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Pediatric Surgery International

, Volume 27, Issue 2, pp 165–173 | Cite as

Abnormal development of the enteric nervous system in rat embryos and fetuses with congenital diaphragmatic hernia

  • Leopoldo Martínez
  • Rosa Aras-López
  • Sara Lancha
  • María Teresa Vallejo-Cremades
  • Federica Pederiva
  • Liu XiaoMei
  • Juan Antonio Tovar
Original Article
  • 153 Downloads

Abstract

Background/Aim

Esophageal dilatation, gastroesophageal reflux, and intestinal obstruction have been demonstrated in CDH survivors. Abnormal esophageal and intestinal innervations were recently found in rats and babies with this disease. Our aim was to further characterize these malformations in embryos and fetal rats exposed to nitrofen.

Methods

Pregnant rats received either 100 mg nitrofen or vehicle on E9.5. Fetuses were recovered at E15, E18, and E21. Sections of esophagus and small bowel were histochemically stained with acetylcholinesterase (AChE) and immunostained for PGP9.5. PGP9.5 gen protein were measured on E21 and PGP9.5 mRNA on E15, E18 and E21. Comparisons between groups were made with non-parametrics tests.

Results

Histochemistry and immunohistochemistry showed deficient innervation in all anatomical areas studied at E15, E18, and E21, and WB confirmed this decrease in E21 fetuses. PGP9.5 messenger was decreased in nitrofen-exposed animals on E18 (esophagus) or E15 (small bowel), and increased on E21 in the esophagus and E18 in small bowel.

Conclusions

Development of the enteric nervous system of the esophagus, stomach, and small bowel is deficient in rat embryos and fetuses exposed to nitrofen. These anomalies could account in part for the long-term gastrointestinal morbidity observed in CDH survivors.

Keywords

Congenital diaphragmatic hernia Nitrofen Enteric nervous system PGP9.5 Histochemistry Acetylcholinesterase Immunohistochemistry Western blot RT-PCR 

Notes

Acknowledgments

Supported in part by FIS (06/0486 and 06/0447), IdiPaz and FMM Grants and by the Spanish Health Institute Carlos III (grant No. RD08/0072: Maternal, Child Health and Development Network).

