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Congenital diaphragmatic hernia
Seminars in Pediatric Surgery (2010) 19, 180-185
Congenital diaphragmatic hernia Richard Keijzer, MD, PhD, MSc,a Prem Puri, MS, FRCS, FRCS (Ed), FACS, FAAP (Hon)b From the aDepartment of Pediatric Surgery, Erasmusmc-Sophia, Rotterdam, The Netherlands; and the b National Children’s Hospital, Tallaght and Children’s Research Centre, Our Lady’s Children’s Hospital, Crumlin, Dublin, Ireland. KEYWORDS Congenital diaphragmatic hernia; Pulmonary hypoplasia; Persistent pulmonary hypertension; Embryology; Pathogenesis
Congenital diaphragmatic hernia (CDH) is a congenital anomaly consisting of a posterolateral defect in the diaphragm also known as a Bochdalek hernia. It occurs in 1 in 2000 to 3000 newborns and is associated with a variable degree of pulmonary hypoplasia (PH) and persistent pulmonary hypertension (PPH). Despite remarkable advances in neonatal resuscitation and intensive care and the new postnatal treatment strategies, many newborns with CDH continue to have high rates of mortality and morbidity as the result of severe respiratory failure secondary to PH and PPH. The pathogenesis of CDH and associated PH and PPH is poorly understood. Herein, we aim to review diaphragm and pulmonary development and correlate this to the abnormalities found in CDH. © 2010 Elsevier Inc. All rights reserved.
In mammals, the diaphragm provides respiratory support because it contracts and thereby induces expansion of the thorax. In this way the diaphragm induces a negative pressure to help draw air into the lungs. In contrast, on exhalation the diaphragm helps to expel air from the lungs by relaxation. To prevent the abdominal organs from moving up and down into the thorax during respiration, the diaphragm physically separates the thorax from the abdomen. This characteristic is disturbed in congenital diaphragmatic hernia (CDH). Approximately 1 in 2000 to 3000 newborns experience CDH.1 Although it is relatively easy to repair the diaphragmatic defect in a newborn either by primary closure or with a patch, the main problem of these children is the associated disturbed development of the lungs, resulting in pulmonary hypoplasia (PH) and persistent pulmonary hypertension (PPH). Recent advancement in the neonatal intensive care unit treatment protocols for these associated pulmonary
problems have significantly reduced the mortality to less than 10% to 20% in tertiary referral centers.2 However, this increase in survival has come with a price—most CDH patients experience a high rate of morbidity from conditions such as bronchopulmonary dysplasia after being in the neonatal intensive care unit because of their exposure to modern treatment modalities, such as high-frequency oscillation and extracorporeal membrane oxygenation. As with other congenital anomalies, an improved understanding of the pathogenesis of CDH and its associated pulmonary problems may help to design new treatment modalities targeted at specific developmental insults. Ultimately, the goal is to positively modulate the natural course of the disease and maybe even help to prevent it from happening at all. Herein we review the normal development of the diaphragm and the lungs and relate this to abnormal development in case of CDH.
Richard Keijzer is the recipient of an ErasmusMC fellowship. Address reprint requests and correspondence: Prem Puri, MS, FRCS, FRCS (Ed), FACS, FAAP (Hon), Children’s Research Centre and Our Lady’s Children’s Hospital, Dublin 12, Ireland. E-mail address: [email protected].
Normal development of the diaphragm
1055-8586/$ -see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1053/j.sempedsurg.2010.03.001
The diaphragm starts to develop at approximately 4 weeks of gestation in humans. It develops from several structures.
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Congenital Diaphragmatic Hernia
The anterior central tendon develops from an infolding of the ventral body wall: the septum transversum. Another infolding on the posterolateral sides establishes the pleuroperitoneal membranes. Closure of the pleuroperitoneal canals occurs when the septum transversum fuses to the structures surrounding the esophagus, the esophageal mesentery, and connects to the pleuroperitoneal membranes. Closure of the pleuroperitoneal canals normally occurs around the 8 week of gestation in humans. The right side of the diaphragm closes before the left side.3 Using different rodent animal models, the group of John Greer demonstrated that during development of the diaphragm in rodents, there is formation of the so-called pleuro-peritoneal folds (PPFs), paired pyramid-shaped transient fusion structures composed of the union of the pleuropericardial folds and the septum transversum.4-6 Subsequently, myogenic cells and phrenic axons target the PPFs, and formation of the diaphragm occurs because of the proliferation and distribution of these structures in the PPFs.7
Normal development of the lungs Mammalian lung development starts with the outgrowth from foregut endoderm of 2 lung-buds into the surrounding splanchnic mesenchyme. Before this, a certain area of the foregut is specified to become the area in which the lung (and not, for instance, the thyroid or pancreas) will develop. The origin and fate of the cells forming the lung anlage within the primitive foregut are unknown. In human, the lung originates as an outpouch of the ventral wall of the posterior end of the laryngotracheal tube and divides into 2 bronchial buds at 3-4 weeks of gestation.8 In mice and rats at 10 and 11.5 days of gestation, respectively (term mice ⫽ 19 days; term rats ⫽ 22 days), the respiratory system develops from paired endodermal buds in the primitive foregut, just proximal to the developing stomach.9,10 As the 2 buds elongate, the primitive tubular foregut tube begins to pinch into 2 tubes, namely, the dorsal esophagus and the ventral trachea.11 Further outgrowth of the lung-buds produces the secondary bronchi. In mice, the right lung characteristically has 4 major lobes (dorsal, caudal, medial, and cranial lobes), whereas the left lung consists of only 1 small lobe. This arrangement also holds true for rats except for the
Table 1
