Neurogastroenterol Motil (2004) 16, 515–531 doi: 10.1111/j.1365-2982.2004.00538.

REVIEW ARTICLE

Human enteric neuropathies: morphology and molecular

pathology
R. DE GIORGIO* & M. CAMILLERI* 

*Department of Internal Medicine & Gastroenterology, University of Bologna, Bologna, Italy

 Clinical Enteric Neuroscience Translational and Epidemiological Research (CENTER) Program, Mayo Clinic College of Medicine,
Rochester, MN, USA

Abstract The aim of this study is to review current
understanding of the molecular and morphological
pathology of the enteric neuropathies affecting motor
function of the human gastrointestinal tract and to
evaluate the described pathological entities in the
literature to assess whether a new nosology may be
proposed. The authors used PUBMED and MEDLINE
searches to explore the literature pertinent to the
molecular events and pathology of gastrointestinal
motility disorders including achalasia, gastroparesis,
intestinal pseudo-obstruction, colonic inertia and
megacolon in order to characterize the disorders
attributable to enteric gut neuropathies. This
scholarly review has shown that the pathological
features are not readily associated with clinical fea-
tures, making it difficult for a patient to be classified
into any specific category. Individual patients may
manifest more than one of the morphological and
molecular abnormalities that include: aganglionosis,
neuronal intranuclear inclusions and apoptosis, neural
degeneration, intestinal neuronal dysplasia, neuronal
hyperplasia and ganglioneuromas, mitochondrial
dysfunction (syndromic and non-syndromic), inflam-
matory neuropathies (caused by cellular or humoral
immune mechanisms), neurotransmitter diseases and
interstitial cell pathology. The pathology of enteric
neuropathies requires further study before an effective
Address for correspondence
Michael Camilleri MD, Clinical Enteric Neuroscience
Translational and Epidemiological Research (CENTER)
Program, Charlton 8-110, Mayo Clinic, 200 First Street S.W.,
Rochester, MN 55905, USA.
Tel: 507-266-2305; e-mail: camilleri.michael@mayo.edu
Received: 22 October 2003
Accepted for publication: 30 December 2003
nosology can be proposed. Carefully studied individ-
ual cases and small series provide the basic frame-
work for standardizing the collection and histological
evaluation of tissue obtained from such patients.
Combined clinical and histopathological studies may
facilitate the translation of basic science to the clin-
ical management of patients with enteric neuropa-
thies.
Keywords Enteric nervous system, neuropathy, proto-
oncogenes, apoptosis, neurodegeneration, biopsy.
INTRODUCTION
The enteric nervous system (ENS) represents a vast
neural network distributed through the entire aliment-
ary tract, biliary tract and pancreas. Based on
histochemical and electrophysiological properties, the
80–100 million enteric neurones can be classified into
functionally distinct subpopulations, including intrin-
sic primary afferent neurones, interneurones, motor
neurones, secretomotor and vasomotor neurones.1
Enteric nerve cells are organized in two main plexuses,
the myenteric (Auerbach’s) and submucosal (Meiss-
ner’s) neurones that are synaptically connected in reflex
circuits. There is modulation of these reflexes by the
central nervous system (CNS). However, the ENS has
the unique ability to control most gut functions, such
as regulating secretion/absorption, vascular tone and
motility.1 Given these important functions of the ENS,
it is not surprising that damage to the ENS results in
digestive disorders and disturbed quality of life.1 The
mechanisms leading to enteric neuropathies remain
incompletely understood. It is also important to
acknowledge that the classification of enteric neurones
is based predominantly on work from laboratory

2004 Blackwell Publishing Ltd 515

R. De Giorgio & M. Camilleri
animals. However, there is increasing evidence that
human ENS neurochemical coding in health mimics
that of laboratory animals such as the guinea-pig.2–4
Although extrinsic neuropathies such as diabetes or
amyloidosis are known to cause gastrointestinal motil-
ity disorders, these are usually well recognized by the
systemic or peripheral features of the disease. A major
clinical diagnostic challenge is presented by the pres-
ence of clinical syndromes such as gastroparesis or
pseudo-obstruction in the absence of such a systemic
disease.5,6 Dilatation of gastrointestinal segments is
not a consistent finding and, hence, the use of function
tests to document the presence of dysmotility. Patho-
logical diagnosis has generally lagged behind func-
tional characterization because the benefit : risk ratio
associated with full-thickness intestinal biopsies has
been unclear.
Nevertheless, the published literature reveals a
variety of morphological abnormalities in enteric neu-
ropathies. With the published data, we posed the
question of whether the described cases, series or
pathological descriptions lend themselves to the devel-
opment of a new nosology, or classification, of the
enteric neuropathies.
Important caveats that must be acknowledged
and limit the outcome of this review
Firstly, smooth muscle disorders result in gut dysmo-
tility; however, the present review focuses on the
structural, cellular or subcellular abnormalities of
neurones rather than muscle cells, except in situations
where the neurones are also affected such as mitoch-
ondrial cytopathy.
Secondly, conditions such as diabetes or amyloidosis
that affect the extrinsic nerves have been associated
with enteric neuropathies, such as the loss of inhibi-
tory nerves or interstitial cells of Cajal in diabetes. We
have elected to focus this review on diseases affecting
the enteric nerves, and have included interstitial cells
of Cajal in this discussion, given the close relationship
with nerves and increasing evidence that they mediate
enteric motor neurotransmission.
Thirdly, the limited motor repertoires and disease
phenotypes (e.g. transfer dysphagia, gastroparesis, con-
stipation, pseudo-obstruction or incontinence) make it
unlikely that individual pathological categories would
be associated with specific clinical features.
Fourthly, pathological features are not mutually
exclusive; there may be an overlap of the mechanisms
that result in damage to the enteric nerves. This
principle is also demonstrated in other diseases such as
chronic inflammatory bowel disease or chronic hepa-
516
Neurogastroenterology and Motility
titis that may be associated with a variety of inflam-
matory cell infiltrates, as well as apoptosis. It is not
surprising that diseased intestinal tissue may demon-
strate several pathological features that may result
from one or more pathobiologies.
The primary aim of this article is to review the
literature on the molecular and morphological pathol-
ogy of enteric neuropathies affecting gut motor function,
and to determine whether a novel nosology, or classifi-
cation, of these conditions can be developed based on the
literature. A secondary aim is to propose the ways in
which to handle and evaluate tissue obtained from
patients with suspected enteric neuropathies in order to
acquire data that will facilitate future attempts to
develop a classification of enteric neuropathies.
PATHOLOGICAL FEATURES OF ENTERIC
NEUROPATHIES
The following section summarizes the reported mor-
phological and molecular features in enteric neuropa-
thies that result in gut dysmotility in the absence of
systemic or easily identified neuromuscular disorders
such as autonomic neuropathies, parkinsonism and
multiple sclerosis. The literature shows that many of
the features may be present in the same tissue
Figure 1 (A) Expression of c-Ret in progenitors of the mam-
malian enteric nervous system (ENS) Whole-mount in situ
hybridization of an E9.5 mouse embryo with a riboprobe
specific for Ret mRNA: note ENS P, RET-expressing precur-
sors of the ENS, entering the gastrointestinal tract. Repro-
duced from Pachnis et al. Am J Physiol 275:G183–6, 1998.
(B) Contribution of vagal and sacral neural crest to formation
of ENS: C-ret dependent sympathoenteric lineage originates
in vagal neural crest of the hindbrain and migrates ventrally
to populate the entire gut and superior cervical ganglion
(SCG); C-ret independent sympathoadrenal lineage originates
in truncal crest and populates foregut and sympathetic chain;
and sacral neural crest is derived from spinal cord and colon-
izes mainly the hindgut.

