Cardiovascular malformations in experimental congenital diaphragmatic hernia

Cardiovascular

Malformations in Experimental Diaphragmatic Hernia

By Paul D. Losty, M. Gwen

Connell, Ralf Freese, Stefan Laval, Bruce 0. Okoye, Dietrich Kluth, and David A. Lloyd Liverpool,

England

and

BackgrouncUPurpose:

Newborns with congenital diaphragmatic hernia (CDH) frequently have associated anomalies that have a major impact on survival rate independent of pulmonary hypoplasia and pulmonary hypertension. Cardiovascular malformations (CVM) represent a major group of lethal extrapulmonary abnormalities that often assume greatest prognostic significance in most CDH studies. Animal models resembling human CDH may aid knowledge of the basic embryology that leads to the coexpression of CDH and CVM. This study, therefore, analyzed the incidence and spectrum of CVM in fetal rats with CDH. Methods: Left-sided CDH (LCDH) was induced in fetal rats by the maternal administration of 100 mg of nitrofen by gavage on day 9.5 gestation (term, day 22). Control animals received olive oil (00) and were used for comparative analysis. Fetal rats were harvested by cesarean section on day 21.5 or day 22, histologically processed and examined for CVM. Resu/ts:A 60 (25%)

significant LCDH rats

number compared

of CVM were observed with 4 of 60 (6.7%)

in 15 of nrtrofen

N

EWBORNS WITH CONGENITAL diaphragmatic hernia (CDH) frequently have associated anomalies that have a major impact on survival independent of pulmonary hypoplasia and pulmonary hypertension.1-9 Cardiovascular malformations (CVM) represent such a group of lethal extrapulmonary abnormalities that often assume prognostic significance in CDH outcome studies.3.6,8Animal models resembling human CDH may aid our knowledge of the basic embryology that leads to the coexpression of CDH and CVM.lon This study therefore analyzed the incidence and spectrum of CVM in fetal rats with CDH to determine whether they occurred with an equal frequency and pathological subtype to that encountered in the human with CDH. MATERIALS

AND

METHODS

Timed-pregnant Sprague-Dawley rats (Charles River UK Ltd; vaginal plug positive. day 0) were given 100 mg nitrofen (ZheJtang Chemicals. Peoples Republic of China) by gavage on gestation day 9.5 (term, day 22) to induce left-sided CDH (LCDH) in fetal rats.loJ* Control animals recteved olive oil (00) and were used for all comparative analysis. Pregnant rats were terminally anesthetised using halothane, and fetal rats were harvested by cesarean section on days 21.5 and day 22. Whole fetuses were dissected to document the presence or absence of a CDH. Specimens were then fixed in 10%

Journal

of Pediatric

Surgery,

Vol34,

Congenital

No 8 (August),

1999: pp 1203-I

207

Hamburg,

Audrey

Smith,

Germany

non-CDH rats (P= .Ol). The spectrum of abnormalities in CDH included ventricular septal (VSD) defects (n = 6), vascular rings (n = 4), anomalous subclavian arteries (n = 3), atrioventricular septal defects (n = 1) and Fallot’s tetralogy (t-r = 1). A VSD (n = I), double-outlet right ventricle VSD (n = 1) and Fallot’s tetralogy (n = 2) were noted in nitrofen non-CDH rats. Control (00) fetal rats (n = 60) displayed no malformations. Conclusions: These results confirm a significant incidence and spectrum of CVM in a teratogenic CDH model similar to that seen in humans with CDH. The findings of this study reinforce the validity of the nitrofen model as a research tool to uncover the genetic and molecular mechanisms responsable for the genesis of CDH and allied malformations. J Pediatr Surg 34:1203-1207. Copyright o 1999 by W.B. Saunders Company. INDEX WORDS: Congenital cular malformations, animal

diaphragmatic hernia, model, nitrofen.

cardiovas-

buffered formalin for 48 to 72 hours. Tissue blocks from the neck region to the upper abdomen were processed histologically, paraffin embedded. lo-pm serial sections taken m a transverse, frontal, or saggital plane and stained with H&E. Adetarled morphological study of the cardiovascular system of each fetus was performed using light mtcroscopy. CVM abnormalittes were documented and photomtcrographs taken of the areas of interest. Statistical analysis was performed using Fisher’s Exact test; a P value less than .05 was considered sigmficant.

