Rescue of the Hypoplastic Lung by Prenatal Cyclical Strain
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美国呼吸和危急护理医学 2005年第6期
Division of Maternal and Child Health Sciences, University of Dundee, Dundee
Department of Paediatric Surgery, Royal Hospital for Sick Children and University of Glasgow
Department of Obstetrics and Fetal Medicine, Queen Mother's Hospital
Department of Veterinary Anatomy, University of Glasgow, Glasgow, Scotland, United Kingdom.
ABSTRACT
We determined the effects of sustained and cyclical prenatal mechanical strain on the hypoplastic lung of the ovine model of congenital diaphragmatic hernia. Over a period of 4 weeks in late gestation, repeated cyclical tracheal occlusion for 23 hours with 1-hour release stimulated minimal growth, but promoted maturation with the development of a saccular lung. In contrast, a cycle consisting of 47 hours with 1-hour release induced optimal lung growth and morphologic maturation of the hypoplastic lung parenchyma. Sustained occlusion resulted in exaggerated lung growth, exceeding that of unaffected controls, and abnormal alveolar development. The extent of induction of lung growth by mechanical strain was inversely proportional to the number of alveolar type II cells remaining in the lung epithelium. These studies show that, although mechanical strain is capable of inducing lung growth and differentiation, cyclical strain is a prerequisite for normal development and that mechanically induced growth occurs at the expense of the alveolar type II cell. We conclude that cyclical strain may allow optimal alveolar development while maintaining a population of alveolar type II cells and may thus facilitate an improvement in postnatal lung function in infants with congenital diaphragmatic hernia.
Key Words: diaphragmatic hernia fetoscopy fetus lung
Despite advances in neonatal care, 50% of infants affected by isolated severe congenital diaphragmatic hernia (CDH) die with respiratory insufficiency secondary to pulmonary hypoplasia (1eC3). Survival is associated with significant long-term morbidity, primarily from pulmonary, neurologic, and gastrointestinal disease (4eC8).
It is well established that lung growth can be modified by manipulating lung liquid volume. Thus drainage of fetal lung fluid retards growth, resulting in pulmonary hypoplasia (9, 10), whereas tracheal occlusion results in pulmonary hyperplasia (9eC16). Tracheal occlusion has been evaluated as an in utero treatment for surgically created CDH in fetal lambs. Although this procedure expands the lung, increases mass, and temporarily improves gas exchange (12), the lungs differentiate abnormally (11), are deplete of type II alveolar cells (17) and surfactant (18), and develop respiratory failure and pulmonary hypertension after birth (19). Further studies have examined the reversal of tracheal occlusion before birth with variable results (20, 21). Prenatal tracheal occlusion has also been applied to human fetuses (22); the initial results were poor (23), but even more recent studies show survival similar to standard postnatal care (2).
Sustained strain causes injury to organotypic lung cultures (24), whereas in vivo sustained strain after tracheal occlusion increases DNA synthesis, which peaks 2 days postocclusion and is largely complete by 4 days (25); subsequently, there is a resumption of growth at control rates (26). The lack of cyclical variation in the degree of mechanical strain, a prerequisite for optimal growth in a number of tissues (27, 28), including lung (24), may be responsible for this plateauing of lung growth (27, 28). In organotypic lung culture, rapid cyclical elongation is essential for proliferation of epithelial cells and fibroblasts (27eC29) and causes a selective increase in steady state mRNA levels of surfactant protein type C (30).
In the latter part of gestation, fetal breathing movements (FBM) occur approximately 30% of the time (31) and, although individually they are virtually isovolumetric (32, 33), continuous periods of FBM are associated with diminished outflow resistance and thus increased egress of fluid ("deflation") (31). Furthermore, the abolition of a cyclical strain to the fetal lung arrests normal differentiation (34). We hypothesized that the periodic loss of volume (and consequent decrease in mechanical strain) during periods of FBM is necessary for normal differentiation (17), and we considered it possible that the fetal lung growth and development achieved by static occlusion could be further improved by applying a cyclical variation in strain. Because DNA synthesis is at its highest during the first 2 days of tracheal occlusion (25), we chose to evaluate cycles of 23 and 47 hours' occlusion, each with 1-hour release, to define which best achieved lung growth with preservation of the alveolar type II cell population.
METHODS
Experimental Design
All animal work was overseen by the Animal Ethics Committee of Glasgow University Veterinary School, and the United Kingdom Home Office approved all procedures under the terms of current United Kingdom animal legislation. Five groups of lamb fetuses from Texel cross ewes (full term 145 ± 1 days) were studied: (1) the CDH group (n = 6) was obtained by creating CDH in utero at 80 days' gestation; (2) the CDH/TO23h group (n = 3) underwent CDH creation at 80 days and tracheal occlusion with a endoluminal silicone balloon between 110 and 138 days—tracheal occlusion was for 23 hours with 1-hour release, and this cyclical regimen was repeated continuously for 28 days; (3) the CDH/TO47h group (n = 4) underwent CDH creation at 80 days and cyclical tracheal occlusion for 47 hours with 1-hour release repeatedly from 110 to 138 days; (4) the CDH/TOstatic group (n = 3) underwent CDH creation at 80 days and sustained tracheal occlusion from 110 to 138 days; and (5) the control group (n = 6), consisted of unoperated fetuses derived from twins of the intervention groups. A total of 58 CDH fetuses were created at 80 days; 33 ewes underwent a second intervention at 110 days for placement of fetal endoluminal balloons. Because of a combination of fetal deaths, abortions, balloon failures, and healed diaphragms, 42 fetuses were excluded from analysis and were not included in the study groups. These losses reduced the numbers of animals in each of the three treatment groups, but further studies were not permitted by the United Kingdom Home Office on the grounds that, in spite of the small numbers, the 47-hour cycle had been shown to be statistically superior for the outcomes studied compared with the CDH, 23-hour cyclical, and static occlusion regimes.
