Intramural ganglion structures in esophageal atresia: a morphologic and immunohistochemical study.

Abstract

Introduction and Aim. Disorders of esophageal motility causing dysphagia and gastroesophageal reflux are frequent in survivors to esophageal atresia (EA) and distal tracheoesophageal fistula (TEF). The aim of the present study was to investigate the histologic and immunohistochemical features in both esophageal atretic segments to further understand the nature of the motor disorders observed in these patients. Material and Methods. Esophageal specimens from 12 newborns with EA/TEF and 5 newborns dead of unrelated causes were examined. The specimens were fixed in 5% buffered formalin, included in paraffin and cut in 5 micron sections that were stained with hematoxilin and eosin (H and E), and immunohistochemical stainings for Actin, S-100 protein, Neurofilament, Neuron-Specific-Enolase, Chromogranin A and Peripherin were evaluated under the microscope. Results. In controls, the distribution of the neural elements was rather homogenous at both levels of the esophagus. In contrast, the atretic segments showed quantitative and qualitative differences between them with sparser nervous tissue in the distal one in comparison with the proximal one and with controls. Conclusions. These results further support the assumption that histomorphological alterations of the muscular and nervous elements within the esophageal wall might contribute to esophageal dysmotility in patients surviving neonatal operations for EA/TEF.

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Hindawi Publishing Corporation
International Journal of Pediatrics
Volume 2009, Article ID 695837, 7 pages
doi:10.1155/2009/695837
Clinical Study
IntramuralGanglionStructures inEsophagealAtresia:
AMorphologicand ImmunohistochemicalStudy
Biagio Zuccarello,1Antonella Spada,1Nunzio Turiaco,1Daniela Villari,1Saveria Parisi,1
IsabellaFrancica,1CarmineFazzari,1FedericaPederiva,2andJuanA.Tovar3
1Policlinico Universitario G.Martino, 98125 Messina, Italy
2Policlinico Universitario, Padova, Italy
3Hospital Universitario La Paz, Madrid, Spain
Correspondence should be addressed to Biagio Zuccarello, bzuccarello@unime.it
Received 27 November 2008; Revised 21 April 2009; Accepted 1 June 2009
Recommended by Waldemar A. Carlo
Introduction and Aim. Disorders of esophageal motility causing dysphagia and gastroesophageal reflux are frequent in survivors to
esophageal atresia (EA) and distal tracheoesophageal fistula (TEF). The aim of the present study was to investigate the histologic
and immunohistochemical features in both esophageal atretic segments to further understand the nature of the motor disorders
observed in these patients. Material and Methods. Esophageal specimens from 12 newborns with EA/TEF and 5 newborns dead
of unrelated causes were examined. The specimens were fixed in 5% buffered formalin, included in paraffin and cut in 5micron
sections that were stained with hematoxilin and eosin (H and E), and immunohistochemical stainings for Actin, S-100 protein,
Neurofilament, Neuron-Specific-Enolase, Chromogranin A and Peripherin were evaluated under the microscope. Results. In
controls, the distribution of the neural elements was rather homogenous at both levels of the esophagus. In contrast, the atretic
segmentsshowed quantitativeandqualitative differences between themwithsparsernervous tissueinthedistaloneincomparison
with the proximal one and with controls. Conclusions. These results further support the assumption that histomorphological
alterationsofthemuscularandnervouselementswithintheesophagealwallmightcontributetoesophagealdysmotilityinpatients
surviving neonatal operations for EA/TEF.
Copyright © 2009 Biagio Zuccarello et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
1.Introduction
Esophageal motor disorders are common following suc-
cessful repair of esophageal atresia and tracheoesophgeal
fistula (EA/TEF) [1–3]. Dysphagia, gastroesophageal reflux,
altered or absent peristaltis in the affected esophagus were
documented by clinical, radiological, isotopic, endoscopic
and pH-manometric observations [4–8]. In the past, this
was attributed to a neurologic defect, probably due to
partial denervation of the esophagus during operative
dissection. Previously, we documented disorders of the
esophageal motor activity in both untouched esophageal
atretic segments on patients with EA/TEF or isolated EA
by preoperative endoluminal esophageal manometry [9].
