The Vagus and Recurrent Laryngeal Nerves in the Rodent Experimental Model of Esophageal Atresia

By Bao Quan Qi, Jamal Merei, Pam Farmer, Suzanne Hasthorpe, Nate A. Myers,

Spencer W. Beasley, and John M. Hutson

Melbourne, Australia

Background: After surgical correction of their esophageal atresia and tracheoesophageal fistula (EA-TEF), many pa- tients exhibit evidence of esophageal dysmotility. Contro- versy exists as to whether the esophageal motility disorders result from denervation caused by surgery or from an inher- ent abnormal innervation of the esophagus.

Methods: The present study used an Adriamycin-induced EA-TEF fetal rat model to trace the course and branching of both the vagus and recurrent laryngeal nerves. Abnormali- ties observed in EA-TEF rat fetuses include: (I) fewer branches from both recurrent laryngeal nerves; (2) deviation of the left vagus from its normal course below the aorta, passing behind the fistula to approach and join with the right vagus to form a single nerve trunk on the right side of the esophagus; (3) relatively few branches from the single vagal nerve trunk

(composed of fibers of the left and the right vagus) on the surface of the lower esophagus.

Cumlosions: Fetuses affected by EA-TEF have inherent abnormalities in the course and branching pattern of the vagus nerves as they descend through the thorax, culminat- ing in a deficient extrinsic nerve fiber plexus in the lower esophagus. These observations may account for the esopha- geal motility disorders seen in patients who have EA-TEF even before surgical intervention. J Pediatr Surg 32: 1580- 1586. Copyright o 1997 by W B. Saunders Company.

INDEX WORDS: Esophageal atresia, tracheoesophageal fis- tula, vagus nerve, recurrent laryngeal nerve, Adriamycin, fetal rat.

LTHOUGH A THE PRIMARY surgical repair of esophageal atresia and tracheoesophageal fistula

(EA-TEF) restores gastrointestinal continuity, it does not guarantee normal esophageal function. It has been demon- strated that a spectrum of esophageal motility abnormali- ties exists in patients after surgical correction of their EA-TEF.1-6 These abnormalities include dysphagia, gas- troesophageal reflux (GER), aperistalsis, and low- amplitude simultaneous contractions in the affected esophagus as documented by radiological findings, mano- metric observations, and intraesophageal pH record- ings.4-7 These may contribute to the development of recurrent respiratory tract infection and anastomotic strictures1z2 However, the etiology of esophageal dysfimc- tion after repair of EA-TEF and the extent to which it is primary (an inherent abnormality) or secondary (from surgical damage during repair) is still controversial. The observation of disorders of the esophageal motor activity in patients who have EA-TEF before surgical interven- tion8zg make it unreasonable to attribute the dysfunction

From the F: Douglas Stephen Surgical Research Laboratory, Royal Children k Hospital, Melbourne, Australia.

Presented at the 30th Annual Meeting of the Pacijic Associatior2 of Pediatric Surgeons, Phoenix, Arizona, May 9-13, 1997.

Address reprint requests to John M. Hutson, Deparhnent of General Surgery, Royal Children k Hospital, Parkville, Victoria 3052, Australia.

Copyright o 1997 by WB. Samders Company 0022-3468/97/3211-0014$03.00/O

entirely to esophageal denervation resulting from surgical mobilization and dissection. It is possible that patients with EA-TEF have a preexisting congenital neuromotor abnormality of the esophagus.