181 right lung lobes, which are termed cranial, medial, accessory and caudal. In humans, the right lung has 3 lobes, whereas the left lung is composed of 2 lobes (upper, middle, and lower on the right; upper and lower on the left). The early branching of the primary bronchial buds tends to be monopodial. Each secondary bronchus subsequently undergoes progressive dichotomous branching as each branch bifurcates repeatedly. Reproducible branching is complete at 16 generations by 16 weeks of gestation in humans.12 The final 7 generations of airways (for a total of 23) in humans are completed during the latter part of gestation. Alveolization begins after approximately 28 to 30 weeks in humans and is completed postnatally. This branching process will ultimately yield a functional lung with its very large surface area for gas exchange after birth. Already in 1967 Reid nicely summarized this process in her laws of development of the human lung13: 1. The bronchial tree is developed by the 16th week of intrauterine life. 2. Alveoli, as commonly understood, develop after birth, increasing in number until the age of 8 years and in size until growth of the chest wall is finished. 3. Blood vessels are remodeled and increase, certainly while new alveoli are forming and probably until growth of the chest is complete. During pulmonary development, 4 histologic stages can be distinguished: the pseudoglandular stage, in which the bronchial tree develops and an undifferentiated primordial system forms; the canalicular stage, in which the terminal sacs develop and vascularization occurs; the saccular or terminal sac stage, in which the number of terminal sacs increases, vascularization proceeds and differentiation of type I and type II cells occur; and finally, the alveolar stage, in which there is a huge multiplication of the alveoli establishing the extensive surface area (see Table 1 for comparison of gestational stages between human, mouse and rat). In humans (as in guinea pigs and sheep), alveolization begins in utero and is completed after birth. In contrast, in rats and mice, alveolization occurs predominantly postnatally. Epithelial cell differentiation is initiated in the saccular stage and occurs in a proximodistal pattern. The trachea and the upper airways are lined with pseudostratified ciliated columnar epithelium with scattered mucus secreting Goblet
Stages of pulmonary development
Stage of pulmonary development
Human age (weeks of gestation)
Mouse age (days of gestation)
Rat age (days of gestation)
Pseudoglandular Canalicular Saccular Alveolar
3-16 16-26 24-38 28-7 Years
10-16.5 16.5-17.5 17.5-19-5 d Days 5-30
11.5-18.5 18.5-19.5 1.9-22-1 wk Week 1-5
In this table, stages of pulmonary development and gestational ages between human, mouse, and rat are compared.
182 cells. In between the epithelial cells, small foci of pulmonary neuroendocrine cells can be found. The lower airways are lined with ciliated columnar epithelium and Clara cells. The alveoli are lined with alveolar type I cells, which are very thin for an efficient gas exchange and type II cells, which produce the surface tension reducing surfactant. The pulmonary interstitial tissue, which contains fibroblasts, myofibroblasts, and smooth muscle cells, surrounds the epithelium. In addition, the pulmonary interstitium comprises the pulmonary vasculature, consisting of arteries, veins and a large capillary network in close apposition to the alveoli. It also includes the lymphatic system and the nervous system.
Abnormal development of the diaphragm in congenital diaphragmatic hernia The pathogenesis of CDH is poorly understood. In humans, 3 different types of hernia can be distinguished: a posterolateral, Bochdalek-type (accounts for approximately 70% of the cases); an anterior, Morgagni-type (accounts for approximately 27% of the cases); and a central hernia, septum transversum-type (accounts for approximately 2%-3% of the cases). The classical picture, a hernia of Bochdalek, consists of a posterolateral defect of approximately 3 cm in diameter; however, complete absent diaphragms are also observed. The posterolateral defect in the diaphragm is most often observed on the left side (85%) but can also occur on the right side (13%) or bilaterally (2%). Abdominal organs herniate into the thorax during development and can stay herniated until after birth.14-16 Animal models have helped to start unravel the process of abnormal diaphragm and pulmonary development. Until recently, mainly 2 animal models for CDH were used: 1 based on the teratogenic effects of nitrofen in rodents and 1 in which a diaphragmatic defect is surgically created in either rabbits or sheep. The herbicide nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether) causes CDH in rats and mice.17,18 When nitrofen is administered to pregnant rat dams on day 9 of gestation, approximately 70% of the offspring will have CDH, and 100% will experience PH.19,20 These percentages are significantly lower in mice.21 The biggest flaw of the nitrofen model is that it is sometimes regarded as a toxicologic model, and more importantly, the use of nitrofen has not been linked to human CDH. One of the possible links between the nitrofen model and human CDH might be a disturbance in the retinoic acid pathway. In the 1950s, vitamin A-deficient rats were demonstrated to have CDH.22,23 Moreover, administration of vitamin A to nitrofen-treated rats reduces the incidence of CDH in the offspring, and different groups have demonstrated that this might function through down-regulation of a retinoic acid-synthesizing enzyme, retinol dehydrogenase-2, in a dose-dependent manner.6,24-30 In addition,
Seminars in Pediatric Surgery, Vol 19, No 3, August 2010 knockout mice for different genes involved in the retinoic acid pathway, such as retinoic acid receptor double knockout mice31 and COUP-TFII,32,33 a downstream target of retinoid signaling, were demonstrated to have diaphragmatic defects and a variable amount of PH. In addition, COUP-TFII was demonstrated to be up-regulated in the early stages of lung development in nitrofen-induced hypoplastic lungs.33 Despite these convincing data from animal studies, the results in humans have been limited. Major et al34 demonstrated more than 15 years ago that retinol blood levels are lower in children with CDH than in control subjects. Comparing human and rat CDH and control cases, our group could not demonstrate a difference in the expression of some of the receptors belonging to the steroid receptor superfamily, including retinoid X receptors.35-37 Under way is an international multicenter study in which the authors are evaluating the retinoic acid status of CDH infants, their mothers, and age-matched