These motor anomalies were confirmed by histological and
immunohistochemicalstudiesinhuman[10–14]andanimal
models [15–21] suggesting that abnormal innervation and
neuromuscular defect of the esophagus are prexisting to the
surgery. The aim of the present study was to investigate the
histologic and immunohistochemical characteristics of the
nerve cells, fibers and bundles in both esophageal atretic
segments to further enlightening the nature of the motor
disorders observed in these patients.
2.MaterialandMethods
Twelve newborns (7 boys and 5 girls, with the average
gestational age 36.1 ± 2.21 days and postnatal age of 3.25
± 1.95 days) with EA/TEF (type C without long gap) were
examined. The specimens were obtained from the proximal
esophageal segment in 9 cases and from the distal segment
in 10 cases at the time (at birth) of the primary anastomosis.

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2 International Journal of Pediatrics
For comparison, 5 esophageal samples were obtained from
autopsiesofbabiesdeadofunrelatedcauses,1to2mmabove
the bifurcation of the trachea.
Histological and immunohistochemical analyses were
performed in all cases on formalin-fixed, paraffin-embedded
tissues. Serial sections (4-5 micron) from the distal end of
the upper esophageal pouch (UEP), the distal esophageal
segment (DES) and it’s the corresponding counterparts from
controls were cut and stained with Hematoxylin and Eosin
(H and E). We also used a immunohistochemical panel
(IHC) with primary monoclonal and polyclonal antibodies.
The sections were heated in citrate buffer (Antigen Retrieval
Citrate buffer pH 6.0) for 30 minutes in a high-power
microwave oven (400W) to perform antigen retrieval. After
rinsing in phosphate buffered saline solution (PBS, pH 7.4),
endogenous peroxidase activity was blocked in 3% hydrogen
peroxide for five minutes. Immunohistochemical procedures
were carried out employing the following primary antibod-
ies.
(1) Anti-A (Actin) mouse monoclonal antibody clone
asm 1 (1A4) Ylem Products S.r.l. Avezzano, Italy). Actin is
a globular structural protein and represents the monomeric
subunit of citosolic microfilaments and of thin filaments
which are part of the contractile apparatus in muscle cells
(actomyosin myofibrils).
(2) Anti S-100 Protein rabbit polyclonal antibody diluted
1 : 600 Novocastra Laboratories Ltd, Belliol Business Park
West, Newcastle (UK). S-100 Protein is a protein that is
present in high concentration in glial and Schwann cells in
which it modulates a wide range of intracellular processes,
including contraction and intracellular signal transduction
and it is involved in promoting axonal growth, glial
proliferation and neuronal differentiation. The Anti S-100
Proteinrepresentsanunequivocalneuroepithelialistogenetic
marker to evaluate the neuroectodermal cell-lines, including
ganglion cells/Schwann cells.
(3) Anti NFs (Neurofilament) mouse monoclonal anti-
body (clone NR4/RT97), diluted 1 : 100 Ylem Products
S.r.l Avezzano, Italy. Neurofilament is a type IV intermediate
filament founded specifically in axons of mature large
neural cells. It probably provides a structural support for
neurons and synapses. The NF immunostaining was used
to evaluate the presence of myenteric plexus in normal and
atretic oesophageal segments and to detect the neurogangliar
structures.
(4) Anti-P (Peripherin) mouse monoclonal antibody
(clone PJM 50), diluted 1 : 100 Novocastra Laboratories
Ltd, Belliol Business Park West, Newcastle (UK). Peripherin
is a type III intermediate filament protein expressed in
matureanddevelopingperipheralneurons,includingenteric
ganglion cells. By revealing biological processes of native
cells and tissue allows to visualize, localize, and quantify
endogenous protein interactions and modifications.
