VISITING GUEST ADDRESS
Congenital
Diaphragmatic
Hernia, What Defect?
By Jan C. Molenaar, Albert P. Bos, Frans W.J. Hazebroek, and Dick Tibboel Rotterdam,
The Netherlands
F
ROM THE DAYS WHEN congenital diaphragmatic hernia (CDH) was first described and treated surgically, it has been interpreted as a purely anatomical defect of the diaphragm, and only occasionally associated with other congenital anatomical defects. Its early repair, preferably before respiratory problems occurred, determined the outcome. Today, there is growing awareness that CDH might be part of a more basic defect afflicting many organs, the defect in the diaphragm probably being the least important. There is a high incidence of other anomalies associated with CDH. Puri and German’ reported a series of 47 cases of CDH among 99,062 births at the National Maternity Hospital in Dublin. There were 15 stillbirths, all showing lethal, nonpulmonary anomalies. Of the 32 liveborn patients, 17 died prior to transfer to the referral pediatric surgical center, 11 of whom showed major nonpulmonary anomalies. In the remaining 15 liveborns, CDH was the only congenital defect, except in the case of one patient with a concomitant heart defect. Very little is known about the etiology and pathogenesis of these serious, often lethal, anatomical anomalies connected with CDH. When fetal surgery for prenatal treatment of CDH is considered, a watertight prenatal diagnosis should be available to prevent the fetal surgeon from closing the defect of a diaphragm in a fetus in whom the anomaly is multifocal. But even in the absence of nonpulmonary anomalies, the survival rate of CDH is very low, and pulmonary hypoplasia and persistent pulmonary hypertension are probably responsible for this. It took considerable time before pediatric surgeons recognized the true nature of the problem. In 1929, Greenwald and Steiner’ published a report on diaphragmatic hernia in infancy and in childhood.
From the Department of Pediatric Surgery, Sophia Children’s Hospital, University Hospital Erasmus University Medical School, Rotterdam, The Netherlands. Visiting Guest Address to the 2lst Annual Meeting of the American Pediatric Surgical Association, Vancouver, British Columbia, May 19-22, 1990. Address reprint requests to .I.C. Molenaar, MD, PhD, Professor of Pediatric Sutgety, Sophia Children’s Hospital, PO Box 70029, 3000 LL, Rofterdam, The Netherlands. Copyright 0 1991 by WB. Saunders Company 0022-3468/91/2603-0002$03.0010
246
They stated: “For the patient in whom the hernia makes its appearance at birth, little or nothing can be done from a surgical standpoint.” However, they emphasized that “for infants several months old, if symptoms are present, it is advisable not to defer surgical treatment because of the danger of increase in the amount of herniated viscus as time goes on.” In 1940, Ladd and Gross3 reported 16 children undergoing operation, with nine survivors. Excluding the hernia of the esophageal hiatus, there were seven survivors, five with a left-sided and two with a right-sided CDH. Ladd and Gross related the poor results to the delay in surgery, and advocated operation immediately after birth. In 1953, Gross4 reported 63 cases of Bochdalek hernia, 53 on the left side and 10 on the right side, with 55 survivors. He emphasized that “surgical therapy is highly satisfactory regardless of the extent of the herniation and the apparent critical condition of the child,” and added “those babies or children should be given operative relief. . . even if asymptomatic, because this is the best way to avoid subsequent complications in the way of intestinal obstruction or incarceration.” But in 1964, Gross’ looked back at experiences of the Boston Children’s Hospital and found it disturbing that over the years the cure rates had dropped. This could be explained by a very much higher proportion of babies less than 24 hours of age coming for treatment. However, the mortality rate did not show any improvement in this category. In 1969, Young6 reported that since 1953 the number of babies admitted to the Hospital for Sick Children, London, within 24 hours of birth had multiplied six times. He showed an inverse relationship between the age on admission and mortality, and concluded that “it is this group which offers the greatest scope for improvement in management” (Table 1). However, over the years improvement did not occur, neither in Great Ormond Street nor in other institutions. When we examine the numbers at this institution, we see no improvement in mortality, and only prolonged artificial ventilation and an increase in hospital stay (Figs 1 and 2). In order to identity the highest risk patients and to treat these more vigorously before the operation, JournalofPediatricSurgety,
Vol26, No 3 (March), 1991:
pp 248-254
CONGENITAL
DIAPHRAGMATIC
249
HERNIA
Table 1. Relationship Between Age at Admission and Mortality of
70-
100 Infants Treated at the Hospital for Sick Children, London, 60.
1951 to 1967 No. of Age on Admission
Patients
MortaliQ
O-24 h
31
19 (61%)
24-48 h
23
5 (22%)
48-72 h
7
2 (29%)
19
3 (16%)
72 h-l mo 1 mo-1 yr
20 100
Total
54
artificial ventilation
hOSDita1 stav
1 (5%) 30
Reprinted with permission.’
scoring systems for risk assessment of newborns with CDH have been developed.‘,’ Arterial blood gas determinations as criteria for survival were introduced in the early seventies. Touloukian and Markowitz’s preoperative scoring system gained much popularity. They related the position of the stomach to mortality.’ The increasing number of neonates suffering from CDH admitted immediately after birth in poor condition, and the observation that emergency repair was often followed by deterioration rather than improvement, stirred neonatologists, anesthetists, and pediatric surgeons to question the doctrine of emergency operation introduced in the near past by Ladd, Gross, and others, and still advocated in the most recent textbooks on pediatric surgery.” More sophisticated ventilatory parameters, such as the difference between alveolar and arterial oxygen pressure, the mean airway pressure, the oxygenation index, and the ventilation index, became more helpful as predictors of mortality in developing a good strategy for treatment. Since 1986 we have been using these in the Sophia Children’s Hospital in a study protocol aimed at improvement of the preoperative condition by delaying surgery. During this period of 50
1 I
bortalitv
1970-1979
1980-1985
1986-1989
Fig 1. Number of emergency patients and number of deaths of CDH (Sophie Children’s Hospital, 1970 to 1989).
Fig 2. Median duration (days) of artificial ventilation and hospital stay (Sophia Children’s Hospital, 1970 to 1989).
delay we try to improve the condition of the baby by providing it all facilities for intensive care available. Only when maximal treatment and thorough evaluation result in stabilization do we perform surgical correction. During the preoperative period of delay and after surgical correction, various ventilatory parameters are registered at regular intervals. In 1988,” we reported our experiences since 1986 and concluded that (1) satisfactory ventilation parameters on admission will remain good during the preoperative stabilization phase and will not be affected by its duration or by subsequent surgery, spelling survival; (2) unsatisfactory ventilation parameters on admission may improve with preoperative stabilization, giving these patients a better chance of survival; and (3) poor ventilation parameters on admission that fail to improve with preoperative stabilization will not improve with surgery or postoperatively, spelling death. Our results show that 16 of 31 patients did not survive (52%). Nine of these deteriorated progressively despite maximal conservative treatment and were no longer given the benefit of the doubt of surgical treatment. Could we have saved some of those who died by other forms of treatment either preoperatively or postoperatively? Could we have improved and stabilized the nine patients who deteriorated and died, and for whom operation was considered not to make any sense? Ten of the 16 nonsurvivors met the criteria accepted by the Extracorporeal Life Support Organization” and would have been allowed to enroll in an extracorporeal membrane oxygenation (ECMO) program. If we consider a best arterial PO, of 100 mm Hg or higher as optimal for capability of oxygen diffusion, then at least six patients were definitely fit for ECMO
250
