pulmonary compliance compared with
AL
the control animals (1.6 + 0.5 for CDH v 8.0 + 0.5 for controls, P = .OOl; Table 1). The mean lung weight (P = .018) and total lung capacity (P < .OOl) of the CDH lambs were also significantly less than the control group. However, there was no difference in birth weights between the two groups (Table 1). Bronchoalveolar Lavage
The amounts of phospholipid and protein in the lavage fluid from control and CDH lambs are shown in Table 2. Figure 2 depicts the same data corrected for lung weight. The total amount of phospholipid in lavage fluid from CDH lambs was significantly reduced (P = .006). In addition, the relative amounts of different surfactant components was altered (Fig 3). Of particular interest is the fact that the proportion of PC, the major surface active component of surfactant, was significantly reduced (P = .02), whereas lysophosphatidylcholine (LPC) and phosphatidylinositol (PI), both minor surfactant factors were significantly increased (P = .Ol and P = .004, respectively) in the CDH lambs. In contrast to the decreased amounts of total lavage phospholipids, the amount of lavage protein was increased in CDH lambs as compared with controls (P = .05). In concordance with these results, dynamic surface activity measurements indicate that the minimum surface tension of the protein-free lavage fluid from CDH lambs was significantly higher than that from control lambs (P < .OOl; Table 2).
150
A
1oc g i! 1 >” 50
0 10
20
Pressure (cm H,O) Fig 1. details).
I
1
I
10
20
30
Pressure (cm H,O)
Quasistatic P-V deflation curves. (A) Mean values for control versus CDH lambs; (B) mean values expressed as % TLC (See text for
THE FETAL LAMB MODEL IS SURFACTANT
DEFICIENT
385
Table 1. Mean Birth Weight, Lung Weight, TLC, emHP’ Compliance COIltrOl Birth Weight (kg)
CDH
3.27 + 0.47
P Value
3.16 + 0.57
Lung Weight(g)
110 & 15
41 z 10.2
TLC,, cmH20(mL) Compliance
130 2 3
43 ? 2
(mL/cm H,O)
NS 0.018 < 0.001 6-
8 +- 0.5
1.6 f 0.5
0.001
NOTE. Data given as mean ? SE.
Phospholipid Synthesis The rate of PC synthesis by isolated, purified type II pneumocytes of control and CDH lambs is shown in Table 2. It appears that the rate of PC synthesis was decreased in the pneumocytes of the CDII lambs, although the difference did not reach statistical significance. DISCUSSION
These results demonstrate that creation of a CDH in the fetal lamb leads to a significant surfactant deficiency in the lungs of the term animal. The presence of CDH was also associated with decreased lung weight, total lung capacity, and compliance. However, this observed pulmonary hypoplasia was not associated with generalized growth retardation as evidenced by the similar birth weights of the experimental and control groups. Decreased lung compliance has been demonstrated in animals with experimental CDH by other investigators and has been thought to be related to the hypoplastic development of these lungs, but these reports did not address the question of surfactant activity.‘.‘.” In the present study, decreased lung compliance was associated with pulmonary hypoplasia as well as a surfactant deficiency, which may be the result of several factors. First, the CDH animals had significantly decreased alveolar phospholipid concentrations as well as grossly abnormal phospholipid composition, particularly with regard to the relative decrease in PC, the primary surface active constituent of surfactant. In addition to the abnormal phospholipid content and composition, a significant increase in lavage protein was also observed, which has been known to result in surfactant inhibition.27.2sThus, in addition to the abnormal surface properties of the Table 2. Results of Bronchoalveolar
Lavage Analysis and PC
Synthesis by Isoleted Type II Pneumocytes
DH
Fig 2. Results of quantitative analysis of bronchoalveolar fluid corrected for lung weight (see text for details).