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specimen, such as inflammation, apoptosis and degen-
eration. A different mechanism is unlikely to be
responsible for each feature; there is a limited mor-
phological phenotype of the histopathology of these
disorders. A combination of molecular derangements
may contribute to the degenerative process that leads
to enteric neuronal loss. These include disorders of
intracellular Ca2+ signalling, mitochondrial dysfunc-
tion, oxidative stress and alterations in signal trans-
duction pathways.
Table 1 lists the pathological features described in
enteric neuropathies in the literature.
Aganglionosis
Aganglionosis occurs most frequently in the congenital
form, Hirschsprung’s disease. This is characterized by
the complete absence of ganglion cells in the sub-
mucosal and myenteric plexuses.7
Morphological and molecular pathology Hirschsprung’s
disease is a polygenic disorder characterized by muta-
tions affecting a wide array of genes that control tyro-
sine kinase function and the neurotrophins that play a
crucial role in neuronal differentiation, maturation and
binding to the tyrosine kinase receptor (Fig. 1). The
genetic disorders affect: (i) the RET proto-oncogene and
the genes encoding for its ligands [glial-derived neuro-
trophic factor (GDNF) and neurturin (NRTN)]; (ii)
endothelin-3 and endothelin-B receptor (EDN3/ED-
NRB), and endothelin-converting enzyme (ECE1); and
(iii) the transcription factors, Sox10 and SMADIP1;8–16
modifiers genes for these transcription factors are as
yet unidentified.
The RET mutations have been found in about 50%
of familial and 17–20% of sporadic forms of Hirsch-
sprung’s disease.8–10 Hirschsprung’s disease or mega-
colon occur either as a sole disease or as part of
syndromes such as multiple endocrine neoplasia type
Table 1 Pathological features of enteric neuromuscular
disease
Aganglionosis
Neuronal intranuclear inclusions and apoptosis
Neural degeneration
Intestinal neuronal dysplasia
Neuronal hyperplasia and ganglioneuromas
Mitochondrial dysfunction: syndromic and non-syndromic
Inflammatory neuropathies: cellular and humoral mechanisms
Neurotransmitter disorders
Interstitial cell pathology

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Neuropathology of gut dysfunction
2B12 or familial thyroid carcinoma or multiple endo-
crine neoplasia 2A13 or Waardenburg–Shah syndrome
in which megacolon is accompanied by pigmentary
disorders and neural deafness.8–10 Deletions or trunca-
ting mutations in SMADIP1 gene are responsible for
syndromic Hirschsprung’s disease with microcephaly,
mental retardation and facial dysmorphism.17
Clinical observations and implications Suction rectal
biopsies that include small amounts of rectal submu-
cosa show the absence of submucosal ganglia and
hypertrophic submucosal nerves, which represent
projections from extrinsic nerve fibres18 into the
muscularis mucosae and lamina propria. Acetylcholi-
nesterase enzyme histochemistry or other markers
facilitate diagnosis in equivocal cases. A physiological
zone of aganglionosis exists in the terminal 1–3 cm of
the rectum and may lead to false-positive diagnosis of
Hirschsprung’s disease;19 conversely, efforts to obtain
biopsies proximal to this zone may ÔmissÕ a very short
segment of clinically significant aganglionosis.20
From these studies, one notes that several differ-
ent genetic disorders or molecular pathologies result
in the same clinical phenotype of Hirschsprung’s
disease.
Neuronal intranuclear inclusions and apoptosis
Morphological and molecular pathology Apoptosis of
myenteric neurones has been described in diseases
associated with myenteric ganglionitis (see below);
earlier literature documented the presence of neuronal
intranuclear inclusions in association with documen-
ted pseudo-obstruction. Analysis of these intranuclear
inclusions showed they were composed of proteina-
ceous material (without evidence of DNA, RNA, or
carbohydrate), and electron microscopy documented
membrane-bounded filaments.
A distinct degenerative process is characterized by
apoptotic bodies, which are features of programmed
cell death.21 These bodies are the result of nuclear
condensation and fragmentation, fragmented DNA,
and subsequent condensation of the cell. Other intra-
cellular organelles may be preserved.
Clinical observations and implications It is unclear
whether intranuclear inclusions are primary abnor-
malities or secondary to an underlying disease. Similar
intranuclear inclusions occur in neurones of patients
with central nervous system diseases which do not
involve the gastrointestinal tract.22–24 Lewy bodies
have also been observed in myenteric neurones
of patients with parkinsonism and experienced
517
R. De Giorgio & M. Camilleri
neuropathological and clinical appraisal of such
patients is needed for correct classification.25
Neuronal degeneration
Morphological and molecular pathology Histological
examinations of the myenteric plexus with silver
staining of sections taken in the long axis of the bowel
from patients with chronic intestinal pseudo-obstruc-
tion showed a reduction in the total number of neu-
rones.26,27 The remaining neurones may be enlarged
with thick, clubbed processes; some show an increase
in the number of Schwann cells and hypertrophy of the
muscularis propria. Neurotrophins, including nerve
growth factor (NGF), brain-derived neurotrophic fac-
tors (BDNF), neurotrophin-3 (NT-3) and other mole-
cules, decrease neuronal death evoked by a number of
noxious agents ranging mechanical (i.e. axotomy),
chemical (i.e. free radicals), or ischaemic injury.
Neurotrophins exert their effects via tyrosine kinase
receptors (Trk-A, -B and -C) and play a crucial role in
neuronal development, differentiation, and survival
and maintenance of the mature ENS.28
Clinical observations and implications Understanding
the mechanisms involved in neuronal degeneration and
death may provide the conceptual basis for treatment of
ENS abnormalities. Neurotrophins may have thera-
peutic potential to heal neuronal injury and prevent
neuronal death and this led to trials in amyotrophic
lateral sclerosis and diabetic neuropathy. Formal studies
of the trophic effects in humans have not been per-
formed to date. A small study patients with chronic
constipation showed that over the short-term, the
neurotrophin NT-3 accelerated small bowel and colonic
transit and relieved constipation.29
Intestinal neuronal dysplasia
Morphological and molecular pathology Two sub-
types of intestinal neuronal dysplasia (IND) defined as
type A and B were recognized.30 IND type A is ex-
tremely rare and is characterized by an immaturity or
hypoplasia of the extrinsic sympathetic nerves sup-
plying the gut. Patients with this extrinsic nerve
abnormality present with diarrhoea and bloody stools.
In contrast, IND type B is more commonly described
and is associated with a variety of changes in the
intrinsic innervation of the gut. These range from in-
creased density of submucosal ganglia (hyperganglio-
nosis) to increased numbers of ganglion cells per
submucosal ganglion (giant ganglia). The latter may be
associated with ectopic neurones localized throughout
518
Neurogastroenterology and Motility
the lamina propria of the colonic mucosa. The
molecular mechanisms associated with these entities
are unclear.
Clinical observations and implications Intestinal
neuronal dysplasia type B is a highly controversial
entity. The reported changes (ectopic ganglia, increased
prominence of submucosal innervation), have been
observed in a variety of clinical contexts including the
transitional zone between ganglionic and aganglionic
gut in patients with Hirschsprung’s disease, proximal
to obstructions, and in patients with idiopathic pseudo-
obstruction. The specificity of the histological features
used to define IND is unclear: some pathologists con-
sider that this finding is within the spectrum of nor-
mality or it represents a secondary event and may have
no aetiological significance.31 Conversely, others place
great emphasis on this disorder, and have recommen-
ded rectal biopsy as a convenient method for diagno-
sis.32 The controversy illustrates the subjective
interpretation of intestinal biopsies, lack of easily ap-
plied methods to quantify ganglion cells, and the need
for better clinical–pathological correlation in this field.
In practice, it is important to note that transit studies
can be abnormal in IND,33 and that the described
neurochemical abnormalities (reduction of substance P
immunoreactive neurones)34 in the circular muscle are
similar to the findings reported in some adults with
slow transit constipation.
Neuronal hyperplasia and ganglioneuromas
Morphological and molecular pathology Ganglioneu-
romas are nodular proliferations of ganglion cells and
abundant nerve fibres with associated glia. They oc-
cur as solitary or diffuse lesions in the myenteric
plexus.35 Diffuse ganglioneuromatosis with massive
proliferations of neural tissue (neurones, supporting
cells and nerve fibres) appears as thickened nerve
trunks among mature nerve cells. This histopatho-
logical appearance is almost pathognomonic of mul-
tiple endocrine neoplasia type 2B (MEN2B), a
heritable disorder associated with tumours of the
neuroendocrine system (Fig. 2).
MEN 2B is a dominantly inherited disorder due in
c. 95% of cases to M918T missense mutation in the
RET proto-oncogene, which encodes a tyrosine kinase
receptor. This is expressed particularly in neural crest-
derived cells including the enteric ganglia.36 The
remaining c. 5% of patients have a point mutation at
codon 883 [A883F].37,38 The mutation alters RET
substrate specificity in a ligand-independent fash-
ion (a gain of function mutation)39–41 that increases