and could have survived. Four of the nine patients who deteriorated and died before surgery could have been undertaken were fit for ECMO. Three even showed best PO, of 100 mm Hg and higher, allowing them to enter an ECMO protocol with good chances of survival. In conclusion, there is evidence that, at least in this hospital, the chances of survival in patients with CDH could be greatly improved with the application of ECMO, provided that strict criteria are applied. At present we are planning the introduction of ECMO in this hospital. However, we must bear in mind that from the 15 survivors without ECMO, two met the criteria for ECMO and would have undergone ECMO and its possible complications, of which massive intracranial bleeding is the worst. The debate on the benefits of ECMO focusses on two main causes of death in these patients: pulmonary hypoplasia and persistent pulmonary hypertension. In 195.5, Campanale and RowlandI called attention to the fact that pathologists like Hunter in 1928, Liebow and Miller in 1940, and Potter in 1952 have been calling attention to pulmonary hypoplasia as the principal cause of mortality of newborns dying from CDH. In a personal communication, Dr Gross informed Campanale and Rowland that he could not recall noting at operation a single instance of definite hypoplasia of the lung, and felt that it must be very rare, or does not occur. And even in his presidential address of 1964,’ Gross states that in many instances expansion of the lung can be accomplished in a few hours, but that in others it might require several days. Nevertheless, Gross remarks, “this process should not be distributed by or hastened by vigorous inflation by the anesthetist nor by vigorous aspiration.” Campanale and Rowland suggest that “the explanation for this totally different experience of the pathologist and surgeon is to be found in the rapid demise of infants with this association of defects.“13 They quote Tolins, who stated in a review of the literature in 1953,14that the youngest infant with CDH to undergo surgery was operated on by Gross at 22 hours of age. Tolins himself reported a patient undergoing operation at the age of 22 hours. He tried to treat the persistent pneumothorax in the postoperative period by repeated thoracentesis and aspiration, but without success. Using suction initially, it took a month before the 50% residual pneumothorax finally diminished. Tolins concluded that this experience bears out the idea of a continuous development from hypoplasia to normal lung. However, several long-term function studies show that although these patients are doing well and
MOLENAAR
ET AL
function normally, the majority, their lung parameters being studied, show a 20% to 30% reduction in lung function when compared with norma1.‘5 Others pointed to a definite risk of the development of lung emphysema, as shown in a histological study by Thurlbeck et a1,16who describe a boy who had undergone operation as a newborn in the Hospital for Sick Children in Toronto for left-sided CDH, but died because of an accident at the age of 5. The study shows normal histology in the right lung, but parts of destructive emphysema in the left lung. To be able to study these late effects, sufficient experimental studies are needed. As early as 1963, Areechon and Reid” stated that “beyond the occasional report that within a few months of operation the radiograph may appear ‘normal,’ little is known of the long-term behaviour of these lungs.” They also said “for an indication of the possible behaviour of these lungs it is necessary to turn to animal experiments which point the importance of the space available to the lung for normal growth as well as growth compensatory to disease.” Areechon and Reid conclude that: (1) in hypoplastic lungs the number of terminal bronchioli and the volume of alveoli are reduced; (2) it would be expected that no correction of the bronchial and bronchiolar deficiency will occur; but that (3) growth within the acini will be great enough to provide the lung with its normal complement of alveoli. These findings were confirmed by Kitagawa et al.” They found not only a reduction in the number of bronchial branches and the number of alveoli, but also a pathological excessive muscularization of preacinar