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The Effect of Mechanical Forces on In Utero Lung Growth in Congenital Diaphragmatic Hernia
CONGENITAL DIAPHRAGMATIC HERNIA
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THE EFFECT OF MECHANICAL FORCES ON IN UTERO LUNG GROWTH IN CONGENITAL DIAPHRAGMATIC HERNIA Kerilyn K. Nobuhara, MD, and Jay M. Wilson, MD, FACS, FAAP
Lung development is a complex process of lung growth and maturation. Although lung maturation appears to be principally regulated by hormonal and biochemical factors, lung growth appears to depend more on physical factors such as lung liquid volume, fetal breathing movements, the size of the intrathoracic space, and the amniotic fluid volume. This article focuses on these physical factors. Specific emphasis is given to the normal mechanisms responsible for lung growth, as well as the consequences of disruption of these normal mechanisms. FETAL BREATHING
Fetal breathing in utero contributes to the movement dynamics of lung liquid and is essential for normal lung growth. Although fetal breathing movements are identical to those of the neonate, owing to the high viscosity of fluid compared with air, changes in volume and pressure within the airway lumen are comparatively small, with pressure changes of 1 to 5 mm Hg and volume changes of less than 5 mL observed. In addition, fetal breathing appears to be episodic, occurring only 20 to 30 minutes out of each hour in late gestation. 9 During these episodes of fetal breathing, lung liquid efflux is increased. Conversely,
From the Department of Surgery, Children's Hospital of Boston and Harvard Medical School, Boston, Massachusetts
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during the intervening episodes of apnea, tracheal fluid efflux does occur but to a lesser degree. 12' 24 Experimental sectioning of the phrenic nerve, which eliminates fetal breathing movements, has been shown to decrease airway fluid volume, lung weight, and total DNA content.1 8 In addition to reducing lung liquid volume, chronic abolition of fetal breathing movements results in delayed lung development. The inhibition of effective fetal breathing movements by rib resection and bilateral thoracoplasty has been shown to create smaller lungs of lower DNA content and diminished compliance, suggesting impaired lung development. 38 Others have confirmed the impairment of lung growth at the histologic level. The lungs from animals subjected to chronic biphrenectomy remain in the saccular phase of lung development, and they do not progress to the alveolar phase that control animals do. 42 Spinal cord transection above the phrenic nucleus also produces less distensible lungs of lower DNA content. 37 The abolition of fetal breathing movements, however, is not the sole cause of delayed lung growth in these models. Physical factors may also contribute, because the denervated diaphragm rises, causing a reduction in thoracic size, thus potentially contributing to delayed lung growth. 32 In support of this is a study by Adzick et al, 3 which showed that in the fetal rabbit model of lung development, cervical cord transection to abolish fetal breathing movements led to pulmonary hypoplasia.
AMNIOTIC FLUID VOLUME
The proper volume of amniotic fluid has also been shown to be fundamental to normal lung development. Conditions that lead to oligohydramnios, including fetal renal agenesis, urinary tract obstruction, and prolonged rupture of membranes, are associated with pulmonary hypoplasia. 31 , 43 , 44, 49 , 57 The exact mechanism by which pulmonary hypoplasia occurs in the setting of oligohydramnios is unclear; however, reductions in both lung liquid volume and thoracic dimension, and, therefore, lung expansion, have been noted in experimental models of oligohydramnios. Experimental oligohydramnios reduces wet and dry lung weights in fetuses 20% to 25%, decreases lung liquid volumes by 65%, decreases lung liquid secretion by 35%, and reduces tracheal flow rates by 40%. 10 Significant but reversible reductions in both transverse thoracic dimension and distance between the manubrium and diaphragm occur within 48 hours of onset of oligohydramnios. 22 In addition, an increase in fetal spinal flexion is noted, with associated pressure increases in the fetal pleural space, intratracheal space, and abdomen. The increased intratracheal pressure accelerates lung liquid efflux, and the upward diaphragmatic displacement causes a further reduction in lung expansion. Therefore, if the oligohydramnios is prolonged, pulmonary hypoplasia results. 25 The postnatal evaluation of lambs exposed to oligohydramnios in utero reveals decreased chest wall compliance, an
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increased respiratory rate, a lower tidal volume, and mild hypercapnia, as compared with control animals. 33 INTRATHORACIC SIZE
Although the in utero maintenance of adequate lung liquid and amniotic volumes is fundamental to normal lung development, the presence of adequate intrathoracic space is also essential. Harrison et al28 • 29 showed that the insertion of an inflatable silicone rubber balloon into the left hemithorax of fetal lambs at 100 days' gestation produced fatal pulmonary hypoplasia. The balloons, which were progressively inflated, resulted in smaller lungs of decreased compliance. Barium gelatin injections revealed a decreased cross-sectional area of the pulmonary vascular bed. The histologic appearance of these lungs was not significantly different from in controls; however, a formal morphometric analysis was not performed. The reversible nature of the underlying pulmonary hypoplasia was shown by balloon deflation at 120 days' gestation, which resulted in an improved lung weight, air capacity, compliance, and cross-sectional area of the pulmonary vascular bed. 28 , 29 Experimental studies first confirmed the reversible nature of the pulmonary hypoplasia associated with CDH. Pringle et al52 created leftsided diaphragmatic hernias at 78 days' gestation. These defects were then repaired in utero at 106 to 124 days' gestation. In utero repair of the diaphragmatic hernia resulted in a more mature histologic appearance of the ipsilateral lung than that of animals without operative correction. In addition, improved pulmonary vascular morphometry has been reported after in utero repair of CDH. 4 LUNG LIQUID
There is now overwhelming evidence that fetal lung liquid volume must be maintained in a narrow range in order for normal lung growth to occur. It was initially believed that fetal lungs were collapsed throughout intrauterine life; however, it was subsequently recognized that fetal lungs were in fact expanded fluid-filled organs. Early investigators believed that lung liquid was derived from the amniotic space51 , 55; however, it is now clear that lung liquid is not derived from amniotic fluid but rather is actively secreted by the pulmonary epithelium,1, 2 , 16, 21 • 34• 45 , 54 58 46 , and that influx of amniotic fluid into the trachea is in fact a , so. 53 , very rare event.11, 17' 23 ' 40 The earliest gestational measurements of lung liquid secretion rate in the sheep fetus are 1.6 mL/kg/hr at 74 days' gestation. 47 This increases to 2.0 mL/kg/hr at 84 days. Secretion is maintained between 2 and 3 mL/kg/hr between 85 and 115 days of gestation. At 115 days' gestation, however, there is a sudden and significant increase in the rate of lung fluid production, with rates increasing to 5.5 mL/kg/hr between