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Volume 16, Number 5, October 2004
Figure 2 Family history and features of
proband with multiple endocrine neoplasia
type 2B: Note the autosomal dominant
inheritance, the blubbery lips and ganglio-
neuromas on the tongue, the massive colon
after resection and the presence of prominent
ganglioneuromas in the muscle layer of the
colon.
susceptibility to endocrine tumours (medullary thyroid
carcinoma, adrenal phaeochromocytoma and parathy-
roid tumours).
Clinical observations and implications MEN 2B pre-
sents with severe constipation or megacolon, diarrhoea
(when associated with enterocolitis), or obstruction,
often in infancy.42,43 Other external stigmata of MEN
2B are: a characteristic facies, Ôblubbery lipsÕ from
mucosal neuromas, marfanoid habitus, medullated
corneal nerve fibres, and medullary thyroid carci-
noma.42,44 The latter develops eventually in almost all
patients. Patients with transmural intestinal ganglio-
neuromatosis should undergo molecular diagnostic
testing by RET mutation analysis,45 prophylactic
thyroidectomy, and adrenal gland surveillance (ultra-
sound scanning and urinary fractionated catecholam-
ines).45
Mitochondrial dysfunction
Mitochondrial dysfunction occurs rarely in syndromic
or genetic mitochondrial cytopathies and more com-
monly as part of a degenerative process, e.g. in ageing.
Mitochondrial neurogastrointestinal
encephalopathy
Morphological and molecular pathology Mitochon-
drial neurogastrointestinal encephalopathy (MNGIE)
forms part of a heterogeneous group of disorders that
result from structural, biochemical or genetic derange-
ments of mitochondria. The literature also refers to
this entity as type II familial visceral myopathy. Mit-
ochondrial DNA contains genes that encode polypep-
tides that are components of the cellular oxidative
phosphorylation system. Nuclear genes, however, also