There is a paucity of information about the nerve supply to the esophagus in the presence of EA-TEF,‘O mainly because of difficulty in obtaining specimens to examine. The Adriamycin-induced EA-TEF fetal rat model” provides us with a useful model to study both the vagus and recurrent laryngeal nerves in animals with EA-TEE The aim of this study was to document whether there is any demonstrable primary abnormality in the morphology, course, or branching of the vagus nerve (VN) and recurrent laryngeal nerve (RLN) in animals with EA-TEE

MATERIALS AND METHODS

Esophageal atresia and tracheoesophageal fistula was induced in fetal rats as described previously. I1 Timed-pregnant Sprague-Dawley rats maintained in the Animal Research Laboratory at the Royal Children’s Hospital were injected intraperitoneally with Adriamycin on gestational days 6 to 9 (four injections) at a dosage of 2 mgikg of body weight. Control rats received saline injections during the corresponding period of gestation. During treatment, the pregnant rats were kept separately in an air-conditioned, 12-hour light/dark cycle environment and fed with commercial rat chow and tap water ad libitum. The fetuses were harvested by cesarian section on day 21 of gestation. Twenty-one treated fetuses and six control fetuses were harvested. They were fixed in 10% formalin or Bouin’s solution for 48 to 72 hours and cut transversely, from neck to upper abdomen, into four blocks. Each tissue block was embedded in paraffin and sectioned at a thickness of 5 m.

1580 Journa/ofPediatricSurgery,Vol 32, No 11 (November), 1997: pp 1580-1586

VAGUS NERVES IN ESOPHAGEAL ATRESIA 1581

Fig 1. These illustrations were reconstructed by observing a series of histological slides of all fetuses examined. (A) Normal course of both vagus nerves and esophageal plexus around the lower esophagus in con- trol fetus. (BI In the EA-TEF fetus, the left vagus nerve, after tracheal bifurcation, passed behind the esophagus and joined with the right vagus nerve on the right side of the esophagus to form a single nerve trunk that descended on the right or posterior-lateral to the esophagus until the cardiac region. No esopha- geal plexus was formed in the lower esophagus.

Right subclavian A.

Diaphragm

The slides were mounted at intervals of 10 sections and stained with Hematoxylin and Eosin.

The histological slides were examined by a single investigator. Initially, all slides from six control animals were observed in the cephalocaudal direction from the level of the cricoid down to the diaphragmatic crura. The course of the VN and RLN and their branches were traced out and mapped to obtain a three-dimensional picture of the normal course of the nerves and their anatomic relationship to other structures such as the trachea, esophagus, and the great vessels. The morphology and caliber of the nerves also were noted. Finally, the same observations and mappings were carried out on the Adriamycin-treated fetuses with or without EA-TEE Photomicrographs were taken from both control and EA-TEF fetuses at the corresponding level to facilitate reconstruction of the three-dimensional diagrams of VNs.

RESULTS

Esophageal atresia with a distal tracheoesophageal fistula was confirmed histologically in 14 of 21 Adriamy- tin-treated fetuses, The upper pouch of the esophagus ended just below the level of the cricoid. The distal esophagus communicated either with the posterior wall of the trachea just near the carina or with the inferior aspect of the left main bronchus near the bifurcation. No such abnormality existed in any control fetuses. In control fetuses and fetuses from Adriamycin-treated dams without EA-TEF, the left RLN originated from the left VN on the left side of the aortic arch and hooked medially around the ductus arteriosus to ascend behind the aortic arch and upward along the left tracheoesopha- geal groove. The right RLN originated from the right VN in front of the right subclavian artery and hooked around this vessel and ascended along the right tracheoesopha- geal groove in the neck (Fig 1A). During their course in the neck, both RLNs divided into two main branches, which from time to time, gave off smaller branches at different levels (Fig 2A).

Right subclavian k

Lower oesophagus

Fig 2. (A) In control fetuses, both RLNs travelled in tracheo- esophageal grooves on each side in the neck. They divided into two main branches (arrowheads) and some small branches (arrows). T, trachea; 0, esophagus. (Original magnification x100.) (B) The trans- verse section in the neck of the fetus with esophageal atresia and tracheoesophageal fistula. The esophagus was absent. Two single trunk RLNs (arrowhead) are located lateral to the trachea (T). Only one branch lay behind the membranous trachea (arrow). (Original magnification X100.)