isolated protein-free surfactant material (Table 2), the presence of increased levels of proteins in the alveolar space would be expected to exacerbate this condition in vivo. The functional, quantitative, and qualitative surfactant abnormalities demonstrated in the lungs of CDH animals are probably yet another consequence of the herniated bowel in the chest during crucial stages of lung development. The exact mechanism of this surfactant abnormality is unclear. Altered metabolic function of the type II pneumocytes was documented by decreased choline incorporation and PC synthesis, but these data did not reach statistical significance. A surfactant deficiency contributing to the pathophysiology of CDH has been considered. Wigglesworth et al have reported that by histological, morphological, and quantitative biochemical criteria, the human newborn with CDH shows many similarities to the premature, surfactant deficient newborn with respiratory distress syndrome.’ Lung immaturity in newborns with CDH has also been reported by Kitawaga et a129and by George et aL3”who noted the 100
5 @ 8o 8% $f ” pd 01 40 6s 2% 8E
Control
Total phospholipid (mg) Total protein (mg) Minimum surface tension (mN/m) PC synthesis (pmol/106 cells/h) NOTE. Data given as mean 2 SE.
60 2 7 185226 8 2 1.6 30.2 -f 9
CDH
821 334r77 35 + 5 18+4
P Value
0.006 0.05
lavage
20
h 0 LPC
SPH
PC
PE
PI
PO
Buffalo, New York 0 The high mortality for congenital diaphragmatic hernia (CDH) has been attributed to a combination of pulmonary hypoplasia and pulmonary hypertension. We hypothesize that a surfactant deficiency may in part be contributing to the pathophysiology of CDH. This study documents the functional, quantitative, and qualitative aspects of the surfactant status of the alveolar air-liquid interface and the type II pneumocyte in the fetal lamb CDH model. Ten lamb fetuses (gestational age, 80 days) had a CDH created via a left thoracotomy and then were allowed to continue in utero development until term. Three litter mates and three nonoperated time-dated fetuses served as controls. At term, pressure-volume curves were performed to measure pulmonary compliance and total lung capacity. Alveolar lavage was then performed to measure the quantitative and the qualitative aspects of pulmonary surfactant. Finally, isolation of type II pneumocytes allowed quantification of phospholipid synthesis. When compared with controls (N = 8). the CDH lambs (N = 5) had significantly smaller lungs (P = .OOg), decreased total lung capacity (P c .OOl) and compliance (P < .OOl), reduced total lavaged phospholipids (P = .008), and decreased percent phosphatidylcholine (P = .02). CDH lambs also had increased total lavaged proteins (P = .05) and higher minimum dynamic surface tension (P -z .OOl). A surfactant deficiency may be contributing to the pathophysiology of CDH. Surfactant replacement therapy in premature infants has been shown to improve lung compliance, decrease morbidity, and improve survival. Exogenous surfactant may also benefit infants with CDH. Copyright o 1992 by W.B. Saunders Company INDEX WORDS: Congenital diaphragmatic diagnosis, fetal therapy; surfactant.