Fetal Surgical Procedures
At 80 days' gestation, maternal and fetal anesthesia was induced and the left-sided diaphragmatic defect created via a laparotomy and hysterotomy as previously described (19, 35). At 110 days' gestation, the ewes underwent a second laparotomy and hysterotomy and a Syntel Latex-Free 4F Arterial Embolectomy Catheter (Applied Medical Resources Corp., Medlink Europe, Eindhoven, The Netherlands) was placed under direct vision into the fetal trachea, secured to the fetal mandible, and tunnelled subcutaneously onto the maternal flank. Externally, the catheter was connected to a three-way tap facilitating balloon inflation and deflation.
At 138 days' gestation, the ewes were killed by an intravenous bolus of sodium pentobarbital and the fetuses retrieved. Each lamb was weighed, the chest wall removed, and the lungs excised and weighed. The isolated left lung was fixed by concurrent immersion and bronchial instillation of 10% buffered formalin at constant pressure of 25 cm H2O.
Histologic and Immunohistochemical Studies
Design-based sampling methods were combined with blinded morphometric assessment of the left lung (36, 37). The data sets and hierarchical equations studied were as previously described (38). Morphometric analysis of the alveolar type II cell population was performed as described previously (39). Additional details on the histologic and stereologic techniques are provided in the online supplement.
Statistical Analyses
Continuous variables were summarized by the mean and SEM. Comparison of three or more means was made using analysis of variance (ANOVA). Multiple comparisons after ANOVA were performed using Tukey's method. Correlation was determined by Pearson correlation coefficient and Spearman's rho. Statistical significance was assumed at the 5% level and analysis was performed using SPSS version 11.0 (SPSS Inc., Chicago, IL).
RESULTS
Body and Lung Weights
In the CDH group, the total (p < 0.0001) and individual right (p < 0.0001) and left (p < 0.0001) lung weights were reduced compared with the control group (Table 1). In the CDH/TO23h group, lung weights and lung weight to body weight ratios were similar to the CDH group. In contrast, the CDH/TO47h group had increased total and individual lung weights (p < 0.0001) compared with the CDH group and were similar to the control group (Table 1). Lung weights were much increased in the CDH/TOstatic group (p < 0.0001) compared with all groups, including the controls (Table 1). The dry:wet ratios did not differ between groups (p = 0.09) (Table 1), although the CDH lung had an increased ratio consistent with a more cellular lung. That progressive occlusions decreased this ratio is suggestive of an increased water content with longer occlusions.
Lung Volumes
The creation of a diaphragmatic defect resulted in a reduction in lung volume (p < 0.001) and parenchyma (p < 0.001) compared with the control group (Table 2). An alteration in lung composition is evident with 58% parenchymal tissue in the CDH group compared with 90% in control fetuses (p < 0.001). The CDH/TO23h group had a small, nonsignificant increase in fixed lung volume and parenchymal tissue, but the CDH/TO47h group had significantly increased lung (p = 0.014) and parenchymal (p < 0.01) volumes compared with control values. The CDH/TOstatic group showed an excessive increase in volume that exceeded control values (p < 0.0001). Pleural (p < 0.05) volumes were significantly increased only in the CDH/TOstatic group. There were no differences in interlobular septal volumes for any group.
Alveolar Morphometry
Alveolar tissue volume fractions, wall thicknesses, and total surface areas differed between the groups (Figure 1). The CDH group had a significantly smaller alveolar surface area compared with the control group, consistent with the decrease in lung volume. This reduction in alveolar total surface area was reversed by the three tracheal occlusion treatments, and longer periods of occlusion were associated with greater increments in alveolar surface area (Figure 1). As the alveolar total surface area increased, the alveolar tissue volume fractions were significantly reduced in the CDH/TO47h and CDH/TOstatic groups compared with the CDH group. Alveolar wall thickness was reduced in the CDH/TO47h group, but not the CDH/TO23h group, compared with the CDH group (p = 0.026) (Figure 1).
Total alveolar number in the CDH group was significantly smaller than the control group (p < 0.001), but the alveolar numerical density did not differ (Figure 2). The total alveolar number was significantly reduced in all three occlusion groups compared with the control group; alveolar numerical density was also decreased but not significantly. Average alveolar volume was only increased in the CDH/TOStatic group compared with the control group (p < 0.001) (Figure 2).
Lung Morphology
The pulmonary parenchyma of the CDH group had a very immature structure resembling the architecture of the normal late canalicular phase with significant heterogeneity resulting from the coexistence of hypoplastic and developmentally advanced parenchyma (Figure 3). Lungs from the CDH/TO23h group were quite markedly different from the CDH group and resembled the saccular phase of lung development (Figure 3). The parenchymal component was characterized by thick-walled saccular airspaces of variable size with little evidence of alveolarization and secondary crest development. Lungs from the CDH/TO47h group were further advanced than either the CDH group or the CDH/TO23h group and were very similar to the control group of 138 days in appearance (Figure 3). There was a dramatic increase in the parenchymal component, with the conducting airways being larger, smoother, and rounder than untreated CDH. The terminal and respiratory bronchioles were well defined, with alveolar ducts frequently originating from them. The alveolar ducts were interrupted by alveolar crests forming a large complex number of thin-walled alveoli. As determined by high-powered light microscopy, the aireCblood barrier was well developed with single capillary complexes, little connective tissue, and attenuated alveolar epithelial type I cells. Similar to the control group, the large bronchi were lined with simple cuboidal cells and contained discrete glands within the underlying wall. Cartilage plates, blood vessels, and ganglia were also enmeshed in a thick fibromuscular coat. The lungs from the CDH/TOstatic group appeared more mature than those of the CDH group, although overall the alveoli were considerably larger than the control group and were abnormal in structure and appearance, with some similarities to an emphysematous lung (Figure 3).