References

  1. 1.
    Arena F, Romeo C, Baldari S, Arena S, Antonuccio P, Campenni A, Zuccarello B, Romeo G (2008) Gastrointestinal sequelae in survivors of congenital diaphragmatic hernia. Pediatr Int 50:76–80CrossRefPubMedGoogle Scholar
  2. 2.
    Bagolan P, Morini F (2007) Long-term follow up of infants with congenital diaphragmatic hernia. Semin Pediatr Surg 16:134–144CrossRefPubMedGoogle Scholar
  3. 3.
    Lally KP, Engle W (2008) Postdischarge follow-up of infants with congenital diaphragmatic hernia. Pediatrics 121:627–632CrossRefPubMedGoogle Scholar
  4. 4.
    Peetsold MG, Kneepkens CF, Heij HA, Ijsselstijn H, Tibboel D, Gemke RJ (2010) Congenital diaphragmatic hernia: long-term risk of gastroesophageal reflux disease. J Pediatr Gastroenterol Nutr 51(4):448–453CrossRefPubMedGoogle Scholar
  5. 5.
    Stolar CJ (1996) What do survivors of congenital diaphragmatic hernia look like when they grow up? Semin Pediatr Surg 5:275–279PubMedGoogle Scholar
  6. 6.
    Vanamo K, Rintala RJ, Lindahl H, Louhimo I (1996) Long-term gastrointestinal morbidity in patients with congenital diaphragmatic defects. J Pediatr Surg 31:551–554CrossRefPubMedGoogle Scholar
  7. 7.
    Fasching G, Huber A, Uray E, Sorantin E, Lindbichler F, Mayr J (2000) Gastroesophageal reflux and diaphragmatic motility after repair of congenital diaphragmatic hernia. Eur J Pediatr Surg 10:360–364PubMedGoogle Scholar
  8. 8.
    Nagaya M, Akatsuka H, Kato J (1994) Gastroesophageal reflux occurring after repair of congenital diaphragmatic hernia. J Pediatr Surg 29:1447–1451CrossRefPubMedGoogle Scholar
  9. 9.
    Qi B, Soto C, Diez-Pardo JA, Tovar JA (1997) An experimental study on the pathogenesis of gastroesophageal reflux after repair of diaphragmatic hernia. J Pediatr Surg 32:1310–1313CrossRefPubMedGoogle Scholar
  10. 10.
    Makhoul IR, Shoshany G, Smolkin T, Epelman M, Sujov P (2001) Transient mega-esophagus in a neonate with congenital diaphragmatic hernia. Eur Radiol 11:867–869CrossRefPubMedGoogle Scholar
  11. 11.
    Stolar CJ, Levy JP, Dillon PW, Reyes C, Belamarich P, Berdon WE (1990) Anatomic and functional abnormalities of the esophagus in infants surviving congenital diaphragmatic hernia. Am J Surg 159:204–207CrossRefPubMedGoogle Scholar
  12. 12.
    Pederiva F, Rodriguez JI, Ruiz-Bravo E, Martinez L, Tovar JA (2009) Abnormal intrinsic esophageal innervation in congenital diaphragmatic hernia: a likely cause of motor dysfunction. J Pediatr Surg 44:496–499CrossRefPubMedGoogle Scholar
  13. 13.
    Mortell A, Montedonico S, Puri P (2006) Animal models in pediatric surgery. Pediatr Surg Int 22:111–128CrossRefPubMedGoogle Scholar
  14. 14.
    Tovar JA (2007) Stephen L Gans distinguished overseas lecture. The neural crest in pediatric surgery. J Pediatr Surg 42:915–926CrossRefPubMedGoogle Scholar
  15. 15.
    Le Douarin N, Kalcheim C (1999) The neural crest. Cambridge University Press, CambridgeGoogle Scholar
  16. 16.
    Le Douarin NM, Smith J (1988) Development of the peripheral nervous system from the neural crest. Annu Rev Cell Biol 4:375–404CrossRefPubMedGoogle Scholar
  17. 17.
    Laranjeira C, Pachnis V (2008) Enteric nervous system development: recent progress and future challenges. Auton Neurosci 151:61–69CrossRefGoogle Scholar
  18. 18.
    Martinez L, Gonzalez-Reyes S, Burgos E, Tovar JA (2004) The vagus and recurrent laryngeal nerves in experimental congenital diaphragmatic hernia. Pediatr Surg Int 20:253–257CrossRefPubMedGoogle Scholar
  19. 19.
    Martinez L, Pederiva F, Martinez-Calonge W, Aras-Lopez R, Tovar JA (2009) The myenteric plexus of the esophagus is abnormal in an experimental congenital diaphragmatic hernia model. Eur J Pediatr Surg 19:163–167CrossRefPubMedGoogle Scholar
  20. 20.
    Chen C, Jeruss S, Chapman JS, Terrin N, Tighiouart H, Glassman E, Wilson JM, Parsons SK (2007) Long-term functional impact of congenital diaphragmatic hernia repair on children. J Pediatr Surg 42:657–665CrossRefPubMedGoogle Scholar
  21. 21.
    Chiu PP, Sauer C, Mihailovic A, Adatia I, Bohn D, Coates AL, Langer JC (2006) The price of success in the management of congenital diaphragmatic hernia: is improved survival accompanied by an increase in long-term morbidity? J Pediatr Surg 41:888–892CrossRefPubMedGoogle Scholar
  22. 22.
    Koivusalo AI, Pakarinen MP, Lindahl HG, Rintala RJ (2008) The cumulative incidence of significant gastroesophageal reflux in patients with congenital diaphragmatic hernia-a systematic clinical, pH-metric, and endoscopic follow-up study. J Pediatr Surg 43:279–282CrossRefPubMedGoogle Scholar
  23. 23.
    Karnak I, Senocak ME, Tanyel FC, Buyukpamukcu N (2001) Abnormal esophageal anatomy associated with a congenital diaphragmatic hernia: report of a case. Surg Today 31:1005–1007CrossRefPubMedGoogle Scholar
  24. 24.
    Sue K, Yamada T, Hirayama Y, Tanaka N, Hirata T, Araki H (1993) Abnormality of lower esophagus associated with congenital diaphragmatic hernia. Pediatr Surg Int 8:14–16CrossRefGoogle Scholar
  25. 25.
    Le Douarin NM, Teillet MA (1973) The migration of neural crest cells to the wall of the digestive tract in avian embryo. J Embryol Exp Morphol 30:31–48PubMedGoogle Scholar
  26. 26.
    Burns AJ (2005) Migration of neural crest-derived enteric nervous system precursor cells to and within the gastrointestinal tract. Int J Dev Biol 49:143–150CrossRefPubMedGoogle Scholar
  27. 27.
    Bondurand N, Kobetz A, Pingault V, Lemort N, Encha-Razavi F, Couly G, Goerich DE, Wegner M, Abitbol M, Goossens M (1998) Expression of the SOX10 gene during human development. FEBS Lett 432:168–172CrossRefPubMedGoogle Scholar
  28. 28.
    Kuhlbrodt K, Herbarth B, Sock E, Hermans-Borgmeyer I, Wegner M (1998) Sox10, a novel transcriptional modulator in glial cells. J Neurosci 18:237–250PubMedGoogle Scholar
  29. 29.
    Young HM, Ciampoli D, Hsuan J, Canty AJ (1999) Expression of Ret-, p75(NTR)-, Phox2a-, Phox2b-, and tyrosine hydroxylase-immunoreactivity by undifferentiated neural crest-derived cells and different classes of enteric neurons in the embryonic mouse gut. Dev Dyn 216:137–152CrossRefPubMedGoogle Scholar
  30. 30.
    Newgreen D, Young HM (2002) Enteric nervous system: development and developmental disturbances–part 1. Pediatr Dev Pathol 5:224–247PubMedGoogle Scholar
  31. 31.
    Newgreen D, Young HM (2002) Enteric nervous system: development and developmental disturbances–part 2. Pediatr Dev Pathol 5:329–349CrossRefPubMedGoogle Scholar
  32. 32.
    Greer JJ, Babiuk RP, Thebaud B (2003) Etiology of congenital diaphragmatic hernia: the retinoid hypothesis. Pediatr Res 53:726–730CrossRefPubMedGoogle Scholar
  33. 33.
    Major D, Cadenas M, Fournier L, Leclerc S, Lefebvre M, Cloutier R (1998) Retinol status of newborn infants with congenital diaphragmatic hernia. Pediatr Surg Int 13:547–549CrossRefPubMedGoogle Scholar
  34. 34.
    Wilson JG, Roth CB, Warkany J (1953) An analysis of the syndrome of malformations induced by maternal vitamin A deficiency. Effects of restoration of vitamin A at various times during gestation. Am J Anat 92:189–217CrossRefPubMedGoogle Scholar
  35. 35.
    Mey J, Babiuk RP, Clugston R, Zhang W, Greer JJ (2003) Retinal dehydrogenase-2 is inhibited by compounds that induce congenital diaphragmatic hernias in rodents. Am J Pathol 162:673–679PubMedGoogle Scholar
  36. 36.
    Niederreither K, Vermot J, Le Roux I, Schuhbaur B, Chambon P, Dolle P (2003) The regional pattern of retinoic acid synthesis by RALDH2 is essential for the development of posterior pharyngeal arches and the enteric nervous system. Development 130:2525–2534CrossRefPubMedGoogle Scholar
  37. 37.
    Sato Y, Heuckeroth RO (2008) Retinoic acid regulates murine enteric nervous system precursor proliferation, enhances neuronal precursor differentiation, and reduces neurite overgrowth in vitro. Dev Biol 310:185–198CrossRefGoogle Scholar
  38. 38.
    Kawaguchi J, Nichols J, Gierl MS, Faial T, Smith A (2010) Isolation and propagation of enteric neural crest progenitor cells from mouse embryonic stem cells and embryos. Development 137:693–704CrossRefPubMedGoogle Scholar
  39. 39.
    Bellusci S, Grindley J, Emoto H, Itoh N, Hogan BL (1997) Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung. Development 124:4867–4878PubMedGoogle Scholar
  40. 40.
    Unger S, Copland I, Tibboel D, Post M (2003) Down-regulation of sonic hedgehog expression in pulmonary hypoplasia is associated with congenital diaphragmatic hernia. Am J Pathol 162:547–555PubMedGoogle Scholar
  41. 41.
    Fu M, Lui VC, Sham MH, Pachnis V, Tam PK (2004) Sonic hedgehog regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut. J Cell Biol 166:673–684CrossRefPubMedGoogle Scholar
  42. 42.
    Reichenbach B, Delalande JM, Kolmogorova E, Prier A, Nguyen T, Smith CM, Holzschuh J, Shepherd IT (2008) Endoderm-derived Sonic hedgehog and mesoderm Hand2 expression are required for enteric nervous system development in zebrafish. Dev Biol 318:52–64CrossRefPubMedGoogle Scholar
  43. 43.
    Goldstein AM, Brewer KC, Doyle AM, Nagy N, Roberts DJ (2005) BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system. Mech Dev 122:821–833CrossRefPubMedGoogle Scholar
  44. 44.