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Neuropathology of gut dysfunction
encode for components of this system. It is now
believed that mutations of nuclear DNA genes that
control the expression of the mitochondrial genome are
the underlying genetic defect of this syndrome.46 The
nuclear DNA genes are located in the long arm of
chromosome 22 (22q13.32-qter), distal to locus
D22S1161.
Clinical observations and implications MNGIE has
an autosomal recessive inheritance and it is charac-
terized by gastrointestinal dysmotility, ophthalmople-
gia and peripheral neuropathy. The ubiquity of
mitochondria explains the association of neuromus-
cular, gastrointestinal and other non-neuromuscular
symptoms that are characteristic of this syndrome. On
skeletal muscle biopsy, there are megamitochondria at
a subsarcolemmal location giving the appearance of
ragged-red fibres, best demonstrated on Gomori tri-
chrome stain.47,48 Additional clinical features include
lactic acidosis, increased cerebrospinal fluid protein,
and leukodystrophy, which is identified by magnetic
resonance imaging of the brain.
Non-syndromic or acquired mitochondrial dysfunc-
tion Two examples of this process are cited from the
literature.
1 Ageing is associated with increased prevalence of
motor disorders of the gut, particularly constipation.
Given the structural and functional similarities of
central and enteric neurones, it has been postulated
that neurodegenerative mechanisms resulting in CNS
diseases may also occur in enteric neuropathies. These
mechanisms include: disorders of intracellular Ca2+
signalling (primary or secondary to autoantibodies
targeting voltage-activated Ca2+ channels, see below),
mitochondrial dysfunction, oxidative stress (i.e. for-
mation and/or reduced scavenging of reactive oxygen
519
R. De Giorgio & M. Camilleri
species including nitric oxide) and alterations in signal
transduction pathway (i.e. mitogen-activated protein
kinase, c-Jun NH2-terminal protein kinase and phos-
phatidylinositol 3-kinase). These and other events can
disrupt enteric neurone homeostasis, thus leading to
either necrosis or apoptosis.49
2 Neuropathic intestinal pseudo-obstruction with
mitochondrial dysfunction: The role of mitochondrial
dysfunction in neural injury was explored through
studies of the mitochondrial membrane proteins enco-
ded by the B-cell lymphoma-2 (BCL-2) gene family.50
In the presence of exogenous noxious agents or stimuli
of apoptosis, Bcl-2 or Bcl-xL expression (anti-apoptotic
agents) decrease while Bax (and related proteins that
are pro-apoptotic) increases. This unbalanced Bcl-2/Bax
ratio triggers the release of cytochrome c, initiating the
cascade of apoptosis.51 Bcl-2 protein expression in the
ENS of patients with neurogenic type of chronic
idiopathic intestinal pseudo-obstruction was reduced
and was associated with increased neuronal apopto-
sis.52
Mitochondrial dysfunction associated with neural
cell death may involve complex mechanisms. For
instance, high concentrations of excitatory neurotrans-
mitters, such as glutamate, may induce neurotoxicity
in the ENS53 by an early process that involves necrosis
and later, by activation of apoptosis. Functional
impairment or damage of mitochondria may also result
in an increased intracytosolic Ca2+ resulting in neur-
onal cell injury, as has been documented in an
inherited form of mitochondrial encephalomyopathy
in humans.54
Inflammatory neuropathies
These forms of neuropathies are characterized by an
inflammatory or immunological insult to the intrinsic
innervation supplying the gastrointestinal tract, and
Figure 3 Micrographs showing both types of T-lymphocytes, CD4 (A) and CD8 (B), detectable within the myenteric plexus of the
small intestine (proximal ileum) of a 20-year old man with chronic intestinal pseudo-obstruction. Note the intense CD4 and CD8
immunoreactivities which represent the predominant component of the immune infiltrate observed in cases of lymphocytic
ganglionitis. Alkaline phosphatase anti-alkaline phosphatase immunohistochemical technique. Original magnification: ·120 in A
and B.
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Neurogastroenterology and Motility
are referred to as enteric ganglionitis.26,55–58 Ganglio-
nitis and axonitis represent derangements in the neuro-
immune interactions occurring within the enteric
neural microenvironment.
Morphological and molecular pathology 1 Cellular
mechanisms in myenteric ganglionitis: immunohisto-
chemical analysis of the inflammatory infiltrate in
cases of myenteric ganglionitis revealed a significant
component of CD3 positive T lymphocytes surround-
ing ganglion cell bodies: the majority were CD4
(T-helper) (Fig. 3A) and CD8 (T-cytotoxic/suppressor)
(Fig. 3B) positive lymphocytes distributed with a
relative ratio of 1 : 1 (instead of the normal 2 : 1). This
suggests predominant T-cytotoxic activity, possibly
directed against proteins expressed by myenteric neu-
rones in chronic intestinal pseudo-obstruction.56, 59–62
Goldblum et al. recently confirmed the predominance
of CD8 positive lymphocytes in achalasia associated
with myenteric ganglionitis.63
Other immunocytes infiltrating the myenteric
plexus include CD79a expressing cells, that is, mature
B-lymphocytes (Fig. 4).59,61,62 In view of the circula-
ting anti-neuronal antibodies in patients with myen-
teric ganglionitis, these B lymphocytes may contribute
to the immune response by synthesizing and releasing
immunoglobulins directed against antigens expressed
by myenteric neurones (see below).
Eosinophils and neutrophils may also affect enteric
neurone function. In a mouse model infected with
Schistosoma mansoni,64 there is mucosal granuloma-
tous ileitis and myenteric ganglionitis with eosinophi-
lic and neutrophilic granulocytes, but no significant
neurodegeneration. Schäppi et al. reported on three
children with intestinal pseudo-obstruction with an
eosinophilic infiltrate and neuronal expression of IL-5,
a potent eosinophil chemoattractant.65 Eosinophilic
ganglionitis was not associated with neuronal cell

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gut motility disorders with an underlying myenteric

Figure 4 Micrograph illustrating lymphocytes immunolabe-
led for CD79a, a marker for mature B cells, surrounding and
infiltrating a myenteric plexus of the small intestine (prox-
imal ileum) of a 20-year-old man with chronic intestinal
pseudo-obstruction. In addition to T lymphocytes, the pres-
ence of B lymphocytes in cases of myenteric ganglionitis
provide the basis for a humoral immune response, which may
contribute to enteric neuron dysfunction. Alkaline phospha-
tase anti-alkaline phosphatase immunohistochemical tech-
nique. Original magnification: ·120.
damage. A similar histopathological pattern was
observed in an adult patient with acute colonic
pseudo-obstruction [i.e. Ogilvie’s syndrome (Barbara
and De Giorgio, unpublished data)].
2 Humoral mechanisms, anti-neuronal antibodies:
myenteric ganglionitis is associated with a wide array
of circulating anti-neuronal antibodies. The detection
of anti-neuronal antibodies is a useful tool to diagnose
ganglionitis.59,66 Anti-neuronal antibodies may be
associated with an underlying disease (mainly a para-
neoplastic syndrome) or to idiopathic forms of myen-
teric ganglionitis.59,62,66 The associated neoplasms
include small cell lung carcinoma, thymoma, gynae-
cological and breast tumours.55,56,66–68 It is hypothes-
ized that these autoantibodies are directed against
antigens shared by tumour cells and by enteric neu-
rones (onconeural antigens).69
In vitro studies show that serum from patients with
circulating antibodies may reduce neuromuscular
function in rat intestinal muscle strips.70 This suggests
that the antibodies are functionally significant.
Anti-neuronal antibodies target a variety of mole-
cules including the RNA binding protein Hu (anti-Hu
or type-1 anti-neuronal nuclear antibodies, ANNA-1),
the Purkinje cell protein Yo (anti-Yo, anti-Purkinje cell
cytoplasmic antibodies), and P/Q- and N-type Ca2+
channels and ganglionic type nicotinic acetylcholine
receptors (Table 2).
(a) Anti-Hu antibodies are the most common type of
anti-neuronal antibody identified in paraneoplastic
conditions.69,71,72 The Hu proteins, including HuC,
HuD, HuR and Hel-N1, are expressed in several cell
types and share sequence homology with RNA-binding
proteins of Drosophila. With the exception of HuR, the
Hu proteins are specifically detected in central, per-
ipheral and enteric neurones, where they are involved
in development and survival mechanisms.73 Anti-Hu
antibodies may also cause neuronal degeneration in

Table 2 Anti-neuronal antibodies in inflammatory neuropathy associated with either paraneoplastic or idiopathic gut dysmotility

Underlying tumours Idiopathic

and related cases
Anti-neuronal Molecular paraneoplastic (unassociated GI motor
autoantibodies target Function syndrome with cancer) disorder Ref. no.