Alveolar Type II Cells
The CDH group alveolar type II cells were more frequent in hypoplastic areas, but in developmentally advanced areas were of similar frequency and distribution to those of the control group. Alveolar type II cells were reduced in both the CDH/TO23h and CDH/TO47h groups when compared with both the control and CDH groups. The distended alveoli of the CDH/TOstatic group were virtually devoid of alveolar type II cells.
The total number of alveolar type II cells was greatest in the control group, with significantly fewer (p = 0.02) and smaller (p = 0.006) cells present in the CDH group (Figure 4). Reductions in alveolar type II cell number, but not size, were observed in the three occlusion groups compared with CDH and control groups (Figure 4). Alveolar type II cell numerical density was negatively correlated with fixed left lung volume (Figure 5); Pearson's correlation coefficient eC0.63, (p = 0.004).
DISCUSSION
The aim of this study was to analyze the growth and maturation of fetal lungs after cyclical or sustained tracheal occlusion in a surgical model of diaphragmatic hernia. CDH results in a reduction in lung weight, volume, airway generation, total number of acini and alveoli, abnormal development of bronchiolar cartilage, abnormal muscularization of the arteriolar wall (40, 41), and decreased cross-sectional area of the pulmonary vascular bed (42). Our surgical model of CDH, with its associated lung hypoplasia that is present by 96 days' gestation and increases in severity toward term (43), is consistent with the known effects of CDH in both animals (35, 44, 45) and humans (40, 46, 47).
During the last third of gestation in humans and sheep, lung development is characterized by expansion of the primitive airways, interstitial thinning with reduction of aireCblood barrier thickness, and the subdivision of terminal saccules to form alveoli, thus increasing the surface area for gas exchange (48). These developmental changes are dependent on adequate lung liquid volumes generated by an active secretory process which gives rise to secretion rates of between 2.7 and 3.7 ml/kg/h over the last trimester (49). Reductions in luminal liquid volume are associated with pulmonary hypoplasia (9, 26); conversely, tracheal occlusion to prevent egress of lung liquid accelerates fetal lung growth (9, 50eC53). Lung liquid volume increases rapidly at the onset of occlusion, doubling within the first 24 hours, and then increasing more slowly to plateau after 7 days (25). Intraluminal tracheal pressure similarly increases after tracheal occlusion but, unlike lung volume, does not correlate with lung growth (25).
We compared the potential of tracheal occlusion for 23 hours with 1-hour deflation (CDH/TO23h) and tracheal occlusion for 47 hours with 1-hour deflation (CDH/TO47h) with static occlusion (CDH/TOstatic) applied over 4 weeks to rescue the hypoplastic lung phenotype. Because of the small numbers in the three treatment groups, we cannot exclude the possibility that Type II errors are responsible for the apparent lack effect of CDH/TO23h, compared with CDH, on any of the outcomes measured. Nevertheless, despite the small numbers, we are confident in our conclusion that CDH/TO47h provides significantly better lung development than CDH/TO23h. The outcomes we found to be significantly different by ANOVA and post hoc analysis remain significant if subjected to nonparametric statistical analysis and, for these outcomes, the CDH/TO47h 95% confidence interval lower boundaries lie well above the CDH/TO23h 95% confidence interval upper boundaries. Our observation that static occlusion (CDH/TOstatic) results in lung hyperplasia and poor differentiation is consistent with previous reports (11, 12).
Our finding that, of the two cyclical protocols, only CDH/TO47h resulted in lung growth, indicates that the cellular mechanisms responsible for transducing the increase of lung volume into lung growth only become effective during the second 23-hour period of obstruction, even though it has been observed that neither fetal lung liquid volume nor tracheal pressure increase during the second half of the 47-hour occlusion period (25). We would expect the growth transduction signal to the lung to be similar after 23 hours or 47 hours of occlusion if lung lumen volume was the only factor underlying the induction of lung growth, but the duration and frequency of exposure to this increased luminal volume may be a key factor in determining the fetal lung response.
After release of tracheal occlusion, we anticipated an immediate egress of fetal lung fluid as a result of the increased pressure gradient between tracheal lumen and amniotic liquid; this was confirmed by postmortem observations. Had there been no volume loss, we would have expected growth to be equivalent to that of the CDH/TOstatic group (i.e., hyperplastic with abnormal differentiation). That this was not the outcome suggests that the periodic loss of volume in our cyclical CDH/TO47h group is essential for lung differentiation.
The periods of increased intraluminal volume and mechanical strain in the cyclical occlusion groups have demonstrable effects on lung maturation that are consistent with in vitro observations (54). The mechanism by which increased intraluminal volume and, consequently, pressure is translated into acceleration of fetal lung growth is not clear. The differences in the maturation of the CDH/TO23h and CDH/TO47h groups suggest that time exposed to mechanical stress is a key determinant of the fetal lung response. The tensegrity model for mechanoregulation based on adhesion molecules and the cytoskeleton (55) would be consistent with exposure time as a key factor (56). In this system, prolonged physical distortion of the lung from tracheal occlusion would change force distributions across adhesion receptors, resulting in both restructuring of the cytoskeleton network and activation of diverse cell signaling activities. Local changes in the cellular force balance may contribute to pattern formation and differentiation during tissue morphogenesis. In support of this, inhibition of Rho-kinase, a GTPase, which promotes myosin light chain kinase phosphorylation and stimulates cytoskeleton contraction, decreases morphogenetic branching of E12 mouse lung rudiments (57). Conversely, treatment to increase cytoskeleton tension with a low dose of a specific activator of Rho, cytotoxic necrotizing factor-1, increases branching (57).