arteries. And Geggel et alI9 concluded that in infants who die after surgical correction of CDH with persistent hypoxemia, prealveolar and intraalveolar arteries have a reduced external diameter or increased medial thickness resulting in a decreased luminal area of these arteries. In other words, pulmonary hypertension in these infants is caused by (1) decreased vascular lumen, and (2) vasoconstriction of the entirely muscularized vascular tree leading to increased pulmonary vascular resistance. But our knowledge of lung morphology in patients with CDH is still very limited. Even more limited is our knowledge of the lung as an endocrine organ, where so many factors, such as innervation, local tumoral mediators, and artificial ventilation, influence the pulmonary circulation and lung function. We studied the effect of surgery on blood levels of thromboxane.20 This local chemical mediator is active in the lung with the smooth muscle cells of the
CONGENITAL
DIAPHRAGMATIC
HERNIA
251
pulmonary vascular tree as their target cells causing vasoconstriction. Figure 3 shows a definite increase of thromboxane levels after surgery for repair of the defect in the diaphragm, going back to normal in most patients. We compared this effect of surgery in patients with CDH with other newborns undergoing operation for other congenital disorders. In these patients surgery did not have this effect (Fig 4). Therefore, the increase of thromboxane levels after surgery might be specific for CDH patients and could explain the further deterioration of these patients after surgery due to triggering of pulmonary hypertension. For a better insight into these phenomena, more experimental animal research is required. In 1967, deLorimier et alzl followed the advice of Areechon and Reid to study the hypoplasia of lung with CDH in experimental animals. They induced CDH in fetal lambs. At term their lungs were hypoplastic, showing a 23% to 75% reduction in lung weight and air capacity compared with normal lungs. Subsequently, a number of other researchers created experimental animal models to study the effects of CDH. What all these experiments have in common is the need for surgical intervention in a relatively late stage of lung development. Only Adzick et al**interfered with pulmonary development early in gestation. Another prospect is to use the experience gained in teratology. Several studies have shown the teratological effects of Nitrofen. In a study in rats, Iritan? used these well-known effects of the administration of Nitrofen to pregnant rats. This herbicide causes CDH and there is a direct relationship between the dose and the incidence of CDH. In studies in this laboratory, CDH was induced in newborn Sprague-Dawley rats by giving 115 mg/kg body weight Nitrofen in a single dose through a gastric tube to the pregnant mother at day 10 of gestation.24 Control animals were given the solvent
d
- lh
operltive procedure
pmol/ml
,
01 -
lh
I
operative procedure
+ lh
Fig 4. The effect of surgery on the development of thromboxane levels in CDH patients (solid line) and controls (broken line) at Sophia Children’s Hospital.
(olive oil) only. Not all newborn rats presented with CDH, but they were all studied after birth. The majority of newborn rats presenting with CDH showed serious respiratory insufficiency. After cesarean section or spontaneous birth, all newborn rats were evaluated for body weight, the presence of a defect in the diaphragm, its position, the nature of intrathoracic organs, and wet lung weight. A right:left ratio of 8:l was noticed, which is the opposite of the ratio in humans. Figure 5 shows the diaphragmatic defect with liver and bowel loops protuding into the thoracic cavity. Often only the liver was present, compressing the
I
+ lh
Fig 3. The effect of surgery on the development levels (Sophia Children’s Hospital).
*ml
of thromboxane
Fig 5. Diaphragmatic defect in rat. Bowel loops (EL) and liver lobe (LL) are closely adjacent to lung (L).
