Abnormal Development of Lung Innervation in Experimental Esophageal Atresia

Abstract

Background/Aim Patients with esophageal atresia and tracheo-esophageal fistula (EA-TEF) have chronic respiratory tract disease and deficient tracheal innervation. This study tests the hypothesis in rats with EA-TEF that deficient lung innervation could be one of the causes of respiratory disease.

Material and Methods Pregnant rats were treated with either 2 mg/kg i.p. adriamycin or vehicle on E7, E8 and E9. Lungs and tracheas were retrieved on E15, E18 and E21 (term: E22). Innervation was examined by regular (PGP 9.5 and GDNF) and whole-mount (PGP 9.5 and α-actin) immunohistochemistry. PGP 9.5 and GDNF mRNA were measured by real-time, quantitative RT-PCR and the levels of PGP 9.5 protein by immunoblot. Embryonic lung primordia harvested on E13 were cultured for 72 h and airway peristalsis was assessed under an inverted microscope. PGP 9.5 expression was then examined in explants by whole-mount immunohistochemistry and RT-PCR. Values were compared with non-parametric tests.

Results Neural networks were present in both EA-TEF and control fetuses on E15, E18 and E21, but the neural network was obviously disorganized in whole-mount immunohistochemistry of EA-TEF. The pan-neural marker PGP 9.5 protein was increased at term whereas the neural chemo-attractant GDNF protein was unchanged. PGP 9.5 mRNA significantly increased from subnormal levels on E15 to very increased ones on E18 compared with controls. GDNF mRNA levels followed the same trend. Airway peristalsis of explanted embryonal lungs was similar in both groups. The neural networks were underdeveloped in these primordia, as assessed by whole-mount PGP 9.5 immunohistochemistry and RT-PCR.

Conclusions The development of respiratory tract innervation in adriamycin-induced EA-TEF was delayed and abnormally controlled in rats compared with controls. However, these deficiencies were apparently compensated for at term and had no effect on airway peristalsis. The possible significance of innervation anomalies for respiratory sequelae in EA-TEF patients deserves further investigation.

Keywords

tracheo-bronchial - lung - innervation - esophageal atresia - tracheo-esophageal fistula