The increase in the alveolar type II cell population in the CDH lung as measured by immunohistochemical computer-assisted stereology is in agreement with previous studies using electron microscopy (58) and in situ hybridization for surfactant-B mRNA (59). The application of a cyclical stretch in the CDH/TO23h and CDH/47h groups caused reductions in the alveolar type II cell population compared with controls, but in each case the reductions were less than in the CDH/TOstatic group. Alveolar type II cell depletion by tracheal occlusion may be attributable to increased transdifferentiation to alveolar type I cells rather than decreased cell proliferation or cell death. Thus distension of alveolar type II cells increases RTI40 mRNA expression, a type I phenotype marker, and decreases mRNA expression of type II markers, surfactant proteins B and C (60). These in vitro observations are supported by in vivo studies that show that sustained tracheal occlusion over a 10-day period decreases alveolar type II cell and increases type I cell numbers, with the transient appearance of an intermediary cell type displaying a mixed phenotype without any evidence of increased proliferation or apoptosis (61). Additionally, it has been shown that tracheal occlusion is associated with reductions in disaturated phosphatidylcholine (62) and surfactant protein expression (62eC64). Deflation of the fetal lung induces differentiation in the opposite direction with an increase in alveolar type II cells and surfactant protein mRNA levels (65). Similar changes are also described in cultured fetal explants (66).
The correlation of increased fetal lung volume with a decreased number of alveolar type II cells is consistent with the concept of differentiation of alveolar type II into type I cells to facilitate the increase in alveolar surface area. The application of a cyclical strain to the alveolar type II cell with its periodic break from growth stimuli may be responsible for the increased number of alveolar type II cells maintained in the CDH/TO23h and CDH/TO47h groups compared with the CDH/TOstatic group. The level of lung transforming growth factor (TGF)-2, one of a family of inhibitory growth factors known to inhibit alveolar type II cell development (67), has been observed to increase in response to tracheal occlusion (68); therefore, the periodic escape from TGF-2 inhibition could explain the link between periodic lung deflation and the retention of a population of alveolar type II cells in the cyclical strain groups. The observed morphologic differences between the cyclical strain groups and the untreated CDH and CDH/TOstatic lung groups suggest that the maintenance of a population of alveolar type II cells with improved postnatal lung function may be achieved in the CDH/TO47h group.
We have demonstrated that mechanical distension by prenatal tracheal occlusion accelerates lung growth and that cyclical strain induces optimal growth and differentiation. We speculate that these findings may also explain the postnatal induction of lung growth by liquid mediated cyclical distension of airspaces. Extracorporeal membrane oxygenation (ECMO) has been extensively used in the management of CDH, principally in the rescue of infants with severe hypoxemia after surgical repair (69eC71). Although pulmonary growth and vascular remodeling do take place in CDH infants after birth, ECMO does not offer enough time for these changes to have a significant impact on outcome (72). Active controlled acceleration of postnatal lung growth would be necessary for the pulmonary hypoplasia to be meaningfully reversed. Lung growth has been achieved in postnatal animal models with continuous intrapulmonary distension by perfluorocarbon for 3 weeks (73). This approach has now been piloted in human patients on ECMO, with survival of unsalvageable infants marooned on ECMO (74, 75). In both of these human studies, a cyclical strain was applied to the lungs as the perfluorocarbons were exchanged daily. However, in contrast to the CDH/TO23h group, which was dependent on the lung liquid secretory mechanism to generate a positive intrapulmonary distending pressure, a continuous positive airway pressure of 4eC7 mm Hg was used in the ECMO patients, a value only achieved after 15eC24 hours of prenatal tracheal occlusion in nonhypoplastic fetal lungs (76). Therefore, these postnatal results achieved with artificial liquid distension of the developing lung bear some resemblance to the CDH/47h prenatal group, in which the lungs were distended by endogenous lung liquid at these pressures for at least 24 hours.
The optimal strategy for rescuing the hypoplastic lung, as measured by morphologic criteria, would appear to be a 47/1-hour cyclical occlusion, performed between 0.76 and 0.95 gestation, given the 145-day gestation of sheep. In contrast, the human fetus has a gestation of 283 days (77), and this intervention period would correspond to 30.7eC38 weeks, much later than previous fetal interventions have been performed (2). It is not clear whether this would provide sufficient time for fetal lung growth to occur. It is similarly unclear whether 47-hour occlusion with 1-hour release would be the optimal regimen in human fetuses because lung liquid production rates in human fetuses have not been quantified, although, as suggested from the postnatal perfluorodecalin studies, this occlusion frequency may be sufficient (75). Moreover, before the application of these experiments to human fetuses, further animal studies are required to confirm that the improved growth, differentiation, and preservation of the small population of alveolar type II cells observed in the CDH/TO47h group correlates with an improved postnatal lung function. These functional studies could be combined usefully with further studies of differentiation by electron microscopy to determine the maturity of the aireCblood barrier. Last, the use of exteriorized fetal catheters is not applicable to humans and significant technologic development is required to create a system comprising a detachable balloon (78) and a miniaturized remotely activated implantable pump (79), which could be used in human gestations.
These caveats notwithstanding, prenatal cyclical tracheal occlusion is a novel potential treatment for the hypoplastic lung of congenital diaphragmatic hernia. It remains to be seen whether additional strategies for increasing the population of alveolar type II cells, such as a period of continuous deflation after a period of cyclical strain or corticosteroids, are effective.
