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Heart hypoplasia in experimental congenital diaphragmatic hernia
Heart Hypoplasia in Experimental Congenital Diaphragmatic Hernia By Lucia Migliazza, Huimin Xia, Jose I. Alvarez, Aranzazu Arnaiz, J u a n A. Diez-Pardo, Luisa F. A l f o n s o , and Juan A. Tovar
Madrid, Spain and Bilbao, Spain
Background/Purpose: Heart hypoplasia is associated with congenital diaphragmatic hernia (CDH) and decisively influences survival rate. This study examines whether nitrofenexposed fetal rats have heart hypoplasia. Methods: Pregnant rats received either 100 mg nitrofen or vehicle on gestational day 9.5. The hearts recovered near full term were either formalin fixed for anatomic studies or snap-frozen for biochemical studies. Heart weight, ventricular chamber diameters and aortic-to-pulmonary root diameter ratios were measured in fixed hearts. Protein and DNA were determined in frozen hearts. Analysis of variance (ANOVA) and correlation-regression studies were used for statistical assessment.
Results: All control fetuses were normal, whereas 61% of those exposed to nitrofen had CDH. Cardiovascular malformations were found in 73% of CDH and in 50% of non-CDH animals. Wet and fixed heart weights in percent of fetal weight, left-to-right ventricular diameter ratio, and aortic-topulmonary root diameter ratio were significantly decreased
U N G H Y P O P L A S I A with biochemical immaturity and arteriolar constriction is the main cause of mortality in congenital diaphragmatic hernia (CDH) and remains one of the challenges of our specialty. Most research on this malformation has been carried out in the fetal lamb model in which the surgical creation of a diaphragmatic defect and the displacement of abdominal viscera into the thorax during fetal life induce 1-3 pulmonary lesions similar to those of CDH patients that can be partially reversed by prenatal lung decompression. 4 The fetal lamb model also reproduces left heart hypoplasia, another component of the CDH spectrum recently recognized as a possible "missing link" capable of explaining the disproportionate lethality despite optimal manage-
in fetuses with CDH in comparison with controls. Only wet heart was significantly decreased in nitrofen-treated fetuses without CDH, although all other variables showed a trend in the same direction. Protein to DNA ratios were similar in the three groups. The structure of the myocytes was histologically similar in all groups.
Conclusions:The spectrum of lesions in the nitrofen model of CDH encompasses heart hypoplasia, further validating its use for research on this condition. Heart hypoplasia is related to cardiopulmonary compression, but its presence in treated animals without CDH demonstrates that the teratogen itself participate directly in its pathogenesis, and this finding invites further research on this line.
J Pediatr Surg 34:706-711. Copyright© 1999by W.B. Saunders Company. INDEX WORDS: Congenital diaphragmatic hernia, rat, nitrofen, heart, hypoplasia.
ment 5 and interpreted also as a probable consequence of fetal mediastinal compression by herniated viscera. Prenatal exposure to teratogenic chemicals like nitrofen also induce CDH and lung lesions very similar to those observed in human disease and in fetal lambs 6-9 providing an equally interesting, easily reproducible, and more affordable model, but this setting has the advantage over the fetal lamb model of potentially reproducing other organ malformations observed in CDH patients. The current study examines whether left heart hypoplasia is a component of the malformative spectrum induced by nitrofen in rats and explores the possible participation of nonmechanical factors in its pathogenesis. MATERIALS A N D METHODS
From the Departments of Surgery, Hospital Infantil "La Paz, "" Madrid, Spain, and the Department of NeonatoIogy, Hospital de Cruces, Bilbao, Spain. Presented at the 1998 Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, San Francisco, California, October 16-19, 1998. Supported by FIS Grants #96/0059-01 and #96/0059-02 Address reprint requests to Dr Juan A. Tovar, Department of Surgery, Hospital lnfantil Universitario "La Paz," R de la Castellana 261, 28046, Madrid, Spain. Copyright © 1999 by W.B. Saunders Company 0022-3468/99/3405-0011 $03.00/0 706
For morphological studies, the following experiments were carried out: two groups of time-mated pregnant Sprague-Dawley rats were treated on day 9.5 of gestation (day 0 = sperm in vaginal smear after overnight mating) either with a dose of 100 mg of nitrofen in 1 mL of olive oil administered intragastrically (experimental group, n = 10) or with an equal volume of oil alone (control group, n = 3). The fetuses were recovered on the 21st gestational day (term, 22nd) and, after assessingunder a binocular surgical microscopethe presence or absence of CDH, they were fixed in 10% buffered formalin for 1 week and subsequently weighed and examined through a wide thoracoabdominal incision. The heart was then removed, separated from the great vessels, and weighed after measuring the aortic and pulmonary artery root diameters. Finally, it was transversely sectioned in an equatorial plane Journal of PediatricSurgery,Vo134, No 5 (May), 1999: pp 706-711
HEART HYPOPLASIA IN EXPERIMENTAL CDH
707
midway between the apex and the aortic root, and the ventricular chamber diameters were measured. For these measurements, that were repeated three times taking the average as the final value, we used an eye-piece micrometer. The hearts of six randomly selected fetuses from each group were embedded in paraffin, sectioned in the equatorial plane, and stained with H&E and Masson trichromic for histological examination. For biochemical studies, another set of experiments was performed: two additional groups of time-mated rats, control (n = 2) and experimental (n = 6), were treated in a similar way until the time of fetal recovery. The pups were then killed and weighed, and, after assessing the presence or absence of CDH, the heart and both lungs were recovered, weighed, and snap-frozen at - 4 0 ° C for further processing. Protein and DNA contents were determined in lung and heart specimens by the methods of Bradford 1° and Labarca and Paigen] 1 respectively and the results expressed in protein to DNA ratios. All experiments were carried out in compliance with the Animal Care Regulations of the European Union and were approved by the Animal Research Institutional Committee. Numerical variables in control and nitrofen-treated animals with or without CDH are described statistically either as proportions or as means _+ SD. Differences among groups were assessed by one-way analysis of variance (ANOVA), and the significance of pairwise comparisons between groups was determined by "post-hoc" Fisher's tests. Correlation and regression analyses were performed displaying the regression lines in graphs with 95% confidence intervals of the means. P values of less than .05 were considered significant throughout.
