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Keywords:

  • foregut duplication;
  • esophageal atresia;
  • tracheoesophageal fistula;
  • rat;
  • embryo;
  • Adriamycin;
  • apoptosis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The pathogenesis of the alimentary tract duplications, including foregut duplications (FgD) remains speculative. The accidental finding of FgD in fetal rats with esophageal atresia and tracheoesophageal fistula (EA-TEF) induced by Adriamycin provided an animal model to investigate a possible relationship between these two entities. Timed-pregnant rats were intraperitoneally injected with Adriamycin (1.75 mg/kg) on gestational Days 6 to 9. Their embryos were harvested by Caesarean section from gestational Days 14 to 21. Forty-six of embryos were processed and serially sectioned in the transverse or sagittal planes. EA-TEF occurred in 43/46 (93%) embryos of which 11 (24%) were found to have an associated FgD located at the level where the esophagus was absent. Six FgDs communicated with the foregut or the trachea. Five noncommunicating FgDs were located between the foregut and the vertebral column. In the control embryo, the notochord was located in the centre of the vertebral column from Day 11 of the gestation. In Day 14, 15 and 16, however, embryos exposed to Adriamycin, an abnormal notochord or branch frequently was located within the mesenchyme of the maldeveloped foregut or attached to the duplication cyst. In some, it appeared that the notochord was drawing the cyst-like structure away from the foregut. The present study confirms that duplications adjacent to the esophagus arise from the foregut and that failure of the foregut to detach from the notochord at the normal time may contribute to the development of foregut duplications. Anat Rec 264:93–100, 2001. © 2001 Wiley-Liss, Inc.

The incidence of alimentary tract duplications (ATD) is estimated to be about 1:4,500 (Bond and Groff, 1998), of which about 33% of ATDs are foregut duplications (FgD) (Bajpai and Mathur, 1994; Bissler and Klein, 1988; Bower et al., 1978; Hocking and Young, 1981). Associated abnormalities include vertebral anomalies, myelomeningocele, double bladder, and bladder or cloacal extrophy (Bond and Groff, 1998; Bower et al., 1978). More recently, the association of esophageal atresia and tracheo-esophageal fistula (EA-TEF) with esophageal duplication has been reported (Kirks and Filston, 1981; Narashimaharao and Mitra, 1987; Snyder et al., 1996), suggesting that these entities may have a common embryologic origin, particularly as they are both derived from the foregut. In clinical practice, however, the association of EA-TEF and FgD is extremely rare, although some small FgD may involute in late embryonic or fetal life. Most hypotheses of the pathogenesis of ATDs (Bremer, 1952; McLetchie et al., 1954; Nathan, 1959; Nobuhara et al., 1997) do not account for the range and variety of ATDs seen in the gastrointestinal tract and none explain the coexistence of EA-TEF and FgD. The observation of esophageal duplication cysts in rat fetuses with EA-TEF induced by intraperitoneal injection of Adriamycin into timed-pregnant rats (Diez-Pardo et al., 1996) prompted us to investigate whether there is any demonstrable relationship between EA-TEF and FgD, and to study the embryogenesis of FgD.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Twenty-one Sprague–Dawley female rats (University of Otago, Dunedin, NZ), with body weights ranging from 250–300 g, were time-mated with male rats. Pregnancy was confirmed by a positive vaginal smear and that day designated as Day 0 of gestation. The pregnant rats were kept separately in an air-conditioned, 12/12 hr light/dark cycle animal laboratory (Christchurch School of Medicine) and fed ad lib. They were divided into control (3 rats) and Adriamycin-treated (18 rats) groups and the latter were subdivided into six different age groups, (3 rats in each age group ranging from gestational days 14–18 and Day 21). The dams were sacrificed from gestational days 14–18, and at Day 21 respectively. Their embryos were fixed in 10% formalin for 24–72 hr according to their size. All specimens were processed, embedded in paraffin and sectioned transversely or sagitally at 5 μm thickness. The number of embryos in each group and the orientation of the sections is shown in Table 1. Sequential sections were mounted at an interval of one in three and stained with haematoxylin and eosin. Slides were examined serially to determine the morphology of trachea and esophagus, with special attention being paid to any abnormal cyst or diverticulum and its relationship to surrounding structures.

Table 1. Distribution of the main malformations in each age group
GestationNo. embryoSectionEA-TEFTAFgDARM
  • EA-TEF, esophageal atresia and tracheo-esophageal fistula; TA, tracheal agenesis; FgD, foregut duplication; ARM, anorectal malformations; NA, embryos were too immature to determine ARM.

  • a

    Externally examined.