Acknowledgments
The authors thank Christine Stirton and her staff for their excellent technical assistance in the care of the animals.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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Department of Paediatric Surgery, Royal Hospital for Sick Children and University of Glasgow
Department of Obstetrics and Fetal Medicine, Queen Mother's Hospital
Department of Veterinary Anatomy, University of Glasgow, Glasgow, Scotland, United Kingdom.
ABSTRACT
We determined the effects of sustained and cyclical prenatal mechanical strain on the hypoplastic lung of the ovine model of congenital diaphragmatic hernia. Over a period of 4 weeks in late gestation, repeated cyclical tracheal occlusion for 23 hours with 1-hour release stimulated minimal growth, but promoted maturation with the development of a saccular lung. In contrast, a cycle consisting of 47 hours with 1-hour release induced optimal lung growth and morphologic maturation of the hypoplastic lung parenchyma. Sustained occlusion resulted in exaggerated lung growth, exceeding that of unaffected controls, and abnormal alveolar development. The extent of induction of lung growth by mechanical strain was inversely proportional to the number of alveolar type II cells remaining in the lung epithelium. These studies show that, although mechanical strain is capable of inducing lung growth and differentiation, cyclical strain is a prerequisite for normal development and that mechanically induced growth occurs at the expense of the alveolar type II cell. We conclude that cyclical strain may allow optimal alveolar development while maintaining a population of alveolar type II cells and may thus facilitate an improvement in postnatal lung function in infants with congenital diaphragmatic hernia.
Key Words: diaphragmatic hernia fetoscopy fetus lung
Despite advances in neonatal care, 50% of infants affected by isolated severe congenital diaphragmatic hernia (CDH) die with respiratory insufficiency secondary to pulmonary hypoplasia (1eC3). Survival is associated with significant long-term morbidity, primarily from pulmonary, neurologic, and gastrointestinal disease (4eC8).
It is well established that lung growth can be modified by manipulating lung liquid volume. Thus drainage of fetal lung fluid retards growth, resulting in pulmonary hypoplasia (9, 10), whereas tracheal occlusion results in pulmonary hyperplasia (9eC16). Tracheal occlusion has been evaluated as an in utero treatment for surgically created CDH in fetal lambs. Although this procedure expands the lung, increases mass, and temporarily improves gas exchange (12), the lungs differentiate abnormally (11), are deplete of type II alveolar cells (17) and surfactant (18), and develop respiratory failure and pulmonary hypertension after birth (19). Further studies have examined the reversal of tracheal occlusion before birth with variable results (20, 21). Prenatal tracheal occlusion has also been applied to human fetuses (22); the initial results were poor (23), but even more recent studies show survival similar to standard postnatal care (2).
Sustained strain causes injury to organotypic lung cultures (24), whereas in vivo sustained strain after tracheal occlusion increases DNA synthesis, which peaks 2 days postocclusion and is largely complete by 4 days (25); subsequently, there is a resumption of growth at control rates (26). The lack of cyclical variation in the degree of mechanical strain, a prerequisite for optimal growth in a number of tissues (27, 28), including lung (24), may be responsible for this plateauing of lung growth (27, 28). In organotypic lung culture, rapid cyclical elongation is essential for proliferation of epithelial cells and fibroblasts (27eC29) and causes a selective increase in steady state mRNA levels of surfactant protein type C (30).
In the latter part of gestation, fetal breathing movements (FBM) occur approximately 30% of the time (31) and, although individually they are virtually isovolumetric (32, 33), continuous periods of FBM are associated with diminished outflow resistance and thus increased egress of fluid ("deflation") (31). Furthermore, the abolition of a cyclical strain to the fetal lung arrests normal differentiation (34). We hypothesized that the periodic loss of volume (and consequent decrease in mechanical strain) during periods of FBM is necessary for normal differentiation (17), and we considered it possible that the fetal lung growth and development achieved by static occlusion could be further improved by applying a cyclical variation in strain. Because DNA synthesis is at its highest during the first 2 days of tracheal occlusion (25), we chose to evaluate cycles of 23 and 47 hours' occlusion, each with 1-hour release, to define which best achieved lung growth with preservation of the alveolar type II cell population.
METHODS
Experimental Design
All animal work was overseen by the Animal Ethics Committee of Glasgow University Veterinary School, and the United Kingdom Home Office approved all procedures under the terms of current United Kingdom animal legislation. Five groups of lamb fetuses from Texel cross ewes (full term 145 ± 1 days) were studied: (1) the CDH group (n = 6) was obtained by creating CDH in utero at 80 days' gestation; (2) the CDH/TO23h group (n = 3) underwent CDH creation at 80 days and tracheal occlusion with a endoluminal silicone balloon between 110 and 138 days—tracheal occlusion was for 23 hours with 1-hour release, and this cyclical regimen was repeated continuously for 28 days; (3) the CDH/TO47h group (n = 4) underwent CDH creation at 80 days and cyclical tracheal occlusion for 47 hours with 1-hour release repeatedly from 110 to 138 days; (4) the CDH/TOstatic group (n = 3) underwent CDH creation at 80 days and sustained tracheal occlusion from 110 to 138 days; and (5) the control group (n = 6), consisted of unoperated fetuses derived from twins of the intervention groups. A total of 58 CDH fetuses were created at 80 days; 33 ewes underwent a second intervention at 110 days for placement of fetal endoluminal balloons. Because of a combination of fetal deaths, abortions, balloon failures, and healed diaphragms, 42 fetuses were excluded from analysis and were not included in the study groups. These losses reduced the numbers of animals in each of the three treatment groups, but further studies were not permitted by the United Kingdom Home Office on the grounds that, in spite of the small numbers, the 47-hour cycle had been shown to be statistically superior for the outcomes studied compared with the CDH, 23-hour cyclical, and static occlusion regimes.