(5) Anti-NSE (Neuron Specific Enolase) mouse mon-
oclonal antibody (clone BBS/NC/VI-H14), diluted 1 :
100 Santa Cruz Biotechnology, CA, USA. The cytosolic
Enolase includes different, widely distributed glycolytic
dimeric enzymes that catalyze the interconversion of 2-
phosphoglycerate and phosphoenolpyruvate. The isoen-
zymes of enolase are mainly founded in neurons and
neuroendocrine cells.
(6) Anti CgA (Chromogranin A) mouse monoclonal
antibody (clone DAK-A3), diluted 1 : 50 Dako A/S,
Glostrup Denmark. Chromogranin A is the major member
of the granin acidic glycoproteins family that plays multiple
roles in the process of regulated neurotransmitters. Cga
immunostaining decores myenteric ganglion cells.
The IHC techniques of S-100 polyclonal and NF mon-
oclonal antibodies were used in both participating institu-
tions.
The sections were incubated with primary antibodies at
4◦C overnight in a moist chamber. After being rinsed in
PBS, the slides were reincubated for 10 minutes at room
temperature with a biotinylated link universal secondary
antibody, and developed in 3.3?-diaminobenzidine-HCl.
After a weak haematoxylin nuclear counterstaining, the
sections were finally dehydrated and mounted in a synthetic
medium.
To evaluate the ganglion cells in each case, five visual
fields that consisted of myenteric and submucous plexuses
were examined under microscope.
3.Results (Table 1)
3.1. Histology. In the control group, the distribution of all
elementswasratherhomogenousinbothproximalanddistal
portions of the esophagus. In contrast, the atretic segments
had hyperplastic lining epithelium, oedema and disarranged
muscle bundles with fibrosis in the UEP. Additionaly, the
DES had mucosal and muscular hypoplasia, abnormal
submuscosal elastic fibers and disorganization of muscularis
mucosae and muscularis propria.
3.2. Immunohistochemistry. (1) Actin. The control exhibited
a well defined muscularis mucosae which was longitudinally
oriented and outer muscle bundles mainly consisted of
skeletal dense fibers by mutual touching. In the UEP
the pathological features displayed agenesia of muscularis
mucosae that was extensively replaced by a soft fibrous
network and an evident disarrangement of muscle bundles.
Moreover,acompletedisorganizationofmuscularismucosae
and muscularis propria was present in the DES. Various
sized muscle bundles were seen, sometimes reaching the
epithelial basal line. The transversal muscular layer was
partially tickened and contained both smooth and striated
contractile cells. In the DES the outer longitudinal muscle
layer was partially loose and thinner than observed in the
control (Figure 1).
(2) S-100 Protein. In this study an increased S-100 cells
expression was evident and prevalent in the muscle layer
of the UEP as computed to the control and distal segment
(Figure 2). Moreover, the atretic segments showed a reduced
number of ganglion cells and the presence, in the DES, of
few small immature neural cells. This feature denotes that
the intrinsic esophageal innervation is deficient.

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International Journal of Pediatrics3
Table 1: Summary of morphologic and immunohistochemical findings of the esophageal segments in human EA/TEF.
Tissue Proximal segment (UEP)
Epithelial hyperplasia
Dysplasia and dystrophy
Reduction of muscularis mucosae
Hyperplasia of elastic fibers
Infiltration of myofibrilles
Dysplasia and dystrophy
Muscular hypoplasia
Fragmentation of the myofibrilles
Connective fibrosis
Endomisial fibrillogenesis
Reduction of neurocells
Increase of Schwann’s cells
Immaturity of neurocells
Muscular hypoplasia
Fragmentation of the myofibrilles
Distal segment (DES)
Epithelial hypoplasia
Dysplasia and dytrophy
Absence of muscularis mucosae
Hypoplasia of elastic fibers
Dysplasia and dystrophy
Mucosa
Submucosa
Circular musculature Muscular hypoplasia
Disorganization of the myofibrilles
Connective fibrosis
Endomisial fibrillogenesis
Reduction of neurocells
Increase of Schwann’s cells
Immaturity of neurocells
Muscular hypoplasia
Disorganization of the myofibrilles
Intermuscular collagen and elastic tissues
Myoenteric plexus of auerbach
Longitudinal musculature
Actin-A control
A
B
C
(a)
Actin-A UEP
B
A
(b)
Actin-A UEP
(c)
Actin-A DES
(d)
Figure 1: Actin-A. (a) Control neonatal esophagus: Anti-A immunostaining marks strongly the muscularis mucosae (A), as well as both the
inner (B) and outer (C) muscle layers; (b) Presence of disarranged and fragmented muscle bundles (A) reaching the epithelial basal line and
segmental agenesia of muscularis mucosae. (B) Anti-A immunostaining is reduced in the smooth musle bundles of the UEP (c), but it is
weaker in (d) the DES.