Day 146Transverse61N/A
Day 1515Transverse (7)1413N/A
Sagittal (8)8
Day 165Sagittal515
Day 174Sagittal4114
Day 184Sagittal314
Day 2112Transverse111412a
Subtotal4643 (93%)3 (6.5%)11 (24%)32 (100%)

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

A total of 18 control and 46 Adriamycin-exposed embryos were harvested.

In normal rat embryos, the trachea and esophagus were completely separate tubes on Day 14 of gestation and the notochord (Nt) was located within the centre of the vertebral column (Fig. 1A), as described in previous studies (Qi and Beasley, 1999). From Day 15 of gestation the Nt expanded between the intervertebral bodies to become the nucleus pulposes, whereas the Nt segments within the vertebral bodies degenerated (Fig. 2A). There were no malformations in the 18 control embryos.

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Figure 1. Normal and abnormal development of the foregut and the notochord on Day 14 of gestation in rat embryos. A: In normal embryo, the foregut has developed into the trachea (Tr) and esophagus (Es). The thin rod-like notochord (Nt) lies within the chondrogenetic centre anterior to the neural tube (NT). B: In Adriamycin-treated rat embryo, the foregut (Fg) remains a single tube. The vastly expanded notochord (Nt) is anteriorly displaced within the foregut mesenchyme, which is more clearly shown in the inset. Scale bar (A, B) = 125 μm. Scale bar (inset) = 25 μ.

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Figure 2. Normal and abnormal development of the foregut and the notochord on Day 15 of gestation in rat embryos. Sagittal sections viewed from the right side. A: Normal embryo: the larynx (La), trachea (Tr) and esophagus (Es) are well developed. The notochord (Nt) lies within the vertebral column centre with the appearance of a string of beads, indicating the formation of the nucleus pulposus in the intervertebral discs. B: Adriamycin-treated embryo: the foregut (Fg) is a single, interrupted tube. A long branch of the notochord (Nt) travels down the mesenchyme of the proximal part of the foregut. Two points of attachment can be observed. A large cyst (Cys) is being distracted from the foregut near the carina. Ao, aorta; Atr, atrium; Liv, liver; LB, lung bud; St, stomach; V, vertebral column. Scale bar = 125 μ.

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In the 46 Adriamycin-exposed embryos, 43 had esophageal atresia with a distal tracheo-esophageal fistula (EA-TEF) of which three also had tracheal agenesis (Fig. 3). In the remaining three, tracheo-esophageal development looked normal (Table 1). Foregut duplications (FgD) of various types were found in 11 of these 43 embryos (Table 1). The FgDs comprised cystic duplications (6) (Fig. 3B and Fig. 4A) and diverticular duplications (5) (Fig. 4B), but there were no tubular duplications (Table 2). In another two embryos a longitudinal mesenchymal mass with a vague lumen in its centre between the foregut and vertebral column was identified (Fig. 5B). One of the six cystic duplications communicated with the trachea. The epithelium of the diverticular FgDs in embryos between gestational days 14–17 was similar to that of the adjacent foregut. In contrast, the epithelium of the cystic duplications in term fetuses was esophageal (2) and intestinal (2), as shown in Figure 3B and in Figure 4A. The location of the FgDs is summarized in Table 2.

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Figure 3. Esophageal atresia with broncho-esophageal fistula and intestinal duplication cyst coexist in an Adriamycin-treated rat fetus. The transverse sections are arranged in cranio-caudal sequence. A: The blind ending of proximal esophagus (Es) is located between the trachea (Tr) and vertebrae (V). B: In the upper thorax, the esophagus is absent between the trachea and vertebra. A large intestinal duplication cyst (Cys) in the right thoracic cavity has a thin attachment to the vertebral body. C: Below the tracheal carina, the distal esophagus (Es) communicates with the right bronchus (RB) by a fistula (arrow). Ao, aorta; Atr, atrium; LB, left bronchus; Liv, liver; Lu, lung; PA, pulmonary artery. Scale bar = 125 μ.

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Figure 4. Different types and locations of intestinal duplications in Adriamycin-treated rat fetuses. A: A large intestinal duplication cyst (Cys) located in front of the vertebral column (V) at the level where the esophagus (Es) is passing through the diaphragm (Diap). There is no gap between them. Arrow indicates ectopic pancreas tissue in the hiatus. B: Within the membranous trachea at the level of the aortic arch (Ao Ar) lies a small cyst (arrow) with squamous epithelium. Ao, aorta; Lu, lung; Tr, trachea; V, vertebrae. Scale bar = 125 μ.