control subjects. In total more than 40 CDH cases have been included in the study. All surgical models are determined on the basis of the surgical creation of a diaphragmatic defect relatively late in gestation. This model has been used in both rabbits and sheep.38-41 Because of the larger size of the animals involved, the surgical model has been especially instructive to investigate interventional therapies. Using this approach, the prenatal administration of corticosteroids, fetal surgery to repair the diaphragmatic defect in utero, and tracheal occlusion to stimulate prenatal pulmonary growth have all been tested before these new treatment modalities were tested in humans with CDH.42-48 The disadvantage of the surgical model is that the diaphragmatic defect is created relatively late in gestation after a period of normal lung and diaphragm development. This makes it less useful to investigate the pathogenetic events of CDH. However, because both the teratogenic and surgical models were the only available models for many years, they were the best to use for investigating this anomaly from a pathogenetic, pathophysiological, and surgical therapeutic point of view. Obviously, to better understand the pathogenesis and etiology of CDH, a different approach is warranted. Recently, genetic studies in which the authors use knockout mice have begun to shed a new light on the pathogenesis of CDH. Although originally investigated for its role in urogenital development, Wilm’s tumor 1 (wt1-null mutant mice display a Bochdalek type of CDH.49 Also, members of the Sonic Hedgehog (Shh) pathway, Shh, Gli2, and Gli3, have been implemented in CDH. Besides its role in the formation of tracheoesophageal fistula, lung branching morphogenesis, and other foregut anomalies,50,51 Shh was demonstrated to be down-regulated in human hypoplastic lungs of CDH patients,52 and Gli2- and Gli3-null mutant mice have diaphragmatic defects.53,54 In mice without the axon guiding gene Slit3, the diaphragmatic defect is located in the central tendon of the diaphragm.55,56 In addition, these mice do not experience PH and do not die of respiratory failure after birth, as in some of the other knockout mice. Another
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Congenital Diaphragmatic Hernia
growth factor-related gene, platelet-derived growth factor receptor-␣ (PDGFR␣), was recently demonstrated to play a role in posterolateral diaphragm and lung development, but these knockout mice displayed features of the human Fryns syndrome, so PDGFR␣ might be involved in nonisolated CDH.57 One of the most elegant approaches to search for genes involved in CDH was used by Ackerman et al. They used a high-throughput analysis based on mutagenesis with the chemical mutagen N-ethyl-N-nitrosourea and identified mice dying from respiratory failure after birth. The pulmonary hypoplasia with or without CDH in these mice could be linked to a mutation in the Fog2 (Friend of GATA) gene. In addition to the mice model, the authors also reported a nonsense mutation in a female patient.57 Besides the previously described COUP-TFII, other binding partners of Fog2, such as Gata4, Gata6, have also been implicated in either abnormal pulmonary development and/or diaphragm development. Gata6 was demonstrated to be essential in lung branching morphogenesis and pulmonary epithelial cell differentiation.58-60 Heart, lung, and diaphragm development are disturbed in 70% of heterozygous Gata4 knockout mice.61 In these mice, the diaphragmatic defect was located ventrally and the hernia was covered by a sac. In the nitrofen model, Gata4 and Gata6 were down-regulated in the hearts of fetal rats treated with nitrofen.62
Abnormal development of the lungs In CDH, respiratory failure at birth is the result of PH, reduced airway branching, and surfactant deficiency. In addition, extensive muscularization of the pulmonary vessels may result in PPH of the newborn. Historically, PH in
183 CDH was believed to be the result of compression of the lungs by the herniating intrathoracic abdominal organs. However, our understanding of abnormal pulmonary development in relation to CDH has significantly improved because of data obtained in the nitrofen model of CDH. Fetal rats and mice from mothers treated with nitrofen experience similar respiratory problems at birth as human babies with CDH. Using this model, others and we were able to demonstrate that pulmonary development is already affected prior to development of the diaphragmatic hernia, implicating that the lungs are primarily disturbed in their development, before mechanical compression can happen.63,64 This led us to postulate the dual-hit hypothesis, which explains PH in CDH as the result of 2 developmental insults. One is affecting both lungs and is occurring before closure of the diaphragm. The second insult is affecting only the ipsilateral lung and is the result of interference of fetal breathing movements of this lung caused by the herniation of the abdominal organs into the thorax (Figure 1).64 Some authors have postulated that abnormal pulmonary development might be negatively influencing diaphragm development, thereby actually causing the diaphragmatic defect.21 However, other studies65,66 in which the authors combined the nitrofen model with transgenic mice studies have demonstrated that this hypothesis is not true. Moreover, transgenic mice carrying a null mutation for Fgf10 do not have any lungs at all and consequently die from respiratory failure at birth.67 However, these null mutant mice have normally developed diaphragms. These results rule out the possibility that abnormal pulmonary development is causing the diaphragmatic defect. Although none of the surgical, transgenic, and toxicologic models for CDH and its associated PH and PPH have succeeded in completely explaining the developmental and
Figure 1 The dual-hit hypothesis: pulmonary hypoplasia in case of CDH is explained by 2 developmental insults to the lungs. The first hit affects both the ipsilateral and contralateral lung and occurs before diaphragm development has started in a background of (unidentified) genetic and environmental factors. The second hit affects only the ipsilateral lung after development of the diaphragmatic defect and herniation of the abdominal organs into the thorax. Interference of the herniated abdominal organs with fetal breathing movements of this lung is responsible for this. (Reprinted with permission from Keijzer et al64).