Fetal Surgical Procedures
At 80 days' gestation, maternal and fetal anesthesia was induced and the left-sided diaphragmatic defect created via a laparotomy and hysterotomy as previously described (19, 35). At 110 days' gestation, the ewes underwent a second laparotomy and hysterotomy and a Syntel Latex-Free 4F Arterial Embolectomy Catheter (Applied Medical Resources Corp., Medlink Europe, Eindhoven, The Netherlands) was placed under direct vision into the fetal trachea, secured to the fetal mandible, and tunnelled subcutaneously onto the maternal flank. Externally, the catheter was connected to a three-way tap facilitating balloon inflation and deflation.
At 138 days' gestation, the ewes were killed by an intravenous bolus of sodium pentobarbital and the fetuses retrieved. Each lamb was weighed, the chest wall removed, and the lungs excised and weighed. The isolated left lung was fixed by concurrent immersion and bronchial instillation of 10% buffered formalin at constant pressure of 25 cm H2O.
Histologic and Immunohistochemical Studies
Design-based sampling methods were combined with blinded morphometric assessment of the left lung (36, 37). The data sets and hierarchical equations studied were as previously described (38). Morphometric analysis of the alveolar type II cell population was performed as described previously (39). Additional details on the histologic and stereologic techniques are provided in the online supplement.
Statistical Analyses
Continuous variables were summarized by the mean and SEM. Comparison of three or more means was made using analysis of variance (ANOVA). Multiple comparisons after ANOVA were performed using Tukey's method. Correlation was determined by Pearson correlation coefficient and Spearman's rho. Statistical significance was assumed at the 5% level and analysis was performed using SPSS version 11.0 (SPSS Inc., Chicago, IL).
RESULTS
Body and Lung Weights
In the CDH group, the total (p < 0.0001) and individual right (p < 0.0001) and left (p < 0.0001) lung weights were reduced compared with the control group (Table 1). In the CDH/TO23h group, lung weights and lung weight to body weight ratios were similar to the CDH group. In contrast, the CDH/TO47h group had increased total and individual lung weights (p < 0.0001) compared with the CDH group and were similar to the control group (Table 1). Lung weights were much increased in the CDH/TOstatic group (p < 0.0001) compared with all groups, including the controls (Table 1). The dry:wet ratios did not differ between groups (p = 0.09) (Table 1), although the CDH lung had an increased ratio consistent with a more cellular lung. That progressive occlusions decreased this ratio is suggestive of an increased water content with longer occlusions.
Lung Volumes
The creation of a diaphragmatic defect resulted in a reduction in lung volume (p < 0.001) and parenchyma (p < 0.001) compared with the control group (Table 2). An alteration in lung composition is evident with 58% parenchymal tissue in the CDH group compared with 90% in control fetuses (p < 0.001). The CDH/TO23h group had a small, nonsignificant increase in fixed lung volume and parenchymal tissue, but the CDH/TO47h group had significantly increased lung (p = 0.014) and parenchymal (p < 0.01) volumes compared with control values. The CDH/TOstatic group showed an excessive increase in volume that exceeded control values (p < 0.0001). Pleural (p < 0.05) volumes were significantly increased only in the CDH/TOstatic group. There were no differences in interlobular septal volumes for any group.
Alveolar Morphometry
Alveolar tissue volume fractions, wall thicknesses, and total surface areas differed between the groups (Figure 1). The CDH group had a significantly smaller alveolar surface area compared with the control group, consistent with the decrease in lung volume. This reduction in alveolar total surface area was reversed by the three tracheal occlusion treatments, and longer periods of occlusion were associated with greater increments in alveolar surface area (Figure 1). As the alveolar total surface area increased, the alveolar tissue volume fractions were significantly reduced in the CDH/TO47h and CDH/TOstatic groups compared with the CDH group. Alveolar wall thickness was reduced in the CDH/TO47h group, but not the CDH/TO23h group, compared with the CDH group (p = 0.026) (Figure 1).
Total alveolar number in the CDH group was significantly smaller than the control group (p < 0.001), but the alveolar numerical density did not differ (Figure 2). The total alveolar number was significantly reduced in all three occlusion groups compared with the control group; alveolar numerical density was also decreased but not significantly. Average alveolar volume was only increased in the CDH/TOStatic group compared with the control group (p < 0.001) (Figure 2).
Lung Morphology
The pulmonary parenchyma of the CDH group had a very immature structure resembling the architecture of the normal late canalicular phase with significant heterogeneity resulting from the coexistence of hypoplastic and developmentally advanced parenchyma (Figure 3). Lungs from the CDH/TO23h group were quite markedly different from the CDH group and resembled the saccular phase of lung development (Figure 3). The parenchymal component was characterized by thick-walled saccular airspaces of variable size with little evidence of alveolarization and secondary crest development. Lungs from the CDH/TO47h group were further advanced than either the CDH group or the CDH/TO23h group and were very similar to the control group of 138 days in appearance (Figure 3). There was a dramatic increase in the parenchymal component, with the conducting airways being larger, smoother, and rounder than untreated CDH. The terminal and respiratory bronchioles were well defined, with alveolar ducts frequently originating from them. The alveolar ducts were interrupted by alveolar crests forming a large complex number of thin-walled alveoli. As determined by high-powered light microscopy, the aireCblood barrier was well developed with single capillary complexes, little connective tissue, and attenuated alveolar epithelial type I cells. Similar to the control group, the large bronchi were lined with simple cuboidal cells and contained discrete glands within the underlying wall. Cartilage plates, blood vessels, and ganglia were also enmeshed in a thick fibromuscular coat. The lungs from the CDH/TOstatic group appeared more mature than those of the CDH group, although overall the alveoli were considerably larger than the control group and were abnormal in structure and appearance, with some similarities to an emphysematous lung (Figure 3).