1582 QI ET AL

In the fetuses with EA-TEE each RLN also originated from its respective VN and ascended in the neck lateral to the trachea or in a groove formed by the trachea and the vertebral column (Fig 1B). In seven of fourteen fetuses with EA-TEF (50%), the RLNs remained as a single trunk, without dividing into two main trunks or giving off smaller branches (Fig 2B). In two of fourteen fetuses with EA-TEF, there was a right-sided aortic arch. In these cases, the right RLN passed from the right VN beneath the right aortic arch and ductus arteriosus and ascended on the right side of the trachea. On the left side, the left RLN hooked around the left subclavian artery and ascended on the left side of the trachea. In another two EA-TEF fetuses, there was an anomalous right subcla- vian artery, which arose from the descending aorta distal to the origin of the left subclavian artery and reached the right side by passing behind the trachea. In both cases, the right RLN could not be identified, and instead the right VN took up a position close to the right side of the trachea during its descent through the neck and upper chest.

In normal fetal rats, both VNs descended through the neck in the carotid sheath accompanied by the carotid artery and internal jugular vein and were not in close contact with the trachea and esophagus. The left VN entered the thorax between the left common carotid and subclavian artery and crossed the left side of the aortic arch. After giving off the left RLN, the left VN ap- proached the esophagus and descended first on its left side (Fig 3A), then gradually on its anterior-lateral or anterior surface (Fig 4A and Fig 1A). The right VN crossed the front of the right subclavian artery to enter the thorax. It descended tirst on the right side of the trachea and then continued onto the right lateral surface of the esophagus after the tracheal bifurcation (Fig 3A). The right VN then descended on the lateral-posterior or posterior surface of the esophagus (Fig 4A and Fig 1A). Between the trachea and the diaphragmatic crura, both VNs gave off some small branches, which formed a plexus on the surface of the esophagus (Fig 1A and Fig 5A). Just before passing through the crura, the VNs became confluent to form the anterior vagal trunk or trunks (mainly from the left VN) and posterior vagal trunk or trunks (mainly from the right VN, Fig 1 A and Fig 6A).

In EA-TEF fetuses, the course of the VNs in the neck and superior thoracic inlet was the same as in the control animals. However, after giving off the RLN, each VN reached the ipsilateral posterior-lateral aspect of the trachea. Distal to the tracheal bifurcation, the right VN continued along the same line, whereas the left VN passed behind the fistula to approach the right VN on the right posterior-lateral aspect of the fistula (Fig 1B and Fig 3B). Soon after separation of the left main bronchus and fistula (esophagus), the left and right VNs joined together

Fig 3. (A) At the level of tracheal bifurcation in control fetuses, left vagus nerves (arrow) lay on the left side of the trachea and the right vagus nerve (arrowhead) in the right trachea-esophageal groove. (6) The tracheal bifurcation and tracheoesophageal fistula are shown. The left vagus nerve (arrow) has approached the right vagus nerve (arrowhead) behind the fistula (F!. T, trachea; o, esophagus; s, spine; Lb, left bronchus: eb, right bronchus; A, aorta. (Original magnification x40.1

to form either a single trunk or two to three trunks, which descended on the right side or posterior-lateral side of the esophagus (Fig 4B). There were very few ramifications observed on the surface of the esophagus and there was no evidence of the esophageal plexus as seen in controls (Fig 5B). The nerve trunk or trunks composed of both the left and right VNs passed through the crura on the right side of the esophagus to enter the abdomen (Fig 6B).