184 biological basis of this congenital anomaly, many new insights into the pathogenesis have been deducted from the results of studies using these models so far. One major improvement in our understanding has been that the lungs are probably affected before the development of a defect in the diaphragm. Most likely, a common mechanism is responsible for the observed phenotype of a diaphragmatic defect, PH, and the frequently observed cardiac anomalies in infants with CDH. It is unlikely that this mechanism is caused by a single gene defect because none of the genetic studies have so far discovered “the” CDH gene. Probably, a fine balanced interaction between environmental factors and maybe even several genes involved in certain developmental pathways will be reported soon to be responsible for CDH. This might then result in new improved treatment strategies custom-made to address this defect to modulate the natural course and maybe even prevent CDH and the associated PH and PPH.
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38. deLorimier AA, Tierney DF, Parker HR. Hypoplastic lungs in fetal lambs with surgically poduced congenital diaphragmatic hernia. Surgery 1967;62:12-7. 39. Flemmer AW, Jani JC, Bergmann F, et al. Lung tissue mechanics predict lung hypoplasia in a rabbit model for congenital diaphragmatic hernia. Pediatr Pulmonol 2007;42:505-12. 40. Lipsett J, Cool JC, Runciman SI, et al. Effect of antenatal tracheal occlusion on lung development in the sheep model of congenital diaphragmatic hernia: A morphometric analysis of pulmonary structure and maturity. Pediatr Pulmonol 1998;25:257-69. 41. Wu J, Yamamoto H, Gratacos E, et al. Lung development following diaphragmatic hernia in the fetal rabbit. Hum Reprod 2000;15:2483-8. 42. De Paepe ME, Johnson BD, Papadakis K, et al. Lung growth response after tracheal occlusion in fetal rabbits is gestational age-dependent. Am J Respir Cell Mol Biol 1999;21:65-76. 43. Deprest JA, Evrard VA, Van Ballaer PP, et al. Tracheoscopic endoluminal plugging using an inflatable device in the fetal lamb model. Eur J Obstet Gynecol Reprod Biol 1998;81:165-9. 44. DiFiore JW, Fauza DO, Slavin R, et al. Experimental fetal tracheal ligation reverses the structural and physiological effects of pulmonary hypoplasia in congenital diaphragmatic hernia. J Pediatr Surg 1994; 29:248-56; discussion: 256-7. 45. Ford WD, Cool J, Derham R. Intrathoracic silo for the potential antenatal repair of diaphragmatic herniae with liver in the chest. Fetal Diagn Ther 1992;7:75-81. 46. Harrison MR, Adzick NS, Longaker MT, et al. Successful repair in utero of a fetal diaphragmatic hernia after removal of herniated viscera from the left thorax. N Engl J Med 1990;322:1582-4. 47. Harrison MR, Keller RL, Hawgood SB, et al. A randomized trial of fetal endoscopic tracheal occlusion for severe fetal congenital diaphragmatic hernia. N Engl J Med 2003;349:1916-24. 48. Roubliova XI, Van der Biest AM, Vaast P, et al. Effect of maternal administration of betamethasone on peripheral arterial development in fetal rabbit lungs. J Neonatol 2008;93:64-72. 49. Kreidberg JA, Sariola H, Loring JM, et al. WT-1 is required for early kidney development. Cell 1993;74:679-91. 50. Motoyama J, Liu J, Mo R, et al. Essential function of Gli2 and Gli3 in the formation of lung, trachea and oesophagus. Nat Genet 1998;20: 54-7. 51. Pepicelli CV, Lewis PM, McMahon AP. Sonic hedgehog regulates branching morphogenesis in the mammalian lung. Curr Biol 1998;8: 1083-6. 52. Unger S, Copland I, Tibboel D, et al. Down-regulation of sonic hedgehog expression in pulmonary hypoplasia is associated with congenital diaphragmatic hernia. Am J Pathol 2003;162:547-55.