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115 and 142 days. Just prior to delivery, lung liquid production precipitously drops back to 2 to 3 mL/kg/hr as the lungs make the transition from a secretory state to an absorptive state in preparation for gas exchange.7 Hormones associated with the stress of labor-namely, epinephrine and arginine vasopressin (AVP)-are known inhibitors of fetal lung liquid secretion.7, 32, 35, 4s, 59, 60 The beta-agonist isoproterenol has also been shown to cause reabsorption of pulmonary liquid in the term sheep fetus. 60 These hormones may act via a synergistic effect with triiodothyronine (T3) and cortisol. Changes in pulmonary epithelial solute permeability at the time of gas ventilation are believed to facilitate lung liquid reabsorption. 56 Intratracheal pressure (ITP) has been reported to be 0,75 to 1.0 mm Hg between 105 and 115 days' gestation, At 115 days' gestation, ITP doubles to 2.0 to 2.5 mm Hg and is maintained at that level until 130 days' gestation. Subsequent studies suggest that ITP is maintained at or above 2.0 mm Hg until term. In summary, therefore, it would appear that at 112 to 115 days' gestation, there is a significant change in fetal lung liquid dynamics, in that the rate of lung liquid production increases substantially, as does the intratracheal pressure, Because Docimo et al1 5 have shown that the greatest increase in lung volume and total alveolar number in fetal lambs occurs between 112 and 124 days' gestation, it is possible that lung growth occurs in response to the up-regulation of fetal lung liquid production, It appears that lung liquid is maintained within a narrow range of volume and pressure by the regulatory activity of the upper airway, This was demonstrated by Dixon and Harding12 in the now classic study in which lung liquid was extracted from fetal sheep, resulting in a spontaneous decrease in the efflux of lung liquid from the trachea to the amniotic fluid until lung liquid volume and intratracheal pressure had been restored to normal. Artificially increasing lung liquid volume by infusing saline resulted in an immediate increase in efflux of lung liquid, until again lung liquid volume and intratracheal pressure were normalized. Harding24 subsequently demonstrated that this regulatory activity was centered within the larynx itself. Sectioning of the recurrent laryngeal nerve as well as spontaneous fetal breathing movements (which resulted in decreases in laryngeal resistance), both led to an increase in the rate of lung liquid efflux. PERTURBATION OF LUNG LIQUID DYNAMICS
Disruptions of the carefully regulated lung liquid dynamics have been shown to dramatically alter lung growth. It has long been recognized that naturally occurring airway occlusions secondary to atresias or stenosis of the larynx, trachea, or bronchus have resulted in large fluid-filled lungs that were histologically normaL2°, zi, 4 o, 42 Experimental tracheal occlusion was first reported by Jost and Policard34 in 1948. In that study, fetal rabbits were decapitated at 19 days' gestation, which resulted in tracheal occlusion, At delivery 9 days later, the alveoli were
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histologically larger and more dilated than control lungs. A second group of animals, in which the trachea was left in open communication with the amniotic cavity, had small collapsed alveoli. Subsequently, Carmel8 ligated the tracheas of fetal rabbits in an attempt to ascertain whether amniotic fluid aspiration was essential for lung growth. Despite the ligation, however, the lungs on delivery were noted to be larger than in controls based on lung weight and lung volume to body weight ratios. Histologically, the lungs were noted to have "obvious thinning of the walls of the alveoli and terminal bronchioles with marked dilation of the structures." The conclusion of Carmel et al 8 was that "normal lung development occurs in the absence of amniotic fluid aspiration." Several additional studies in fetal lambs were performed that essentially confirmed the findings of the earlier studies in the fetal rabbit. 6• 36 Alcorn, 5 a in 1977, showed in fetal sheep that tracheal occlusion led to increased lung growth, whereas chronic drainage of lung liquid from the trachea led to pulmonary hypoplasia. Alcorn's study was subsequently confirmed by Fewell19 in 1983, who noted that tracheotomies in fetal lambs led to hypoplastic lungs. In 1984, Adzick3 demonstrated that tracheal ligation in fetal lambs could prevent pulmonary hypoplasia associated with experimental oligohydramnios. All studies to date, although demonstrating that lung liquid drainage leads to pulmonary hypoplasia and lung liquid retention leads to pulmonary hyperplasia, did not suggest a mechanism. In 1990, however, Moessinger 41 a in an elegant experiment chronically drained fetal lung liquid from the right lung while performing a bronchial ligation in the left lung. Analysis of wet and dry lung weights and DNA content suggested hypoplasia of the drained lung and hyperplasia of the ligated lung. From this study, the authors concluded that fetal lung cell multiplication was influenced by local distention of the pulmonary cells by retained lung liquid rather than by a humoral substance. Had a circulating humoral substance been responsible for the lung growth, the ligated left lung would be expected to have positively influenced the growth of the drained lung. FETAL LUNG LIQUID DYNAMICS AND CDH
An experiment of nature described by Potter et al 50 in 1941 provided the insight to utilize the disruption of normal lung liquid dynamics to prevent the pulmonary hypoplasia associated with CDH. An infant with a left-sided diaphragmatic hernia was noted on postmortem examination to have a left lower accessory lobe with no connection to the airway. The remainder of the left lung was hypoplastic, as would be expected with CDH; however, the sequestered left lobe was fluid-filled and expanded, with a normal histologic appearance. This observation suggested that the maintenance of fetal lung liquid volume, and therefore lung expansion in utero, would prevent, or perhaps even reverse, the pulmonary hypoplasia associated with CDH. Wigglesworth and Desai61 reported similar observations in three postmortem cases of laryngeal atresia. A marked increase in surface area and lung volume for age,
n alveolar number and advance in elastin te two cases of isolated laryngeal atresia. ;e of pulmonary hypoplasia secondary to a ume, was present in the third case. Because ;enesis and resulting retention of fetal lung •as found to have an alveolar number eight ts with renal agenesis of similar gestational ere of normal histologic appearance. lployed by Wilson62 to prevent the pulmowith fetal nephrectomy in fetal lambs, as ural and physiologic effects of pulmonary al nephrectomy is an established model of 1bly operating via a mechanism of reduced ; volume to body weight ratios were inover that of normal controls in animals l ligation or in utero tracheal ligation with 11, both tracheal ligation groups were found r total alveolar surface area. Total alveolar l the fetal nephrectomy I tracheal ligation e fetal nephrectomy, normal control, and A and protein contents were significantly
ipen via median sternotomy. Scissor passes through = heart; s = stomach; i = intestine; d = diaphragm; racheal ligation. B, DH/TL animal with chest open via ng has completely reduced the herniated viscera and ty. (From Difiore JW, Fauza DO, Slavin R, et al: reverses the structural and physiological effects of diaphragmatic hernia. J Pediatr Surg 29:248-257,
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higher in both groups subjected to tracheal ligation. The relatively constant DNA to protein content ratio suggested that growth occurred by cell proliferation, rather than by cellular hypertrophy. To assess the ability of tracheal ligation to reverse the pulmonary hypoplasia associated with CDH, a left-sided diaphragmatic hernia was created in utero at 90 days' gestation and tracheal ligation performed during the same operation in some animals. At delivery near term, animals subjected to the creation of diaphragmatic hernia alone were noted to have abdominal viscera in the left chest and hypoplastic lungs. In contrast, the group subjected to both tracheal ligation and creation of a diaphragmatic hernia were found to have the abdominal viscera reduced by the enlarged fluid-filled lung (Fig. 1). This group showed significant increases in lung volume to body weight ratio, alveolar surface area, and alveolar number. Histologically, the lungs of the diaphragmatic hernia/tracheal ligation (DH/TL) group appeared normal (Fig. 2). In addition, the airspace fraction and the alveolar numerical density remained normal. Again, the growth was attributed to cell proliferation,
Figure 2. A, Normal fetal lamb lung at 135 days' gestation. Note the thin alveolar septa and minimal amount of interstitial tissue. B, DH lamb lung at 135 days' gestation. When compared with A, alveolar walls are markedly thickened, interstitial tissue is markedly increased, and alveolar air space is markedly diminished. C, DHffl lamb lung at 135 days' gestation. Alveolar septa, interstitial tissue, and alveolar airspace have returned to normal. C = control. (toluidine blue stained, original magnification x 400). (From 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 29:248-257, 1994; with permission.)
