Human Enteric Neuropathies Morphology and Molecular

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REVIEW ARTICLE

Human enteric neuropathies: morphology and molecularpathology

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 currentunderstanding 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 entericneuropathies requires further study before an effective

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

80100 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 (Auerbachs) and submucosal (Meiss-

ners) 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

Address for correspondence

Michael Camilleri MD, Clinical Enteric NeuroscienceTranslational 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: [email protected]: 22 October 2003

Accepted for publication: 30 December 2003

Neurogastroenterol Motil (2004) 16, 515531 doi: 10.1111/j.1365-2982.2004.00538.x

2004 Blackwell Publishing Ltd 515


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animals. However, there is increasing evidence that

human ENS neurochemical coding in health mimics

that of laboratory animals such as the guinea-pig.24

Although extrinsic neuropathies such as diabetes or

amyloidosis are known to cause gastrointestinal motil-

ity disorders, these are usually well recognized by thesystemic 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 acknowledgedand 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 ofneurones 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-

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 theliterature on the molecular and morphological pathol-

ogy of enteric neuropathies affectinggut motorfunction,

and to determine whether a novel nosology, or classifi-

cation,of these conditions canbe developedbasedon 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

NEUROPATHIESThe 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 riboprobespecific 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:G1836, 1998.(B) Contribution of vagal and sacral neural crest to formationof ENS: C-ret dependent sympathoenteric lineage originatesin vagal neural crest of the hindbrain and migrates ventrallyto populate the entire gut and superior cervical ganglion(SCG); C-ret independent sympathoadrenal lineage originatesin truncal crest and populates foregut and sympathetic chain;and sacral neural crest is derived from spinal cord and colon-izes mainly the hindgut.

516 2004 Blackwell Publishing Ltd

R. De Giorgio & M. Camilleri Neurogastroenterology and Motility


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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 leadsto 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, Hirschsprungs disease. This is characterized by

the complete absence of ganglion cells in the sub-

mucosal and myenteric plexuses.7

Morphological and molecular pathology Hirschsprungs

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 RETproto-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;816

modifiers genes for these transcription factors are as

yet unidentified.

The RET mutations have been found in about 50%

of familial and 1720% of sporadic forms of Hirsch-

sprungs disease.810 Hirschsprungs disease or mega-

colon occur either as a sole disease or as part of

syndromes such as multiple endocrine neoplasia type

2B12 or familial thyroid carcinoma or multiple endo-

crine neoplasia 2A13 or WaardenburgShah syndrome

in which megacolon is accompanied by pigmentary

disorders and neural deafness.810 Deletions or trunca-

ting mutations in SMADIP1 gene are responsible for

syndromic Hirschsprungs 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 13 cm of

the rectum and may lead to false-positive diagnosis of

Hirschsprungs disease;19 conversely, efforts to obtain

biopsies proximal to this zone may miss 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 Hirschsprungs

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.2224 Lewy bodies

have also been observed in myenteric neurones

of patients with parkinsonism and experienced

Table 1 Pathological features of enteric neuromuscular

disease

AganglionosisNeuronal intranuclear inclusions and apoptosisNeural degenerationIntestinal neuronal dysplasiaNeuronal hyperplasia and ganglioneuromasMitochondrial dysfunction: syndromic and non-syndromicInflammatory neuropathies: cellular and humoral mechanismsNeurotransmitter disordersInterstitial cell pathology


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neuropathological and clinical appraisal of such

patients is needed for correct classification.25

Neuronal degeneration

Morphological and molecular pathology Histologicalexaminations 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 sclerosisand diabetic neuropathy. Formal studiesof 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

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 controversialentity. 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 Hirschsprungs 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 clinicalpathological 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)3941 that increases




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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, blubbery lips 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

prophylacticthyroidectomy, 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

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. Onskeletal 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

Figure 2 Family history and features ofproband with multiple endocrine neoplasiatype 2B: Note the autosomal dominantinheritance, the blubbery lips and ganglio-neuromas on the tongue, the massive colonafter resection and the presence of prominentganglioneuromas in the muscle layer of thecolon.




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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 encephalomyopathyin 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

are referred to as enteric ganglionitis.26,5558 Ganglio-

nitis and axonitis represent derangements in the neuro-

immune interactions occurring within the enteric

neural microenvironment.

Morphological and molecular pathology 1 Cellularmechanisms 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, 5962

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. Schappi 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

Figure 3 Micrographs showing both types of T-lymphocytes, CD4 (A) and CD8 (B), detectable within the myenteric plexus of thesmall intestine (proximal ileum) of a 20-year old man with chronic intestinal pseudo-obstruction. Note the intense CD4 and CD8immunoreactivities which represent the predominant component of the immune infiltrate observed in cases of lymphocyticganglionitis. Alkaline phosphatase anti-alkaline phosphatase immunohistochemical technique. Original magnification: 120 in Aand B.




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damage. A similar histopathological pattern was

observed in an adult patient with acute colonic

pseudo-obstruction [i.e. Ogilvies 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

gut motility disorders with an underlying myenteric

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.55,56,6668 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

Anti-neuronalautoantibodies

Moleculartarget Function

Underlying tumoursand relatedparaneoplasticsyndrome

Idiopathiccases(unassociatedwith cancer)

GI motordisorder Ref. no.

ANNA-1(or Anti-Hu)

HuD, HuC,HuR Hel-N1

Family of RNAbinding proteins

SCLC/opsoclonusmyoclonus; ataxia

Yes Gastroparesis,CIP,megacolon

6974

Anti-VGCC Voltage-gatedCa2+ channels,including P/Qand N-typechannels

Regulation of Ca2+

influx andsignalling

SCLC/Lambert-Eatonsyndrome

Unknown CIP 71,75

Anti-ganglionicacetylcholireceptors

Nicotinicreceptors

Acetylcholinesignalling

Thymoma, SCLC/dysautonomia

Yes Gastroparesis,CIP,constipation

76

Anti-Yo Cdr2 Transductionsignal protein

Gynaecological tumours(i.e. ovary)/cerebellarparaneoplasticdegeneration

Unknown CIP 66,77

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

Figure 4 Micrograph illustrating lymphocytes immunolabe-led for CD79a, a marker for mature B cells, surrounding andinfiltrating a myenteric plexus of the small intestine (prox-imal ileum) of a 20-year-old man with chronic intestinalpseudo-obstruction. In addition to T lymphocytes, the pres-

ence of B lymphocytes in cases of myenteric ganglionitisprovide the basis for a humoral immune response, which maycontribute to enteric neuron dysfunction. Alkaline phospha-tase anti-alkaline phosphatase immunohistochemical tech-nique. Original magnification: 120.




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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- andN-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),66,7981 infectious (e.g.

Chagas disease),8284 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 Crohns disease).87 Some cases

have no underlying cause identified (idiopathic).5962

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

clinical manifestations and include oesophageal and

lower oesophageal sphincter (LOS) dysmotility,88

gastroparesis,61 intestinal pseudo-obstruction and

colonic inertia or megacolon.5862,66,7981 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 tosensorimotor abnormalities in functional bowel syn-

dromes unassociated with bowel dilatation. The find-

ing of few lymphocytes (1.97.1 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.5862,79,80,84,89 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

individuals genetic background. A detailed study of

the inflammatory infiltrate of IBS patients focused

on the mucosa and lamina propria, although no

Figure 5 Representative micrographs taken from the colon ofa 23-year-old woman with long-standing history of chronicidiopathic constipation and megacolon due to an underlyinglymphocytic myenteric ganglionitis. The two photomicro-graphs show the absence of the immunolabelling for thegeneral neuronal marker neuron-specific enolase in themyenteric plexus in (A), which is in contrast with the normalappearance of the same marker immunoreactivity identifiedin the submucous plexus of the same section, as illustrated in(B). Streptavidinbiotin complex peroxidase immunohisto-chemical technique. Original magnification: 160 in A and B.




9/17

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.

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).5862,65 How-

ever, there is a need for controlled studies comparinganti-inflammatory agents including immunosuppres-

sives and plasma exchange before recommendations

are made to treat patients with potentially harmful

therapies.

Neurotransmitter disorders

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

Patients anddysmotilitytype Pathology

Inflammatoryinfiltrate

Anti-neuronalantibodies

Anti-inflammatory therapy

Ref. no.Treatment givenOutcome oftreatment

4 F patients withCIP

Lymphoid infiltratein the LP, MP,MyP; noneuromusculardegeneration

PolyclonalT and B cells

Not tested 3 antibiotic1 cyclophosphamideand prednisone

Mildsymptomaticimprovement

59

Two patients withCIP (1 M, 1 F)

Myentericganglionitis,neuronal loss, oraganglionosis

Predominanceof T cells (CD4and CD8positive)

Anti-Hu(orANNA-1)in both cases

Steroids atdifferent doses

1 improved;1 SB transplant

60

1 M patient withgastroparesis

Myentericganglionitis,neurodegeneration,marked decrease ofSP-containing nerves


Not tested Methylprednisolonein a taperingfashion

Markedimprovement

61

2 F patients withcolonic inertia ormegacolon and1 M patientwith CIP

Myentericganglionitis,neuronal loss, oraganglionosis


Anti-Hu(orANNA-1) inthe male patient;not tested inthe 2 F patients

Short-coursemethyl-prednisolonetreatment inthe male patient

Significantlyameliorated

62

3 F patients withCIP

Myentericganglionitis, noneurodegeneration

Predominanteosinophilicinfiltrate

Not tested Steroids azathioprine;continuedlong-term fashion

Markedimprovementin all cases

65

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

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 smallintestine (proximal ileum) of a 20-year-old man with chronicintestinal pseudo-obstruction. Alkaline phosphatase

anti-alkaline phosphatase immunohistochemical technique.Original magnification: 120.




10/17

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-

ture92108 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 theconstipation 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 Cajal112115 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

Table 4 Colonic neuropathology in slow transit constipation

Histological and immunohistochemical findings Ref. no.

Decreased number or abnormal appearance of silver staining neurones or axonsIncreased number of variably sized nuclei within ganglia

92

Decreased colonic VIP nerves 93Decreased neurofilament staining in myenteric plexus in 75% patients17/29 entire colon affected12/29 segmental involvement

94

Increased number of PGP 9.5 reactive nerve fibres in muscularis layer of ascending and descending colon 95Decreased total nerve density in myenteric plexusDecreased VIP and increased NO positive neurones

96

Decreased substance P nerves in 7/10 patientsDecreased VIP nerves in four of seven patients

97

Decreased substance P in mucosa and submucosa of rectal biopsies 98Increased VIP, substance P and galanin in ascending colonIncreased VIP and galanin in transverse colonIncreased VIP and neuropeptide Y in descending colon myenteric plexusDecreased VIP in submucosa

99

Decreased tachykinin (substance P) and enkephalin fibres in circular muscle 100

Decreased colonic total neuron densityDecreased VIP and NO neurones in myentericdecreased VIP neurones in submucous plexus

101

Decreased enteroglucagon and 5-HT cells in mucosaDecreased cell secretory indices of enteroglucagon and somatostatin cells

102

Decreased volume of interstitial cells of Cajal and neurones in circular muscle 103108




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receptors, including neurokinin 1 and somatostatin

receptor, and they synthesize andrelease 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,121123 hypertrophic pyloric steno-

sis,124 idiopathic gastroparesis,125 Hirschsprungs

disease,126 chronic intestinal pseudo-obstruc-

tion,127130 slow transit constipation103106 and

Chagasic107 and idiopathic megacolon.105,108 Electron

microscopy evaluation and/or Kit immunolabelling

coupled with quantitative confocal microscopy with

image analysis103105, 108, 124126,128,129 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 causeeffect 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 re-differentiation120 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.

POTENTIAL IMPLICATIONS OF ENTERICNEUROPATHOLOGY TO THE PRACTICALMANAGEMENT OF ENTERICNEUROPATHIES

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

(030 min) postprandial, effortless regurgitation of

undigested food after virtually every meal.

Current steps in the clinical evaluation ofpatients 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 Crohns 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 intestine137139 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.




12/17

(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 inneuropathic 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-

bilityof a brainstemlesion. In patients with a myopathic

disorderof unclear cause, the evaluationshould consider

amyloidosis (immunoglobulin electrophoresis, fat aspir-

ate, or rectal biopsy), systemic sclerosis (SCL-70) and

thyroid disease. In appropriate settings, porphyria and

Chagas 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 andwhat 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: primum non

nocere.

The advantages of obtaining tissue are first, to

provide patients with information on the diagnosis

and, in some cases, its potential genetic implications.

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 developfurther 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-




13/17

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

patients 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 36 pertain to patients previously reported in

De Giorgio et al.62

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

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General neuronal markers PGP9.5, NSE, MAP-2, NFs Useful to map the general architecture of the ENS;neurofilaments (NFs) markers include antibodies

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Human Enteric Neuropathies Morphology and Molecular

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