RESULTS
The diaphragms and the cardiovascular systems were normal in control fetuses allocated for morphological studies (n = 21). Conversely, there was CDH in 80 of 130 or 61% of treated animals used for this purpose (72 left sided, four right sided, and four bilateral) and cardiovascular malformations in 84 of 130 or 64% (59 with CDH and 25 without CDH) including 24 narrow outflow pulmonary tracts, 21 Fallots, 18 VSD, 20 truncus arteriosus, 56 aortic arches anomalies, and others. Table 1 shows that the heart of animals with CDH was hypoplastic because its weight expressed as percentage of body weight after fixation was significantly decreased in comparison with controls. The left-to-right ventricular chamber diameter ratio and the aortic-to-pulmonary root diameter ratio (AO-PA) of CDH fetuses were signifiTable 1. Heart Measurements in Control and Nitrofen-Exposed Fetuses After Formalin Fixation Nitrofen Control (n = 21)
No-CDH (n = 50)
CDH (n = 80)
Heart w e i g h t (% o f BW)
0.893 ± 0.083
0.838 _+ 0.15
1.168 -+ 0.042
1.135 + 0.5
0.963 ± 0,049
0.801 _+ 0.462
0.749 ± 0 . 1 * t
Left to right ventricular c h a m b e r diameter
0.9 _+ 0.102"¢
AO/PA d i a m e t e r ratio
NOTE: Data are expressed as mean -+ SD. * P < .05 c o m p a r e d w i t h control group. tP<
.05 c o m p a r e d w i t h no-CDH group.
0,668 ± 0.6*
cantly decreased in comparison with controls demonstrating smaller left chambers and relatively narrower aortic outlet. These variables showed a trend in the same direction in nitrofen-exposed fetuses without CDH after fixation (Table 1 and Fig 1). The myocardial structure appeared histologically normal on microscopic examination in all groups, although the mass obviously was reduced in CDH animals and, to a lesser extent, in nitrofen-exposed ones without CDH. This mass reduction was biventricular, but it was more conspicuous on the left side. Table 2 shows that, in the set of experiments directed to biochemical studies, all animals treated with nitrofen had lung and heart hypoplasia as shown by decreased organ wet weight in percent of body weight irrespective of the presence or absence of CDH. Protein to DNA ratios were similar in all groups showing that cell size was similar in them. Figure 2 depicts the plots of lung against wet heart weights in control and nitrofen-treated animals without and with CDH. It can be appreciated that there was no correlation between both magnitudes in control animals (r -- 0.14, not significant) but that there was significant correlation between them in animals with CDH (r = 0.41, P < .01) and also in those without CDH (r = 0.54, P < .05) allowing prediction of lung hypoplasia when heart weight is taken as independent variable. DISCUSSION
Despite all refinements of the current management, survival rates of patients born with CDH remains frustratingly low because of pulmonary structural hypoplasia, biochemical immaturity, and arteriolar thickening and constriction with persistence of the fetal circulation pattern. In addition, associated anomalies are frequent 12 and undoubtedly account for many of the deaths, particularly when the cardiovascular system is involved. 13,14 Although it was pointed out several years ago, 15 the decisive role of the hypoplastic left heart in survival of these patients was acknowledged only recently and has been used as an ultrasonographic predictor of bad prognosis in prenatally diagnosed fetuses. 16-2° Echocardiographic studies and autopsy records found variable degrees of reduction in left-to-right ventricular sizes and in aortic-to-pulmonary diameter ratios. 17,2°,21 Intrauterine compression and decreased right-to-left shunting through the foramen ovale have been considered as the most likely explanations for this left heart underdevelopment, and all interpretations have been made in the sense of a purely mechanical mechanism. 15,18 This issue has been investigated extensively in the fetal lamb model of CDH in which hypoplastic heart is constantly found at term. Karamanoukian et aF 2 demonstrated that the reduction in size corresponded to hypoplasia rather than to atrophy because wet-dry weight and
708
MIGLIAZZA ET AL
Table 2. Lung and Heart Wet Weights and Protein to DNA Ratios in Control and Nitrofen-Exposed Fetuses Nitrofen Control (n - 21)
No-CDH (n = 21)
CDH (n - 46)
Lung w e i g h t (% of BW)
Z916 _+ 0.304
2.32 + 0.334"
1.708 _+ 0.315"t
0.634 _+ 0.053
0.585 _+ 0.087*
0.521 ± 0.078"t
Heart w e i g h t (% of BW)
Heart protein to DNA ratio 27.261 _+ 2.997 27.234 ± 2.998
31.819 -- 17,073
NOTE: Data are expressed as mean _+ SD. * P < .05 compared with control group. f P < .05 compared with no-CDH group.
Fig 1. Formalin-fixed fetal rat hearts near full term transversally sectioned in an equatorial plane midway between the base and the apex. In control fetuses (A), ventricular muscle mass is w e l l developed in both sides and the chambers are relatively wide, Nitrofenexposed fetuses with CDH (C) have obvious underdevelopment of muscle in both sides with reduced chamber diameters, whereas the lesions in nitrofen-exposed fetuses without CDH (B) are intermediate between A and C. RV, right ventricle; LV, left ventricle, Units are millimeters and minor marks represent 0.1 mm.
DNA to protein ratios were similar in CDH and control groups. They also showed significant positive correlation between heart and lung weights in CDH lambs that allowed prediction of the degree of lung hypoplasia as a function of heart size. This correlation was absent in controls.23 Furthermore, these investigators elegantly demonstrated that intrauterine correction of surgically created CDH late in gestation reversed heart hypoplasia in fetal lambs but, surprisingly, that tracheal ligation did not. This was interpreted as probably caused by the ongoing heart compression by the expanding lung in ligated animals. 24 These investigations leave little doubt about the participation of mechanical compression of the heart as the leading mechanism accounting for heart hypoplasia in CDH. However, although the fetal surgical model of CDH mimics accurately hypoplasia of both the lung and the heart and has largely contributed to our current understanding of the pathophysiology of the disease, it involves fetal manipulation and does not reproduce the whole spectrum of anomalies of embryonic origin and particularly the considerable proportions of neurological, cardiovascular, craneofacial, and costovertebral defects that burden CDH patients. 14,25,26 Conversely, teratogens administered to pregnant rats on the appropriate gestational days disturb organogenesis during early embryonal life and induce malformations in their offspring that provide good models of several of the most investigated ones like CDH and anorectal or esophageal atresias. However, these teratogens act simultaneously on many tissues and often produce malformations of so many organs that the appropriatedness of such models for investigating isolated anomalies can be questioned. We believe that human CDH and anorectal or esophageal atresias are precisely multiorgan malformations and that the more severe ends of the spectrum in each of them encompasses many of the components present in the teratogenic rodent models that could be more appropriate for their research. For instance, in CDH, nitrofen-
HEART HYPOPLASIA IN EXPERIMENTAL CDH
Control i
LW (g) .2
I
i
I
709
(n=21) i
I
i
I
.15 .1
.O5 p=n.s.