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rather than cellular hypertrophy, by the elevated total lung DNA and protein contents, with maintenance of normal DNA to protein ratios. 13 After establishing that alveolar growth had been stimulated, the pulmonary vasculature was evaluated. Large vessel analysis was carried out by computer digital evaluation of angiogram lung slices, which showed that the total area of large vessels was increased in the DH/TL group, as compared with normal control animals, or the DH group (Fig. 3). The ratio of large vessel area to lung area was similar in all groups, however. Microscopic morphometric analysis revealed that the total number of capillaries was increased in the DH/TL lungs over both DH and control groups; however, the number of capillaries per alveolus was
Figure 3. Pulmonary arteriograms of fetal lamb lungs at delivery. A, Normal control; B, DH lung; C, DH/TL lung. Note the decrease in the overall size of the pulmonary vascular bed when DH lung is compared with control lung. In DH/TL lung, the size of the pulmonary vascular bed is increased over both DH and control lungs. (From DiFiore JW, Fauza DO, Slavin R, et al: Experimental fetal tracheal ligation and congenital diaphragmatic hernia: A pulmonary vascular morphometric analysis. J Pediatr Surg 30:917-924, 1995; with permission.)
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similar in all groups. In addition, the percentage of vessels less than 100 µmin diameter that were fully muscularized in the DH group was 29%, whereas no vessels less than 100 µm in diameter were muscularized in either the DH/TL or the control lungs. At the ultrastructural level, the capillary wall and capillary-alveolar interface appeared normal in the DH/TL group as opposed to the DH group 14 (Fig. 4). To evaluate pulmonary function, animals underwent CDH creation at 90 days' gestation and tracheal ligation at 110 days. This was done to avoid herniation of lung into the abdomen. These animals were ventilated via tracheostomy. The tracheal ligation group was noted to have a significantly higher Pao 2 , significantly lower Paco2, and significantly
Figure 4. A, Transmission electron micrograph of normal fetal lamb lung at 135 days' gestation (original magnification x 3400). Note thickened alveolar-capillary interface in B, with return to normal in the DHffL animal in C. Alv = alveolus; Cap = capillary; BM = basement membrane. (From DiFiore JW, Fauza DO, Slavin R, et al: Experimental fetal tracheal ligation and congenital diaphragmatic hernia: A pulmonary vascular morphometric analysis. J Pediatr Surg 30:917-924, 1995; with permission.)
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improved lung compliance, when compared with animals that were subjected to diaphragmatic hernia creation alone. 13 FUTURE DIRECTIONS
The reversal of pulmonary hypoplasia associated with CDH in the human has been attempted both in utero and in the postnatal period. Harrison et al27 • 30 reported the first successful in utero CDH repair in 1990. Although still limited by intractable labor and in utero fetal demise, techniques in antenatal diagnosis and fetal surgery continue to evolve. Currently, fetoscopic tracheal occlusion, with its subsequent increase in intratracheal pressure, appears to be the most promising of the fetal interventions (R. Jennings, personal communication). 26• 27• 30 The intratracheal instillation of perfluorocarbon in the postnatal period, as a means of reversing the pulmonary hypoplasia associated with CDH, is also now under investigation. References 1. Adams FH, Fujiwara T: Surfactant in fetal lamb tracheal fluid. J Pediatr 63:537-542, 1963 2. Adams FH, Fujiwara T, Rowshan G: The Nature and origin of the fluid in the fetal lamb lung. J Pediatr 63:881-888, 1963 3. Adzick NS, Harrison MR, Glick PL, et al: Experimental pulmonary hypoplasia and oligohydramnios: Relative contributions of lung fluid and fetal breathing movements. J Pediatr Surg 19:658-663, 1984 4. Adzick NS, Outwater KM, Harrison MR, et al: Correction of congenital diaphragmatic hernia in utero: IV. An early gestational fetal lamb model for pulmonary vascular morphometric analysis. J Pediatr Surg 20:673-680, 1985 5. Bealer JF, Skarsgard ED, Hedrick MH: The "PLUG" odyssey: Adventures in experimental fetal tracheal occlusion. J Pediatr Surg 30:361-365, 1995 5a. Alcorn D, Adamson TM, Lambert TF, et al: Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat 123:649-660, 1977 6. Berton JP: Effects de! la ligature de la trachee chez le foetus de mouton a la fine du premier et au deuxieme tiers de! la gestation: Accumulation de liquide dans les ramifications bronchique primitive et anasarque foeto-placentaire. Comptes Rendus de l' Association des Anatomistes 145:122-131, 1969 7. Brown MJ, Olver RE, Ramsden CA, et al: Effects of adrenaline and spontaneous labour on the secretion and absorption of lung liquid in the fetal lamb. J Physiol (Lond) 344:137-152, 1983 8. Carmel JA, Friedman F, Adams EH: Fetal tracheal ligation and lung development. Am J Dis Child 109:452-456, 1965 9. Dawes GS, Fox HE, Leduc BM, et al: Respiratory movements and rapid eye movement sleep in the foetal lamb. J Physiol 220:119-143, 1970 10. Dickson KA, Harding R: Decline in lung liquid volume and secretion rate during oligohydramnios in fetal sheep. J Appl Physiol 67:2401-2407, 1989 11. Dickson KA, Harding R: Restoration of lung liquid volume following its acute alteration in fetal sheep. J Physiol 385:531-543, 1987 12. Dickson KA, Maloney JE, Berger PJ: State-related changes in lung liquid secretion and tracheal flow rate in fetal lambs.J Appl Physiol 62:34-38, 1987 13. 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 29:248-257, 1994
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14. DiFiore JW, Fauza DO, Slavin R, et al: Experimental fetal tracheal ligation and congenital diaphragmatic hernia: A pulmonary vascular morphometric analysis. J Pediatr Surg 30:917-924, 1995 15. Docimo SG, Crone RI<, Davies P, et al: Pulmonary development in the fetal lamb: Morphometric study of the alveolar phase. Anat Rec 229:495-498, 1991 16. Enhorning G: Surface tension of amniotic fluid. Am J Obstet Gynecol 88:519, 1964 17. Fewell JE, Johnson P: Upper airway dynamics during breathing and during apnea in fetal lambs. J Physiol 339:495-504, 1983 18. Fewell JE, Lee C, Kitterman JA: Effects of phrenic nerve section on the respiratory system of fetal lambs. J Appl Physiol 51:293-297, 1981 19. Fewell JE, Hislop AA, Kitterman JA, et al: Effect of tracheostomy on lung development in fetal lambs. J Appl Physiol 55:1103-1108, 1983 20. Goodlin R, Lloyed D: Fetal tracheal excretion of bilirubin. Biol Neonate 12:1, 1968 21. Griscom NT, Harris C, Wohl ME, et al: Fluid-filled lung due to airway obstruction in the newborn. Pediatrics 43:383-390, 1969 22. Harding R, Liggins GC: The influence of oligohydramnios on thoracic dimensions of fetal sheep. J Dev Physiol 16:355-361, 1991 23. Harding R, Bocking AD Sigger JN: Influence of upper respiratory tract on liquid flow to and from fetal lungs. J Appl Physiol 61:68-74, 1986 24. Harding R, Sigger JN, Wickham PJD, Bocking AD: The regulation of flow of pulmonary fluid in fetal sheep. Respir Physiol 57:47-59, 1984 25. Harding R, Hooper SB, Dickson KA: A mechanism leading to reduced lung expansion and lung hypoplasia in fetal sheep during oligohydrarrmios. Am J Obstet Gynecol 163:1904-1913, 1990 26. Harrison MR, Adzick NS, Flake AW: Correction of congenital diaphragmatic