Alveolar Type II Cells
The CDH group alveolar type II cells were more frequent in hypoplastic areas, but in developmentally advanced areas were of similar frequency and distribution to those of the control group. Alveolar type II cells were reduced in both the CDH/TO23h and CDH/TO47h groups when compared with both the control and CDH groups. The distended alveoli of the CDH/TOstatic group were virtually devoid of alveolar type II cells.
The total number of alveolar type II cells was greatest in the control group, with significantly fewer (p = 0.02) and smaller (p = 0.006) cells present in the CDH group (Figure 4). Reductions in alveolar type II cell number, but not size, were observed in the three occlusion groups compared with CDH and control groups (Figure 4). Alveolar type II cell numerical density was negatively correlated with fixed left lung volume (Figure 5); Pearson's correlation coefficient eC0.63, (p = 0.004).
DISCUSSION
The aim of this study was to analyze the growth and maturation of fetal lungs after cyclical or sustained tracheal occlusion in a surgical model of diaphragmatic hernia. CDH results in a reduction in lung weight, volume, airway generation, total number of acini and alveoli, abnormal development of bronchiolar cartilage, abnormal muscularization of the arteriolar wall (40, 41), and decreased cross-sectional area of the pulmonary vascular bed (42). Our surgical model of CDH, with its associated lung hypoplasia that is present by 96 days' gestation and increases in severity toward term (43), is consistent with the known effects of CDH in both animals (35, 44, 45) and humans (40, 46, 47).
During the last third of gestation in humans and sheep, lung development is characterized by expansion of the primitive airways, interstitial thinning with reduction of aireCblood barrier thickness, and the subdivision of terminal saccules to form alveoli, thus increasing the surface area for gas exchange (48). These developmental changes are dependent on adequate lung liquid volumes generated by an active secretory process which gives rise to secretion rates of between 2.7 and 3.7 ml/kg/h over the last trimester (49). Reductions in luminal liquid volume are associated with pulmonary hypoplasia (9, 26); conversely, tracheal occlusion to prevent egress of lung liquid accelerates fetal lung growth (9, 50eC53). Lung liquid volume increases rapidly at the onset of occlusion, doubling within the first 24 hours, and then increasing more slowly to plateau after 7 days (25). Intraluminal tracheal pressure similarly increases after tracheal occlusion but, unlike lung volume, does not correlate with lung growth (25).
We compared the potential of tracheal occlusion for 23 hours with 1-hour deflation (CDH/TO23h) and tracheal occlusion for 47 hours with 1-hour deflation (CDH/TO47h) with static occlusion (CDH/TOstatic) applied over 4 weeks to rescue the hypoplastic lung phenotype. Because of the small numbers in the three treatment groups, we cannot exclude the possibility that Type II errors are responsible for the apparent lack effect of CDH/TO23h, compared with CDH, on any of the outcomes measured. Nevertheless, despite the small numbers, we are confident in our conclusion that CDH/TO47h provides significantly better lung development than CDH/TO23h. The outcomes we found to be significantly different by ANOVA and post hoc analysis remain significant if subjected to nonparametric statistical analysis and, for these outcomes, the CDH/TO47h 95% confidence interval lower boundaries lie well above the CDH/TO23h 95% confidence interval upper boundaries. Our observation that static occlusion (CDH/TOstatic) results in lung hyperplasia and poor differentiation is consistent with previous reports (11, 12).
Our finding that, of the two cyclical protocols, only CDH/TO47h resulted in lung growth, indicates that the cellular mechanisms responsible for transducing the increase of lung volume into lung growth only become effective during the second 23-hour period of obstruction, even though it has been observed that neither fetal lung liquid volume nor tracheal pressure increase during the second half of the 47-hour occlusion period (25). We would expect the growth transduction signal to the lung to be similar after 23 hours or 47 hours of occlusion if lung lumen volume was the only factor underlying the induction of lung growth, but the duration and frequency of exposure to this increased luminal volume may be a key factor in determining the fetal lung response.
After release of tracheal occlusion, we anticipated an immediate egress of fetal lung fluid as a result of the increased pressure gradient between tracheal lumen and amniotic liquid; this was confirmed by postmortem observations. Had there been no volume loss, we would have expected growth to be equivalent to that of the CDH/TOstatic group (i.e., hyperplastic with abnormal differentiation). That this was not the outcome suggests that the periodic loss of volume in our cyclical CDH/TO47h group is essential for lung differentiation.
The periods of increased intraluminal volume and mechanical strain in the cyclical occlusion groups have demonstrable effects on lung maturation that are consistent with in vitro observations (54). The mechanism by which increased intraluminal volume and, consequently, pressure is translated into acceleration of fetal lung growth is not clear. The differences in the maturation of the CDH/TO23h and CDH/TO47h groups suggest that time exposed to mechanical stress is a key determinant of the fetal lung response. The tensegrity model for mechanoregulation based on adhesion molecules and the cytoskeleton (55) would be consistent with exposure time as a key factor (56). In this system, prolonged physical distortion of the lung from tracheal occlusion would change force distributions across adhesion receptors, resulting in both restructuring of the cytoskeleton network and activation of diverse cell signaling activities. Local changes in the cellular force balance may contribute to pattern formation and differentiation during tissue morphogenesis. In support of this, inhibition of Rho-kinase, a GTPase, which promotes myosin light chain kinase phosphorylation and stimulates cytoskeleton contraction, decreases morphogenetic branching of E12 mouse lung rudiments (57). Conversely, treatment to increase cytoskeleton tension with a low dose of a specific activator of Rho, cytotoxic necrotizing factor-1, increases branching (57).