• References

• 1 Spitz L. Oesophageal atresia. Orphanet J Rare Dis 2007; 2: 24
• 2 Dudley NE, Phelan PD. Respiratory complications in long-term survivors of oesophageal atresia. Arch Dis Child 1976; 51: 279-282
• 3 Lister J. Oesophageal atresia and respiratory complications. Matti Sulamaa lecture - 30^th May 1983. Ann Chir Gynaecol 1985; 74: 52-59
• 4 LeSouef PN, Myers NA, Landau LI. Etiologic factors in long-term respiratory function abnormalities following esophageal atresia repair. J Pediatr Surg 1987; 22: 918-922
• 5 Beardsmore CS, MacFadyen UM, Johnstone MS , et al. Clinical findings and respiratory function in infants following repair of oesophageal atresia and tracheo-oesophageal fistula. Eur Respir J 1994; 7: 1039-1047
• 6 Agrawal L, Beardsmore CS, MacFadyen UM. Respiratory function in childhood following repair of oesophageal atresia and tracheoesophageal fistula. Arch Dis Child 1999; 81: 404-408
• 7 Cozzi DA, Capocaccia P, Roggini M , et al. Respiratory status of infants with esophageal atresia. Pediatr Surg Int 2001; 17: 92-96
• 8 Usui N, Kamata S, Ishikawa S , et al. Anomalies of the tracheobronchial tree in patients with esophageal atresia. J Pediatr Surg 1996; 31: 258-262
• 9 Chen CY, Tsao PN, Chou HC , et al. Esophageal atresia associated with tracheal stenosis and right lung agenesis: report of one case. Acta Paediatr Taiwan 2002; 43: 348-350
• 10 Downard CD, Kim HB, Laningham F , et al. Esophageal atresia, duodenal atresia, and unilateral lung agenesis: a case report. J Pediatr Surg 2004; 39: 1283-1285
• 11 Montedonico S, Diez-Pardo JA, Lassaletta L , et al. Malformaciones respiratorias asociadas a la atresia de esófago. Cir Pediatr 1999; 12: 61-64
• 12 Wales PW, Drab SA, Connolly B , et al. Horseshoe lung in association with other foregut anomalies: what is the significance?. J Pediatr Surg 2002; 37: 1205-1207
• 13 Becker J, Hernandez A, Dipietro M , et al. Identical twins concordant for pulmonary sequestration communicating with the esophagus and discordant for the VACTERL association. Pediatr Surg Int 2005; 21: 541-546
• 14 Liu X, Aras-Lopez R, Martinez L , et al. Lung hypoplasia in rats with esophageal atresia and tracheoesophageal fistula. Pediatr Research 2011; (Accepted)
• 15 Davies M. Anatomy of the extrinsic nerve supply in oesophageal atresia of the common type. Pediatr Surg Int 1996; 11: 230-233
• 16 Nakazato Y, Wells TR, Landing BH. Abnormal tracheal innervation in patients with esophageal atresia and tracheoesophageal fistula: study of the intrinsic tracheal nerve plexuses by a microdissection technique. J Pediatr Surg 1986; 21: 838-844
• 17 Boleken M, Demirbilek S, Kirimiloglu H , et al. Reduced neuronal innervation in the distal end of the proximal esophageal atretic segment in cases of esophageal atresia with distal tracheoesophageal fistula. World J Surg 2007; 31: 1512-1517
• 18 Pederiva F, Burgos E, Francica I , et al. Intrinsic esophageal innervation in esophageal atresia without fistula. Pediatr Surg Int 2008; 24: 95-100
• 19 Shono T, Suita S. Motility studies of the esophagus in a case of esophageal atresia before primary anastomosis and in experimental models. Eur J Pediatr Surg 1997; 7: 138-142
• 20 Li K, Zheng S, Xiao X , et al. The structural characteristics and expression of neuropeptides in the esophagus of patients with congenital esophageal atresia and tracheoesophageal fistula. J Pediatr Surg 2007; 42: 1433-1438
• 21 Qi BQ, Uemura S, Farmer P , et al. Intrinsic innervation of the oesophagus in fetal rats with oesophageal atresia. Pediatr Surg Int 1999; 15: 2-7
• 22 Cheng W, Bishop AE, Spitz L , et al. Abnormal enteric nerve morphology in atretic esophagus of fetal rats with adriamycin-induced esophageal atresia. Pediatr Surg Int 1999; 15: 8-10
• 23 Weichselbaum M, Sparrow MP. A confocal microscopic study of the formation of ganglia in the airways of fetal pig lung. Am J Respir Cell Mol Biol 1999; 21: 607-620
• 24 Sparrow MP, Weichselbaum M, McCray PB. Development of the innervation and airway smooth muscle in human fetal lung. Am J Respir Cell Mol Biol 1999; 20: 550-560
• 25 Sparrow MP, Warwick SP, Everett AW. Innervation and function of the distal airways in the developing bronchial tree of fetal pig lung. Am J Respir Cell Mol Biol 1995; 13: 518-525