hernia, prenatal
T
HE MORTALITY for congenital diaphragmatic hernia (CDH) remains between 50% and 88% despite recent advances in prenatal diagnosis, mater-
nal transport, delivery room resuscitation, expeditious repair, and modern intensive care nursery care.“’ The high mortality in these infants has been attributed to the combination of pulmonary hypoplasia and pulmonary hypertension, both of which are consequences of herniation of the viscus into the chest during critical stages of pulmonary development.1’3,4In addition, studies in animal models and clinical reports have demonstrated that newborns with CDH have lung compliance, pressure-volume (P-V) curves, and hyaline membrane formation resembling the changes seen in surfactant-deficient premature newborns.5-7 Also similar to premature newborns, amniotic fluid (AF) analysis has shown that full-term fetuses with prenatally diagnosed CDH have immature AF lecithin-sphingomyelin (L/S) ratios and absent AF phosphatidylglycerol (PG).‘.lo Surfactant replacement therapy in premature infants with immature L/S ratios and absent PG has been shown to improve lung compliance, decrease morbidity, and improve survival.‘* However, controlled experimental studies have not been done to determine the surfactant status in CDH. This study documents the status of the pulmonary surfactant system in the fetal lamb CDH model. Specifically, pulmonary mechanics, alveolar surfactant levels, surfactant biophysical activity, and type II pneumocyte function were measured in newborn lambs with CDH. MATERIALS AND METHODS
Animal Model From the Buffalo Institute of Fetal Therapy; The Children’s Hospital of Buffalo Perinatal Center; and the Departments of Surgery, Pediatrics, and ObstetticslGynecology, School of Medicine and Biomedical Sciences, The University at Buffalo, State University of New York, The Children S Hospital of Buffalo, Buffalo, NY. Supported in part by The Women’s and Children’s Health Research Foundation of the Children’s Hospital of Buffalo, Buffalo, NY, BSRGIRDF Award, State University of New York at Buffalo, NIH Grant HL40896, and The United States Surgical Corporation, Norwalk, CT. Presented at the 22nd Annual Meeting of the American Pediatric SutgkalAssociation, Lake Buena Vuta, Florida, May 15-18, 1991. Address reprint requests to Philip L. Click, MD, The Buffalo Institute of Fetal Therapy, The Children’s Hospital of Buffalo, 219 Btyant St, Buffalo, NY 14222 Copyright o I992 by W B. Saunders Company 0022-3468/92/2703-0020$03.00/O 392
Ten time-dated pregnant ewes at 78 days of gestation were fasted for 48 hours, induced with ketamine (2 to 4 mg/kg), intubated, and maintained with halothane-oxygen (1.0% to 2.0%) on a Harvard ventilator (Billerica, MA). This gestational age was specifically chosen to create the CDHs during the crucial glandular stage of lung development.‘~” Similar to human newborns with CDH, this model has documented that there is a decrease in the total size of the pulmonary vascular bed, a decrease in the number of vessels per unit lung, and an increase in the muscularization of the pulmonary arterial tree when compared with the controls.) One gram of ampicillin and 40 mg of gentamicin were given to the ewe intramuscularly, and 500 mg of ampicillin and 20 mg of gentamicin were instilled into the AF prior to closing the uterus. We used the GIA stapling device to open the uterus and the TA-90 stapler (US Surgical Corp, Norwalk, CT) to close the hysterotomy.13 The diaphragmatic hernias were created as originally described by deLorimier et alI4 and later modified by Adzick et al,’ Pringle et Journalof Pediatric Surgery, Vol27, No 3 (March), 1992: pp 382-388
THE FETALLAMBMODELIS SURFACTANTDEFICIENT al,” and Harrison et al.” A hysterotomy was performed over the fetal left hemithorax. The upper torso of the fetus was carefully brought out of the uterus and a posterolateral left thoracotomy was made in the 11th intercostal space. The lung was retracted superiorly and the posterior-lateral diaphragm was incised widely; the stomach, omentum, and small intestine were then gently pulled up into the chest. The thoracotomy was closed in one layer, followed by closure of the hysterotomy. At 136 to 141 days gestation, the pregnant ewes were fasted for 48 hours and underwent cesarean section as described previously. Via a hysterotomy, the fetal head and neck were exposed with the umbilical cord intact. An endotracheal tube was placed proximal to the right upper lobe bronchus and secured with an circumferential umbilical tape placed via a transverse neck incision, before any spontaneous breaths occurred. The lambs were killed by intravenous potassium chloride injection as the cord was divided. The lambs were delivered, dried, and weighed. The sternal portion of the thoracic wall of each lamb was removed to expose the lungs, mediastinum and diaphragmatic hernia. Three sham-operated (ie, uterus opened and closed) litter mates and three additional time-bred ewes underwent cesarean section at 139 to 145 days gestation and the lambs were prepared for study as described previously. These animals served as gestational agematched controls.