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Table 2. Summary of 11 rat embryos with foregut duplication induced by adriamycin
Embryo/FetusGestationFg malformationFgDCommunicationEpitheliumLocation of FgD
  1. Fg, foregut; EA-TEF, esophageal atresia and tracheo-esophageal fistula; TA, tracheal agenesis; FgD, foregut duplication; diverti, diverticulum.

1Day 14EA-TEFDiverti (small)With foregut?Above carina, posterior to foregut
2Day 15EA-TEFDiverti (small)With esophagusEsophagealJust above diaphragm
3Day 15EA-TEFDiverti (large)With foregut?Posterior to carina
4Day 15EA and TADiverti (large)With lower foregut?Above carina, posterior to foregut
5Day 16EA-TEFCyst (large)No?Above carina, posterior to foregut
6Day 17TACyst (small)NoIntestinalL1–L2 level
7Day 18EA-TEFDiverti (small)With tracheaTrachealNear the cricoid
8Day 21EA-TEFCyst (large)NoIntestinalRight chest, below aortic arch
9Day 21EA-TEFCyst (large)NoIntestinalAbove the diaphragm, right to aorta
10Day 21EA-TEFCyst (small)NoEsophagealPosterior to trachea, near aortic arch
11Day 21EA-TEFCyst (small)With tracheaEsophagealPosterior to trachea, near the cricoid
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Figure 5. Relationship between an abnormal notochord and cyst formation in adriamycin-treated rat embryo. A: Sagittal section of a day 16 Adriamycin-treated rat embryo with esophageal atresia and intestinal duplication cyst. Abnormal notochord (arrow) is situated above the cyst (Cys). B: Transverse section of a day 17 Adriamycin-treated rat embryo. An isolated cyst-like mass (cys) exists between the trachea (Tr) and the vertebral body (V). There are some degenerating epithelial-like cells within its centre. The mass is in contact with an abnormal notochord branch (arrow) that is in the process of degeneration itself and located ventral to the rest of the notochord (Nt). Ao, aorta; AoAr, aortic arch; Br, bronchus; Lu, lung; V, vertebral column; Vag, Vagus nerve. Scale bar (A) = 125 μ. Scale bar (B) = 25 μ.

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In the 24 embryos with EA-TEF ranging from gestational days 14–18, delayed degeneration of the Nt within the vertebral column and abnormal Nt branching or mass was consistent with earlier reports (Qi and Beasley, 1999). The abnormal Nt or its branches often joined or became closely applied to the FgD cyst or diverticulum (Figs. 2, 5 and 6). On day 17 of gestation, the abnormally-positioned Nt and its branches degenerated by apoptosis (Fig. 5B). By day 21 there was no Nt tissue in the vicinity of the esophageal upper pouch or the FgD. The attachment of the duplicate cysts to the vertebral column was narrow or broad (Fig. 3B and 4A).

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Figure 6. Relationship between an abnormal notochord, cyst formation and foregut malformation in the adriamycin-treated rat embryo. Sagittal sections are viewed from the right side. A: A convoluted (double folded) notochord (Nt) is displacing a foregut cyst away from the lower foregut. B: A section slightly towards the left shows that the abnormal notochord segment (Nt) is interposed between the interrupted upper segment of the foregut and the cyst. C: The contact of the abnormal notochord segment (Nt) with the foregut (Fg) and the cyst is shown (Arrows) in under higher power. Scale bar (A,B) = 125 μ. Scale bar (C) = 25 μ.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Alimentary tract duplications (ATD) have three main characteristics: (1) the presence of a well-developed coat of smooth muscle; (2) an epithelial lining typical of some portion of the alimentary tract; and (3) (usually) an intimate attachment to some portion of the gastrointestinal tract (A). The morphological features of the duplication cysts or diverticula observed in the present study, especially in near-term fetuses, had these features. In young embryos (younger than day 16) the smooth muscle had not yet developed, and the epithelium in the duplication and the foregut at the same level was similar. The present study confirmed that these duplication cysts were of foregut origin. Only one of six communicating duplication cysts communicated with the esophagus; the remainder joined with the trachea or with the undifferentiated foregut (Fig. 2 and 6). For this reason, we prefer to call them foregut duplications (FgD). We also observed that the esophagus in adriamycin-treated fetuses with EA-TEF was absent between the cricoid and carina, the same region where most of the FgDs were located.