• 26 Weichselbaum M, Everett AW, Sparrow MP. Mapping the innervation of the bronchial tree in fetal and postnatal pig lung using antibodies to PGP 9.5 and SV2. Am J Respir Cell Mol Biol 1996; 15: 703-710
• 27 Sparrow MP, Weichselbaum M. Structure and function of the adventitial and mucosal nerve plexuses of the bronchial tree in the developing lung. Clin Exp Pharmacol Physiol 1997; 24: 261-268
• 28 Tollet J, Everett AW, Sparrow MP. Spatial and temporal distribution of nerves, ganglia, and smooth muscle during the early pseudoglandular stage of fetal mouse lung development. Dev Dyn 2001; 221: 48-60
• 29 Burns AJ, Delalande JM. Neural crest cell origin for intrinsic ganglia of the developing chicken lung. Dev Biol 2005; 277: 63-79
• 30 Crimi N, Mistretta A. Non-adrenergic non-cholinergic nervous control of airways. Eur Respir J Suppl 1989; 6: 508s-511s
• 31 Undem BJ, Kollarik M. The role of vagal afferent nerves in chronic obstructive pulmonary disease. Proc Am Thorac Soc 2005; 2: 355-360
• 32 Tollet J, Everett AW, Sparrow MP. Development of neural tissue and airway smooth muscle in fetal mouse lung explants: a role for glial-derived neurotrophic factor in lung innervation. Am J Respir Cell Mol Biol 2002; 26: 420-429
• 33 Towers PR, Woolf AS, Hardman P. Glial cell line-derived neurotrophic factor stimulates ureteric bud outgrowth and enhances survival of ureteric bud cells in vitro. Exp Nephrol 1998; 6: 337-351
• 34 Heuckeroth RO, Lampe PA, Johnson EM , et al. Neurturin and GDNF promote proliferation and survival of enteric neuron and glial progenitors in vitro. Dev Biol 1998; 200: 116-129
• 35 Sanchez MP, Silos-Santiago I, Frisen J , et al. Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 1996; 382: 70-73
• 36 Harding R, Hooper SB. Regulation of lung expansion and lung growth before birth. J Appl Physiol 1996; 81: 209-224
• 37 Kitano Y, Flake AW, Quinn TM , et al. Lung growth induced by tracheal occlusion in the sheep is augmented by airway pressurization. J Pediatr Surg 2000; 35: 216-221
• 38 Jesudason EC, Smith NP, Connell MG , et al. Developing rat lung has a sided pacemaker region for morphogenesis-related airway peristalsis. Am J Respir Cell Mol Biol 2005; 32: 118-127
• 39 Schittny JC, Miserocchi G, Sparrow MP. Spontaneous peristaltic airway contractions propel lung liquid through the bronchial tree of intact and fetal lung explants. Am J Respir Cell Mol Biol 2000; 23: 11-18
• 40 Featherstone NC, Jesudason EC, Connell MG , et al. Spontaneous propagating calcium waves underpin airway peristalsis in embryonic rat lung. Am J Respir Cell Mol Biol 2005; 33: 153-160
• 41 Booth RJ, Sparrow MP, Mitchell HW. Early maturation of force production in pig tracheal smooth muscle during fetal development. Am J Respir Cell Mol Biol 1992; 7: 590-597
• 42 van der Velden VH, Hulsmann AR. Autonomic innervation of human airways: structure, function, and pathophysiology in asthma. Neuroimmunomodulation 1999; 6: 145-159
• 43 Belmonte KE. Cholinergic pathways in the lungs and anticholinergic therapy for chronic obstructive pulmonary disease. Proc Am Thorac Soc 2005; 2: 297-304
• 44 Chetcuti P, Phelan PD, Greenwood R. Lung function abnormalities in repaired oesophageal atresia and tracheo-oesophageal fistula. Thorax 1992; 47: 1030-1034
• 45 Schier F, Korn S, Michel E. Experiences of a parent support group with the long-term consequences of esophageal atresia. J Pediatr Surg 2001; 36: 605-610
• 46 Malmstrom K, Lohi J, Lindahl H , et al. Longitudinal follow-up of bronchial inflammation, respiratory symptoms, and pulmonary function in adolescents after repair of esophageal atresia with tracheoesophageal fistula. J Pediatr 2008; 153: 396-401
• 47 Couriel JM, Hibbert M, Olinsky A , et al. Long term pulmonary consequences of oesophageal atresia with tracheo-oesophageal fistula. Acta Paediatr Scand 1982; 71: 973-978
• 48 Sistonen S, Malmberg P, Malmstrom K , et al. Repaired oesophageal atresia: respiratory morbidity and pulmonary function in adults. Eur Respir J 2010; 36: 1106-1112
• 49 Delius RE, Wheatley MJ, Coran AG. Etiology and management of respiratory complications after repair of esophageal atresia with tracheoesophageal fistula. Surgery 1992; 112: 527-532