252
developing lung. On gross examination the lung at the defected side was definitely smaller in all affected newborns. Wet lung weights of both lungs were reduced in Nitrofen rats with or without a diaphragmatic defect, those in rats with the defect being lowest. Body weight was also reduced. Lung weight/body weights ratios, both prepartum and postpartum, showed a reduction in all Nitrofen rats without or with a diaphragmatic defect, those in rats with a defect being lowest. To prove pulmonary hypoplasia, the number of aIveoli should also be reduced. In 1960, Eme$ introduced the radial alveolar count, a special technique to assess the alveolar space in lungs. We found a definite reduction in newborn rats effected by Nitrofen, with or without a defect in the diaphragm. But the reduction was largest in rats with a congenital diaphragmatic defect. In other words, we were able to induce CDH early in gestation, resulting in pulmonary hypoplasia resembling that in the human situation. To evaluate the pulmonary vessels the pulmonary arterial trunk was perfused with a heated bariumgelatine solution under constant pressure.26 This method facilitates the identification of the smallest branches of the pulmonary vascular tree and their accompanying airways as far as the alveoIar waI1. After identification of the artery and its accompanying airway, the medial wall thickness and external diameter are measured. The muscularity of the arteries was assessed in three ways: (1) completely muscular, when the artery was completely surrounded by a muscular coat; (2) partially muscular when this was only partial; and (3) nonmuscular. At the level of the conducting airways the external diameter in CDH rats was smaller than that in controls. More peripherally, at the level of the terminal bronchioli, there was no difference. To evaluate the cross-sectional vascular lumen, we calculated the percentage of wall thickness. The animals with CDH showed pulmonary arteries at all levels with an increased wall thickness resulting in a reduced vessel lumen. This is in full agreement with what is found in humans with CDH. At the level of the conducting airways we found an increased muscularization of the vessel walls. However, we also found increased muscularization more peripherally at the level of the terminal bronchi. As in humans, the animal model shows serious lung hypoplasia, with a lower air space capacity, a reduced vascular space, and a pathological extension of muscularization into the peripheral pulmonary arteries. These findings stirred us to ask more questions. Fortunately, much basic research has been done in
MOLENAAR ET AL
80
%@J m g
k40 20
0
m lam-1300
Fig 6. Incidence of BPD related to birth weight in premature infants (data from BancalarP) and CDH survivors (Sophia Children’s Hospital, 1980 to 1989).
the normal rat. This provides sufficiently methodology to start further studies using this experimental model. It is common experience that the artificially ventilated hypoplastic lung is very susceptible to bronchopulmonary dysplasia (BPD). A retrospective study of 105 patients treated for CDH over the last 20 years showed an increase in artificial ventilation time without any decrease in the mortality rate (Fig 2). Bancalari and Gerhardt*’ defined BPD as a dependency on oxygen for more than 28 days following mechanical ventilation during the first week of life, in combination with persistent increased densities on chest radiographs. Comparing the CDH survivors at this institution over the last 10 years with the premature infants included in Bancalari’s series2’ we find an astonishing number of patients with BPD resulting in nearly 40% of all survivors (Fig 6). The development of BPD is related to tissue damage by oxygen free radicals and barotrauma. The defense mechanism against pulmonary oxygen toxicity is based on the formation of antioxydant enzyme activity.” To study this mechanism in hypoplastic lungs, newborn rats with CDH were ventilated after pregnancies interfered with Nitrofen, and compared
_L
I
*
1
Fig 7. Reaction of CuZn ouperoxidedismutase in control rats (white box) and CDH rats (black box) after artificial ventilation for 5 hours. lP < .05.
CONGENITAL
DIAPHRAGMATIC
253
HERNIA
T
Fig 8. Reaction of glutathione peroxidase in control rats (white box) and CDH rats (black box) after artificial ventilation for 5 hours. ‘P < .05.
Fig 10. (controls, rats).