Lung Mechanics To measure the functional status of the alveolar air-liquid interface, pulmonary compliance of this system was measured. Pulmonary compliance in the absence of chest wall compliance was assessed by removing the anterior thoracic cage prior to performing quasistatic P-V measurements. The P-V measurements were made by the method described by Bermel et alI6 and modified by Holm et al.” Transpulmonary pressure (P,,) was measured between the trachea and the atmosphere with a P23AC Statham transducer connected to a Gould 2800s g-channel recorder (Cleveland, OH). The airless lungs were slowly inflated with air introduced by a Harvard syringe pump from 0 to 30 cm H,O P,, at a rate of 7.64 mlimin. Total lung capacity (TLC) was defined when P,, of 30 cm H,O was achieved and maintained for 1 minute. At this point stress adaptation of the lungs was performed by intermittently delivering air to maintain P,, at the plateau value. Absence of air leakage was verified by observing a constant P,, for at least 1 minute. Deflation was at a rate of 4.0 mL/min and continued until P,, of zero to define a quasistatic P-V deflation curve.
Bronchoalveolar Lavage Analysis To measure the quantitative and qualitative aspects of the pulmonary surfactant status the lungs were carefully removed from the lamb and weighed, after completion of the P-V measurements. The intact lungs were gently lavaged via the endotracheal tube with 30-mL volumes of normal saline (0.15 mol/L NaCl). This was repeated for a total lavage volume of 1 L. The lavage fluid was immediately centrifuged at 250 g for 30 minutes to remove ceils. Aliquots of the supernatant were saved for protein and phospholipid analysis. The remaining cell free supernatant was centrifuged at 40,000 g for 30 minutes to isolate surfactant phospholipids for surface activity measurements. The phospholipids were extracted from lavages with chloroform and methanol as described by Bligh and Dyer. ‘” The microphosphorus technique of Chen et al” 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.” Protein content in the reserved aliquots was determined by a
modification of the method addition, dynamic surface surfactant was measured at phospholipid per milliliter, described by Enhorning.”
originally described by Lowry et al.” In activity of the isolated protein-free a standardized concentration of 2 mg of using the oscillating bubble apparatus
Type II Pneumocyte Isolation After completion of bronchoalveolar lavage, the lungs were further processed to isolate the type 11 alveolar cell using the enzymatic dissociation technique of Finklestein and Shapiro.” Briefly, the lungs were inflated with Ca2+free balanced salt solution (BSS) containing 5 mg of BaSO, particles and incubated for 10 minutes at 37°C. After a second lavage to remove bariumcontaining macrophages, the lungs were reinflated with warmed Joklik modified minimum essential medium (JMEM) containing 10 ug/mL DNase I, 17.5 ug/mL trypsin, and 1.0 U/mL elastase and incubated at 37°C for 35 minutes. The digestion was stopped by the addition of cold JMEM with 50 u.g/mL DNase I, 2.5 mg/mL trypsin inhibitor, and 10% fetal bovine serum. At this point, the major airways were dissected from the lung tissue, which was minced with sharp scissors. All minced tissue was stirred at 4°C in retained media for 10 minutes. The resulting cell suspension was filtered through three consecutive Nitex nylon gauze filters (160, 41, and 15 urn; Tetko, Lancaster, NY) and washed free of protease. Type II cells were isolated from the crude cell preparation by centrifugation on a discontinuous density gradient consisting of 2 mL of Percoll (density, 1.08) and 10 mL of Percoll (density, 1.04). Five milliliters of crude cell suspension at 1 x IO’ were added to each gradient and the tubes centrifuged at 2,SOOgfor 20 minutes at 4°C. The cell band between the JMEM and Percoll (density, 1.04) was collected and washed. Cell counts were determined with a hemocytometer and viability assessed by trypan blue exclusion. This methodology yielded type II cells of 80% purity and >90% viability. There were no apparent differences in cell yield or viability between control and experimental animals.