(3) In our study NF-positive reaction was localized in the
myenteric plexus of normal esophagus and was less intensely
reactiveintheUEPandweakorabsentintheDES(Figure 3):
this weak or negative NF immunostaining reveals a diffuse
neural dismaturity inside the esophageal wall.
(4) In this study myoenteric ganglion cells strongly
express P in the DES, as compared to esophageal controls;
the immunoreactivity for P is less marked in the UEP.
This finding denotes regenerative events of enteric ganglion
cells. This finding is coherent with a parallel defective NF
expression by the same neuroepithelial cell line (Figure 4).
(5) In our cases the NSE immunoreactivity showed
poor or negative espression in both atretic segments in

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4 International Journal of Pediatrics
S-100 P UEP
B
A
(a)
C
S-100 P DES
(b)
Figure 2: S-100 Protein (S-100). (a) The UEP shows fewer ganglion cells in the myenteric plexus (A) and an increased immunoreactivity for
S-100 (B) than the control; (b) The DES shows few small immature cells (C) with less marked immunostaining than in the UEP.
NF UEP-DES
Figure 3: Neurofilament (NF). A diffuse weak immunostaining
expression is obvious at myoenteric site of both atretic esophageal
segments. In some cases the NF immunoreactivity is absent in the
DES.
EA/TEF group (Figure 5). This negative immunoreactivity is
significant for a deficit of the neoglycogenesis.
(6) In the present study we observed, in atretic segments,
a positive immunoreactivity more marked in the UES than
in the DES (Figure 6). A reduced expression of immunore-
active CgA in ganglion could be significant for a defective
neurotransmitter release.
4.Discussion
Despite a significant progressive improvement in the man-
agement of EA/TEF has been achieved, cause of refinements
of surgical and anesthesiologic techniques and neonatal
intensivecare[1–3],thesymptomsrelatedtoesophagealdys-
motility (dysphagia, gastroesophageal reflux disease, altered
or absent peristaltis) are common following successful repair
of EA/TEF [4]. In the past, several authors have pointed out
the presence of motor disorders in the esophageal body and
sphyncters on EA/TEF symptomatic or asymptomatic sur-
vivors:cinefluorographicandisotopicstudiesshowedaltered
esophageal motility also in the long-term follow-up [5, 6];
pH-metry revealed the presence of gastroesophageal reflux
[7]; manometry showed abnormal spastic esophageal zones,
incomplete relaxation of the upper esophageal sphincter,
bifasic peristaltic waves in the esophageal body and, also,
hypotension of the lower esophageal sphincter [8, 9].
Esophageal innervation has been investigated in EA/TEF
to clarify the altered esophageal peristalsis. Nakazato et
al. studying the Auerbach plexus in EA/TEF with H and
E, showed a markedly lower relative amount of neural
tissue, especially in the DES, larger ganglia and ticker
interganglionic fibers in the UEP, a looser than normal
AuerbachplexusintheDESandtoalesserdegreeintheUEP,
suggesting the existence of congenital functional impairment
due to abnormal development of the myenteric plexus [10].
Immunohistochemical studies with Actin showed agen-
esia of muscularis mucosae and an evident disarrangement
of muscle bundles in the UEP [11] and, histologically, com-
plete disorganization of muscularis mucosae and muscularis
propria in the DES [12]. From the above findings, it can
be inferred that EA/TEF is accompanied by a development
failure of some mesodermal-derived buds in connection
with a diagonal deviation of the esophago-tracheal septum.