RESULTS

A summary of the findings are listed in the Table. CVM abnormalities were noted in 15 of 60 (25%) LCDH rats compared with 4 of 60 (6.7%) nitrofen non-CDH rats

From the Departments of Paediatric Surgery and Cardzac Anatomy, Institute of Child Health, Alder Hey Children’s Hospital. The ihversity of Liverpool, Liverpool, England, arid the Department of Pediatric Surgery Universlfy Hospital Hamburg, Hambwg, Germany. This study was supported by grants from the Chddren’s Research Furld and The Royal Liverpool Children’s Hospital NHS Trust (Alder Hey) Endowment Fund. Address reprint requests to Paul D. Losty, Institute of Child Health, Alder Hey Children‘s Hospital, Eaton Rd, Liverpool L1-7 2AP. England. Copyright 0 I999 by WB. Saunders Compalzy 0022-3468/99/3408-0003$03.00/O

1203

1204

LOST?’

Table 1. Incidence LCDH,

and Spectrum Nitrofen

Total (X) cardiac malformations Cardiac defect

of Cardiovascular

Non-CDH

Rats,

Malformations

Nitrofen Non-CDH (n = 60)

15 (25)*

4 (6.7)

4Vascular

3 Anomalous subclavian arteries

00

COfltrOlS (n = 60)

0 (0)

1 VSD 1 DORVNSD

rings

in

and Controls

LCDH (n = 60)

6 VSD

ET AL

-

2 Fallots

1 AVSD 1 Fallots Abbreviations: DORV, tricular septal defect. *P = .Ol LCDH versus

double nitrofen

outlet

right

non-CDH,

ventricle; Fisher’s

AVSD,

atrioven-

Exact test.

(P = .Ol, Fisher’s Exact test). The abnormalities in CDH included ventricular septal defects (n = 6; Fig l), vascular rings of varying complexity (n = 4; Fig 2), anomalous right subclavian arteries (n = 3; Fig 3), atrioventricular septal defects (n = 1; Fig 4), and Fallot’s tetralogy (n = 1; Figs 5A and B). Nitrofen non-CDH rats had a significantly lower incidence of CVM (6.7%). However, the spectrum was similar and included a ventricular septal defect (n = l), double outlet right ventricleventricular septal defect (n = 1; Figs 6A and B), and Fallot’s tetralogy (n = 2; Figs 7A and B). Control fetal (00) rats (n = 60) displayed no malformations. All hearts examined had usual atrial arrangements and concordant atrioventricular connections.

DISCUSSION

Cardiovascular malformations (CVM) form a group of lethal extrapulmonary abnormalities that often assume

Fig 1. Ventricular septal defect (arrow) ventricle; LV, left ventricle; AV, aortic valve. tion x25.)

in a CDH rat. RV, right (H&E, original magnifica-

Fig 2. Representative example of a vascular ring anomaly in a CDH rat. In this section, a right(R), and left(L) arterial duct (bilateral ductus arteriosus) is shown. A ligamentous (Lig) portion of the vascular ring is also seen compressing the trachea (TR) and esophagus (OES). Ao, aorta. (H&E, original magnification x40.)