hernia in utero: VI. Hard-earned lessons. J Pediatr Surg 28:1411-1418, 1993 27. Harrison MR, Adzick NS, Longaker MT: Successful repair in utero of a fetal diaphragmatic hernia after removal of herniated viscera from the left thorax. N Engl J Med 322:1582-1584, 1990 28. Harrison MR, Jester JA, Ross NA: Correction of congenital diaphragmatic hernia in utero. The model: Intra thoracic balloon produces fatal pulmonary hypoplasia. Surgery 88:174-182, 1980 29. Harrison MR, Bressack MA, Churg AM, et al: Correction of congenital diaphragmatic hernia in utero: II. Simulated correction permits fetal lung growth with survival at birth. Surgery 88:260-268, 1980 30. Harrison MR, Langer JC, Adzick NS: Correction of congenital diaphragmatic hernia in utero. V. Initial clinical experience. J Pediatr Surg 25:47-57, 1990 31. Hislop A, Hey E, Reid L: The lungs in congenital bilateral renal agenesis and dysplasia. Arch Dis Child 54:32-35, 1979 32. Hooper SB, Harding R: Fetal lung liquid: A major determinant of the growth and functional development of the fetal lung. Clin Exp Pharmacol Physiol 22:235-247, 1995 33. Jakubowska AE, Billings K, Dohns DP, et al: Respiratory function in lambs after prolonged oligohydramnios during late gestation. Pediatr Res 34:611-617, 1993 34. Jost PA, Policard A: Contribution experimentale a l'etude du developpement prenatal du poumon chez le lapin. Archives d'Anatomic Microscopique 37:323-332, 1948 35. Kindler PM, Chuang DC, Perks AM: Fluid production by in vitro lungs from nearterm fetal guinea pigs: Effects of cortisol and aldosterone. Acta Endorcrinologica 129:169-177, 1993 36. Lanman JT, Schaffer A, Herod L, et al: Distensibility of the fetal lung with fluid in sheep. Pediatr Res 5:586-590, 1971 37. Liggins GC, Vilas GA, Campos GA, et al: The effect of spinal cord transection on lung development in fetal sheep. J Dev Physiol 3:267-274, 1981 38. Liggins GC, Vilas GA, Campos GA, et al: The effect of bilateral thoracoplasty on lung development in fetal sheep. J Dev Physiol 3:275-282, 1981 39. MacGregor AR: Pathology of infancy and childhood. London, Churchill Livingstone, 1960, pp 172-173 40. Maloney JE, Adamson TM, Brodecky V, et al: Diaphragmatic activity and lung liquid flow in the unanesthetized fetal sheep. J Appl Physiol 39:423-428, 1975
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41. Milles G, Dorsey DB: Intra-uterine respiration-like movements in relation to development of the fetal vascular system: A discussion of intra-uterine physiology based upon cases of congenital absence of the trachea, abnormal vascular development and other anomalies. Am J Pathol 26:411, 1950 41a. Moessinger AC, Harding R, Adamson TM, et al: Role of lung fluid volume in growth and maturation of the fetal sheep lung. J Clin Invest 86:1270-1277, 1990 42. Nagai A, Thurlbeck WM, Jansen AH, et al: The effect of chronic biphrenectomy on lung growth and maturation in fetal lambs. Am Rev Respir Dis 137:167-172, 1988 43. Nicolini U, Fisk NM, Rodeck CH, et al: Low amniotic pressure in oligohydramnios: Is this the cause of pulmonary hypoplasia? Am J Obstet Gynecol 161:1098-1101, 1989 44. Nimrod C, Varela-Gittings F, Machin G, et al: The effect of very prolonged membrane rupture on fetal development. Am J Obstet Gynecol 148:540-543, 1984 45. Olver RE: Fetal lung liquids. Fed Proc 36:2669-2775, 1977 46. Olver RE, Strang LB: Ion fluxes across the pulmonary epithelium and the secretion of lung liquid in the fetal lamb. J Physiol (Lond) 241:327-357, 1974 47. Olver RE, Schneeberger EE, Walters DV: Epithelial solute permeability, ion transport and tight junction morphology in the developing lung of the fetal lamb. J Physiol 315:395-412, 1981 48. Perks AM, Cassin S: The effects of arginine vasopressin and other factors on the production of lung fluid in fetal goats. Chest 8l:S63-65, 1982 49. Potter EL: Bilateral renal agenesis. J Pediatr 29:68-76, 1946 50. Potter EL, Bohlender GP: Intrauterine respiration in relation to development of the fetal lung. Am J Obstet Gynecol 42:14-22, 1941 51. Preyer W: Specielle physiologie des embryo. Leipzig, Greeben, 1885, p 149 53. Pringle KC, Turner JW, Schofield JE, et al: Creation and repair of diaphragmatic hernia in the fetal lamb: Lung development and morphology. J Pediatr Surg 19:131140, 1984 54. Ross BB: Comparison of fetal pulmonary fluid with fetal plasma and amniotic fluid. Nature 199:1100, 1963 55. Setnikar I, Agostini E, Taglietti A: Fetal lung: Source of amniotic fluid. Soc Exp Biol Med 101:842, 1959 56. Snyder FF, Rosenfeld M: Direct observation of intrauterine respiratory movements and the role of carbon dioxide and oxygen in their regulation. Am J Physiol 119:153166, 1937 57. Strang LB: Fetal lung liquid: Secretion and reabsorption. Physiol Rev 71:991-1016, 1991 58. Thiebeault DW, Beatty EC, Hall RT, et al: Neonatal pulmonary hypoplasia with premature rupture of fetal membranes and oligohydramnios. J Pediatr 107:273-277, 1985 59. Vilos GA, Liggins GC: Intrathoracic pressures in fetal sheep. J Dev Physiol 4:247256, 1982 60. Wallace MJ, Hooper SB, Harding R: Regulation of lung liquid secretion by arginine vasopressin in fetal sheep. Am J Physiol 258:R104-lll, 1990 61. Walters DV, Olver RE: The role of catecholamines in lung liquid absorption at birth. Pediatr Res 12:239-242, 1978 62. Wigglesworth JS, Desai R, Hislop AA: Fetal lung growth in congenital laryngeal atresia. Pediatr Pathol 7:515-525, 1987 63. Wilson JM, DiFiore JW, Peters CA: Experimental fetal tracheal ligation prevents the pulmonary hypoplasia associated with fetal nephrectomy: Possible application for congenital diaphragmatic hernia. J Pediatr Surg 28:1433-1440, 1993
Address reprint requests to Jay M. Wilson, MD, FACS, FAAP Children's Hospital, Fegan 3 300 Longwood Avenue Boston, MA 02115 e-mail: [email protected]
0095-5108/96 $0.00 + .20
THE EFFECT OF MECHANICAL FORCES ON IN UTERO LUNG GROWTH IN CONGENITAL DIAPHRAGMATIC HERNIA Kerilyn K. Nobuhara, MD, and Jay M. Wilson, MD, FACS, FAAP
Lung development is a complex process of lung growth and maturation. Although lung maturation appears to be principally regulated by hormonal and biochemical factors, lung growth appears to depend more on physical factors such as lung liquid volume, fetal breathing movements, the size of the intrathoracic space, and the amniotic fluid volume. This article focuses on these physical factors. Specific emphasis is given to the normal mechanisms responsible for lung growth, as well as the consequences of disruption of these normal mechanisms. FETAL BREATHING
Fetal breathing in utero contributes to the movement dynamics of lung liquid and is essential for normal lung growth. Although fetal breathing movements are identical to those of the neonate, owing to the high viscosity of fluid compared with air, changes in volume and pressure within the airway lumen are comparatively small, with pressure changes of 1 to 5 mm Hg and volume changes of less than 5 mL observed. In addition, fetal breathing appears to be episodic, occurring only 20 to 30 minutes out of each hour in late gestation. 9 During these episodes of fetal breathing, lung liquid efflux is increased. Conversely,
From the Department of Surgery, Children's Hospital of Boston and Harvard Medical School, Boston, Massachusetts