ANNA-1 HuD, HuC, Family of RNA SCLC/opsoclonus Yes Gastroparesis, 69–74

(or Anti-Hu) HuR Hel-N1 binding proteins myoclonus; ataxia CIP,
megacolon
Anti-VGCC Voltage-gated Regulation of Ca2+ SCLC/ Unknown CIP 71,75
Ca2+ channels, influx and Lambert-Eaton
including P/Q signalling syndrome
and N-type
channels
Anti-ganglionic Nicotinic Acetylcholine Thymoma, SCLC/ Yes Gastroparesis, 76
acetylcholi receptors signalling dysautonomia CIP,
receptors constipation
Anti-Yo Cdr2 Transduction Gynaecological tumours Unknown CIP 66,77
signal protein (i.e. ovary)/cerebellar
paraneoplastic
degeneration

SCLC, small cell lung cancer; VGCC, voltage gated calcium channel; CIP, chronic intestinal pseudo-obstruction.

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R. De Giorgio & M. Camilleri
the ENS. Serum containing high titres of anti-Hu
antibodies applied in vitro to neuroblastoma cell line
and cultured enteric neurones evoked apoptosis74 with
expression of activated, pro-apoptotic messengers,
including caspase-3 and apaf-1.
(b) Anti-voltage-gated Ca2+ channel (P/Q- and
N-type) antibodies are primarily identified in Lam-
bert-Eaton myasthenic syndrome related to small cell
lung carcinoma75 and may evoke autonomic nervous
system dysfunction. With anti-Hu, autoantibodies
targeting the N-type Ca2+ channels are the most
prevalent in patients with paraneoplastic dysmotili-
ty.71
(c) Anti-ganglionic acetylcholine receptor antibodies
have been reported in patients with a wide array of
idiopathic or secondary (including paraneoplastic) dys-
autonomic diseases with involvement of the gastroin-
testinal tract,76 although the ability to block the
receptor is infrequent. Titre fluctuation appears to
correlate with disease severity, suggesting a contribu-
tion to dysautonomia and gut dysmotility.76
(d) Anti-Yo antibodies occur in rare cases of para-
neoplastic gastrointestinal dysmotility as a manifesta-
tion of ovarian carcinoma.66 The molecular target is
the Yo antigen recently re-defined as the cerebellar-
degeneration-related (Yo/cdr) transduction signal pro-
tein. This inhibits c-myc transcriptional activity and
may lead to neuronal degeneration via apoptosis.77
Other antibodies directed against neuronal proteins
may be generated in response to neuronal degeneration
in myenteric ganglionitis.78
Clinical observations and implications Enteric gan-
glionitis is characterized by a dense infiltrate of
lymphocytes and plasma cells involving either the two
major ganglionated plexuses and related axonal
processes of the ENS or, more commonly, only the
myenteric plexus (i.e. myenteric ganglionitis). This can
be secondary to a wide variety of conditions including
paraneoplastic (e.g. small cell carcinoma, carcinoid,
neuroblastoma and thymoma),66,79–81 infectious (e.g.
ChagasÕ disease),82–84 immune-mediated degenerative
processes of the central nervous system (e.g. encephalo-
myeloneuropathy),85 connective tissue disorders (e.g.
scleroderma)86 and inflammatory bowel diseases (e.g.
ulcerative colitis and Crohn’s disease).87 Some cases
have no underlying cause identified (idiopathic).59–62
Myenteric ganglionitis is often associated with degen-
eration and neuronal loss, which, in its rarer extreme
form, is called acquired aganglionosis60 (Fig. 5A, B). The
resulting impairment of enteric neurone reflexes leads
to dysmotility and delayed transit. The segment of
the gastrointestinal tract affected determines the
522
Neurogastroenterology and Motility
Figure 5 Representative micrographs taken from the colon of
a 23-year-old woman with long-standing history of chronic
idiopathic constipation and megacolon due to an underlying
lymphocytic myenteric ganglionitis. The two photomicro-
graphs show the absence of the immunolabelling for the
general neuronal marker neuron-specific enolase in the
myenteric plexus in (A), which is in contrast with the normal
appearance of the same marker immunoreactivity identified
in the submucous plexus of the same section, as illustrated in
(B). Streptavidin–biotin complex peroxidase immunohisto-
chemical technique. Original magnification: ·160 in A and B.
clinical manifestations and include oesophageal and
lower oesophageal sphincter (LOS) dysmotility,88
gastroparesis,61 intestinal pseudo-obstruction and
colonic inertia or megacolon.58–62,66,79–81 The clinical
implication is that in patients with idiopathic motility
disorders should have tests for a panel of antibodies,
and underlying malignancy should be sought when the
serology is positive.
Recently, Tornblom et al. described low-grade
lymphocytic myenteric ganglionitis in the proximal
jejunum in nine of 10 patients with severe irritable
bowel syndrome (IBS)88 and proposed that an inflam-
matory neuropathy of the ENS may contribute to
sensorimotor abnormalities in functional bowel syn-
dromes unassociated with bowel dilatation. The find-
ing of few lymphocytes (1.9–7.1 per ganglion) within
enteric ganglia of IBS patients raises the question about
the specificity of such an inflammatory infiltrate
within the ENS, particularly because some element
of neuronal degeneration was also demonstrable mor-
phologically. In contrast, virtually all cases of myen-
teric ganglionitis described in the literature are
consistently associated with a dense lymphocytic
infiltrate (Fig. 6), neuronal degeneration and loss, and
severe gut motor impairment sometimes with bowel
dilatation.58–62,79,80,84,89 It is unclear whether the
severity of inflammatory infiltration predicts the
degree of neuromuscular dysfunction in the con-
tinuum between IBS and more overt motility disorders
(i.e. pseudo-obstruction with dilated segments of
bowel), or whether the consequences of the inflamma-
tory insult to the myenteric plexus depends on the
individual’s genetic background. A detailed study of
the inflammatory infiltrate of IBS patients focused
on the mucosa and lamina propria, although no

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Volume 16, Number 5, October 2004 Neuropathology of gut dysfunction

Understanding these interactions may provide new

perspectives in the pathophysiology of motility disor-
ders, and, with early diagnosis, may provide a rationale
for immunosuppressive treatment as demonstrated by
several small clinical reports (Table 3).58–62,65 How-
ever, there is a need for controlled studies comparing
anti-inflammatory agents including immunosuppres-
sives and plasma exchange before recommendations
are made to treat patients with potentially harmful
therapies.