Y = .135 + .824 * X
0
.01'
0
" .0'2
.03 '
. 0 '4
.05
HW (g)
Nitrofen. LW (g)
.2
i
I
no CDH (n=21) I
.15' ~
I
i
~
I
I
I
I
o ~..~
•1'
.05 • 0
• /, 0
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/
.01
, Y ~ .o2,1 + 3.~79 * x •02
•03
.04
.o5
XW (g)
Nitrofen. l
LW (g) .2
CDH (n=46) I
I
l
i
.15" .1
.05 0
p
0
• .01'
" . 0"2
" .03 '
.05 " .04 ' HW (O)
Fig 2. Lung wet weight plotted against heart wet weight in control, nitrofen-exposed fetuses w i t h o u t CDH, and nitrofen-exposed fetuses with CDH. Regression lines are drawn with 95% confidence intervals for means. Correlation between both variables was significant in both groups of nitrofen-treated fetuses in which lung weight can be predicted taking heart weight as independent variable.
exposed rat fetuses have diaphragmatic defects, lung hypoplasia with biochemical immaturity, and arteriolar anomalies in all similar to those of the human and ovine diseases, 68,27 but they also have neural crest-derived cardiovascular anomalies 28 and costovertebral malforma-
tions (unpublished observations) that closely reproduce those described in babies with CDH and that are certainly not caused by intrathoracic space conflicts? 5,29,3° Furthermore, often it is understated that the lungs of the few fetuses of the nitrofen-exposed litters that do not have CDH also have significant degrees of lung hypoplasia6 and a trend toward biochemical immaturity 7 that strongly suggest that disturbed organogenesis of the lung itself occurs before compression jeopardizes its development. The present study confirms that, like in human CDH and in the fetal lamb model, heart size is markedly reduced in nitrofen-treated rats with CDH, that this reduction of muscle mass is more marked in the left chambers, and that it corresponds to organ hypoplasia. Even the lack of correlation between heart and lung weights in control animals and its significant correlation in those with CDH are similar to those described in fetal lambs? 3 Therefore, the nature of heart size reduction in the rat model must be very similar to that seen in human and fetal lamb CDH, and this further validates this model for the study of these particular aspects of the disease. Other investigators had previously pointed out that the heart is one of the target organs for nitrofen 31 and that prenatal exposure to lower doses of the chemical caused heart hypoplasia with preservation of protein to DNA ratio 32 and functional disturbances 33 in the offspring, but, unfortunately, no distinction was made in those studies between animals with and without CDH precluding analysis of the possible influence of the diaphragmatic defect on the heart. Our current study adds the concept of the teratogen causing directly some degree of heart hypoplasia even in fetuses without CDH and hence without mediastinal space conflict. We acknowledge that heart hypoplasia in these fetuses was only demonstrated by significant decrease of the heart to body wet weight ratio, but all other variables, although failing to reach statistical significance, showed trends in the same direction. These findings, along with the demonstration of lung hypoplasia in this group of nitrofen-exposed rats without CDH and the previous observation in them of pulmonary lesions, 6 biochemical immaturity] and cardiovascular malformations 28 of intermediate severity between controls and fetuses with CDH suggest that nonmechanical factors play a role in these components of the disease. We believe that investigation of the molecular and genetic regulation of organogenesis in this model should be pursued on this line.
REFERENCES 1. Harrison MR, Jester JA, Ross NA: Correction of congenital diaphragmatic hernia in utero. I. The model: Intrathoracic balloon produces fatal pulmonary bypoplasia. Surgery 88:174-182, 1980
2. Pringle KC, Turner JW, Schofield JC, et al: Creation and repair of diaphragmatic hernia in the fetal lamb: Lung development and morphology. J Pediatr Surg 19:131-139, 1984
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3. Hashimoto E, Pringle C, Super R, et al: The creation and repair of diaphragmatic hernia in fetal lambs: Morphology of the type II alveolar cell. J Pediatr Surg 20:354-356, 1985 4. Harrison MR, Bressack MA, Churg AM: Correction of congenital diaphragmatic hernia in utero. II. Simulated correction permits fetal lung growth with survival at birth. Surgery 88:260-268, 1980 5. Karamanoukian HL, Wilcox DT, Glick PL: The "missing link" in congenital diaphragmatic hemia. J Pediatr Surg 29:954-956, 1994 (letter) 6. Alfonso LF, Vilanova J, Aldaz~tbal E et al: Lung growth and maturation in the rat model of experimentally induced congenital diaphragmatic hernia. Eur J Pediatr Surg 3:6-11, 1993 7. Alfonso LF, Amaiz A, Alvarez FJ, et al: Lung hypoplasia and suffactant immaturity induced in the fetal rat by prenatal exposure to nitrofen. Biol Neonate 69:94-100, 1996 8. Tenbrinck R, Tibboel D, Gaillard JLJ, et al: Experimentally induced congenital diaphragmatic hernia in rats. J Pediatr Surg 25:426429, 1990 9. Iritani I: Experimental study on embryogenesis of congenital diaphragmatic hernia. Anat Embryol 169:133-139, 1984 10. Bradford MM: A rapid and sensitive method for the qnantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254, 1976 11. Labarca C, Paigen K: A simple, rapid and sensitive DNA assay procedure. Anal Biochem 72:344-352, 1980 12. Martinez-Frias ML, Prieto L, Urioste M, et al: Clinical/ epidemiological analysis of congenital anomalies associated with diaphragmatic hernia. Am J Med Genet 62:71-76, 1996 13. Greenwood RD, Rosenthal