The increase in the alveolar type II cell population in the CDH lung as measured by immunohistochemical computer-assisted stereology is in agreement with previous studies using electron microscopy (58) and in situ hybridization for surfactant-B mRNA (59). The application of a cyclical stretch in the CDH/TO23h and CDH/47h groups caused reductions in the alveolar type II cell population compared with controls, but in each case the reductions were less than in the CDH/TOstatic group. Alveolar type II cell depletion by tracheal occlusion may be attributable to increased transdifferentiation to alveolar type I cells rather than decreased cell proliferation or cell death. Thus distension of alveolar type II cells increases RTI40 mRNA expression, a type I phenotype marker, and decreases mRNA expression of type II markers, surfactant proteins B and C (60). These in vitro observations are supported by in vivo studies that show that sustained tracheal occlusion over a 10-day period decreases alveolar type II cell and increases type I cell numbers, with the transient appearance of an intermediary cell type displaying a mixed phenotype without any evidence of increased proliferation or apoptosis (61). Additionally, it has been shown that tracheal occlusion is associated with reductions in disaturated phosphatidylcholine (62) and surfactant protein expression (62eC64). Deflation of the fetal lung induces differentiation in the opposite direction with an increase in alveolar type II cells and surfactant protein mRNA levels (65). Similar changes are also described in cultured fetal explants (66).
The correlation of increased fetal lung volume with a decreased number of alveolar type II cells is consistent with the concept of differentiation of alveolar type II into type I cells to facilitate the increase in alveolar surface area. The application of a cyclical strain to the alveolar type II cell with its periodic break from growth stimuli may be responsible for the increased number of alveolar type II cells maintained in the CDH/TO23h and CDH/TO47h groups compared with the CDH/TOstatic group. The level of lung transforming growth factor (TGF)-2, one of a family of inhibitory growth factors known to inhibit alveolar type II cell development (67), has been observed to increase in response to tracheal occlusion (68); therefore, the periodic escape from TGF-2 inhibition could explain the link between periodic lung deflation and the retention of a population of alveolar type II cells in the cyclical strain groups. The observed morphologic differences between the cyclical strain groups and the untreated CDH and CDH/TOstatic lung groups suggest that the maintenance of a population of alveolar type II cells with improved postnatal lung function may be achieved in the CDH/TO47h group.
We have demonstrated that mechanical distension by prenatal tracheal occlusion accelerates lung growth and that cyclical strain induces optimal growth and differentiation. We speculate that these findings may also explain the postnatal induction of lung growth by liquid mediated cyclical distension of airspaces. Extracorporeal membrane oxygenation (ECMO) has been extensively used in the management of CDH, principally in the rescue of infants with severe hypoxemia after surgical repair (69eC71). Although pulmonary growth and vascular remodeling do take place in CDH infants after birth, ECMO does not offer enough time for these changes to have a significant impact on outcome (72). Active controlled acceleration of postnatal lung growth would be necessary for the pulmonary hypoplasia to be meaningfully reversed. Lung growth has been achieved in postnatal animal models with continuous intrapulmonary distension by perfluorocarbon for 3 weeks (73). This approach has now been piloted in human patients on ECMO, with survival of unsalvageable infants marooned on ECMO (74, 75). In both of these human studies, a cyclical strain was applied to the lungs as the perfluorocarbons were exchanged daily. However, in contrast to the CDH/TO23h group, which was dependent on the lung liquid secretory mechanism to generate a positive intrapulmonary distending pressure, a continuous positive airway pressure of 4eC7 mm Hg was used in the ECMO patients, a value only achieved after 15eC24 hours of prenatal tracheal occlusion in nonhypoplastic fetal lungs (76). Therefore, these postnatal results achieved with artificial liquid distension of the developing lung bear some resemblance to the CDH/47h prenatal group, in which the lungs were distended by endogenous lung liquid at these pressures for at least 24 hours.
The optimal strategy for rescuing the hypoplastic lung, as measured by morphologic criteria, would appear to be a 47/1-hour cyclical occlusion, performed between 0.76 and 0.95 gestation, given the 145-day gestation of sheep. In contrast, the human fetus has a gestation of 283 days (77), and this intervention period would correspond to 30.7eC38 weeks, much later than previous fetal interventions have been performed (2). It is not clear whether this would provide sufficient time for fetal lung growth to occur. It is similarly unclear whether 47-hour occlusion with 1-hour release would be the optimal regimen in human fetuses because lung liquid production rates in human fetuses have not been quantified, although, as suggested from the postnatal perfluorodecalin studies, this occlusion frequency may be sufficient (75). Moreover, before the application of these experiments to human fetuses, further animal studies are required to confirm that the improved growth, differentiation, and preservation of the small population of alveolar type II cells observed in the CDH/TO47h group correlates with an improved postnatal lung function. These functional studies could be combined usefully with further studies of differentiation by electron microscopy to determine the maturity of the aireCblood barrier. Last, the use of exteriorized fetal catheters is not applicable to humans and significant technologic development is required to create a system comprising a detachable balloon (78) and a miniaturized remotely activated implantable pump (79), which could be used in human gestations.
These caveats notwithstanding, prenatal cyclical tracheal occlusion is a novel potential treatment for the hypoplastic lung of congenital diaphragmatic hernia. It remains to be seen whether additional strategies for increasing the population of alveolar type II cells, such as a period of continuous deflation after a period of cyclical strain or corticosteroids, are effective.
Acknowledgments
The authors thank Christine Stirton and her staff for their excellent technical assistance in the care of the animals.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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