with normal control newborn rats. In this laboratory we are able to ventilate newborn rats with a modified Servo ventilator for at least 18 hours. For lung compliance studies a heated body plethysmograph is used. We studied the effects of artificial ventilation on the production of enzymatic antioxidants in the lung. The developmental pattern during gestation of CuZn superoxidedismutase, catalase, and glutathione peroxidase, was equal in all rats. We found that lungs of newborn rats with CDH were unable to produce normal lung antioxidant enzyme activity in response to either exposure to normal air ventilation or exposure to pure oxygen. This could be shown for the enzyme CuZn superoxidedismutase and also for glutathione peroxidase (Figs 7 and 8). In another study we found that lungs of newborn rats showing hypoplasia due to Nitrofen, both with and without a diaphragmatic defect, have a lower compliance (Fig 9) and need a higher pressure to open them (Fig 10). These effects might be related to insufficient surfactant production in affected lungs. From these studies it may be concluded that a defective antiolcydant system and defective surfactant
production in hypoplastic lungs are probably related to the development of BPD during artificial ventilation. We did not discuss the exponential growth in knowledge and its possible significance for CDH patients of the endothelium in modulating the tone of the underlying smooth muscle in response to pharmacological agents, physiological stimuli, and disease.3” In 1989, Adzick et a131published the experiences of the Boston Children’s Hospital with the clinical outcome in 38 consecutive cases with CDH, diagnosed in utero. Again, a high incidence of associated lethal anomalies was noted (Fig 11). Only nine of the 27 potentially salvageable patients survived, three of whom needed ECMO. Because of the high percentage of nonsurvivors, the authors suggest early fetal surgical repair. This has to be done very early, because we know that by the 16th week the embryonic bronchial tree has developed. And in one of the patients, who ultimately died, they seriously considered neonatal lung transplantation, using ECMO as “a bridge to transplant.” We believe that this might be a bridge too far. There is still so much research ahead that should be done before we have to decide on such drastic measures. Surfactant therapy, the
B
2.50
m
Nltrofen cardiac
R I
2.00
6 > 5
1.50
C! .z
1.00
Opening pressures in artificially ventilated newborn rats Nitrofen rats without a diaphragmatic defect, and CDH
neural
Fig 9. Compliance in artificially ventilated newborn rats (controls, Nitrofen rats without a diaphragmatic defect. and CDH rats).
rlsomy
anomal
18.
21.
12p
(41
tube 14)
defec
Fig 11. Associated anomalies tients (data from Adzick et al”).
in prenatally
diagnosed
CDH pa-
254
MOLENAAR
effect of antioxidant supply, and the manipulation of vasoactive substances are still ail open for further research. Some of the words of John Hunteq3’ the founding father of modern surgery, are still relevant to our times. This last part of surgery, namely, operations, is a reflection on the healing art; it is a tacit acknowledgement of the insufficiency of surgery. It is like an armed savage who attempts to get that by force which a civilized man would get by stratagem. No surgeon should approach the victim of his operation without a sacred dread and reluctance, and should
ET AL
be superior to that popular &zt generally attending painful operations, often only because they are so, or because they are expensive to the patient.
His critical remarks related to the art of surgery may sound somewhat rude and old-fashioned to us, but his words should also ring a bell, which might lead us to recall another of Hunter’s principles: surgeons have been too much satisfied with considering and treating the effects of diseases only, and without the knowledge of the causes of disease a man cannot be a surgeon.