Surfactant Synthesis Phospholipid synthesis in the purified type II cells was evaluated by measuring the rate of incorporation of [methyl-‘H] choline (75 Ciimmol; Amersham, Arlington Heights, IL) into phosphatidylcholine (PC) according to the method of Finkelstein and Mavi? as modified by Holm et al.” Isolated type II cells in suspension medium were incubated at 37°C in 12 x 75 mm polypropylene tubes. The suspension medium consisted of minimal essential media (MEM) ph 7.4,0.1 mmol/L choline chloride, and 2 p,Ci/mL [methyl-ZH] choline. The tubes were preincubated for 60 minutes to allow equilibration and the reactions subsequently stopped at 0, 30, and 60 minutes by the addition of 3 mL of cold 0.15 mol/L NaCl. The cells were washed to remove unincorporated label and resuspended in 0.8 mL of normal saline. Three milliliters of chloroform:methanol(1:2vol/vol) were added and the phospholipids extracted by the method of Bligh and Dyer.‘” The solvent system of Touchstone et al’” was used to separate the extracted phospholipids, which were visualized with rhodamine and compared with PC, phosphoglyceroi, sphingomyelin, and phosphatidylethanolamine standards (Sigma chemicals, St Louis, MO). The PC spot was isolated and its associated radioactivity measured in a scintillation counter.
Statistical Analysis Results are expressed as the mean + SEM. All animals were equally weighted statistically; significant differences compared with controls were determined using an unpaired Student’s f test for
GUCK ET
384
equal or unequal variances. Compliance was calculated by taking the slope of the regression line from 0 cm H,O to 5 cm H,O. RESULTS One ewe aborted her fetus shortly after the initial operation. The remainder apparently continued to term and the lambs were delivered by cesarean section. Five of the operated lambs had large diaphragmatic hernias evident on opening the thoracic cavity. In each the lungs were markedly hypoplastic, the left lung being completely obscured by herniated viscera that consisted of dilated stomach, small bowel, and liver. It was also noted that each of these animals had a paucity of lung fluid at the time of intubation. In the remaining four ewes the operated lamb was found to be dead or resorbed at cesarean section for an overall fetal wastage per in utero surgery of 50%. In three cases a viable litter mate was present and studied as a control animal. The three unoperated lambs were also delivered near term and were used as control animals. Therefore, a total of 11 lambs were evaluated, consisting of 5 CDH lambs and 6 normal lambs.
Lung Mechanics
The deflation limbs of the quasistatic P-V curves for the control and CDH lambs are shown in Figs 1A and 1B. It is apparent that there is a significant difference between the two curves, which still remains after correction for the differences in total lung capacity (Fig 1B). The CDH lambs had a marked decrease in pulmonary compliance compared with
AL
the control animals (1.6 + 0.5 for CDH v 8.0 + 0.5 for controls, P = .OOl; Table 1). The mean lung weight (P = .018) and total lung capacity (P < .OOl) of the CDH lambs were also significantly less than the control group. However, there was no difference in birth weights between the two groups (Table 1). Bronchoalveolar Lavage
The amounts of phospholipid and protein in the lavage fluid from control and CDH lambs are shown in Table 2. Figure 2 depicts the same data corrected for lung weight. The total amount of phospholipid in lavage fluid from CDH lambs was significantly reduced (P = .006). In addition, the relative amounts of different surfactant components was altered (Fig 3). Of particular interest is the fact that the proportion of PC, the major surface active component of surfactant, was significantly reduced (P = .02), whereas lysophosphatidylcholine (LPC) and phosphatidylinositol (PI), both minor surfactant factors were significantly increased (P = .Ol and P = .004, respectively) in the CDH lambs. In contrast to the decreased amounts of total lavage phospholipids, the amount of lavage protein was increased in CDH lambs as compared with controls (P = .05). In concordance with these results, dynamic surface activity measurements indicate that the minimum surface tension of the protein-free lavage fluid from CDH lambs was significantly higher than that from control lambs (P < .OOl; Table 2).