The finding of disarranged transversal muscle layer for the
immunoreactive to actinin antibodies support this point of
view and it is also in agreement with ganglion and nerve
thinning out.
Recently, other authors studied EA/TEF by histology and
immunohistochemistry in human and in an animal model.
Histopathologic studies with H and E were conducted on
an Adriamycin treated animal model to determine how
closely it resembles the human pattern. In the rat model
immunohistopathological studies found there were signifi-
cant abnormalities of the intramural nervous components of
the oesophagus, involving both the excitatory and inhibitory
intramural nerves and the distribution of nerve fibers in the
plane of the myenteric plexus in the atretic esophagus is

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International Journal of Pediatrics5
P UEP
(a)
P DES
(b)
Figure 4: Peripherin (P). Immunoreactivity is less marked in the myoenteric ganglion cells of (a) the UEP than in (b) the DES.
NSE UEP-DES
Figure 5: Neuron Specific Enolase (NSE). Weak cytoplasmic
positivity in both proximal and distal segments.
deficient [15–21]. These patterns suggested that these abnor-
malities may underlie the esophageal dismotility clinically
showed by infant with EA. Boleken et al. showed, in the
UEP of human EA/TEF, a deficiency of distribution of neural
tissue and elevated expression of S100, as a fine fibrillary
pattern, in the muscular layer and myenteric plexus which
stains for glial cells; moreover, the NF immunoreactivity
was significantly reduced [13]. As this finding was not
detected with NF stain, the increased cells were thought to
be Schwannian in origin, and the hypertrophied glial tissue
might signify a compensation for defective neuronal tissue.
InthepresentstudyweconfirmedthefindingsofBoleken
et al. evidenced in the UEP, while we showed that the
deficiency of distribution of neural tissue is more marked
in the DES. We observed the similar defective expression
for NF in the UEP but it, in some instances, is absent in
the DES. The NF immunostaining is a specific stain for
mature neurofilament, which means a defective or absent
maturation of nerve cells.
For this purpose, we used the Peripherin (P) stain
observing an immunoreactivity more marked in the DES
than in the UEP; this finding denotes an early maturation
levelofentericganglioncells(neuroblasts).Itiscoherentwith
aparalleldefectiveNFexpressionbythesameneuroepithelial
cell line. The specificity of the P indicates that this inter-
mediate filament protein has potential as a marker for this
related neural crest neuropathy. Li et al. showed, in the DES,
decreased staining of NSE (and substance P) and increased
staining of VIP (and NOS): the imbalanced excretion of
neurotrasmitters in the intramuscular motor nerve endings
and the abnormal intrinsic dysplasia of myenteric nerve
plexus, were the main characteristics of abnormal intrinsic
innervation, which may be responsible for the postoperative
dysfunction of EA/TEF [14].
In the present study, we also confirmed the previous
innervation patterns described by Li et al. for the NSE
immnoreactivity and we observed a decreased or negative
cytoplasmic positivity in both proximal and distal segments.
This negative immunoreactivity is significant for a deficit of
neoglycogenesis.
CgA has been used as an immunohistochemical marker
for normal and pathological neuroendocrine tissues: the
physiologic role of this protein has not been fully elucidated,
but it has been hypothesized that it may serve as a precursor
to other biologically active peptides or may serve as a
function in the intracellular production of hormones and
neuropeptides. We used the CgA stain observing a positive
immunoreactivity more marked in the UEP than in the DES;
the reduced Cga expression in the DES might be significant
for incomplete activity of neurotransmitter release.
In conclusion, our histological and immunohistochem-
ical studies partially confirmed the previous observations,
adding at the same time new possibility to study in
depth the UEP and DES segments. In fact, the present
investigation on the atretic segments of EA/TEF, specially
the DES, showed, with the P-immunostaining, the presence
of immature neural cells (neuroblasts) and, with the CgA
immunoreactivity, a granin deficit with subsequent altered
activity of neurotransmitter release. These findings indicate
a delay in neuronal differentiation and myenteric plexus
organization that helps to explain the esophageal motility
disorders seen before surgery in patients with EA/TEF, and