A, Nadas AS: Cardiovascular abnormalities associated with congenital diaphragmatic hernia. Pediatrics 57:92-97, 1976 14. Sweed Y, Puri P: Congenital diaphragmatic hernia: Influence of associated malformations on survival. Arch Dis Child 69:68-70, 1993 15. Siebert JR, Haas JE, Beckwith JB: Left ventricular hypoplasia in congenital diaphragmatic liemia. J Pediatr Surg 19:567-571, 1984 16. Crawford DC, Wright VM, Drake DE et al: Fetal diaphragmatic hernia: The value of fetal echocardiography in the prediction of postnatal outcome. Br J Obstet Gyneco196:705-710, 1989 17. Sharland GK, Lockhart SM, Heward AJ, et al: Prognosis in fetal diaphragmatic hernia. Am J Obstet Gynecol 166:9-13, 1992 18. Schwartz SM, Vermillion RE Hirschl RB: Evaluation of left ventricular mass in children with left congenital diaphragmatic hernia. J PediaU 125:447-451, 1994
19. Allan LD, Irish MS, Glick PL: The fetal heart in diaphragmatic hernia. Clin Perinato123:795-812, 1996 20. Thebaud B, Azancot A, De Lagausie P, et al: Congenital diaphragmatic hernia: Antenatal prognostic factors. Int Care Med 23:1062-1069, 1997 21. VanderWall KJ, Kohl T, Adzick NS, et al: Fetal diaphragmatic hernia: Echocardiography and clinical outcome. J Pediatr Surg 32:223225; discussion 225-226, 1997 22. Karamanoukian HL, Glick PL, Wilcox DT, et al: Pathophysiology of congenital diaphragmatic hernia. 11. Anatomic and biochemical characterization of the heart in the fetal lamb CDH model. J Pediatr Surg 30:925-929, 1995 23. Karamanoukian HL, O'Toole SJ, Rossman JR, et al: Can cardiac weight predict lung weight in patients with congenital diaphragmatic hernia? J Pediatr Surg 31:823-825, 1996 24. Karamanoukian HL, O'Toole SJ, Rossman MS, et al: Fetal surgical interventions and the development of the heart in congenital diaphragmatic hernia. J SurgRes 65:5-8, 1996 25. Benjamin DR, Juul S, Siebert JR: Congenital posterolateral diaphragmatic hernia: Associated malformations. J Pediatr Surg 23:899903, 1988 26. Losty PD, Suen HC, Manganaro TF, et al: Prenatal hormonal therapy improves pulmonary compliance in the nitrofen-induced CDH rat model. J Pediatr Surg 30:420-426, 1995 27. Tenbrinck R, Gaillard JLJ, Tibboel D, et al: Pulmonary vascular abnormalities in experimentally induced congenital diaphragmatic hernia in rats. J Pediatr Surg 27:862-865, 1992 28. Migliazza L, Otken C, Xia H, et al: Cardiovascular malformations in experimental diaphragmatic hernia. J Pediatr Surg (in press) 29. David TJ, Illingworth CA: Diaphragmatic hernia in the southwest of England. J Med Genet 13:253-262, 1976 30. Losty PD, Vanamo K, Rintala RJ, et al: Congenital diaphragmatic hernia--Does the side of the defect influence the incidence of associated malformations? J Pediatr Surg 33:507-510, 1998 31. Costlow RD, Manson JM: The heart and diaphragm: Target organs in the neonatal death induced by nitrofen (2,4-Dichlorophenyl-pnitrophenyl Ether). Toxicology 20:209-227, 1981 32. Zeman FJ, Heng H, Hoogenboom ER, et al: Cell number and size in selected organs of fetuses of rats malnourished and exposed to nitrofen. Teratog Carcinog Mutagen 6:339-347, 1986 33. Lau C, Cameron AM, Irsula O, et al: Effects of prenatal nitrofen exposure on cardiac structure and function in the rat. Toxicol Appl Pharmacol 86:22-32, 1986
Discussion A. Coran (Ann Arbor, MI): In this p a r t i c u l a r m o d e l d i d
s t u d y t h e l u n g s in this m o d e l a n d see e v i d e n c e o f
you detect evidence of pulmonary hypertension histologically, s u c h as t h i c k e n i n g o f t h e p u l m o n a r y arterial m e d i a ?
pulmonary hypertension? J. Tovar (response): I n this p a r t i c u l a r e x p e r i m e n t w e
A n d d o y o u t h i n k t h a t h a s a n y r e l a t i o n s h i p to the c a r d i a c
d i d n o t s t u d y that, b u t f o r m e r l y w e h a v e d o n e it s e v e r a l
findings?
times. O t h e r i n v e s t i g a t o r s , s o m e o f t h e m i n this r o o m ,
L. Migliazza (response): W e h a v e f o u n d t h a t h e a r t
h a v e i l l u s t r a t e d t h a t t h e r e are v a s c u l a r a n o m a l i e s i n the
h y p o p l a s i a is p r e s e n t also in t h e fetus w i t h o u t hernia. So
l u n g s t h a t are c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t t h e y
w e c a n h y p o t h e s i z e t h a t t h e r e is also s o m e o t h e r f a c t o r
have pulmonary hypertension.
i m p l i c a t e d in this h y p o p l a s i a o t h e r t h a n the a n a t o m i c c h a n g e s or t h e h y p o p l a s i a o f the l u n g a n d m e c h a n i c a l compression.
A. Coran (Ann Arbor, MI): D i d y o u h i s t o l o g i c a l l y
A. DeLormier (San Francisco, CA): It is c l e a r t h a t n i t r o f e n is a s y s t e m i c p o i s o n . I w o n d e r a b o u t u s i n g this or l e a n i n g o n this m o d e l a n d e x t e n d i n g this to the s i t u a t i o n in a h u m a n d i a p h r a g m a t i c hernia. It w o u l d b e h e l p f u l i f
HEART HYPOPLASIA IN EXPERIMENTAL CDH
you could just mechanically create the diaphragmatic hernia and study the heart effects. I would not be surprised if it was not a mechanical effect on heart development. It would be a purer model. J. Tovar (response): It is very well known that this heart hypoplasia has been produced in the surgical model
711
of fetal diaphragmatic hernia. I think that it has been very consistently proven that it can be related to pressure itself. But the point of this paper is to show that perhaps there are other factors involved, not only pressure, but also some nonmechanical factors that can act on the heart.