185 53. Kim J, Kim P, Hui CC, the VACTERL Association: Lessons from the sonic hedgehog pathway. Clin Genet 2001;59:306-15. 54. Kim PC, Mo R, Hui CC. Murine models of VACTERL syndrome: Role of sonic hedgehog signaling pathway. J Pediatr Surg 2001;36: 381-4. 55. Liu J, Zhang L, Wang D, et al. Congenital diaphragmatic hernia, kidney agenesis and cardiac defects associated with Slit3-deficiency in mice. Mech Dev 2003;120:1059-70. 56. Yuan W, Rao Y, Babiuk RP, et al. A genetic model for a central (septum transversum) congenital diaphragmatic hernia in mice lacking Slit3. Proc Natl Acad Sci U S A 2003;100:5217-22. 57. Bleyl SB, Moshrefi A, Shaw GM, et al. Candidate genes for congenital diaphragmatic hernia from animal models: Sequencing of FOG2 and PDGFRalpha reveals rare variants in diaphragmatic hernia patients. Eur J Hum Genet 2007;15:950-8. 58. Keijzer R, van Tuyl M, Meijers C, et al. The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development. Development 2001;128:503-11. 59. Koutsourakis M, Keijzer R, Visser P, et al. Branching and differentiation defects in pulmonary epithelium with elevated Gata6 expression. Mech Dev 2001;105:105-14. 60. Yang H, Lu MM, Zhang L, et al. GATA6 regulates differentiation of distal lung epithelium. Development 2002;129:2233-46. 61. Ackerman KG, Wang J, Luo L, et al. Gata4 is necessary for normal pulmonary lobar development. Am J Respir Cell Mol Biol 2007;36: 391-7. 62. Takayasu H, Sato H, Sugimoto K, et al. Downregulation of GATA4 and GATA6 in the heart of rats with nitrofen-induced diaphragmatic hernia. J Pediatr Surg 2008;43:362-6. 63. Jesudason EC, Connell MG, Fernig DG, et al. Early lung malformations in congenital diaphragmatic hernia. J Pediatr Surg 2000;35: 124-7; discussion: 128. 64. Keijzer R, Liu J, Deimling J, et al. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia. Am J Pathol 2000;156:1299-306. 65. Babiuk RP, Greer JJ. Diaphragm defects occur in a CDH hernia model independently of myogenesis and lung formation. Am J Physiol Lung Cell Mol Physiol 2002;283:L1310-4. 66. Guilbert TW, Gebb SA, Shannon JM. Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development. Am J Physiol Lung Cell Mol Physiol 2000;279:L1159-71. 67. Sekine K, Ohuchi H, Fujiwara M, et al. Fgf10 is essential for limb and lung formation. Nat Genet 1999;21:138-41.
Congenital diaphragmatic hernia Richard Keijzer, MD, PhD, MSc,a Prem Puri, MS, FRCS, FRCS (Ed), FACS, FAAP (Hon)b From the aDepartment of Pediatric Surgery, Erasmusmc-Sophia, Rotterdam, The Netherlands; and the b National Children’s Hospital, Tallaght and Children’s Research Centre, Our Lady’s Children’s Hospital, Crumlin, Dublin, Ireland. KEYWORDS Congenital diaphragmatic hernia; Pulmonary hypoplasia; Persistent pulmonary hypertension; Embryology; Pathogenesis
Congenital diaphragmatic hernia (CDH) is a congenital anomaly consisting of a posterolateral defect in the diaphragm also known as a Bochdalek hernia. It occurs in 1 in 2000 to 3000 newborns and is associated with a variable degree of pulmonary hypoplasia (PH) and persistent pulmonary hypertension (PPH). Despite remarkable advances in neonatal resuscitation and intensive care and the new postnatal treatment strategies, many newborns with CDH continue to have high rates of mortality and morbidity as the result of severe respiratory failure secondary to PH and PPH. The pathogenesis of CDH and associated PH and PPH is poorly understood. Herein, we aim to review diaphragm and pulmonary development and correlate this to the abnormalities found in CDH. © 2010 Elsevier Inc. All rights reserved.
In mammals, the diaphragm provides respiratory support because it contracts and thereby induces expansion of the thorax. In this way the diaphragm induces a negative pressure to help draw air into the lungs. In contrast, on exhalation the diaphragm helps to expel air from the lungs by relaxation. To prevent the abdominal organs from moving up and down into the thorax during respiration, the diaphragm physically separates the thorax from the abdomen. This characteristic is disturbed in congenital diaphragmatic hernia (CDH). Approximately 1 in 2000 to 3000 newborns experience CDH.1 Although it is relatively easy to repair the diaphragmatic defect in a newborn either by primary closure or with a patch, the main problem of these children is the associated disturbed development of the lungs, resulting in pulmonary hypoplasia (PH) and persistent pulmonary hypertension (PPH). Recent advancement in the neonatal intensive care unit treatment protocols for these associated pulmonary
problems have significantly reduced the mortality to less than 10% to 20% in tertiary referral centers.2 However, this increase in survival has come with a price—most CDH patients experience a high rate of morbidity from conditions such as bronchopulmonary dysplasia after being in the neonatal intensive care unit because of their exposure to modern treatment modalities, such as high-frequency oscillation and extracorporeal membrane oxygenation. As with other congenital anomalies, an improved understanding of the pathogenesis of CDH and its associated pulmonary problems may help to design new treatment modalities targeted at specific developmental insults. Ultimately, the goal is to positively modulate the natural course of the disease and maybe even help to prevent it from happening at all. Herein we review the normal development of the diaphragm and the lungs and relate this to abnormal development in case of CDH.
Richard Keijzer is the recipient of an ErasmusMC fellowship. Address reprint requests and correspondence: Prem Puri, MS, FRCS, FRCS (Ed), FACS, FAAP (Hon), Children’s Research Centre and Our Lady’s Children’s Hospital, Dublin 12, Ireland. E-mail address: [email protected].
Normal development of the diaphragm
1055-8586/$ -see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1053/j.sempedsurg.2010.03.001
The diaphragm starts to develop at approximately 4 weeks of gestation in humans. It develops from several structures.