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during the intervening episodes of apnea, tracheal fluid efflux does occur but to a lesser degree. 12' 24 Experimental sectioning of the phrenic nerve, which eliminates fetal breathing movements, has been shown to decrease airway fluid volume, lung weight, and total DNA content.1 8 In addition to reducing lung liquid volume, chronic abolition of fetal breathing movements results in delayed lung development. The inhibition of effective fetal breathing movements by rib resection and bilateral thoracoplasty has been shown to create smaller lungs of lower DNA content and diminished compliance, suggesting impaired lung development. 38 Others have confirmed the impairment of lung growth at the histologic level. The lungs from animals subjected to chronic biphrenectomy remain in the saccular phase of lung development, and they do not progress to the alveolar phase that control animals do. 42 Spinal cord transection above the phrenic nucleus also produces less distensible lungs of lower DNA content. 37 The abolition of fetal breathing movements, however, is not the sole cause of delayed lung growth in these models. Physical factors may also contribute, because the denervated diaphragm rises, causing a reduction in thoracic size, thus potentially contributing to delayed lung growth. 32 In support of this is a study by Adzick et al, 3 which showed that in the fetal rabbit model of lung development, cervical cord transection to abolish fetal breathing movements led to pulmonary hypoplasia.
AMNIOTIC FLUID VOLUME
The proper volume of amniotic fluid has also been shown to be fundamental to normal lung development. Conditions that lead to oligohydramnios, including fetal renal agenesis, urinary tract obstruction, and prolonged rupture of membranes, are associated with pulmonary hypoplasia. 31 , 43 , 44, 49 , 57 The exact mechanism by which pulmonary hypoplasia occurs in the setting of oligohydramnios is unclear; however, reductions in both lung liquid volume and thoracic dimension, and, therefore, lung expansion, have been noted in experimental models of oligohydramnios. Experimental oligohydramnios reduces wet and dry lung weights in fetuses 20% to 25%, decreases lung liquid volumes by 65%, decreases lung liquid secretion by 35%, and reduces tracheal flow rates by 40%. 10 Significant but reversible reductions in both transverse thoracic dimension and distance between the manubrium and diaphragm occur within 48 hours of onset of oligohydramnios. 22 In addition, an increase in fetal spinal flexion is noted, with associated pressure increases in the fetal pleural space, intratracheal space, and abdomen. The increased intratracheal pressure accelerates lung liquid efflux, and the upward diaphragmatic displacement causes a further reduction in lung expansion. Therefore, if the oligohydramnios is prolonged, pulmonary hypoplasia results. 25 The postnatal evaluation of lambs exposed to oligohydramnios in utero reveals decreased chest wall compliance, an
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increased respiratory rate, a lower tidal volume, and mild hypercapnia, as compared with control animals. 33 INTRATHORACIC SIZE
Although the in utero maintenance of adequate lung liquid and amniotic volumes is fundamental to normal lung development, the presence of adequate intrathoracic space is also essential. Harrison et al28 • 29 showed that the insertion of an inflatable silicone rubber balloon into the left hemithorax of fetal lambs at 100 days' gestation produced fatal pulmonary hypoplasia. The balloons, which were progressively inflated, resulted in smaller lungs of decreased compliance. Barium gelatin injections revealed a decreased cross-sectional area of the pulmonary vascular bed. The histologic appearance of these lungs was not significantly different from in controls; however, a formal morphometric analysis was not performed. The reversible nature of the underlying pulmonary hypoplasia was shown by balloon deflation at 120 days' gestation, which resulted in an improved lung weight, air capacity, compliance, and cross-sectional area of the pulmonary vascular bed. 28 , 29 Experimental studies first confirmed the reversible nature of the pulmonary hypoplasia associated with CDH. Pringle et al52 created leftsided diaphragmatic hernias at 78 days' gestation. These defects were then repaired in utero at 106 to 124 days' gestation. In utero repair of the diaphragmatic hernia resulted in a more mature histologic appearance of the ipsilateral lung than that of animals without operative correction. In addition, improved pulmonary vascular morphometry has been reported after in utero repair of CDH. 4 LUNG LIQUID
There is now overwhelming evidence that fetal lung liquid volume must be maintained in a narrow range in order for normal lung growth to occur. It was initially believed that fetal lungs were collapsed throughout intrauterine life; however, it was subsequently recognized that fetal lungs were in fact expanded fluid-filled organs. Early investigators believed that lung liquid was derived from the amniotic space51 , 55; however, it is now clear that lung liquid is not derived from amniotic fluid but rather is actively secreted by the pulmonary epithelium,1, 2 , 16, 21 • 34• 45 , 54 58 46 , and that influx of amniotic fluid into the trachea is in fact a , so. 53 , very rare event.11, 17' 23 ' 40 The earliest gestational measurements of lung liquid secretion rate in the sheep fetus are 1.6 mL/kg/hr at 74 days' gestation. 47 This increases to 2.0 mL/kg/hr at 84 days. Secretion is maintained between 2 and 3 mL/kg/hr between 85 and 115 days of gestation. At 115 days' gestation, however, there is a sudden and significant increase in the rate of lung fluid production, with rates increasing to 5.5 mL/kg/hr between
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115 and 142 days. Just prior to delivery, lung liquid production precipitously drops back to 2 to 3 mL/kg/hr as the lungs make the transition from a secretory state to an absorptive state in preparation for gas exchange.7 Hormones associated with the stress of labor-namely, epinephrine and arginine vasopressin (AVP)-are known inhibitors of fetal lung liquid secretion.7, 32, 35, 4s, 59, 60 The beta-agonist isoproterenol has also been shown to cause reabsorption of pulmonary liquid in the term sheep fetus. 60 These hormones may act via a synergistic effect with triiodothyronine (T3) and cortisol. Changes in pulmonary epithelial solute permeability at the time of gas ventilation are believed to facilitate lung liquid reabsorption. 56 Intratracheal pressure (ITP) has been reported to be 0,75 to 1.0 mm Hg between 105 and 115 days' gestation, At 115 days' gestation, ITP doubles to 2.0 to 2.5 mm Hg and is maintained at that level until 130 days' gestation. Subsequent studies suggest that ITP is maintained at or above 2.0 mm Hg until term. In summary, therefore, it would appear that at 112 to 115 days' gestation, there is a significant change in fetal lung liquid dynamics, in that the rate of lung liquid production increases substantially, as does the intratracheal pressure, Because Docimo et al1 5 have shown that the greatest increase in lung volume and total alveolar number in fetal lambs occurs between 112 and 124 days' gestation, it is possible that lung growth occurs in response to the up-regulation of fetal lung liquid production, It appears that lung liquid is maintained within a narrow range of volume and pressure by the regulatory activity of the upper airway, This was demonstrated by Dixon and Harding12 in the now classic study in which lung liquid was extracted from fetal sheep, resulting in a spontaneous decrease in the efflux of lung liquid from the trachea to the amniotic fluid until lung liquid volume and intratracheal pressure had been restored to normal. Artificially increasing lung liquid volume by infusing saline resulted in an immediate increase in efflux of lung liquid, until again lung liquid volume and intratracheal pressure were normalized. Harding24 subsequently demonstrated that this regulatory activity was centered within the larynx itself. Sectioning of the recurrent laryngeal nerve as well as spontaneous fetal breathing movements (which resulted in decreases in laryngeal resistance), both led to an increase in the rate of lung liquid efflux. PERTURBATION OF LUNG LIQUID DYNAMICS