Neurotransmitter disorders

Figure 6 Representative photomicrograph showing an infil-
trate of CD3 positive lymphocytes (T cells) (red-brown colour)
densely packed within a myenteric plexus of the small
intestine (proximal ileum) of a 20-year-old man with chronic
intestinal pseudo-obstruction. Alkaline phosphatase
anti-alkaline phosphatase immunohistochemical technique.
Original magnification: ·120.
evaluation of neuronal injury or enteric plexuses was
undertaken.90 More quantitative studies of the mor-
phology of the myenteric plexus, neurotransmitter and
receptor expression are required.
Morphological and molecular pathology Neurotrans-
mitter disorders are described in dysfunction of
sphincteric regions and include achalasia and congen-
ital hypertrophic pyloric stenosis, in which loss of
intrinsic inhibitory neurones (NO, VIP, somatostatin)
has been described. The deficiency may be attributable
to a variety of genetic defects91 but more importantly,
these deficiencies (e.g. in achalasia) may result from
inflammatory or degenerative processes.
Of the acquired neurotransmitter disorders affecting
non-sphincteric regions, the neurotransmitter disor-

Table 3 Clinicopathological features and outcome of patients with primary forms of myenteric ganglionitis

Anti-inflammatory therapy
Patients and
dysmotility Inflammatory Anti-neuronal Outcome of
type Pathology infiltrate antibodies Treatment given treatment Ref. no.

4 F patients with Lymphoid infiltrate Polyclonal Not tested 3 ¼ antibiotic Mild 59

CIP in the LP, MP, T and B cells 1 ¼ cyclophosphamide symptomatic
MyP; no and prednisone improvement
neuromuscular
degeneration
Two patients with Myenteric Predominance Anti-Hu Steroids at 1 improved; 60
CIP (1 M, 1 F) ganglionitis, of T cells (CD4 (orANNA-1) different doses 1 SB transplant
neuronal loss, or and CD8 in both cases
aganglionosis positive)
1 M patient with Myenteric Predominance Not tested Methyl Marked 61
gastroparesis ganglionitis, of T cells (CD4 prednisolone improvement
neurodegeneration, and CD8 in a tapering
marked decrease of positive) fashion
SP-containing nerves
2 F patients with Myenteric Predominance Anti-Hu Short-course Significantly 62
colonic inertia or ganglionitis, of T cells (CD4 (orANNA-1) in methyl- ameliorated
megacolon and neuronal loss, or and CD8 the male patient; prednisolone
1 M patient aganglionosis positive) not tested in treatment in
with CIP the 2 F patients the male patient
3 F patients with Myenteric Predominant Not tested Steroids ± Marked 65
CIP ganglionitis, no eosinophilic azathioprine; improvement
neurodegeneration infiltrate continued in all cases
long-term fashion

CIP, chronic intestinal pseudo-obstruction; F, female; M, male; SB, small bowel; LP, lamina propria; MP, muscularis propria; MyP,
myenteric plexus.

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R. De Giorgio & M. Camilleri Neurogastroenterology and Motility

Table 4 Colonic neuropathology in slow transit constipation

Histological and immunohistochemical findings Ref. no.

Decreased number or abnormal appearance of silver staining neurones or axons 92

Increased number of variably sized nuclei within ganglia
Decreased colonic VIP nerves 93
Decreased neurofilament staining in myenteric plexus in 75% patients 94
17/29 entire colon affected
12/29 segmental involvement
Increased number of PGP 9.5 reactive nerve fibres in muscularis layer of ascending and descending colon 95
Decreased total nerve density in myenteric plexus 96
Decreased VIP and increased NO positive neurones
Decreased substance P nerves in 7/10 patients 97
Decreased VIP nerves in four of seven patients
Decreased substance P in mucosa and submucosa of rectal biopsies 98
Increased VIP, substance P and galanin in ascending colon 99
Increased VIP and galanin in transverse colon
Increased VIP and neuropeptide Y in descending colon myenteric plexus
Decreased VIP in submucosa
Decreased tachykinin (substance P) and enkephalin fibres in circular muscle 100
Decreased colonic total neuron density 101
Decreased VIP and NO neurones in myenteric
decreased VIP neurones in submucous plexus
Decreased enteroglucagon and 5-HT cells in mucosa 102
Decreased cell secretory indices of enteroglucagon and somatostatin cells
Decreased volume of interstitial cells of Cajal and neurones in circular muscle 103–108

ders that have been best characterized are in colonic
inertia patients. While recent literature has focused on
the interstitial cells of Cajal (see below), which are
reduced in number and are morphologically abnormal,
the precise mechanism and neurotransmitter deficien-
cies of this disorder are unclear. Table 4 summarizes
information from a number of studies in the litera-
ture92–108 regarding histopathological changes found in
patients with slow transit constipation severe enough
to warrant subtotal colectomy. There are, however, a
number of limitations in these studies, which are
characterized by relatively small numbers and, in
some, lack of observer blinding. More stringent stud-
ies, exemplified by the study of Wedel et al.,105 provide
insights on optimizing future studies by using whole
mounts of the human myenteric plexus and accurately
counting enteric neurones. In general, it appears that
reduced substance P and increased nitrergic neurones
are associated with constipation.
Clinical observations and implications Idiopathic
slow transit constipation is easily recognized and
managed in clinical practice in the vast majority of
patients.109 It is unclear whether severity of the slow
transit is related to the deficiency of transmitters.
Presumably, the functional reserve provided by the
surviving enteric neurones accounts for the response to
therapy, for example, with 5-HT4 agonists. In parkin-
sonism associated with constipation, there is a select-
ive reduction of dopaminergic neurones, with normal
populations of extrinsic adrenergic and intrinsic
VIPergic neurones.110 The pathophysiology of the
constipation is complicated by disturbances of evacu-
ation in this condition. Hence, colonic prokinetics are
not always effective in the relief of constipation in
these patients, and failure to respond to these agents
should lead to a search for a defecation disorder.111
Interstitial cell pathology
The interstitial cells of Cajal112–115 are specialized cells
that have been thoroughly investigated; there are at
least three types of functionally distinct ICCs:116 (i)
those forming a plexus around myenteric ganglia (also
termed ICCs-MY). These are involved in the generation
and propagation of slow waves, and are regarded as
pacemaker cells of the gastrointestinal tract; (ii) those
localized throughout the muscle layer (i.e. ICCs-IM);
and (iii) those between the inner surface of the circular
muscle and the submucosa. The latter is called the deep
muscular plexus (termed ICCs-DMP). Both ICCs-CM
and ICCs-DMP contribute to neurotransmission, trans-
ducing inputs from enteric motor neurones to the
smooth muscle syncytium. ICCs express transmitter