REFERENCES 1. Puri P, Gorman WA: Natural history of congenital diaphragmatic hernia: Implications for management. Pediatr Surg Int 2:327-330, 1987 2. Greenwald HM, Steiner M: Diaphragmatic hernia in infancy and childhood. Am J Dis Child 38:361-392,1929 3. Ladd W, Gross RE: Congenital diaphragmatic hernia. N Engl J Med 223:917-924,194O 4. Gross RE: The Surgery of Infancy and Childhood, Its Principles and Techniques. Philadelphia, PA, Saunders, 1953, pp 428-444 5. Gross RE: Thoracic surgery for infants (presidential address). J Thorac Cardiovasc Surg 48:152-176,1964 6. Young D: Diaphragmatic hernia in infancy, in Wilkinson AW (ed): Recent Advances in Paediatric Surgery. London, England, Churchill, 1969, pp 142-151 7. Raphaely RC, Downes JJ: Congenital diaphragmatic hernia; prediction of survival. J Pediatr Surg 8:815-823,1973 8. Boix-Ochoa J, Perguero G, Seijo G, et al. Acid-base balance and blood gases in prognosis and therapy of congenital diaphragmatic hernia. J Pediatr Surg 9:49-57, 1974 9. Touloukian RJ, Markowitz RI: A preoperative x-ray scoring system for risk assessment of newborns with congenital diaphragmatic hernia. J Pediatr Surg 19:252-257,1984 10. Anderson KD: Congenital diaphragmatic hernia, in Welch KJ, Randolph JG, Ravitch MN, et al (eds): Pediatric Surgery. Chicago, IL, Year Book, 1986, pp 589601 11. Hazebroek FWJ, Tibboel D, Bos AP, et al: Congenital diaphragmatic hernia: Impact of preoperative stabilization. A prospective pilot study in 13 patients. J Pediatr Surg 23:1139-1146, 1988 12. Bartlett R, Gazzaniga A, Toomasian J, et al: Extracorporeal membrane oxygenation (ECMO) in neonatal respiratory failure. Ann Surg 204:236-243,1986 13. Campanale RP, Rowland RH: Hypoplsia of the lung associated with congenital diaphragmatic hernia. Ann Surg 142:176-189, 1955 14. Tolins SH: Congenital diaphragmatic hernia in the newborn. Ann Surg 137:276-281, 1953 15. Freyschuss U, Lannergren K, Frencker B: Lung function after repair of congenital diaphragmatic hernia. Acta Pediatr Stand 73:589-593,1984 16. Thurlbeck WM, Kida K, Langston C, et al: Postnatal lung growth after repair of diaphragmatic hernia. Thorax 34:338-342, 1979 17. Areechon W, Reid L: Hypoplasia of lung with congenital diaphragmatic hernia. Br Med J 1:230-233,1963
18. Kitagawa M, Hislop A, Boyden E, et al: Lung hypoplasia in congenital diaphragmatic hernia-A quantative study of airway, artery, and alveolar development. Br J Surg 58:342-346, 1971 19. Geggel R, Murphy J, Langleben D, et al: Congenital diaphragmatic hernia. Arterial structural changes and persistent pulmonary hypertension after surgical repair. J Pediatr 107:457464,1985 20. Bos AP, Tibboel D, Hazebroek FWJ, et al: Congenital diaphragmatic hernia: The impact of prostanoids in the perioperative period. Arch Dis Child 65:994-995, 1990 21. deLorimier AA, Tierney OF, Parker HF: Hypoplastic lungs in fetal lambs with surgically produced congenital diaphragmatic hernia. Surgery 62:12-17,1967 22. Adzick NS, Harrison MR, Outwater KM, 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 23. Iritani I: Experimental study on embryogenesis of congenital diaphragmatic hernia. Anat Embryo1 169:133-139,1984 24. Tenbrinck R, Tibboel D, Gaillard JLJ, et al: Experimentally induced congenital diaphragmatic hernia in rats. J Pediatr Surg 25:426-429, 1990 25. Emery JL: The number of alveoli in the terminal respiratory unit of man during late intrauterine life and childhood. Arch Dis Child 35544-547, 1960 26. Tibboel D, Tenbrinck R, Bos AP, et al: An experimental model of congenital diaphragmatic hernia, in Beaufils F, Aigrain Y, Nivoche Y (eds): Defauts Congenitaux de Laparoi Abdominale. Paris, France, Arnette, 1990, pp 99-105 27. Bancalari E, Gerhardt T: Bronchopulmonary dysplasia. Pediatr Clin North Am 33:1-23,1986 28. Bancalari E: Pathogenesis of bronchopulmonary dysplasia: An overview, in Bancalari E, Stocker JT (eds): Bronchopulmonary Dysplasia. Washington, DC, Hemisphere, 1988, pp 3-15 29. Heffner JE, Repine JE: Pulmonary strategies of antioxydant defense. State of the art. Am Rev Respir Dis 140:531-554, 1989 30. Vanhoutte PM: The endothelium-modulator of vascular smooth-muscle tone. N Engl J Med 319:512-513,1988 31. Adzick NS, Vacanti JP, Lillehei CW, et al: Fetal diaphragmatic hernia: Ultrasound diagnosis and clinical outcome in 38 cases. J Pediatr Surg 24:654-658,1989 32. Palmer JF: The works of John Hunter F.R.S., ~011. London, England, Longman, Rees, Orme, Brown, Green, and Longman, 1835, p 210