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The anterior central tendon develops from an infolding of the ventral body wall: the septum transversum. Another infolding on the posterolateral sides establishes the pleuroperitoneal membranes. Closure of the pleuroperitoneal canals occurs when the septum transversum fuses to the structures surrounding the esophagus, the esophageal mesentery, and connects to the pleuroperitoneal membranes. Closure of the pleuroperitoneal canals normally occurs around the 8 week of gestation in humans. The right side of the diaphragm closes before the left side.3 Using different rodent animal models, the group of John Greer demonstrated that during development of the diaphragm in rodents, there is formation of the so-called pleuro-peritoneal folds (PPFs), paired pyramid-shaped transient fusion structures composed of the union of the pleuropericardial folds and the septum transversum.4-6 Subsequently, myogenic cells and phrenic axons target the PPFs, and formation of the diaphragm occurs because of the proliferation and distribution of these structures in the PPFs.7
Normal development of the lungs Mammalian lung development starts with the outgrowth from foregut endoderm of 2 lung-buds into the surrounding splanchnic mesenchyme. Before this, a certain area of the foregut is specified to become the area in which the lung (and not, for instance, the thyroid or pancreas) will develop. The origin and fate of the cells forming the lung anlage within the primitive foregut are unknown. In human, the lung originates as an outpouch of the ventral wall of the posterior end of the laryngotracheal tube and divides into 2 bronchial buds at 3-4 weeks of gestation.8 In mice and rats at 10 and 11.5 days of gestation, respectively (term mice ⫽ 19 days; term rats ⫽ 22 days), the respiratory system develops from paired endodermal buds in the primitive foregut, just proximal to the developing stomach.9,10 As the 2 buds elongate, the primitive tubular foregut tube begins to pinch into 2 tubes, namely, the dorsal esophagus and the ventral trachea.11 Further outgrowth of the lung-buds produces the secondary bronchi. In mice, the right lung characteristically has 4 major lobes (dorsal, caudal, medial, and cranial lobes), whereas the left lung consists of only 1 small lobe. This arrangement also holds true for rats except for the
Table 1
181 right lung lobes, which are termed cranial, medial, accessory and caudal. In humans, the right lung has 3 lobes, whereas the left lung is composed of 2 lobes (upper, middle, and lower on the right; upper and lower on the left). The early branching of the primary bronchial buds tends to be monopodial. Each secondary bronchus subsequently undergoes progressive dichotomous branching as each branch bifurcates repeatedly. Reproducible branching is complete at 16 generations by 16 weeks of gestation in humans.12 The final 7 generations of airways (for a total of 23) in humans are completed during the latter part of gestation. Alveolization begins after approximately 28 to 30 weeks in humans and is completed postnatally. This branching process will ultimately yield a functional lung with its very large surface area for gas exchange after birth. Already in 1967 Reid nicely summarized this process in her laws of development of the human lung13: 1. The bronchial tree is developed by the 16th week of intrauterine life. 2. Alveoli, as commonly understood, develop after birth, increasing in number until the age of 8 years and in size until growth of the chest wall is finished. 3. Blood vessels are remodeled and increase, certainly while new alveoli are forming and probably until growth of the chest is complete. During pulmonary development, 4 histologic stages can be distinguished: the pseudoglandular stage, in which the bronchial tree develops and an undifferentiated primordial system forms; the canalicular stage, in which the terminal sacs develop and vascularization occurs; the saccular or terminal sac stage, in which the number of terminal sacs increases, vascularization proceeds and differentiation of type I and type II cells occur; and finally, the alveolar stage, in which there is a huge multiplication of the alveoli establishing the extensive surface area (see Table 1 for comparison of gestational stages between human, mouse and rat). In humans (as in guinea pigs and sheep), alveolization begins in utero and is completed after birth. In contrast, in rats and mice, alveolization occurs predominantly postnatally. Epithelial cell differentiation is initiated in the saccular stage and occurs in a proximodistal pattern. The trachea and the upper airways are lined with pseudostratified ciliated columnar epithelium with scattered mucus secreting Goblet
Stages of pulmonary development
Stage of pulmonary development
Human age (weeks of gestation)
Mouse age (days of gestation)
Rat age (days of gestation)
Pseudoglandular Canalicular Saccular Alveolar
3-16 16-26 24-38 28-7 Years
10-16.5 16.5-17.5 17.5-19-5 d Days 5-30
11.5-18.5 18.5-19.5 1.9-22-1 wk Week 1-5
In this table, stages of pulmonary development and gestational ages between human, mouse, and rat are compared.
182 cells. In between the epithelial cells, small foci of pulmonary neuroendocrine cells can be found. The lower airways are lined with ciliated columnar epithelium and Clara cells. The alveoli are lined with alveolar type I cells, which are very thin for an efficient gas exchange and type II cells, which produce the surface tension reducing surfactant. The pulmonary interstitial tissue, which contains fibroblasts, myofibroblasts, and smooth muscle cells, surrounds the epithelium. In addition, the pulmonary interstitium comprises the pulmonary vasculature, consisting of arteries, veins and a large capillary network in close apposition to the alveoli. It also includes the lymphatic system and the nervous system.