Disruptions of the carefully regulated lung liquid dynamics have been shown to dramatically alter lung growth. It has long been recognized that naturally occurring airway occlusions secondary to atresias or stenosis of the larynx, trachea, or bronchus have resulted in large fluid-filled lungs that were histologically normaL2°, zi, 4 o, 42 Experimental tracheal occlusion was first reported by Jost and Policard34 in 1948. In that study, fetal rabbits were decapitated at 19 days' gestation, which resulted in tracheal occlusion, At delivery 9 days later, the alveoli were
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histologically larger and more dilated than control lungs. A second group of animals, in which the trachea was left in open communication with the amniotic cavity, had small collapsed alveoli. Subsequently, Carmel8 ligated the tracheas of fetal rabbits in an attempt to ascertain whether amniotic fluid aspiration was essential for lung growth. Despite the ligation, however, the lungs on delivery were noted to be larger than in controls based on lung weight and lung volume to body weight ratios. Histologically, the lungs were noted to have "obvious thinning of the walls of the alveoli and terminal bronchioles with marked dilation of the structures." The conclusion of Carmel et al 8 was that "normal lung development occurs in the absence of amniotic fluid aspiration." Several additional studies in fetal lambs were performed that essentially confirmed the findings of the earlier studies in the fetal rabbit. 6• 36 Alcorn, 5 a in 1977, showed in fetal sheep that tracheal occlusion led to increased lung growth, whereas chronic drainage of lung liquid from the trachea led to pulmonary hypoplasia. Alcorn's study was subsequently confirmed by Fewell19 in 1983, who noted that tracheotomies in fetal lambs led to hypoplastic lungs. In 1984, Adzick3 demonstrated that tracheal ligation in fetal lambs could prevent pulmonary hypoplasia associated with experimental oligohydramnios. All studies to date, although demonstrating that lung liquid drainage leads to pulmonary hypoplasia and lung liquid retention leads to pulmonary hyperplasia, did not suggest a mechanism. In 1990, however, Moessinger 41 a in an elegant experiment chronically drained fetal lung liquid from the right lung while performing a bronchial ligation in the left lung. Analysis of wet and dry lung weights and DNA content suggested hypoplasia of the drained lung and hyperplasia of the ligated lung. From this study, the authors concluded that fetal lung cell multiplication was influenced by local distention of the pulmonary cells by retained lung liquid rather than by a humoral substance. Had a circulating humoral substance been responsible for the lung growth, the ligated left lung would be expected to have positively influenced the growth of the drained lung. FETAL LUNG LIQUID DYNAMICS AND CDH
An experiment of nature described by Potter et al 50 in 1941 provided the insight to utilize the disruption of normal lung liquid dynamics to prevent the pulmonary hypoplasia associated with CDH. An infant with a left-sided diaphragmatic hernia was noted on postmortem examination to have a left lower accessory lobe with no connection to the airway. The remainder of the left lung was hypoplastic, as would be expected with CDH; however, the sequestered left lobe was fluid-filled and expanded, with a normal histologic appearance. This observation suggested that the maintenance of fetal lung liquid volume, and therefore lung expansion in utero, would prevent, or perhaps even reverse, the pulmonary hypoplasia associated with CDH. Wigglesworth and Desai61 reported similar observations in three postmortem cases of laryngeal atresia. A marked increase in surface area and lung volume for age,
n alveolar number and advance in elastin te two cases of isolated laryngeal atresia. ;e of pulmonary hypoplasia secondary to a ume, was present in the third case. Because ;enesis and resulting retention of fetal lung •as found to have an alveolar number eight ts with renal agenesis of similar gestational ere of normal histologic appearance. lployed by Wilson62 to prevent the pulmowith fetal nephrectomy in fetal lambs, as ural and physiologic effects of pulmonary al nephrectomy is an established model of 1bly operating via a mechanism of reduced ; volume to body weight ratios were inover that of normal controls in animals l ligation or in utero tracheal ligation with 11, both tracheal ligation groups were found r total alveolar surface area. Total alveolar l the fetal nephrectomy I tracheal ligation e fetal nephrectomy, normal control, and A and protein contents were significantly
ipen via median sternotomy. Scissor passes through = heart; s = stomach; i = intestine; d = diaphragm; racheal ligation. B, DH/TL animal with chest open via ng has completely reduced the herniated viscera and ty. (From Difiore JW, Fauza DO, Slavin R, et al: reverses the structural and physiological effects of diaphragmatic hernia. J Pediatr Surg 29:248-257,
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higher in both groups subjected to tracheal ligation. The relatively constant DNA to protein content ratio suggested that growth occurred by cell proliferation, rather than by cellular hypertrophy. To assess the ability of tracheal ligation to reverse the pulmonary hypoplasia associated with CDH, a left-sided diaphragmatic hernia was created in utero at 90 days' gestation and tracheal ligation performed during the same operation in some animals. At delivery near term, animals subjected to the creation of diaphragmatic hernia alone were noted to have abdominal viscera in the left chest and hypoplastic lungs. In contrast, the group subjected to both tracheal ligation and creation of a diaphragmatic hernia were found to have the abdominal viscera reduced by the enlarged fluid-filled lung (Fig. 1). This group showed significant increases in lung volume to body weight ratio, alveolar surface area, and alveolar number. Histologically, the lungs of the diaphragmatic hernia/tracheal ligation (DH/TL) group appeared normal (Fig. 2). In addition, the airspace fraction and the alveolar numerical density remained normal. Again, the growth was attributed to cell proliferation,
Figure 2. A, Normal fetal lamb lung at 135 days' gestation. Note the thin alveolar septa and minimal amount of interstitial tissue. B, DH lamb lung at 135 days' gestation. When compared with A, alveolar walls are markedly thickened, interstitial tissue is markedly increased, and alveolar air space is markedly diminished. C, DHffl lamb lung at 135 days' gestation. Alveolar septa, interstitial tissue, and alveolar airspace have returned to normal. C = control. (toluidine blue stained, original magnification x 400). (From 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 29:248-257, 1994; with permission.)