DISCUSSION

Although the rat fetus is too small and the VNs with their branches are too minor to be appreciated by microdissection, the VNs, the RLN, and even their small branches in the vertical direction, can be traced clearly in the histological slides, as this study showed, by their characteristic histological appearance and anatomy. Our findings, supported by other work,12 show that the course of VNs and RLNs and their anatomic relationship to the other organs in the normal rat are very similar to those

VAGUS NERVES IN ESOPHAGEAL ATRESIA 1583

Fig 4. (A) Below the tracheal bifurcation in control fetuses, the left vagus nerve (arrowhead) moved to the front of the esophagus (0) and the right vagus nerve (arrow) to the right posterior-lateral side. (B) The single nerve trunk (arrowhead) composed of both the left and right vagus nerve was located on the right side of the esophagus (0) in a fetus with esophageal atresia and tracheoesophageal fistula. A fistula (F) existed between the oesophagus and the left bronchus Lb). Rb, right bronchus; A; aorta; s, spine. (Original magnification x40.1

seen in humans.13-I6 This similarity means that the EA-TEF fetal rat model is likely to be a useful tool to investigate the extrinsic innervation of the esophagus in the presence of EA-TEE

Numerous studies have documented that esophageal motility is abnormal after surgical repair of EA-TEEI Kirkpatrick et al’* demonstrated that the esophagus lacked propagative peristalsis and coordinated activity by showing that the contrast medium remained in the esophagus for many minutes or moved in both antegrade and retrograde directions. In severe cases, the barium even entered the pharynx and spilled over into the larynx and airway. There was also free GER when intraabdomi- nal pressure was increased in some cases.l* Later, Orrin- ger et al6 and Shermeta et al4 used esophageal manometry and intraesophageal pH recording to substantiate abnor- mal esophageal motor function including lack of progres- sive peristalsis, simultaneous esophageal contraction ini- tiated by swallow, and incompetence of the lower esophageal sphincter.4-6.19 They concluded that dissection and mobilization of the esophagus during surgery leads to damage to the esophageal branches of the VNs, resulting

in postoperative esophageal motor disfunction.5-7 How- ever, controversy still remains as to whether the main cause of abnormal esophageal motility is surgical damage to the nerve fibers that innervate the esophagus or an inherent abnormality of neuromuscular components.

Interestingly, preoperative esophagography and mano- metric studies on patients who have H type TEF also showed gastroesophageal reflux and uncoordinated peri- stalsis below the fistula.8.1*,20 Romeo et al9 investigated esophageal motility in 20 newborns who had EA-TEF by preoperatively monitoring intraluminal pressure of both proximal and distal segments. They found incomplete relaxation of the upper esophageal sphincter, a positive resting pressure in the esophageal body in both segments, and total motor incoordination (synchronous waves) in the distal segment.9 These findings suggest that esopha- geal dysmotility may be caused by some congenital abnormality of its neromuscular components, which may be exacerbated by the operative dissection. There is little information about the innervation of the esophagus in patients who have EA-TEE

The present study suggested that there might be an

1584 01 ET AL

Fig 5. LA) In the lower esophagus of control fetuses, the left vagus nerve (LV) is in front of the esophagus (0) and the right vagus nerve (RV) behind it. Some small nerve branches (arrows) were attached to the surface of the esophagus. (B) The lower esophagus Lo) of a fetus with EA-TEF had abnormal epithelium and a thin muscle layer. A single nerve trunk (arrow) was located on its right side and only one nerve ramulus (small arrow) existed on the esophageal surface. (Original magnification x 100.)

abnormality of the extrinsic innervation of the esophagus in the presence of EA-TEF, especially in the lower segment of esophagus. In normal fetal rats, below the aortic arch, the left VN approaches the esophagus on the left side and shifts gradually anteriorly during its descent. The right VN, however, moves more posteriorly or

lateroposteriorly while descending (Fig 1). Although the distribution of each VN to the thoracic portion of the esophagus is not confined to its own side, the left VN supplies mainly the anterior surface and the right VN mainly the posterior surface of this part of the esophagus. The vagi with their intercommunicating ramifications constitute a plexus surrounding the esophagus (periesopha- geal plexus). Branches arising from this plexus penetrate the esophageal wall to innervate the esophagus.*l How- ever, in EA-TEF fetal rats, below the aortic arch the left VN rotated behind the esophagus to the right lateroposte- rior or right side of the esophagus. In most cases, the left VN joined with the right to form a single nerve trunk. Very few ramifications or branches from both vagi could be found (Fig 1B) and no evidence of the periesophageal plexus could be seen on the surface of the lower segment of the esophagus (Fig 5). Because the left VN changed from its normal course to reach the right side without forming the usual periesophageal plexus, we suspect