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Volume 16, Number 5, October 2004
receptors, including neurokinin 1 and somatostatin
receptor, and they synthesize and release nitric oxide.117
In all types of ICCs, the proto-oncogene c-kit
encodes for a tyrosine kinase receptor whose natural
ligand is stem cell factor (SCF). The interaction
between SCF and Kit is fundamental for ICCs devel-
opment, survival and maintenance.118 c-Kit knockout
mice show gut dilatation and absence of peristalsis
provides further evidence on the critical role played by
ICCs in regulating gut motility.119,120
Morphological and molecular pathology The ICC
networks are markedly reduced in several gut dysmo-
tilities: achalasia,121–123 hypertrophic pyloric steno-
sis,124 idiopathic gastroparesis,125 Hirschsprung’s
disease,126 chronic intestinal pseudo-obstruc-
tion,127–130 slow transit constipation103–106 and
Chagasic107 and idiopathic megacolon.105,108 Electron
microscopy evaluation and/or Kit immunolabelling
coupled with quantitative confocal microscopy with
image analysis103–105, 108, 124–126,128,129 quantify loss of
processes of ICCs and damage to the intracellular
cytoskeleton and organelles in ICCs.
Delayed maturation or maldevelopment of ICCs
may also be associated with severe, but reversible
dysmotility.131 ICC loss in diabetic neuropathy in
animal132 and human133 studies was associated with
marked decrease of inhibitory innervation (i.e. NOS-,
VIP- and PACAP-containing neurones) and increased
neuronal substance P-immunoreactivity.133
Clinical observations and implications The ICCs are
decreased in a wide spectrum of primary enteric neu-
ropathies, and in secondary motility disorders, such as
diabetes. However, as with IND (see above), most of
the studies dealing with ICCs are mainly descriptive
(i.e. either reduced Kit-immunostaining or ultrastruc-
tural alterations) and were not associated with thor-
ough evaluation of the neuromuscular tissues.
Moreover, it is unclear whether dilatation associated
with severe gut dysmotility significantly affects the
ICC network. A second pitfall is that virtually all
studies on ICC abnormalities are unavoidably per-
formed long after the onset of a motor disorder, and a
clear cause–effect relationship cannot be established.
Thirdly, the mechanisms leading to ICC degeneration
and loss in disease states are still unclear. The ICCs do
not progress to apoptosis or necrosis, but they undergo
phenotypic changes. This Ôre-differentiationÕ120 of ICCs
into smooth muscle cells is mediated by Kit signal-
ling.134 Knowing the factors controlling ICC phenotype
in several disease states133 may lead to treatment of gut
motor disorders related to ICC abnormalities.

2004 Blackwell Publishing Ltd
Neuropathology of gut dysfunction
POTENTIAL IMPLICATIONS OF ENTERIC
NEUROPATHOLOGY TO THE PRACTICAL
MANAGEMENT OF ENTERIC
NEUROPATHIES
The differential diagnosis of motility disorders5,6
includes mechanical obstruction, functional gastroin-
testinal disorders, anorexia nervosa and the rumination
syndrome,135,136 which typically presents as early
(0–30 min) postprandial, effortless regurgitation of
undigested food after virtually every meal.
Current steps in the clinical evaluation of
patients with suspected motility disorders
A motility disorder of the stomach or small bowel is
usually suspected when undigested solid food or large
volumes of liquids are observed during an oesophago-
gastroduodenoscopy performed to investigate upper
abdominal symptoms. The goals of the evaluation
are to determine what regions of the digestive tract are
malfunctioning and whether the symptoms are
because of a neuropathy or a myopathy.
Key steps in evaluation include: (a) Exclusion of
mechanical obstruction by upper gastrointestinal
endoscopy and barium studies, including a small bowel
follow-through. A motor disorder may be suspected if
there is gross dilatation, dilution of barium or retained
solid food within the stomach. However, these studies
rarely identify the cause except in patients with
systemic sclerosis.
(b) Assessment of motility. After mechanical obstru-
ction and alternative diagnoses such as Crohn’s disease
have been excluded, a transit profile of the stomach,
small bowel and colon should be performed.5,6 If the
cause of a disturbance of transit is unclear, manometry
using a multilumen tube with sensors in the distal
stomach and proximal small intestine137–139 can dif-
ferentiate a neuropathic process (normal amplitude
contractions, but abnormal patterns of contractility)
from a myopathic process (low amplitude of contrac-
tions in the affected segments). Colonic manometry
and tone measurement140,141 facilitate identification of
colonic inertia by the poor contractile response to
feeding or to medications such as neostigmine or
bisacodyl.
However, there are important caveats in the appli-
cation of manometry to diagnosis. For example, an
absent rectoanal inhibitory reflex is typically the result
of megarectum rather than the expression of congenital
aganglionosis. Secondly, there are no large series that
validated manometry of the small bowel and histopa-
thology of the nerves and muscles of the intestine.
525
R. De Giorgio & M. Camilleri
(c) Identify complications of the motility disorder,
including bacterial overgrowth, dehydration, malnu-
trition. In patients presenting with diarrhoea, it is
important to assess nutritional status and to exclude
bacterial overgrowth by culture of small bowel aspir-
ates. Bacterial overgrowth is relatively uncommon in
neuropathic disorders but is more often found in
myopathic conditions, such as scleroderma, that are
more often associated with dilatation or low amplitude
contractions. An empiric trial of antibiotics (see below)
is often used instead of formal testing.
(d) Identify the pathogenesis. In patients with neur-
opathic causes of uncertain aetiology, tests should
assess autonomic dysfunction, measure ANNA-1 asso-
ciated with paraneoplastic syndromes, and consider
imaging the brain and brainstem to exclude the possi-
bility of a brainstem lesion. In patients with a myopathic
disorder of unclear cause, the evaluation should consider
amyloidosis (immunoglobulin electrophoresis, fat aspir-
ate, or rectal biopsy), systemic sclerosis (SCL-70) and
thyroid disease. In appropriate settings, porphyria and
ChagasÕ disease may need to be excluded. In refractory
cases, referral to a specialized centre for genetic testing
and/or full-thickness biopsy of the small intestine may
be indicated to identify metabolic muscle disorders and
mitochondrial myopathies.
When these steps identify the cause of the motility
disorder, no further testing or intestinal biopsies are
indicated. The next section describes the indications
for intestinal biopsy and the recommended evalua-
tions.
When should the intestine be biopsied and
what should be done with the tissue?
At present, there are no definite or clear indications to
biopsy gastrointestinal tissues of patients with severe
dysmotility. With improvements in surgical equip-
ment and techniques (i.e. laparoscopic surgery) biop-
sies may be indicated in patients with severe
derangements of gut motility of unknown origin not
responsive to therapy, or when mechanical obstruction
cannot be excluded with preoperative tests, or when a
permanent feeding tube is being placed. For the
physician managing the patient, there is a continual
tension: does the full-thickness biopsy directly benefit
this individual patient? After all, the risks associated
with laparoscopy and biopsy are greater than zero, and
we are charged with the responsibility: Ôprimum non
nocereÕ.
The advantages of obtaining tissue are first, to
provide patients with information on the diagnosis
and, in some cases, its potential genetic implications.
526
Neurogastroenterology and Motility
Secondly, tissue diagnosis is important to address
prognosis and organize appropriate long-term support
(i.e. home enteral or parenteral nutrition). Thirdly, it is
important for researchers to investigate the underlying
mechanisms leading to neuromuscular dysfunction
and correlate these with pathophysiology to develop
further understanding of enteric neuropathies and
useful therapeutic strategies.
Small intestine and/or colonic full-thickness speci-
mens require appropriate tissue handling, including
rapid transfer of tissue from the operating room to the
laboratory, careful processing (i.e. removal of faeces,
blood, other secretions and fat), fixation with appro-
priate fixatives [including buffered formalin (for rout-
ine histopathology), 4% paraformaldehyde (commonly
used for immunohistochemical evaluation) and 2.5%
glutaraldehyde (for electron microscopy analysis)].
When possible, whole mount preparations (i.e. entirely
fixed-mucosa side up) should be used, allowing for a
better analysis of the neuromuscular layer. Following
overnight fixation (avoiding excessive exposure which
may interfere with immunohistochemical analysis),
tissue is embedded and sections cut and analysed for a
variety of markers, some of which are listed in Table 5.
Small specimens of tissue should also be frozen
quickly in liquid nitrogen and preserved in RNAse-
free tubes at )80 C for molecular biology assessments.
All histological and immunolabelled materials
should be analysed with conventional or confocal
microscopy equipped with image analysis to facilitate
morphometric evaluation.
SUMMARY AND A LOOK TO THE FUTURE
Advances in the understanding of the ontogeny, basic
mechanisms and molecular pathology of enteric neur-
opathy reflect the application of the new biology and
imaging to animal models and, subsequently, to
human disease. To date, the relatively unstructured
(often non-quantitative) neuropathological assessment
of tissue biopsies has led to limited mechanistic
insights regarding the enteric neuropathies and a new
nosology is not yet possible. However, the observations
recorded in the literature provide the basis for more
structured assessment of tissue. This is essential for
the greater understanding of these conditions and for
the development of a new classification in the future,
based on pathological mechanisms. We have identified
genetic molecular disorders, acquired selective neuro-
transmitter deficiencies, degenerative disorders with
inflammation (with predominant humoral or cellular
responses) or without predominant inflammation (e.g.
apoptosis), hyperplastic disorders (including ganglio-

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Volume 16, Number 5, October 2004 Neuropathology of gut dysfunction

Table 5 Markers for immunohistochemical analysis of tissue specimens obtained from patients with severe dysmotility

Marker Example Comment

General neuronal markers
Enteroglial cells
Interstitial cells of Cajal
Neuropeptides/transmitters
and related receptors
Other neuronal markers
Neurotrophins and related
receptors
Apoptosis and related pathways Bcl-2, TUNEL technique,
Smooth muscle markers
Immune cells, chemokines and CD3, CD4, CD8, CD79a, CD68;
cytokines
PGP9.5, NSE, MAP-2, NFs
S-100, GFAP
Kit
SP, VIP, PACAP, CGRP, NPY,
Galanin, 5-HT, NOS, ChAT,
somatostatin, NK1, NK2 and NK3
Calbindin, NeuN
NGF, NT-3, BDNF, Trk-A,
Trk-B and Trk-C
Caspase-3, Caspase-8, Apaf-1
Actin, myosin, desmin
MIP-1a, TNF-a, IFN-c
Useful to map the general architecture of the ENS;
neurofilaments (NFs) markers include antibodies
targeting different isoforms
S-100 is expressed by Schwann cells; GFAP detect
astrocyte-like cells within enteric ganglia
Identifies the ICC networks
Characterization of neurochemical coding and enteric
neuron subclasses
In combination with staining for transmitters and related
receptors, these markers better define subclasses of
enteric neurones (i.e. intrinsic afferent neurones)
Identification of neurotrophic factors
Detection of apoptosis and related mechanisms
Supplement light and electron microscopy to diagnose
gut myopathy
Identification of B (CD79a) and T-lymphocytes (CD3),
T-helper (CD4), T-suppressor (CD8), macrophages (CD68)
in enteric ganglionitis; MIP-1a is a chemokine; TNF-a
and IFN-c are pro-inflammatory cytokines

Bcl-2, B cell lymphoma-2 protein; BDNF, brain-derived neurotrophic factor; ChAT, choline acetyltransferase; CGRP, calcitonin
gene-related peptide; ENS, enteric nervous system; GFAP, glial fibrillary acidic protein; IFN-c, interferon c; MAP-2, microtubule
associated protein-2; MIP1-a, macrophage inflammatory protein-1a; NeuN, neuronal-specific nuclear protein; NGF, nerve growth
factor; NFs, neurofilaments; NK1, NK2, NK3, neurokinin1, neurokinin2, neurokinin3; NOS, nitic oxide synthase; NPY, neuro-
peptide Y; NSE, neuron-specific enolase; NT-3, neurotrophin-3; PACAP, pituitary adenylate cyclase activating polypeptide;
PGP9.5, protein gene product 9.5; 5-HT, 5-hydroxytryptamine (serotonin); SP, substance P; TNF-a, tumour necrosis factor a; Trk-A,
-B, -C, tyrosine kinase receptors A, B and C; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate
nick-end labelling; VIP, vasoactive intestinal polypeptide.

neuromatosis), and mitochondrial disease (respiratory
enzymopathies). Future classifications may start here
and expand on this framework.
In the past, acquisition of full-thickness intestinal
biopsies was typically not recommended because the
benefits from the limited pathological evaluations did
not outweigh the risk of recurrent obstruction from
adhesions, that often led to the need for repeat
laparotomy in these patients. Recent advances suggest
that in the future, tissue should be obtained in patients
in whom the diagnosis is unclear. This is justifiable as
long as formal, in-depth and quantitative studies
proposed are used to garner maximum information
and benefit from the full thickness tissue biopsies. The
patientsÕ phenotype must be thoroughly appraised, and
the tissue should be regarded as a unique resource for
the structured and multidisciplinary studies needed to
advance the field, develop an effective nosology based
on disease mechanisms, and develop novel treatments.
Formally trained enteric neuroscientists including
clinicians and pathologists are required to implement
the advances in basic science of the enteric nervous
system. The rarity of these disorders and the complex-
ity of the analyses required call for multicentre studies
or the development of tissue registries that have also
been strongly recommended in the past.
ACKNOWLEDGMENTS
This work is supported in part by National Institutes of
Health grants R01 DK54681 (MC), K24 DK02638 (MC),
and General Clinical Research Center grant RR00585,
as well as by grants to RDG from the Italian Ministry
of University, Research, Science and Technology
and National Research Council (CNRC0008-02).
Figures 3–6 pertain to patients previously reported in
De Giorgio et al.62
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