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rather than cellular hypertrophy, by the elevated total lung DNA and protein contents, with maintenance of normal DNA to protein ratios. 13 After establishing that alveolar growth had been stimulated, the pulmonary vasculature was evaluated. Large vessel analysis was carried out by computer digital evaluation of angiogram lung slices, which showed that the total area of large vessels was increased in the DH/TL group, as compared with normal control animals, or the DH group (Fig. 3). The ratio of large vessel area to lung area was similar in all groups, however. Microscopic morphometric analysis revealed that the total number of capillaries was increased in the DH/TL lungs over both DH and control groups; however, the number of capillaries per alveolus was
Figure 3. Pulmonary arteriograms of fetal lamb lungs at delivery. A, Normal control; B, DH lung; C, DH/TL lung. Note the decrease in the overall size of the pulmonary vascular bed when DH lung is compared with control lung. In DH/TL lung, the size of the pulmonary vascular bed is increased over both DH and control lungs. (From DiFiore JW, Fauza DO, Slavin R, et al: Experimental fetal tracheal ligation and congenital diaphragmatic hernia: A pulmonary vascular morphometric analysis. J Pediatr Surg 30:917-924, 1995; with permission.)
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similar in all groups. In addition, the percentage of vessels less than 100 µmin diameter that were fully muscularized in the DH group was 29%, whereas no vessels less than 100 µm in diameter were muscularized in either the DH/TL or the control lungs. At the ultrastructural level, the capillary wall and capillary-alveolar interface appeared normal in the DH/TL group as opposed to the DH group 14 (Fig. 4). To evaluate pulmonary function, animals underwent CDH creation at 90 days' gestation and tracheal ligation at 110 days. This was done to avoid herniation of lung into the abdomen. These animals were ventilated via tracheostomy. The tracheal ligation group was noted to have a significantly higher Pao 2 , significantly lower Paco2, and significantly
Figure 4. A, Transmission electron micrograph of normal fetal lamb lung at 135 days' gestation (original magnification x 3400). Note thickened alveolar-capillary interface in B, with return to normal in the DHffL animal in C. Alv = alveolus; Cap = capillary; BM = basement membrane. (From DiFiore JW, Fauza DO, Slavin R, et al: Experimental fetal tracheal ligation and congenital diaphragmatic hernia: A pulmonary vascular morphometric analysis. J Pediatr Surg 30:917-924, 1995; with permission.)
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improved lung compliance, when compared with animals that were subjected to diaphragmatic hernia creation alone. 13 FUTURE DIRECTIONS
The reversal of pulmonary hypoplasia associated with CDH in the human has been attempted both in utero and in the postnatal period. Harrison et al27 • 30 reported the first successful in utero CDH repair in 1990. Although still limited by intractable labor and in utero fetal demise, techniques in antenatal diagnosis and fetal surgery continue to evolve. Currently, fetoscopic tracheal occlusion, with its subsequent increase in intratracheal pressure, appears to be the most promising of the fetal interventions (R. Jennings, personal communication). 26• 27• 30 The intratracheal instillation of perfluorocarbon in the postnatal period, as a means of reversing the pulmonary hypoplasia associated with CDH, is also now under investigation. References 1. Adams FH, Fujiwara T: Surfactant in fetal lamb tracheal fluid. J Pediatr 63:537-542, 1963 2. Adams FH, Fujiwara T, Rowshan G: The Nature and origin of the fluid in the fetal lamb lung. J Pediatr 63:881-888, 1963 3. Adzick NS, Harrison MR, Glick PL, et al: Experimental pulmonary hypoplasia and oligohydramnios: Relative contributions of lung fluid and fetal breathing movements. J Pediatr Surg 19:658-663, 1984 4. Adzick NS, Outwater KM, Harrison MR, et al: Correction of congenital diaphragmatic hernia in utero: IV. An early gestational fetal lamb model for pulmonary vascular morphometric analysis. J Pediatr Surg 20:673-680, 1985 5. Bealer JF, Skarsgard ED, Hedrick MH: The "PLUG" odyssey: Adventures in experimental fetal tracheal occlusion. J Pediatr Surg 30:361-365, 1995 5a. Alcorn D, Adamson TM, Lambert TF, et al: Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat 123:649-660, 1977 6. Berton JP: Effects de! la ligature de la trachee chez le foetus de mouton a la fine du premier et au deuxieme tiers de! la gestation: Accumulation de liquide dans les ramifications bronchique primitive et anasarque foeto-placentaire. Comptes Rendus de l' Association des Anatomistes 145:122-131, 1969 7. Brown MJ, Olver RE, Ramsden CA, et al: Effects of adrenaline and spontaneous labour on the secretion and absorption of lung liquid in the fetal lamb. J Physiol (Lond) 344:137-152, 1983 8. Carmel JA, Friedman F, Adams EH: Fetal tracheal ligation and lung development. Am J Dis Child 109:452-456, 1965 9. Dawes GS, Fox HE, Leduc BM, et al: Respiratory movements and rapid eye movement sleep in the foetal lamb. J Physiol 220:119-143, 1970 10. Dickson KA, Harding R: Decline in lung liquid volume and secretion rate during oligohydramnios in fetal sheep. J Appl Physiol 67:2401-2407, 1989 11. Dickson KA, Harding R: Restoration of lung liquid volume following its acute alteration in fetal sheep. J Physiol 385:531-543, 1987 12. Dickson KA, Maloney JE, Berger PJ: State-related changes in lung liquid secretion and tracheal flow rate in fetal lambs.J Appl Physiol 62:34-38, 1987 13. 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 29:248-257, 1994