Abnormal development of the diaphragm in congenital diaphragmatic hernia The pathogenesis of CDH is poorly understood. In humans, 3 different types of hernia can be distinguished: a posterolateral, Bochdalek-type (accounts for approximately 70% of the cases); an anterior, Morgagni-type (accounts for approximately 27% of the cases); and a central hernia, septum transversum-type (accounts for approximately 2%-3% of the cases). The classical picture, a hernia of Bochdalek, consists of a posterolateral defect of approximately 3 cm in diameter; however, complete absent diaphragms are also observed. The posterolateral defect in the diaphragm is most often observed on the left side (85%) but can also occur on the right side (13%) or bilaterally (2%). Abdominal organs herniate into the thorax during development and can stay herniated until after birth.14-16 Animal models have helped to start unravel the process of abnormal diaphragm and pulmonary development. Until recently, mainly 2 animal models for CDH were used: 1 based on the teratogenic effects of nitrofen in rodents and 1 in which a diaphragmatic defect is surgically created in either rabbits or sheep. The herbicide nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether) causes CDH in rats and mice.17,18 When nitrofen is administered to pregnant rat dams on day 9 of gestation, approximately 70% of the offspring will have CDH, and 100% will experience PH.19,20 These percentages are significantly lower in mice.21 The biggest flaw of the nitrofen model is that it is sometimes regarded as a toxicologic model, and more importantly, the use of nitrofen has not been linked to human CDH. One of the possible links between the nitrofen model and human CDH might be a disturbance in the retinoic acid pathway. In the 1950s, vitamin A-deficient rats were demonstrated to have CDH.22,23 Moreover, administration of vitamin A to nitrofen-treated rats reduces the incidence of CDH in the offspring, and different groups have demonstrated that this might function through down-regulation of a retinoic acid-synthesizing enzyme, retinol dehydrogenase-2, in a dose-dependent manner.6,24-30 In addition,
Seminars in Pediatric Surgery, Vol 19, No 3, August 2010 knockout mice for different genes involved in the retinoic acid pathway, such as retinoic acid receptor double knockout mice31 and COUP-TFII,32,33 a downstream target of retinoid signaling, were demonstrated to have diaphragmatic defects and a variable amount of PH. In addition, COUP-TFII was demonstrated to be up-regulated in the early stages of lung development in nitrofen-induced hypoplastic lungs.33 Despite these convincing data from animal studies, the results in humans have been limited. Major et al34 demonstrated more than 15 years ago that retinol blood levels are lower in children with CDH than in control subjects. Comparing human and rat CDH and control cases, our group could not demonstrate a difference in the expression of some of the receptors belonging to the steroid receptor superfamily, including retinoid X receptors.35-37 Under way is an international multicenter study in which the authors are evaluating the retinoic acid status of CDH infants, their mothers, and age-matched control subjects. In total more than 40 CDH cases have been included in the study. All surgical models are determined on the basis of the surgical creation of a diaphragmatic defect relatively late in gestation. This model has been used in both rabbits and sheep.38-41 Because of the larger size of the animals involved, the surgical model has been especially instructive to investigate interventional therapies. Using this approach, the prenatal administration of corticosteroids, fetal surgery to repair the diaphragmatic defect in utero, and tracheal occlusion to stimulate prenatal pulmonary growth have all been tested before these new treatment modalities were tested in humans with CDH.42-48 The disadvantage of the surgical model is that the diaphragmatic defect is created relatively late in gestation after a period of normal lung and diaphragm development. This makes it less useful to investigate the pathogenetic events of CDH. However, because both the teratogenic and surgical models were the only available models for many years, they were the best to use for investigating this anomaly from a pathogenetic, pathophysiological, and surgical therapeutic point of view. Obviously, to better understand the pathogenesis and etiology of CDH, a different approach is warranted. Recently, genetic studies in which the authors use knockout mice have begun to shed a new light on the pathogenesis of CDH. Although originally investigated for its role in urogenital development, Wilm’s tumor 1 (wt1-null mutant mice display a Bochdalek type of CDH.49 Also, members of the Sonic Hedgehog (Shh) pathway, Shh, Gli2, and Gli3, have been implemented in CDH. Besides its role in the formation of tracheoesophageal fistula, lung branching morphogenesis, and other foregut anomalies,50,51 Shh was demonstrated to be down-regulated in human hypoplastic lungs of CDH patients,52 and Gli2- and Gli3-null mutant mice have diaphragmatic defects.53,54 In mice without the axon guiding gene Slit3, the diaphragmatic defect is located in the central tendon of the diaphragm.55,56 In addition, these mice do not experience PH and do not die of respiratory failure after birth, as in some of the other knockout mice. Another
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growth factor-related gene, platelet-derived growth factor receptor-␣ (PDGFR␣), was recently demonstrated to play a role in posterolateral diaphragm and lung development, but these knockout mice displayed features of the human Fryns syndrome, so PDGFR␣ might be involved in nonisolated CDH.57 One of the most elegant approaches to search for genes involved in CDH was used by Ackerman et al. They used a high-throughput analysis based on mutagenesis with the chemical mutagen N-ethyl-N-nitrosourea and identified mice dying from respiratory failure after birth. The pulmonary hypoplasia with or without CDH in these mice could be linked to a mutation in the Fog2 (Friend of GATA) gene. In addition to the mice model, the authors also reported a nonsense mutation in a female patient.57 Besides the previously described COUP-TFII, other binding partners of Fog2, such as Gata4, Gata6, have also been implicated in either abnormal pulmonary development and/or diaphragm development. Gata6 was demonstrated to be essential in lung branching morphogenesis and pulmonary epithelial cell differentiation.58-60 Heart, lung, and diaphragm development are disturbed in 70% of heterozygous Gata4 knockout mice.61 In these mice, the diaphragmatic defect was located ventrally and the hernia was covered by a sac. In the nitrofen model, Gata4 and Gata6 were down-regulated in the hearts of fetal rats treated with nitrofen.62