Fig 6. (A) At the level of the diaphragmatic crura (DC) in control fetuses. the left (arrowhead) and right vagus nerves (arrow) of a normal fetus travelled on the anterior and posterior surface of the esophagus lo), respectively. IB) The single nerve trunk (arrow) still lays to the right of the esophagus lo) and broke into nerve bundles in the fetus with EA-TEF. DC, diaphragmatic crura. (Original magnifica- tion x 100.1

VAGUS NERVES IN ESOPHAGEAL ATRESIA

relatively few branches from both VN trunks have penetrated the esophagus to innervate it. Specifically, the left wall of the esophagus may have a relatively sparse innervation compared with normal.

Many early studies have documented the important role that the VNs play in regulating esophageal motil- ity.22-24 Carveth et a125 found that after vagotomy or resection of esophageal branches of the VN, dysphagia developed in dogs, they retched after eating meals, and there was dilatation of the lower portion of the esophagus with retention of barium. There was only a rare degluti- tive response in the body of the esophagus, and when it occurred, it was weak, with repetitive simultaneous contractions. Even the relaxation and contraction func- tions of lower esophageal sphincter were damaged.23,25 These observations imply that normal esophageal func- tions rely-apart from the integral intrinsic intramural nervous system-on an intact extrinsic innervation (the VNs). However, our results showed that the presence of EA-TEF in fetal rats was accompanied by an abnormal extrinsic nerve supply. The course and branching of the VNs were affected and the usual periesophageal plexus was deficient. It is conjectured that the anomalous VNs in EA-TEF entity may contribute to abnormal regulation of esophageal motility. Although one has to be cautious extrapolating the findings in this animal study to the human situation, esophageal motility disorders are found in so many patients who have an H fistula or EA-TEF before surgical intervention. A reasonable explanation is that these patients may have an abnormality in extrinsic

nerve supply to the esophagus. In addition, surgical mobilization and dissection, which would cause damage to the vagi, may worsen the degree of esophageal dysfunction.

We have no explanation why the left VN consistently follows an abnormal course below the aortic arch. The observation that the fistula in many fetuses who have EA-TEF connects from the proximal part of the left main bronchus in this model suggests that the presence of a fistula originating from the left bronchus may prevent the left VN from moving from the left to the anterior surface of the esophagus as it descends.

The findings of this study show congenital abnormali- ties in the course and branching pattern of the VNs as they descend through the thorax. This study also has demonstrated a relative deficiency in the nerve fiber plexus in the lower esophagus in rat fetuses affected by EA-TEE The results may be helpful in explaining the esophageal motility disorders seen in patients who have EA-TEF before surgical correction. Further research is needed to determine more precisely the exact nature of the innervation and neuromotor abnormalities in patients who have EA-TEE

ACKNOWLEDGMENTS

The authors sincerely thank the Esophageal Atresia Research Auxil- iary and the Education and Research Fund of the Department of General Surgery, Royal Children’s Hospital, Melbourne, for supporting this project.

REFERENCES

1. Parker AF, Christie DL, Cahill JL: Incidence and significance of gastresophageal reflux following repair of esophageal atresia and tracheesophageal fistula and the need for anti-reflux procedures. .I Pediatr Surg 145-8, 1979

2. Leendertse-Verloop K, Tibboel D, Hazebroek FWJ, et al: Postop- erative morbidity in patients with eosphageal atresia. Pediatr Surg Int 212-5, 1987

3. Spitz L, Keily E, Brereton R: Esophageal atresia: Five year experience with 148 cases. .I Pediatr Surg 22:103-108, 1987

4. Shermeta DW, Whitington PF, Seto DS, et al: Lower esophageal sphincter dysfunction in esophageal atresia: Nocturnal regurgitation and aspiration pneumonia. J Pediatr Surg 12:871-876, 1977