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14. DiFiore JW, Fauza DO, Slavin R, et al: Experimental fetal tracheal ligation and congenital diaphragmatic hernia: A pulmonary vascular morphometric analysis. J Pediatr Surg 30:917-924, 1995 15. Docimo SG, Crone RI<, Davies P, et al: Pulmonary development in the fetal lamb: Morphometric study of the alveolar phase. Anat Rec 229:495-498, 1991 16. Enhorning G: Surface tension of amniotic fluid. Am J Obstet Gynecol 88:519, 1964 17. Fewell JE, Johnson P: Upper airway dynamics during breathing and during apnea in fetal lambs. J Physiol 339:495-504, 1983 18. Fewell JE, Lee C, Kitterman JA: Effects of phrenic nerve section on the respiratory system of fetal lambs. J Appl Physiol 51:293-297, 1981 19. Fewell JE, Hislop AA, Kitterman JA, et al: Effect of tracheostomy on lung development in fetal lambs. J Appl Physiol 55:1103-1108, 1983 20. Goodlin R, Lloyed D: Fetal tracheal excretion of bilirubin. Biol Neonate 12:1, 1968 21. Griscom NT, Harris C, Wohl ME, et al: Fluid-filled lung due to airway obstruction in the newborn. Pediatrics 43:383-390, 1969 22. Harding R, Liggins GC: The influence of oligohydramnios on thoracic dimensions of fetal sheep. J Dev Physiol 16:355-361, 1991 23. Harding R, Bocking AD Sigger JN: Influence of upper respiratory tract on liquid flow to and from fetal lungs. J Appl Physiol 61:68-74, 1986 24. Harding R, Sigger JN, Wickham PJD, Bocking AD: The regulation of flow of pulmonary fluid in fetal sheep. Respir Physiol 57:47-59, 1984 25. Harding R, Hooper SB, Dickson KA: A mechanism leading to reduced lung expansion and lung hypoplasia in fetal sheep during oligohydrarrmios. Am J Obstet Gynecol 163:1904-1913, 1990 26. Harrison MR, Adzick NS, Flake AW: Correction of congenital diaphragmatic hernia in utero: VI. Hard-earned lessons. J Pediatr Surg 28:1411-1418, 1993 27. Harrison MR, Adzick NS, Longaker MT: Successful repair in utero of a fetal diaphragmatic hernia after removal of herniated viscera from the left thorax. N Engl J Med 322:1582-1584, 1990 28. Harrison MR, Jester JA, Ross NA: Correction of congenital diaphragmatic hernia in utero. The model: Intra thoracic balloon produces fatal pulmonary hypoplasia. Surgery 88:174-182, 1980 29. Harrison MR, Bressack MA, Churg AM, et al: Correction of congenital diaphragmatic hernia in utero: II. Simulated correction permits fetal lung growth with survival at birth. Surgery 88:260-268, 1980 30. Harrison MR, Langer JC, Adzick NS: Correction of congenital diaphragmatic hernia in utero. V. Initial clinical experience. J Pediatr Surg 25:47-57, 1990 31. Hislop A, Hey E, Reid L: The lungs in congenital bilateral renal agenesis and dysplasia. Arch Dis Child 54:32-35, 1979 32. Hooper SB, Harding R: Fetal lung liquid: A major determinant of the growth and functional development of the fetal lung. Clin Exp Pharmacol Physiol 22:235-247, 1995 33. Jakubowska AE, Billings K, Dohns DP, et al: Respiratory function in lambs after prolonged oligohydramnios during late gestation. Pediatr Res 34:611-617, 1993 34. Jost PA, Policard A: Contribution experimentale a l'etude du developpement prenatal du poumon chez le lapin. Archives d'Anatomic Microscopique 37:323-332, 1948 35. Kindler PM, Chuang DC, Perks AM: Fluid production by in vitro lungs from nearterm fetal guinea pigs: Effects of cortisol and aldosterone. Acta Endorcrinologica 129:169-177, 1993 36. Lanman JT, Schaffer A, Herod L, et al: Distensibility of the fetal lung with fluid in sheep. Pediatr Res 5:586-590, 1971 37. Liggins GC, Vilas GA, Campos GA, et al: The effect of spinal cord transection on lung development in fetal sheep. J Dev Physiol 3:267-274, 1981 38. Liggins GC, Vilas GA, Campos GA, et al: The effect of bilateral thoracoplasty on lung development in fetal sheep. J Dev Physiol 3:275-282, 1981 39. MacGregor AR: Pathology of infancy and childhood. London, Churchill Livingstone, 1960, pp 172-173 40. Maloney JE, Adamson TM, Brodecky V, et al: Diaphragmatic activity and lung liquid flow in the unanesthetized fetal sheep. J Appl Physiol 39:423-428, 1975
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41. Milles G, Dorsey DB: Intra-uterine respiration-like movements in relation to development of the fetal vascular system: A discussion of intra-uterine physiology based upon cases of congenital absence of the trachea, abnormal vascular development and other anomalies. Am J Pathol 26:411, 1950 41a. Moessinger AC, Harding R, Adamson TM, et al: Role of lung fluid volume in growth and maturation of the fetal sheep lung. J Clin Invest 86:1270-1277, 1990 42. Nagai A, Thurlbeck WM, Jansen AH, et al: The effect of chronic biphrenectomy on lung growth and maturation in fetal lambs. Am Rev Respir Dis 137:167-172, 1988 43. Nicolini U, Fisk NM, Rodeck CH, et al: Low amniotic pressure in oligohydramnios: Is this the cause of pulmonary hypoplasia? Am J Obstet Gynecol 161:1098-1101, 1989 44. Nimrod C, Varela-Gittings F, Machin G, et al: The effect of very prolonged membrane rupture on fetal development. Am J Obstet Gynecol 148:540-543, 1984 45. Olver RE: Fetal lung liquids. Fed Proc 36:2669-2775, 1977 46. Olver RE, Strang LB: Ion fluxes across the pulmonary epithelium and the secretion of lung liquid in the fetal lamb. J Physiol (Lond) 241:327-357, 1974 47. Olver RE, Schneeberger EE, Walters DV: Epithelial solute permeability, ion transport and tight junction morphology in the developing lung of the fetal lamb. J Physiol 315:395-412, 1981 48. Perks AM, Cassin S: The effects of arginine vasopressin and other factors on the production of lung fluid in fetal goats. Chest 8l:S63-65, 1982 49. Potter EL: Bilateral renal agenesis. J Pediatr 29:68-76, 1946 50. Potter EL, Bohlender GP: Intrauterine respiration in relation to development of the fetal lung. Am J Obstet Gynecol 42:14-22, 1941 51. Preyer W: Specielle physiologie des embryo. Leipzig, Greeben, 1885, p 149 53. Pringle KC, Turner JW, Schofield JE, et al: Creation and repair of diaphragmatic hernia in the fetal lamb: Lung development and morphology. J Pediatr Surg 19:131140, 1984 54. Ross BB: Comparison of fetal pulmonary fluid with fetal plasma and amniotic fluid. Nature 199:1100, 1963 55. Setnikar I, Agostini E, Taglietti A: Fetal lung: Source of amniotic fluid. Soc Exp Biol Med 101:842, 1959 56. Snyder FF, Rosenfeld M: Direct observation of intrauterine respiratory movements and the role of carbon dioxide and oxygen in their regulation. Am J Physiol 119:153166, 1937 57. Strang LB: Fetal lung liquid: Secretion and reabsorption. Physiol Rev 71:991-1016, 1991 58. Thiebeault DW, Beatty EC, Hall RT, et al: Neonatal pulmonary hypoplasia with premature rupture of fetal membranes and oligohydramnios. J Pediatr 107:273-277, 1985 59. Vilos GA, Liggins GC: Intrathoracic pressures in fetal sheep. J Dev Physiol 4:247256, 1982 60. Wallace MJ, Hooper SB, Harding R: Regulation of lung liquid secretion by arginine vasopressin in fetal sheep. Am J Physiol 258:R104-lll, 1990 61. Walters DV, Olver RE: The role of catecholamines in lung liquid absorption at birth. Pediatr Res 12:239-242, 1978 62. Wigglesworth JS, Desai R, Hislop AA: Fetal lung growth in congenital laryngeal atresia. Pediatr Pathol 7:515-525, 1987 63. Wilson JM, DiFiore JW, Peters CA: Experimental fetal tracheal ligation prevents the pulmonary hypoplasia associated with fetal nephrectomy: Possible application for congenital diaphragmatic hernia. J Pediatr Surg 28:1433-1440, 1993
Address reprint requests to Jay M. Wilson, MD, FACS, FAAP Children's Hospital, Fegan 3 300 Longwood Avenue Boston, MA 02115 e-mail: [email protected]