Madrid, Spain and Bilbao, Spain
Background/Purpose: Heart hypoplasia is associated with congenital diaphragmatic hernia (CDH) and decisively influences survival rate. This study examines whether nitrofenexposed fetal rats have heart hypoplasia. Methods: Pregnant rats received either 100 mg nitrofen or vehicle on gestational day 9.5. The hearts recovered near full term were either formalin fixed for anatomic studies or snap-frozen for biochemical studies. Heart weight, ventricular chamber diameters and aortic-to-pulmonary root diameter ratios were measured in fixed hearts. Protein and DNA were determined in frozen hearts. Analysis of variance (ANOVA) and correlation-regression studies were used for statistical assessment.
Results: All control fetuses were normal, whereas 61% of those exposed to nitrofen had CDH. Cardiovascular malformations were found in 73% of CDH and in 50% of non-CDH animals. Wet and fixed heart weights in percent of fetal weight, left-to-right ventricular diameter ratio, and aortic-topulmonary root diameter ratio were significantly decreased
U N G H Y P O P L A S I A with biochemical immaturity and arteriolar constriction is the main cause of mortality in congenital diaphragmatic hernia (CDH) and remains one of the challenges of our specialty. Most research on this malformation has been carried out in the fetal lamb model in which the surgical creation of a diaphragmatic defect and the displacement of abdominal viscera into the thorax during fetal life induce 1-3 pulmonary lesions similar to those of CDH patients that can be partially reversed by prenatal lung decompression. 4 The fetal lamb model also reproduces left heart hypoplasia, another component of the CDH spectrum recently recognized as a possible "missing link" capable of explaining the disproportionate lethality despite optimal manage-
in fetuses with CDH in comparison with controls. Only wet heart was significantly decreased in nitrofen-treated fetuses without CDH, although all other variables showed a trend in the same direction. Protein to DNA ratios were similar in the three groups. The structure of the myocytes was histologically similar in all groups.
Conclusions:The spectrum of lesions in the nitrofen model of CDH encompasses heart hypoplasia, further validating its use for research on this condition. Heart hypoplasia is related to cardiopulmonary compression, but its presence in treated animals without CDH demonstrates that the teratogen itself participate directly in its pathogenesis, and this finding invites further research on this line.
J Pediatr Surg 34:706-711. Copyright© 1999by W.B. Saunders Company. INDEX WORDS: Congenital diaphragmatic hernia, rat, nitrofen, heart, hypoplasia.
ment 5 and interpreted also as a probable consequence of fetal mediastinal compression by herniated viscera. Prenatal exposure to teratogenic chemicals like nitrofen also induce CDH and lung lesions very similar to those observed in human disease and in fetal lambs 6-9 providing an equally interesting, easily reproducible, and more affordable model, but this setting has the advantage over the fetal lamb model of potentially reproducing other organ malformations observed in CDH patients. The current study examines whether left heart hypoplasia is a component of the malformative spectrum induced by nitrofen in rats and explores the possible participation of nonmechanical factors in its pathogenesis. MATERIALS A N D METHODS
From the Departments of Surgery, Hospital Infantil "La Paz, "" Madrid, Spain, and the Department of NeonatoIogy, Hospital de Cruces, Bilbao, Spain. Presented at the 1998 Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, San Francisco, California, October 16-19, 1998. Supported by FIS Grants #96/0059-01 and #96/0059-02 Address reprint requests to Dr Juan A. Tovar, Department of Surgery, Hospital lnfantil Universitario "La Paz," R de la Castellana 261, 28046, Madrid, Spain. Copyright © 1999 by W.B. Saunders Company 0022-3468/99/3405-0011 $03.00/0 706
For morphological studies, the following experiments were carried out: two groups of time-mated pregnant Sprague-Dawley rats were treated on day 9.5 of gestation (day 0 = sperm in vaginal smear after overnight mating) either with a dose of 100 mg of nitrofen in 1 mL of olive oil administered intragastrically (experimental group, n = 10) or with an equal volume of oil alone (control group, n = 3). The fetuses were recovered on the 21st gestational day (term, 22nd) and, after assessingunder a binocular surgical microscopethe presence or absence of CDH, they were fixed in 10% buffered formalin for 1 week and subsequently weighed and examined through a wide thoracoabdominal incision. The heart was then removed, separated from the great vessels, and weighed after measuring the aortic and pulmonary artery root diameters. Finally, it was transversely sectioned in an equatorial plane Journal of PediatricSurgery,Vo134, No 5 (May), 1999: pp 706-711
HEART HYPOPLASIA IN EXPERIMENTAL CDH
707
midway between the apex and the aortic root, and the ventricular chamber diameters were measured. For these measurements, that were repeated three times taking the average as the final value, we used an eye-piece micrometer. The hearts of six randomly selected fetuses from each group were embedded in paraffin, sectioned in the equatorial plane, and stained with H&E and Masson trichromic for histological examination. For biochemical studies, another set of experiments was performed: two additional groups of time-mated rats, control (n = 2) and experimental (n = 6), were treated in a similar way until the time of fetal recovery. The pups were then killed and weighed, and, after assessing the presence or absence of CDH, the heart and both lungs were recovered, weighed, and snap-frozen at - 4 0 ° C for further processing. Protein and DNA contents were determined in lung and heart specimens by the methods of Bradford 1° and Labarca and Paigen] 1 respectively and the results expressed in protein to DNA ratios. All experiments were carried out in compliance with the Animal Care Regulations of the European Union and were approved by the Animal Research Institutional Committee. Numerical variables in control and nitrofen-treated animals with or without CDH are described statistically either as proportions or as means _+ SD. Differences among groups were assessed by one-way analysis of variance (ANOVA), and the significance of pairwise comparisons between groups was determined by "post-hoc" Fisher's tests. Correlation and regression analyses were performed displaying the regression lines in graphs with 95% confidence intervals of the means. P values of less than .05 were considered significant throughout.
RESULTS
The diaphragms and the cardiovascular systems were normal in control fetuses allocated for morphological studies (n = 21). Conversely, there was CDH in 80 of 130 or 61% of treated animals used for this purpose (72 left sided, four right sided, and four bilateral) and cardiovascular malformations in 84 of 130 or 64% (59 with CDH and 25 without CDH) including 24 narrow outflow pulmonary tracts, 21 Fallots, 18 VSD, 20 truncus arteriosus, 56 aortic arches anomalies, and others. Table 1 shows that the heart of animals with CDH was hypoplastic because its weight expressed as percentage of body weight after fixation was significantly decreased in comparison with controls. The left-to-right ventricular chamber diameter ratio and the aortic-to-pulmonary root diameter ratio (AO-PA) of CDH fetuses were signifiTable 1. Heart Measurements in Control and Nitrofen-Exposed Fetuses After Formalin Fixation Nitrofen Control (n = 21)
No-CDH (n = 50)
CDH (n = 80)
Heart w e i g h t (% o f BW)
0.893 ± 0.083
0.838 _+ 0.15
1.168 -+ 0.042
1.135 + 0.5
0.963 ± 0,049
0.801 _+ 0.462
0.749 ± 0 . 1 * t
Left to right ventricular c h a m b e r diameter
0.9 _+ 0.102"¢
AO/PA d i a m e t e r ratio
NOTE: Data are expressed as mean -+ SD. * P < .05 c o m p a r e d w i t h control group. tP<
.05 c o m p a r e d w i t h no-CDH group.
0,668 ± 0.6*
cantly decreased in comparison with controls demonstrating smaller left chambers and relatively narrower aortic outlet. These variables showed a trend in the same direction in nitrofen-exposed fetuses without CDH after fixation (Table 1 and Fig 1). The myocardial structure appeared histologically normal on microscopic examination in all groups, although the mass obviously was reduced in CDH animals and, to a lesser extent, in nitrofen-exposed ones without CDH. This mass reduction was biventricular, but it was more conspicuous on the left side. Table 2 shows that, in the set of experiments directed to biochemical studies, all animals treated with nitrofen had lung and heart hypoplasia as shown by decreased organ wet weight in percent of body weight irrespective of the presence or absence of CDH. Protein to DNA ratios were similar in all groups showing that cell size was similar in them. Figure 2 depicts the plots of lung against wet heart weights in control and nitrofen-treated animals without and with CDH. It can be appreciated that there was no correlation between both magnitudes in control animals (r -- 0.14, not significant) but that there was significant correlation between them in animals with CDH (r = 0.41, P < .01) and also in those without CDH (r = 0.54, P < .05) allowing prediction of lung hypoplasia when heart weight is taken as independent variable. DISCUSSION
Despite all refinements of the current management, survival rates of patients born with CDH remains frustratingly low because of pulmonary structural hypoplasia, biochemical immaturity, and arteriolar thickening and constriction with persistence of the fetal circulation pattern. In addition, associated anomalies are frequent 12 and undoubtedly account for many of the deaths, particularly when the cardiovascular system is involved. 13,14 Although it was pointed out several years ago, 15 the decisive role of the hypoplastic left heart in survival of these patients was acknowledged only recently and has been used as an ultrasonographic predictor of bad prognosis in prenatally diagnosed fetuses. 16-2° Echocardiographic studies and autopsy records found variable degrees of reduction in left-to-right ventricular sizes and in aortic-to-pulmonary diameter ratios. 17,2°,21 Intrauterine compression and decreased right-to-left shunting through the foramen ovale have been considered as the most likely explanations for this left heart underdevelopment, and all interpretations have been made in the sense of a purely mechanical mechanism. 15,18 This issue has been investigated extensively in the fetal lamb model of CDH in which hypoplastic heart is constantly found at term. Karamanoukian et aF 2 demonstrated that the reduction in size corresponded to hypoplasia rather than to atrophy because wet-dry weight and
708
MIGLIAZZA ET AL
Table 2. Lung and Heart Wet Weights and Protein to DNA Ratios in Control and Nitrofen-Exposed Fetuses Nitrofen Control (n - 21)
No-CDH (n = 21)
CDH (n - 46)
Lung w e i g h t (% of BW)
Z916 _+ 0.304
2.32 + 0.334"
1.708 _+ 0.315"t
0.634 _+ 0.053
0.585 _+ 0.087*
0.521 ± 0.078"t
Heart w e i g h t (% of BW)
Heart protein to DNA ratio 27.261 _+ 2.997 27.234 ± 2.998
31.819 -- 17,073
NOTE: Data are expressed as mean _+ SD. * P < .05 compared with control group. f P < .05 compared with no-CDH group.
Fig 1. Formalin-fixed fetal rat hearts near full term transversally sectioned in an equatorial plane midway between the base and the apex. In control fetuses (A), ventricular muscle mass is w e l l developed in both sides and the chambers are relatively wide, Nitrofenexposed fetuses with CDH (C) have obvious underdevelopment of muscle in both sides with reduced chamber diameters, whereas the lesions in nitrofen-exposed fetuses without CDH (B) are intermediate between A and C. RV, right ventricle; LV, left ventricle, Units are millimeters and minor marks represent 0.1 mm.
DNA to protein ratios were similar in CDH and control groups. They also showed significant positive correlation between heart and lung weights in CDH lambs that allowed prediction of the degree of lung hypoplasia as a function of heart size. This correlation was absent in controls.23 Furthermore, these investigators elegantly demonstrated that intrauterine correction of surgically created CDH late in gestation reversed heart hypoplasia in fetal lambs but, surprisingly, that tracheal ligation did not. This was interpreted as probably caused by the ongoing heart compression by the expanding lung in ligated animals. 24 These investigations leave little doubt about the participation of mechanical compression of the heart as the leading mechanism accounting for heart hypoplasia in CDH. However, although the fetal surgical model of CDH mimics accurately hypoplasia of both the lung and the heart and has largely contributed to our current understanding of the pathophysiology of the disease, it involves fetal manipulation and does not reproduce the whole spectrum of anomalies of embryonic origin and particularly the considerable proportions of neurological, cardiovascular, craneofacial, and costovertebral defects that burden CDH patients. 14,25,26 Conversely, teratogens administered to pregnant rats on the appropriate gestational days disturb organogenesis during early embryonal life and induce malformations in their offspring that provide good models of several of the most investigated ones like CDH and anorectal or esophageal atresias. However, these teratogens act simultaneously on many tissues and often produce malformations of so many organs that the appropriatedness of such models for investigating isolated anomalies can be questioned. We believe that human CDH and anorectal or esophageal atresias are precisely multiorgan malformations and that the more severe ends of the spectrum in each of them encompasses many of the components present in the teratogenic rodent models that could be more appropriate for their research. For instance, in CDH, nitrofen-
HEART HYPOPLASIA IN EXPERIMENTAL CDH
Control i
LW (g) .2
I
i
I
709
(n=21) i
I
i
I
.15 .1
.O5 p=n.s.
Y = .135 + .824 * X
0
.01'
0
" .0'2
.03 '
. 0 '4
.05
HW (g)
Nitrofen. LW (g)
.2
i
I
no CDH (n=21) I
.15' ~
I
i
~
I
I
I
I
o ~..~
•1'
.05 • 0
• /, 0
p
/
.01
, Y ~ .o2,1 + 3.~79 * x •02
•03
.04
.o5
XW (g)
Nitrofen. l
LW (g) .2
CDH (n=46) I
I
l
i
.15" .1
.05 0
p
0
• .01'
" . 0"2
" .03 '
.05 " .04 ' HW (O)
Fig 2. Lung wet weight plotted against heart wet weight in control, nitrofen-exposed fetuses w i t h o u t CDH, and nitrofen-exposed fetuses with CDH. Regression lines are drawn with 95% confidence intervals for means. Correlation between both variables was significant in both groups of nitrofen-treated fetuses in which lung weight can be predicted taking heart weight as independent variable.
exposed rat fetuses have diaphragmatic defects, lung hypoplasia with biochemical immaturity, and arteriolar anomalies in all similar to those of the human and ovine diseases, 68,27 but they also have neural crest-derived cardiovascular anomalies 28 and costovertebral malforma-
tions (unpublished observations) that closely reproduce those described in babies with CDH and that are certainly not caused by intrathoracic space conflicts? 5,29,3° Furthermore, often it is understated that the lungs of the few fetuses of the nitrofen-exposed litters that do not have CDH also have significant degrees of lung hypoplasia6 and a trend toward biochemical immaturity 7 that strongly suggest that disturbed organogenesis of the lung itself occurs before compression jeopardizes its development. The present study confirms that, like in human CDH and in the fetal lamb model, heart size is markedly reduced in nitrofen-treated rats with CDH, that this reduction of muscle mass is more marked in the left chambers, and that it corresponds to organ hypoplasia. Even the lack of correlation between heart and lung weights in control animals and its significant correlation in those with CDH are similar to those described in fetal lambs? 3 Therefore, the nature of heart size reduction in the rat model must be very similar to that seen in human and fetal lamb CDH, and this further validates this model for the study of these particular aspects of the disease. Other investigators had previously pointed out that the heart is one of the target organs for nitrofen 31 and that prenatal exposure to lower doses of the chemical caused heart hypoplasia with preservation of protein to DNA ratio 32 and functional disturbances 33 in the offspring, but, unfortunately, no distinction was made in those studies between animals with and without CDH precluding analysis of the possible influence of the diaphragmatic defect on the heart. Our current study adds the concept of the teratogen causing directly some degree of heart hypoplasia even in fetuses without CDH and hence without mediastinal space conflict. We acknowledge that heart hypoplasia in these fetuses was only demonstrated by significant decrease of the heart to body wet weight ratio, but all other variables, although failing to reach statistical significance, showed trends in the same direction. These findings, along with the demonstration of lung hypoplasia in this group of nitrofen-exposed rats without CDH and the previous observation in them of pulmonary lesions, 6 biochemical immaturity] and cardiovascular malformations 28 of intermediate severity between controls and fetuses with CDH suggest that nonmechanical factors play a role in these components of the disease. We believe that investigation of the molecular and genetic regulation of organogenesis in this model should be pursued on this line.
REFERENCES 1. Harrison MR, Jester JA, Ross NA: Correction of congenital diaphragmatic hernia in utero. I. The model: Intrathoracic balloon produces fatal pulmonary bypoplasia. Surgery 88:174-182, 1980
2. Pringle KC, Turner JW, Schofield JC, et al: Creation and repair of diaphragmatic hernia in the fetal lamb: Lung development and morphology. J Pediatr Surg 19:131-139, 1984
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3. Hashimoto E, Pringle C, Super R, et al: The creation and repair of diaphragmatic hernia in fetal lambs: Morphology of the type II alveolar cell. J Pediatr Surg 20:354-356, 1985 4. Harrison MR, Bressack MA, Churg AM: Correction of congenital diaphragmatic hernia in utero. II. Simulated correction permits fetal lung growth with survival at birth. Surgery 88:260-268, 1980 5. Karamanoukian HL, Wilcox DT, Glick PL: The "missing link" in congenital diaphragmatic hemia. J Pediatr Surg 29:954-956, 1994 (letter) 6. Alfonso LF, Vilanova J, Aldaz~tbal E et al: Lung growth and maturation in the rat model of experimentally induced congenital diaphragmatic hernia. Eur J Pediatr Surg 3:6-11, 1993 7. Alfonso LF, Amaiz A, Alvarez FJ, et al: Lung hypoplasia and suffactant immaturity induced in the fetal rat by prenatal exposure to nitrofen. Biol Neonate 69:94-100, 1996 8. Tenbrinck R, Tibboel D, Gaillard JLJ, et al: Experimentally induced congenital diaphragmatic hernia in rats. J Pediatr Surg 25:426429, 1990 9. Iritani I: Experimental study on embryogenesis of congenital diaphragmatic hernia. Anat Embryol 169:133-139, 1984 10. Bradford MM: A rapid and sensitive method for the qnantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254, 1976 11. Labarca C, Paigen K: A simple, rapid and sensitive DNA assay procedure. Anal Biochem 72:344-352, 1980 12. Martinez-Frias ML, Prieto L, Urioste M, et al: Clinical/ epidemiological analysis of congenital anomalies associated with diaphragmatic hernia. Am J Med Genet 62:71-76, 1996 13. Greenwood RD, Rosenthal A, Nadas AS: Cardiovascular abnormalities associated with congenital diaphragmatic hernia. Pediatrics 57:92-97, 1976 14. Sweed Y, Puri P: Congenital diaphragmatic hernia: Influence of associated malformations on survival. Arch Dis Child 69:68-70, 1993 15. Siebert JR, Haas JE, Beckwith JB: Left ventricular hypoplasia in congenital diaphragmatic liemia. J Pediatr Surg 19:567-571, 1984 16. Crawford DC, Wright VM, Drake DE et al: Fetal diaphragmatic hernia: The value of fetal echocardiography in the prediction of postnatal outcome. Br J Obstet Gyneco196:705-710, 1989 17. Sharland GK, Lockhart SM, Heward AJ, et al: Prognosis in fetal diaphragmatic hernia. Am J Obstet Gynecol 166:9-13, 1992 18. Schwartz SM, Vermillion RE Hirschl RB: Evaluation of left ventricular mass in children with left congenital diaphragmatic hernia. J PediaU 125:447-451, 1994