Abnormal development of the lungs In CDH, respiratory failure at birth is the result of PH, reduced airway branching, and surfactant deficiency. In addition, extensive muscularization of the pulmonary vessels may result in PPH of the newborn. Historically, PH in
183 CDH was believed to be the result of compression of the lungs by the herniating intrathoracic abdominal organs. However, our understanding of abnormal pulmonary development in relation to CDH has significantly improved because of data obtained in the nitrofen model of CDH. Fetal rats and mice from mothers treated with nitrofen experience similar respiratory problems at birth as human babies with CDH. Using this model, others and we were able to demonstrate that pulmonary development is already affected prior to development of the diaphragmatic hernia, implicating that the lungs are primarily disturbed in their development, before mechanical compression can happen.63,64 This led us to postulate the dual-hit hypothesis, which explains PH in CDH as the result of 2 developmental insults. One is affecting both lungs and is occurring before closure of the diaphragm. The second insult is affecting only the ipsilateral lung and is the result of interference of fetal breathing movements of this lung caused by the herniation of the abdominal organs into the thorax (Figure 1).64 Some authors have postulated that abnormal pulmonary development might be negatively influencing diaphragm development, thereby actually causing the diaphragmatic defect.21 However, other studies65,66 in which the authors combined the nitrofen model with transgenic mice studies have demonstrated that this hypothesis is not true. Moreover, transgenic mice carrying a null mutation for Fgf10 do not have any lungs at all and consequently die from respiratory failure at birth.67 However, these null mutant mice have normally developed diaphragms. These results rule out the possibility that abnormal pulmonary development is causing the diaphragmatic defect. Although none of the surgical, transgenic, and toxicologic models for CDH and its associated PH and PPH have succeeded in completely explaining the developmental and
Figure 1 The dual-hit hypothesis: pulmonary hypoplasia in case of CDH is explained by 2 developmental insults to the lungs. The first hit affects both the ipsilateral and contralateral lung and occurs before diaphragm development has started in a background of (unidentified) genetic and environmental factors. The second hit affects only the ipsilateral lung after development of the diaphragmatic defect and herniation of the abdominal organs into the thorax. Interference of the herniated abdominal organs with fetal breathing movements of this lung is responsible for this. (Reprinted with permission from Keijzer et al64).
184 biological basis of this congenital anomaly, many new insights into the pathogenesis have been deducted from the results of studies using these models so far. One major improvement in our understanding has been that the lungs are probably affected before the development of a defect in the diaphragm. Most likely, a common mechanism is responsible for the observed phenotype of a diaphragmatic defect, PH, and the frequently observed cardiac anomalies in infants with CDH. It is unlikely that this mechanism is caused by a single gene defect because none of the genetic studies have so far discovered “the” CDH gene. Probably, a fine balanced interaction between environmental factors and maybe even several genes involved in certain developmental pathways will be reported soon to be responsible for CDH. This might then result in new improved treatment strategies custom-made to address this defect to modulate the natural course and maybe even prevent CDH and the associated PH and PPH.
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185 53. Kim J, Kim P, Hui CC, the VACTERL Association: Lessons from the sonic hedgehog pathway. Clin Genet 2001;59:306-15. 54. Kim PC, Mo R, Hui CC. Murine models of VACTERL syndrome: Role of sonic hedgehog signaling pathway. J Pediatr Surg 2001;36: 381-4. 55. Liu J, Zhang L, Wang D, et al. Congenital diaphragmatic hernia, kidney agenesis and cardiac defects associated with Slit3-deficiency in mice. Mech Dev 2003;120:1059-70. 56. Yuan W, Rao Y, Babiuk RP, et al. A genetic model for a central (septum transversum) congenital diaphragmatic hernia in mice lacking Slit3. Proc Natl Acad Sci U S A 2003;100:5217-22. 57. Bleyl SB, Moshrefi A, Shaw GM, et al. Candidate genes for congenital diaphragmatic hernia from animal models: Sequencing of FOG2 and PDGFRalpha reveals rare variants in diaphragmatic hernia patients. Eur J Hum Genet 2007;15:950-8. 58. Keijzer R, van Tuyl M, Meijers C, et al. The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development. Development 2001;128:503-11. 59. Koutsourakis M, Keijzer R, Visser P, et al. Branching and differentiation defects in pulmonary epithelium with elevated Gata6 expression. Mech Dev 2001;105:105-14. 60. Yang H, Lu MM, Zhang L, et al. GATA6 regulates differentiation of distal lung epithelium. Development 2002;129:2233-46. 61. Ackerman KG, Wang J, Luo L, et al. Gata4 is necessary for normal pulmonary lobar development. Am J Respir Cell Mol Biol 2007;36: 391-7. 62. Takayasu H, Sato H, Sugimoto K, et al. Downregulation of GATA4 and GATA6 in the heart of rats with nitrofen-induced diaphragmatic hernia. J Pediatr Surg 2008;43:362-6. 63. Jesudason EC, Connell MG, Fernig DG, et al. Early lung malformations in congenital diaphragmatic hernia. J Pediatr Surg 2000;35: 124-7; discussion: 128. 64. Keijzer R, Liu J, Deimling J, et al. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia. Am J Pathol 2000;156:1299-306. 65. Babiuk RP, Greer JJ. Diaphragm defects occur in a CDH hernia model independently of myogenesis and lung formation. Am J Physiol Lung Cell Mol Physiol 2002;283:L1310-4. 66. Guilbert TW, Gebb SA, Shannon JM. Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development. Am J Physiol Lung Cell Mol Physiol 2000;279:L1159-71. 67. Sekine K, Ohuchi H, Fujiwara M, et al. Fgf10 is essential for limb and lung formation. Nat Genet 1999;21:138-41.