150
A
1oc g i! 1 >” 50
0 10
20
Pressure (cm H,O) Fig 1. details).
I
1
I
10
20
30
Pressure (cm H,O)
Quasistatic P-V deflation curves. (A) Mean values for control versus CDH lambs; (B) mean values expressed as % TLC (See text for
THE FETAL LAMB MODEL IS SURFACTANT
DEFICIENT
385
Table 1. Mean Birth Weight, Lung Weight, TLC, emHP’ Compliance COIltrOl Birth Weight (kg)
CDH
3.27 + 0.47
P Value
3.16 + 0.57
Lung Weight(g)
110 & 15
41 z 10.2
TLC,, cmH20(mL) Compliance
130 2 3
43 ? 2
(mL/cm H,O)
NS 0.018 < 0.001 6-
8 +- 0.5
1.6 f 0.5
0.001
NOTE. Data given as mean ? SE.
Phospholipid Synthesis The rate of PC synthesis by isolated, purified type II pneumocytes of control and CDH lambs is shown in Table 2. It appears that the rate of PC synthesis was decreased in the pneumocytes of the CDII lambs, although the difference did not reach statistical significance. DISCUSSION
These results demonstrate that creation of a CDH in the fetal lamb leads to a significant surfactant deficiency in the lungs of the term animal. The presence of CDH was also associated with decreased lung weight, total lung capacity, and compliance. However, this observed pulmonary hypoplasia was not associated with generalized growth retardation as evidenced by the similar birth weights of the experimental and control groups. Decreased lung compliance has been demonstrated in animals with experimental CDH by other investigators and has been thought to be related to the hypoplastic development of these lungs, but these reports did not address the question of surfactant activity.‘.‘.” In the present study, decreased lung compliance was associated with pulmonary hypoplasia as well as a surfactant deficiency, which may be the result of several factors. First, the CDH animals had significantly decreased alveolar phospholipid concentrations as well as grossly abnormal phospholipid composition, particularly with regard to the relative decrease in PC, the primary surface active constituent of surfactant. In addition to the abnormal phospholipid content and composition, a significant increase in lavage protein was also observed, which has been known to result in surfactant inhibition.27.2sThus, in addition to the abnormal surface properties of the Table 2. Results of Bronchoalveolar
Lavage Analysis and PC
Synthesis by Isoleted Type II Pneumocytes
DH
Fig 2. Results of quantitative analysis of bronchoalveolar fluid corrected for lung weight (see text for details).
isolated protein-free surfactant material (Table 2), the presence of increased levels of proteins in the alveolar space would be expected to exacerbate this condition in vivo. The functional, quantitative, and qualitative surfactant abnormalities demonstrated in the lungs of CDH animals are probably yet another consequence of the herniated bowel in the chest during crucial stages of lung development. The exact mechanism of this surfactant abnormality is unclear. Altered metabolic function of the type II pneumocytes was documented by decreased choline incorporation and PC synthesis, but these data did not reach statistical significance. A surfactant deficiency contributing to the pathophysiology of CDH has been considered. Wigglesworth et al have reported that by histological, morphological, and quantitative biochemical criteria, the human newborn with CDH shows many similarities to the premature, surfactant deficient newborn with respiratory distress syndrome.’ Lung immaturity in newborns with CDH has also been reported by Kitawaga et a129and by George et aL3”who noted the 100
5 @ 8o 8% $f ” pd 01 40 6s 2% 8E
Control
Total phospholipid (mg) Total protein (mg) Minimum surface tension (mN/m) PC synthesis (pmol/106 cells/h) NOTE. Data given as mean 2 SE.
60 2 7 185226 8 2 1.6 30.2 -f 9
CDH
821 334r77 35 + 5 18+4
P Value
0.006 0.05
lavage
20
h 0 LPC
SPH
PC
PE
PI
PO