    Groenman F, Unger S, Post M (2005) The molecular basis for abnormal human lung development. Biol Neonate 87:164–177CrossRefPubMedGoogle Scholar
  45. 45.
    Gershon MD, Ratcliffe EM (2004) Developmental biology of the enteric nervous system: pathogenesis of Hirschsprung’s disease and other congenital dysmotilities. Semin Pediatr Surg 13:224–235CrossRefPubMedGoogle Scholar
  46. 46.
    Pederiva F, Aras-Lopez R, Rodriguez JI, Martinez L, Tovar JA (2010) Bronchopulmonary innervation defects in infants and rats with congenital diaphragmatic hernia. J Pediatr Surg 45:360–365CrossRefPubMedGoogle Scholar
  47. 47.
    Aubin J, Chailler P, Menard D, Jeannotte L (1999) Loss of Hoxa5 gene function in mice perturbs intestinal maturation. Am J Physiol 277:C965–C973PubMedGoogle Scholar
  48. 48.
    Chinoy MR, Nielsen HC, Volpe MV (2002) Mesenchymal nuclear transcription factors in nitrofen-induced hypoplastic lung. J Surg Res 108:203–211CrossRefPubMedGoogle Scholar
  49. 49.
    Fuchs S, Herzog D, Sumara G, Buchmann-Moller S, Civenni G, Wu X, Chrostek-Grashoff A, Suter U, Ricci R, Relvas JB, Brakebusch C, Sommer L (2009) Stage-specific control of neural crest stem cell proliferation by the small rho GTPases Cdc42 and Rac1. Cell Stem Cell 4:236–247CrossRefPubMedGoogle Scholar
  50. 50.
    Teramoto H, Yoneda A, Puri P (2003) Gene expression of fibroblast growth factors 10 and 7 is downregulated in the lung of nitrofen-induced diaphragmatic hernia in rats. J Pediatr Surg 38:1021–1024CrossRefPubMedGoogle Scholar
  51. 51.
    Guarino N, Solari V, Shima H, Puri P (2004) Upregulated expression of EGF and TGF-alpha in the proximal respiratory epithelium in the human hypoplastic lung in congenital diaphragmatic hernia. Pediatr Surg Int 19:755–759CrossRefPubMedGoogle Scholar
  52. 52.
    Kavanagh M, Seaborn T, Crochetiere J, Fournier L, Battistini B, Piedboeuf B, Major D (2005) Modulating effect of a selective endothelin A receptor antagonist on pulmonary endothelin system protein expression in experimental diaphragmatic hernia. J Pediatr Surg 40:1382–1389CrossRefPubMedGoogle Scholar
  53. 53.
    Lee HO, Levorse JM, Shin MK (2003) The endothelin receptor-B is required for the migration of neural crest-derived melanocyte and enteric neuron precursors. Dev Biol 259:162–175CrossRefPubMedGoogle Scholar
  54. 54.
    Kapur RP (2009) Practical pathology and genetics of Hirschsprung’s disease. Semin Pediatr Surg 18:212–223CrossRefPubMedGoogle Scholar
  55. 55.
    Gonzalez-Reyes S, Fernandez-Dumont V, Calonge WM, Martinez L, Tovar JA (2006) Vitamin A improves Pax3 expression that is decreased in the heart of rats with experimental diaphragmatic hernia. J Pediatr Surg 41:327–330CrossRefPubMedGoogle Scholar
  56. 56.
    Yu J, Gonzalez S, Martinez L, Diez-Pardo JA, Tovar JA (2003) Effects of retinoic acid on the neural crest-controlled organs of fetal rats. Pediatr Surg Int 19(5):355–358CrossRefPubMedGoogle Scholar
  57. 57.
    Yu J, Gonzalez S, Rodriguez JI, Diez-Pardo JA, Tovar JA (2001) Neural crest-derived defects in experimental congenital diaphragmatic hernia. Pediatr Surg Int 17:294–298CrossRefPubMedGoogle Scholar
  58. 58.
    Pederiva F, Aras-Lopez R, Martinez L, Tovar JA (2009) Tracheal innervation is abnormal in rats with experimental congenital diaphragmatic hernia. J Pediatr Surg 44:1159–1164CrossRefPubMedGoogle Scholar
  59. 59.
    Burns AJ, Delalande JM (2005) Neural crest cell origin for intrinsic ganglia of the developing chicken lung. Dev Biol 277:63–79CrossRefPubMedGoogle Scholar
  60. 60.
    Wilson PO, Barber PC, Hamid QA, Power BF, Dhillon AP, Rode J, Day IN, Thompson RJ, Polak JM (1988) The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. Br J Exp Pathol 69:91–104PubMedGoogle Scholar
  61. 61.
    Schofield JN, Day IN, Thompson RJ, Edwards YH (1995) PGP9.5, a ubiquitin C-terminal hydrolase; pattern of mRNA and protein expression during neural development in the mouse. Brain Res Dev Brain Res 85:229–238CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Leopoldo Martínez
    • 1
  • Rosa Aras-López
    • 2
  • Sara Lancha
    • 2
  • María Teresa Vallejo-Cremades
    • 2
  • Federica Pederiva
    • 1
  • Liu XiaoMei
    • 3
    • 4
  • Juan Antonio Tovar
    • 1
  1. 1.Department of Pediatric SurgeryHospital Universitario La PazMadridSpain
  2. 2.Research Laboratory IdipazHospital Universitario La PazMadridSpain
  3. 3.Central LaboratoryShengjing Hospital of China Medical UniversityShenyangChina
  4. 4.Research Laboratory IdiPazHospital Universitario La PazMadridSpain

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