19. Allan LD, Irish MS, Glick PL: The fetal heart in diaphragmatic hernia. Clin Perinato123:795-812, 1996 20. Thebaud B, Azancot A, De Lagausie P, et al: Congenital diaphragmatic hernia: Antenatal prognostic factors. Int Care Med 23:1062-1069, 1997 21. VanderWall KJ, Kohl T, Adzick NS, et al: Fetal diaphragmatic hernia: Echocardiography and clinical outcome. J Pediatr Surg 32:223225; discussion 225-226, 1997 22. Karamanoukian HL, Glick PL, Wilcox DT, et al: Pathophysiology of congenital diaphragmatic hernia. 11. Anatomic and biochemical characterization of the heart in the fetal lamb CDH model. J Pediatr Surg 30:925-929, 1995 23. Karamanoukian HL, O'Toole SJ, Rossman JR, et al: Can cardiac weight predict lung weight in patients with congenital diaphragmatic hernia? J Pediatr Surg 31:823-825, 1996 24. Karamanoukian HL, O'Toole SJ, Rossman MS, et al: Fetal surgical interventions and the development of the heart in congenital diaphragmatic hernia. J SurgRes 65:5-8, 1996 25. Benjamin DR, Juul S, Siebert JR: Congenital posterolateral diaphragmatic hernia: Associated malformations. J Pediatr Surg 23:899903, 1988 26. Losty PD, Suen HC, Manganaro TF, et al: Prenatal hormonal therapy improves pulmonary compliance in the nitrofen-induced CDH rat model. J Pediatr Surg 30:420-426, 1995 27. Tenbrinck R, Gaillard JLJ, Tibboel D, et al: Pulmonary vascular abnormalities in experimentally induced congenital diaphragmatic hernia in rats. J Pediatr Surg 27:862-865, 1992 28. Migliazza L, Otken C, Xia H, et al: Cardiovascular malformations in experimental diaphragmatic hernia. J Pediatr Surg (in press) 29. David TJ, Illingworth CA: Diaphragmatic hernia in the southwest of England. J Med Genet 13:253-262, 1976 30. Losty PD, Vanamo K, Rintala RJ, et al: Congenital diaphragmatic hernia--Does the side of the defect influence the incidence of associated malformations? J Pediatr Surg 33:507-510, 1998 31. Costlow RD, Manson JM: The heart and diaphragm: Target organs in the neonatal death induced by nitrofen (2,4-Dichlorophenyl-pnitrophenyl Ether). Toxicology 20:209-227, 1981 32. Zeman FJ, Heng H, Hoogenboom ER, et al: Cell number and size in selected organs of fetuses of rats malnourished and exposed to nitrofen. Teratog Carcinog Mutagen 6:339-347, 1986 33. Lau C, Cameron AM, Irsula O, et al: Effects of prenatal nitrofen exposure on cardiac structure and function in the rat. Toxicol Appl Pharmacol 86:22-32, 1986
Discussion A. Coran (Ann Arbor, MI): In this p a r t i c u l a r m o d e l d i d
s t u d y t h e l u n g s in this m o d e l a n d see e v i d e n c e o f
you detect evidence of pulmonary hypertension histologically, s u c h as t h i c k e n i n g o f t h e p u l m o n a r y arterial m e d i a ?
pulmonary hypertension? J. Tovar (response): I n this p a r t i c u l a r e x p e r i m e n t w e
A n d d o y o u t h i n k t h a t h a s a n y r e l a t i o n s h i p to the c a r d i a c
d i d n o t s t u d y that, b u t f o r m e r l y w e h a v e d o n e it s e v e r a l
findings?
times. O t h e r i n v e s t i g a t o r s , s o m e o f t h e m i n this r o o m ,
L. Migliazza (response): W e h a v e f o u n d t h a t h e a r t
h a v e i l l u s t r a t e d t h a t t h e r e are v a s c u l a r a n o m a l i e s i n the
h y p o p l a s i a is p r e s e n t also in t h e fetus w i t h o u t hernia. So
l u n g s t h a t are c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t t h e y
w e c a n h y p o t h e s i z e t h a t t h e r e is also s o m e o t h e r f a c t o r
have pulmonary hypertension.
i m p l i c a t e d in this h y p o p l a s i a o t h e r t h a n the a n a t o m i c c h a n g e s or t h e h y p o p l a s i a o f the l u n g a n d m e c h a n i c a l compression.
A. Coran (Ann Arbor, MI): D i d y o u h i s t o l o g i c a l l y
A. DeLormier (San Francisco, CA): It is c l e a r t h a t n i t r o f e n is a s y s t e m i c p o i s o n . I w o n d e r a b o u t u s i n g this or l e a n i n g o n this m o d e l a n d e x t e n d i n g this to the s i t u a t i o n in a h u m a n d i a p h r a g m a t i c hernia. It w o u l d b e h e l p f u l i f
HEART HYPOPLASIA IN EXPERIMENTAL CDH
you could just mechanically create the diaphragmatic hernia and study the heart effects. I would not be surprised if it was not a mechanical effect on heart development. It would be a purer model. J. Tovar (response): It is very well known that this heart hypoplasia has been produced in the surgical model
711
of fetal diaphragmatic hernia. I think that it has been very consistently proven that it can be related to pressure itself. But the point of this paper is to show that perhaps there are other factors involved, not only pressure, but also some nonmechanical factors that can act on the heart.