5. Duranceau A, Fisher SR, Flye MW, et al: Motor function of the esophagus after repair of esophageal atresia and tracheebophageal fistula. Surgery 82:116-123, 1977

6. Orringer MB, Kirsh MM, Sloan HS: Long-term esophageal function following repair of esophageal atresia. Ann Surg 186:436-443, 1977

7. Shono T, Suita S, Arima T, et al: Motility function of the esophagus before primary anastomosis in esophageal atresia. J Pediatr Surg 28673-676, 1993

8. Gundry SR, Orringer MB: Esophageal motor dysfunction in an adult with a congenital tracheesophageal fistula. Arch Surg 120: 1082- 1083,1985

9. Romeo G, Zuccarello B, Proietto F, et al: Disorders of the

esophageal motor activity in anesia of the esophagus. J Pediatr Surg 22:120-124, 1987

10. Davies MRQ: Anatomy of the extrinsic nerve supply of the esophagus in esophageal atresia of the common type. Pediatr Surg Int 11:230-233, 1996

11. Diez-Pardo JA, Qi BQ, Tovar JA: A new rodent experimental model of esophageal atresia and tracheesophageal fistula: Preliminary report. J Pediatr Surg 31:498-502, 1996

12. Greene EC: Nervous system, in Greene EC (ed): Anatomy of the rat. New York, NY, Hafner Publishing Company, 1963, pp 115-173

13. Skandalakis LJ, Gray SW, Skandalakis JE: The history and surgical anatomy of the vagus nerve. Surg Gynecol Obstet 162:75-85, 1986

14. Williams PL: Nervous system, in Williams PL, Bannister LH, Berry MM, et al (eds): Gray’s Anatomy. (ed 38). New York, NY, Churchhill Livingstone, 1995, pp 901-1369

15. Katz AD, Nemiroff P: Anastomoses and bifurcations of the recurrent laryngeal nerve-Report of 1177 nerves visualized. Am Surg 59:188-191, 1993

16. Steinburg JL, Khane GJ, Fernandes CMC, et al: Anatomy of the recurrent laryngeal nerve: A redescription. J Laryngol Otol 100:919- 927, 1986

17. Stokes KB: Pathophysiology, in Beasley SW, Myers NA, Auldist AW (eds): Oesophageal Atresia. London, Chapman and Hall Medical, 1991, pp 59-73

Ql ET AL

18. Kirkpatrick JA, Cresson SL, Pilling GP: The motor activity of the esophagus in association with esophageal atresia and tracheesopha- geal listula. Am J Radio1 86884-887, 1966

19. Takano K, Iwafuchi M, Uchiyama M. et al: Evaluation of lower esophageal sphincter function in infants and children following esopha- geal surgery. J Pediatr Surg 23:410-414, 1988

20. Johnston PW, Hastings N: Congenital trachea-esophageal fistuia without esophageal atresia. Am J Surg 112:233-240, 1966

21. Kuntz A: Innervation of the digestive tube, in Kuntz A (ed): The autonomic nervous system. 3rd ed, Philadelphia, PA, Lea & Febiger, 1947, pp 216239

22. Paterson WG: Neuromuscular mechanisms of esophageal re-

sponses at and proximal to a distending balloon. Am J Physiol 260:G148-G155, 1991

23. Rossiter CD, Norman WP, Jain M, et al: Control of lower esophageal sphincter pressure by two sites in dorsal motor nucleus of the vagus. Am J Physiol259:G899-G906, 1990

24. Haller JA, Brooker AF, Talbert JL, et al: Esophageal function following resection-Studies in newborn puppies. Ann Thorac Surg 2:180-187, 1966

25. Carveth SW, Schlegel JF, Code CF, et al: Esophageal motility after vagotomy, phrenicotomy, myotomy, and myomectomy in dogs. Surg Gynecol Obstet 114:31-42, 1962