prognostic significance in CDH outcome studies.3,6*8In an effort to gain insight into the pathogenesis of these associated anomalies in humans, the current study analyzed CVM in fetal rats with nitrofen-induced CDH. We primarily consider it instructive to determine whether they occurred with an equal frequency and pathological subtype to that encountered in the human with a CDH. Our findings have illustrated a significant incidence and spectrum of cardiovascular abnormalities in the nitrofen model similar to that reported in malformation studies on humans with CDH.3-9 Nitrofen is widely known to induce multiple abnormalities in many organ systems in the rodent, notably, renal, central nervous system, gastrointestinal, lung malformations, and diaphragmatic defects. 10~12-16 However, the heart and diaphragm generally are believed to be the principle primary

CARDIOVASCULAR

ABNORMALITIES

IN EXPERIMENTAL

CDH

1205

Fig 3. Anomalous right-sided subclavian artery (WA) in a CDH rat. AA, aortic arch; TR, trachea; OES, esophagus. (H&E, original magnification X40.)

targets for the teratogen.r3 The pathology encountered in the rodent seems to be critically regulated by the dosage schedule and timing of prenatal nitrofen exposure.*“J3.15 Using protocols designed to generate an animal phenotype typical of the most common lethal form of human CDH. namely the left-sided congenital diaphragmatic defect (LCDH), it was of interest that we observed a comparable spectrum and frequency of cardiovascular malformations in CDH rats. Moreover, these CVM abnormalities were encountered in significantly greater numbers in nitrofen CDH versus nitrofen non-CDH rats, leading us to speculate a specific aberration in the genetic phenotype of the CDH animals. The cardiovascular malformations in CDH and nitrofen non-CDH rats included a wide range of abnormalities namely ventricular septal defects, atrio-ventricular septal defects, Fallot’s tetralogy, double outlet right ventricle, vascular rings, and anomalous right subclavian arteries.

Fig 4. Atrioventricular septal defect in a CDH rat. Asterisk denotes a common junction between all four heart chambers. RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle. The atria are filled with blood, and the ventricles appear slitlike. (H&E, original magnification x40.)

Fig 5. (A) Tetralogy of Fallot malformation in a CDH rat. This section shows the origins of an overiding aortic valve (AV). RV, right ventricle; LV, left ventricle. A ventricular septal defect (arrow) also is seen. (H&E, original magnification x40.) (B) Tetralogy of Fallot malformation in a CDH rat. This section, taken at valvar level, shows a pulmonary arterial valvar stenosis (Pv). AV, aor-tic valve. (H&E, original magnification, x40.)

The majority of these malformations are believed to occur embryologically through a generalized disturbance in migration of neural crest cells destined to form the cardiac outflow tract and great vessels.r7.rs It may be assumed; therefore, that nitrofen in some way interferes with this migration process. Further studies are required with neural crest cell markers to fully answer these unresolved questions. However, the concept of an embryological “field defect” theory in the pathogenesis of CDH and associated cardiovascular malformations is increasingly appealing given these observations. The findings of this experimental study further strengthen the validity of the nitrofen CDH model as a research tool to uncover the molecular mechanisms responsible for the genesis of human CDH and allied malformations. Differential display polymerase (ddPCR) gene techniques may now be used to screen for genetic abnormalities, which, in turn, could unravel potential new therapies for this highly lethal human anomaly.5J9-21

1206

LOSTY

Fig 6. Double outlet right ventricle in a nitrofen non-CDH rat. Both great arteries pulmonary valve. (H&E, original magnification x25.) (B) Anomaly as above in a nitrofen (arrow). RV, right ventricle; *v, aortic valve; LV, left ventricle. (H&E, original magnification

Fig 7. RV, right

section x40.)

(A) Tetralogy of Fallot malformation in a nitrofen non-CDH rat. A ventricular ventricle; LV, left ventricle. (H&E, original magnification x40.) (B) Tetralogy a small and narrow pulmonary root and pulmonary valve (PV) is illustrated.

arise from the right ventricle non-CDH rat shows in addition x25.)