Theories of the pathogenesis of ATDs, (including FgDs) have not been substantiated by observations on either human or animal embryos. The concept of the split notochord syndrome (Bentley and Smith, 1960; Fairs and Crowe, 1975) and accessory neurenteric canal (Bremer, 1952) imply coexistence of some kinds of ATDs and vertebral defects. In essence, these theories suggest that abnormal adherence of the endoderm (from the primitive gut) to the ectoderm (from the neural tube) and herniation through a sagittally split notochord dorsally may lead to the development of posterior neurenteric fistula. If the fistula is not closed or obliterated by subsequent differential growth of the nearby tissues, a variety of abnormalities may ensue, including pre- or post-vertebral enteric cyst or diverticula, and spina bifida or posterior neurenteric fistula (Bentley and Smith, 1960; Bremer, 1952; Fairs and Crowe, 1975). The split notochord theory, however, cannot explain why the majority of small and large bowel duplications are located on the antimesenteric border (Bissler and Klein, 1988; Bond and Groff, 1998; Bower et al., 1978).

Aberrant esophageal luminal revacuolization has been suggested as an etiological explanation for FgD (Bremer, 1944), and may account for the development of a diverticulum or cyst, but not a tubular duplication (Mikaelian et al., 1981). In our animal model there was no solid phase and no subsequent recanalization phase in the esophagus between gestational days 11 to 16, the period when it might be expected. Although the esophageal lumen was extremely tiny on day 13 of gestation, it never became occluded. Likewise, Chen and Tam (1998) did not observe any proliferative occlusion or recanalization phase in the duodenum of rat embryos.

During normal early embryonic development, the notochord primordium fuses with the (gut) endoderm. The notochord then separates from the endoderm and later consolidates to form a continuous rod-like structure after separation (Jurand, 1974). If the notochord were not to separate from the foregut or primitive esophagus, the adherent part of the esophagus might be distracted by this adherence and separated from the rest of the developing esophagus, thus leading to the formation of a cyst or diverticulum (Veeneklass, 1952). This theory proposed by Veeneklass (1952) is consistent with our observations. Certainly, the present study confirms that abnormal development of the notochord accompanies the occurrence of foregut duplications in rat fetuses exposed to Adriamycin.

Several studies have now demonstrated that adriamycin-exposed rat embryos with EA-TEF have associated notochord abnormalities and this may explain the relationship between vertebral defects and EA-TEF (Merei, 1998; Possoegel et al., 1999; Qi and Beasley, 1999). No single sagittally split or branched notochord had been observed by these authors; instead, the notochord was always displaced ventrally or anterior to the vertebral mesenchyme. The abnormally displaced and branched notochord often extended into the foregut mesenchyme and was frequently attached to the endoderm of the foregut (Fig. 2B and Fig. 6). In several specimens from day 15 embryos, a phenomenon of displacement of part of the foregut lumen by the abnormal notochord branch was obvious as seen in Figure 6. The usual level at which the notochord was affected ranged between C1 and L2, and corresponded to the distribution of the vertebral defects (Abu-Hijleh et al., 2000). The FgDs observed were also distributed in the same region. Because the notochord has been considered the central organiser of axial organogenesis, it may inhibit endodermal development when it remains attached or is in close proximity to the endoderm (Elliott et al., 1970). This study perhaps implicates the abnormally-positioned notochord or its attachment to the foregut in the development of EA-TEF, foregut duplications and vertebral defects.

Yet, in three embryos with small foregut diverticula, there were no abnormal notochord branches in the vicinity. Kirks and Filston (1981) have raised the possibility that esophageal duplication cysts are due to abnormal outpouching of the endoderm from the primitive foregut wall. If this abnormal budding loses its communication with the foregut or esophagus lumen, a noncommunicating cyst will ensue (Kirks and Filston, 1981). Abnormal budding in the dorsal wall of the foregut might explain the displacement of the foregut caused by the unseparated (or attached) notochord. In one fetus, however, the small epithelial diverticulum “budded” from the ventral wall of the foregut in the absence of direct contact with an abnormal notochord. We suspect that an abnormal epithelium–mesenchyme interaction may be one of the causes of ATD, although the biochemical nature of any such interaction is yet to be verified.

The different types and locations of ATD may reflect differences in the timing and severity of the causative factors that may also influence whether they retain or lose their connections with the gastrointestinal tract.

Our observations suggest that duplication cysts and diverticula adjacent to the esophagus arise from the foregut and provide further evidence to support Veeneklass' theory (1952) that failure of the foregut to detach from the notochord at the correct time may contribute to the development of foregut duplications. The coexistence of EA-TEF, FgD and cervical-thoracic vertebral defects with abnormal development of the Nt may imply a common cause for these malformations.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors sincerely thank the Robert McClelland Trust, RG Bell Foundation, for supporting this study.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED