Transcript Document

The Enteric Nervous
System in Chagasic
and
Idiopathic Megacolon
Guido Iantorno, MD,* Gabrio Bassotti, MD, PhD, w Zulema
Kogan, MD, z Carlos Miguel Lumi, MD, y Ana Maria Cabanne,
MD, z Simona Fisogni, MD,J
Liliana Monastra Varrica, MD,* Claudio R. Bilder, MD,* Juan
Pablo Munˇoz, MD, y
Barbara Liserre, MD,J Antonio Morelli, MD, w and Vincenzo
Villanacci, MDJ
(Am J Surg Pathol 2007;31:460–468)
Int 林睿禹
Introduction
• Chagas disease in humans is due to the
infection with the protozoan parasite
Trypanosoma cruzi
• The clinical picture of this disease is
dominated by cardiologic and
gastrointestinal manifestations
• Digestive involvement in Chagas disease
primarily involves the esophagus
• often causing a megacolon, even though the
entire gut may be involved
• The involvement of the enteric nervous system
is pivotal in the pathogenesis of the
gastrointestinal disorders in Chagas disease
• Previous studies on chagasic colonic
involvement
– degeneration and decreased number of intrinsic
myenteric neurons
– reduced number of nitric oxidecontaining,myenteric
neurons
– deficiency of interstitial cells of Cajal (ICC)
– ganglion cell damage by T lymphocytes,
– increased mast cell count
Purpose of the present study
• to assess simultaneously several
aspects of the enteric nervous system
in Argentinian patients with chagasic
megacolon
• and compare them with those found in
patients with idiopathic megacolon and
in controls.
PATIENTS
• Specimens from 12 patients with
megacolon due to Chagas disease (1
woman, 11 men, age range 41 to 72 y)
• 9 patients with idiopathic megacolon
(3 women,6 men, age range 39 to 68 y)
• all undergoing surgery for constipation
refractory to medical treatment
• All patients were living in metropolitan
Buenos Aires at the time of surgery
PATIENTS
• A diagnosis of Chagas disease was
made on the basis of 3 standard
serologic reactions against T. cruzi:
– passive hemagglutination
– indirect immuno.uorescence
– and enzyme immunoassay
• The idiopathic megacolon group
patients had the 3 tests negative
PATIENTS
• Patients with idiopathic megacolon had a
normal sphincteric response to anorectal
manometry
• excluding Hirschsprung disease,46 whereas
chagasic patients displayed an impairment
of the sphincteric response to rectal
distention.
• No patient in both groups had evidence of
cardiovascular, neurologic, metabolic,
and/or systemic disease.
CONTROLS
• Ten patients (9 women, 1 man, age range
43 to 75 y) undergoing left hemicolectomy
for nonobstructing colorectal cancer were
used as controls
• the distribution of ICC is relatively uniform
throughout the human colon.
• The control specimens were taken at least 5
cm from the resection margin in tumor-free
areas.
METHODS
• After removal, the surgical specimens were
immediately fixed in 10% neutral-bu.ered
formalin for 24hours
• then 4 to 8 full-thickness samples from the
resected colon were taken and transversal
sections obtained.
• For conventional histology, 5 mm paraffin
sections were stained with hematoxylineosin, periodic acid-Schi., and trichrome
stain. Immunohistochemistry
METHODS-markers
• monoclonal antibodies toward neuronspecific enolase(NSE, NCL-NSE2,
Novocastra laboratories, dilution1:50) acting
as a marker of ganglion cells
• protein S100 (S-100, Dako, Carpinteria, CA,
dilution 1:50) a marker of Schwann cells,
localized exclusively in the glial cells in the
gastrointestinal tract.16,31
• Because ICC express Kit,63 an anti-Kit
antibody (rabbit polyclonal antibody,IgG,
Dako, dilution 1:50) was used to detect
these cells
METHODS-markers
• The presence of lymphocytes was assessed by
means of a monoclonal mouse antihuman CD 3
antibody (Dako Cytomation, dilution 1:40).
• The colonic smooth muscle was evaluated by
means of anti–a-actin monoclonal antibody (MAb)
(dilution 1:100; Biogenex, San Ramon, CA),
• muscle-specific actin
• MAb (clone HHF35, Dako)
• vimentin (mouse monoclonal
• antibody, Biogenex)
• desmin (monoclonal antibody, Biogenex).
• As a marker of apoptosis in the enteric
nervous system we employed the MAb to
single-stranded DNA,20
• the formamide-MAb method (Mab F7-26
BMS156, Bender MedSystem), which
detects apoptotic cells in tissue processed
with routine histologic techniques
• allows discrimination of apoptosis from
necrosis
• NSE and S-100 immunostaining was carried
out using a peroxidase-based visualization
kit (Dako LSAB),following the
manufacturer’s recommendations.
• Diaminobenzidine tetrahydrochloride was
used as chromogen.
• The slides were then counterstained with
Mayer’s hematoxylin for 5 seconds,
dehydrated and mounted in Clarion
(Biomeda).
• To account for nonspecific staining, peptides
that blocked polyclonal antibody bindings
(passage with normal goat serum) were
used, or sections were incubated in the
absence of primary antibody
• In these cases, no immunostaining was
detected. Expression of Kit Consecutive
formalin-fixed, paraffin sections were
dewaxed and rehydrated through
decreasing alcohol series up to distilled
water. .
• Sections were then subjected to heatinduced epitope retrieval by immersion
in a heat resistant container filled with
citrate buffer solution (pH 6.0) placed
in a pressure cooker
• and microwaved for 20 minutes.
Endogenous peroxidase activity was
suppressedby incubation with 3%
solution of H2O2 for 5 minutes
• Kit immunostaining was carried out
using a peroxidasebased visualization
kit (Dako EnVision), following the
manufacturer’s recommendations
Anti–single-stranded DNA
Immunohistochemistry
• Sections 2 to 3 mm thick were warmed
overnight at 601C, then dewaxed and
rehydrated through decreasing alcohol
series up to distilled water.
• Thereafter, the sections were incubated for
5 minutes in phosphate buffered saline with
the addition of 20% Tween 20
• followed by a passage with proteinase K
(Dako) for 20 minutes. The sections were
then rinsed with distilled water and heated in
50% formamide prewarmed to 601C for 20
minutes
• After cooling, endogenous peroxidase
activity was suppressed by incubation with
3% solution of H2O2 for 5 minutes.
• Normal serum diluted 1:50 was applied for
10 minutes to room temperature, followed
by anti-DNA MAb for 30 minutes, according
to the manufacturer’s recommendations.
• After that, the sections were incubated at
room temperature with secondary polymeric
antibody for 20 minutes and ABC (Kit super
sensitive nonbiotin detection system,
Menarini) for 30 minutes
• a 5-minute reaction in the dark with
diaminobenzidine (Bio-Optica) was
carried out, and the sections were then
counterstained with Mayer’s
hematoxylin for 5 seconds, dehydrated,
and mounted in Clarion (Biomeda).
• Positivity was observed under the
microscope as an intense brown
reaction.
DATA ANALYSIS
• All slides were coded and analyzed
blind by 2 pathologists. For NSE, S100,
and formamide-MAb positive cells both
the submucosal and the myenteric
plexuses were taken into account by
optical microscopy at 40 magnification
(Olympus BX 40)
• To be considered as positive, the intensity of
cell immunostaining had to be from
moderate to strong
• The density of ICC was graded, according
to a previously described method,26 after
the evaluation of 10 well-stained and welloriented fields at _20 magnification
• Not only nucleated cells but also Kit-positive
labeled elongated structures were
considered for analysis
STATISTICAL ANALYSIS
• Nonparametric tests were employed to
analyze the data. The Kruskall-Wallis
test, the Wilcoxon’s signed rank test,
and the w2 test were employed, where
appropriate.
• Values of P<0.05 were chosen for
rejection of the null hypothesis.
• Data are presented as median (95%
CI).
ETHICAL CONSIDERATIONS
• The study was carried out in accordance to
local ethical rules, following the
recommendations of the Declaration of
Helsinki (Edinburgh revision, 2000).
• Because no individual patient identi.cation
was involved and no study-driven clinical
intervention was performed, a simplified
Institutional Review Board approval was
obtained and no patient consent was
considered necessary.
RESULTS
Conventional Histology
• One chagasic and one idiopathic megacolon
patients had sporadic small diverticula in the
resected specimen.
• The presence of pseudomelanosis coli was
found in one patient with Chagas disease
• Compared to idiopathic megacolon and
controls, patients with Chagas disease had
an increased amount of fibrotic tissue in the
smooth muscle and within and around the
myenteric ganglia
(A) and in the colonic smooth muscle
(B) in a patient with Chagas disease (trichrome stain, original magnifications: A,
100; B, 20). Colonic myenteric ganglion of a patient
with idiopathic megacolon, showing the presence of enteric neurons
• The presence of myenteric neurons
was documented in all controls and
patients with idiopathic megacolon (Fig.
1C)
• 7 (58%) chagasic patients no
myenteric neurons were identified at
several .elds on conventional staining
(Fig. 1D).
(C), compared to that of a chagasic patient
(D), showing no enteric neurons (hematoxylin and eosin,
original magnifications: C, 20; D, 40).
Immunohistochemistry
• Compared to controls, the number of
NSE-positive and S100-positive cells
was significantly decreased in
chagasic patients
• in patients with idiopathic megacolon
in both the submucosal and the
myenteric plexus (Table 1, Figs. 2A–F).
• No differences were found between
the 2 groups of patients concerning
these markers.
• Concerning ICC, ICC-MY were
signi.cantly reduced in the 2
megacolon groups compared to
controls
(A)and their decrease in a chagasic patient
(B) ICC-IM in a control
(C) and in a chagasic patient
(D) showing an increase in the latter (A-D: CD 117, original magnification 40).
• It is worth noting that in 10 chagasic patients
an increase of mast cells in the circular layer
of the colon smooth muscle was detected
Presence of mast cells
(arrow) in the circular colon
musculature of a patient with
Chagas disease (CD117
, original magnification 40).
• No di.erences between megacolon
patients and controls were found
concerning the number of apoptotic
neurons
Apoptotic neurons
(arrows) in
the colonic
submucosal plexus
of a idiopathic
megacolon patient
(formamide-Ab, original
magnification 100).
• No apoptotic phenomena were detected in ICC, in
both controls and patients
• All patients and controls showed strong intensity for
a-actin, muscle-speci.c actin, vimentin, and desmin
immunostaining, so that the colonic smooth muscle
was judged to display normal characteristics
• Concerning CD3 assessment, lymphocytes were
absent in all controls at muscular, submucosal
plexus,and myenteric plexus level.
• In megacolon patients, an infiltration of these cells
was found in 75% of chagasic and 67% of
idiopathic megacolon (P=0.13)
• In chagasic patients lymphocytes were
usually more numerous (8 to 15) than in
idiopathic megacolon, and were found in the
submucosal (2 cases) and the myenteric (4
cases) plexus
• in the remaining 3 cases they were located
in the smooth muscle. In idiopathic
megacolon patients the lymphocytes (no
more than 3 to 4) were equally found
– submucosal (3 cases)
– The myenteric (3 cases) plexus, but not in the
smooth muscle.
Presence of lymphocytes (arrows) within a colonic
myenteric ganglion
of a chagasic patient (CD3: C, original magnification 40; D, 100).
DISCUSSION
• This is the first study to assess in a
comprehensive way and in a
reasonable group of subjects the
enteric nervous system and smooth
muscle pathology of megacolon,
• one of the most frequent
gastrointestinal manifestations of
Chagas disease
•
• previous studies on the pathologic
aspects of the neuromuscular aspects
of chagasic megacolon usually only
focused on one aspect, such as
– the enteric neurons
– the ICC
– The inflammatory response in the
myenteric plexus
– The role of mast cells and fibrosis.
• Compared to controls, we found a
significant reduction of enteric neurons
• to a lesser extent, of ICC (ICC-SM and ICCMY) in chagasic patients; similar
• findings were also found, in a milder form, in
patients with idiopathic megacolon.
• A finding never described before in
megacolon patients was the significant
reduction of enteric glial cells, in both the
submucosal and the myenteric plexus
• The smooth muscle was apparently intact, as
shown by conventional stainings and
immunohistochemistry,
• Except for an increased amount of fibrosis in
chagasic patients
• The increased amount of .brosis found in
chagasic patients could be related to the
increased mastocytosis, as previously
hypothesized
• A mild/moderate lymphocytic infiltrate, more
prominent in patients with Chagas disease, was
also demonstrated, mainly in the myenteric
plexus
• The prevalent disturbance of intestinal
function in Chagas disease is represented
by a progressive loss of motor activity,
ending in megaviscera formation
• The primary target of injury is the neuron, in
both the intrinsic (myenteric and submucous
plexuses) and extrinsic (autonomic) nervous
system
• The abnormalities found in chagasic
patients are due to a molecular mimicry
leading to immune cross-reactivity between
T. cruzi and the enteric neurons
• In fact, a flagellar antigen of the parasite
antigenically mimics a protein expressed by
myenteric neurons,attracting immune cells
within the ganglia and causing an acute
myenteric ganglionitis
• It is worth noting that in experimental animal
models intestinal in.ammation, even when
mild and limited to the mucosa, can cause
significant alterations of gut motility
• On the other hand, the parasite is becoming
increasingly detected in chronically infected hosts
and may also be the cause of pathology either
directly or through parasite-specific mediated
inflammatory responses.
• The enteric infection due to T. cruzi in turn leads to
neuronal destruction in the long-term period.
• This extensive neuronal damage has been also
reproduced in experimental animal models, and it
has been calculated that about 95% of the neurons
in the myenteric plexus
• The neuronal damage, associated to the presence
of autoantibodies against muscarinic acetylcholine
receptors demonstrated in chagasic patients with
megacolon
• occurring as a result of denervation
• In addition, the motor responses evoked by agents
acting primarily through enteric nerves are altered
or absent in these circumstances, as shown by
pentagastrin
• cholecystokinin’s failure in stimulating rectosigmoid
motility in patients with megacolon due to Chagas
disease.
• it is likely that changes in colonic epithelial
function secondary to the damage of the
submucous plexus may occur
• These changes may further aggravate
colonic motility due to a decrease of the
content of important neurotransmitters
– vasoactive intestinal peptide
– substance P
• The role of ICC as intestinal pacemakers has
been clearly established in experimental animal
models
• A decrease or loss of ICC function might
therefore impair the electrical slow wave activity
of the colon, reducing the contractile response in
chagasic patients
• The preservation of some of these cells in the
submucosal and the myenteric plexuses
suggests that a residual pacemaker activity is
still present in these patients
• the increase of the ICC-IM, the ICC
subpopulation more distant from the main
in.ammatory process (and from the parasite
location), may act as a vicariating
emergency mechanism to supply slow wave
activity to the viscus
• These discrepancies may be due to the fact
that their patients’ series was half that
reported in the present study, and to the
semiquantitative assessment of ICC they
adopted.
• The loss of enteric glial cells is also
important, because these cells not only
provide support for neuronal elements but
also have a role as modulators needed for
the homeostasis of enteric neurons
• the reduction of enteric glial cells could
synergically act with the above
abnormalities to further impairing colonic
motility in chagasic patients with megacolon
• These findings were not significantly different
between the 2 megacolon groups, this might be
due to a sample bias,
• In that the idiopathic group included only 9
patients.
• Previous studies in patients with idiopathic
megacolon have shown normal architecture of
the enteric nervous system
• in a small subset of patients with idiopathic
megacolon we have recently described the
presence of myenteric ganglionitis with
important lymphoid infiltration of the enteric
plexuses and neuronal loss
• A decreased number of enteric neurons and ICC
has been also reported in patients with idiopathic
megacolon without ganglionitis
• It is worth noting that the findings we and others
found with modern immunohistochemistry
techniques in idiopathic megacolon patients are
similar to those described in patients with severe
slow transit constipation
• differently than in the latter patients, we did not
found an increased apoptosis of enteric neurons as
contributing factor to their reduction.
• Important abnormalities of the enteric
nervous system are present in patients
with chagasic and idiopathic
megacolon.