ET AL

(RV). AV, aortic valve; PV, a ventricular septal defect

septal defect (arrow) and overiding aortic valve (AV) is seen. of Fallot malformation in a nitrofen non-CDH rat. In this Ao, aorta; RV, right ventricle. (H&E, original magnification

CARDIOVASCULAR

ABNORMALITIES

IN EXPERIMENTAL

1207

CDH

REFERENCES I. Adzick NS, Harrison MR. Ghck PL, et al: Diaphragmatic hernia m the fetus: Prenatal diagnosis and outcome m 94 cases. J Pediatr Surg 20:357-361,1985 2. Adzick NS, Vacanti JP, Lillehei CW, et al: Fetal diaphragmatic hernia: Ultrasound diagnosis and clmical outcome in 38 cases. J Pediatr Surg 24:654-658. 1989 3. Fauza DO, Wilson JM: Congenital diaphragmatic hernia and associated anomalies, Their inctdence. identification and impact on prognosis. J Pediatr Surg 29:1113-1117,1994 4. Fitzgerald RT: Congenital diaphragmatic hernia: A study of mortality factors. Ir J Med Sci 146:280-284, 1977 5. Harrison MR. Adzick NS. Flake AW: Congenital diaphragmatic hernia: An unsolved problem. Semin Pediatr Surg 2:109-112, 1993 6. Sweed Y, Puri P: Congenital diaphragmatic hernia: Influence of associated malformations on survival. Arch Dis Child 69:76-70. 1993 7. Losty PD, Vanamo K, Rmtala RJ, et al: Congenital diaphragmatic hernia-Does the side of the defect influence the incidence of associated malformations? J Pediatr Sug 33:507-510. 1998 8. Cuniff C, Jones KL, Jones MC: Patterns of malformation in children with congemtal diaphragmatic defects. J Pediatr 116:258-261; 1990 9. Butler N, Claireaux AE: Congenital diaphragmatic hernia as a cause of perinatal mortality. Lancet 1 659-661, 1962 10. Kluth D, Kangah P. Reich P, et al: Nitrofen-induced diaphragmatic hernias in rats: An animal model. J Pediatr Sug 25:850-854.1990 11. Tenbrinck R, Tibboel D, Gaillard JLJ. et al: Experimentally induced congenital diaphragmatic hernia m rats. J Pediatr Surg 25:426429.1990

12. Nakao Y. Intani I: Experimental animal model of congenital dtaphragmatic hernia induced chemically. Teratology 24: 1 IA, 198 1 (abstr) 13. Costlow R, Manson J: The heart and diaphragm: Target organs in the neonatal death induced by mtrofen (2.4~dichlorophenyl-p-nmophenyl ether). Toxicology 20.209-227, 1981 14. Iritani I: Experimental study on embryogenesis of congenital diaphragmatic hernia. Anat Embryo1 169:133-139, 1984 15. Wickman D, Siebert J. Benjamm D: Nitrofen-induced congenital diaphragmatic defects in CD-l mice. Teratology 47:119-125, 1993 16. Baoquan Q. Diez-Pardo JA, Tovar JA: Intestinal rotation m experimental congenital diaphragmatic hernia. J Pedtatr Surg 30:14571462,1995 17. LeLievre CS, Le Douarin NM: Mesenchymal derivatives of the neural crest: Analysis of chimaeric quail and chick embryos. J Embryo1 Exp Morph01 34:125-154, 1976 18. Kirby ML, Waldo KL: Role of neural crest in congemtal heart disease. Circulation 82:332-340, 1990 19. Liang P, Pardee AB: Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction Science 257:967-971, 1992 20. Liang l? Zhu W, Zhang X, et al: Differentral display using one-base anchored ohgo-dT primers. Nucleic Acids Res 225763-5764, 1994 21. Zhang L. Zgleszewski SE, Cilley RE. et al: Differential display of genes in normal and hypoplastic fetal murine lungs. J Surg Res 75:66-73, 1998