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The Enteric Nervous System in Chagasic and Idiopathic Megacolon

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Title: The Enteric Nervous System in Chagasic and Idiopathic Megacolon


1
The Enteric Nervous System in Chagasic
andIdiopathic 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 Mun?oz, MD, y
  • Barbara Liserre, MD,J Antonio Morelli, MD, w and
    Vincenzo Villanacci, MDJ

(Am J Surg Pathol 200731460468)
Int ???
2
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

3
  • 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

4
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.

5
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

6
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

7
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.

8
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.

9
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 hematoxylin-eosin,
    periodic acid-Schi., and trichrome stain.
    Immunohistochemistry

10
METHODS-markers
  • monoclonal antibodies toward neuron-specific
    enolase(NSE, NCL-NSE2, Novocastra laboratories,
    dilution150) acting as a marker of ganglion
    cells
  • protein S100 (S-100, Dako, Carpinteria, CA,
    dilution 150) 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
    150) was used to detect these cells

11
METHODS-markers
  • The presence of lymphocytes was assessed by means
    of a monoclonal mouse antihuman CD 3 antibody
    (Dako Cytomation, dilution 140).
  • The colonic smooth muscle was evaluated by means
    of antia-actin monoclonal antibody (MAb)
    (dilution 1100 Biogenex, San Ramon, CA),
  • muscle-specific actin
  • MAb (clone HHF35, Dako)
  • vimentin (mouse monoclonal
  • antibody, Biogenex)
  • desmin (monoclonal antibody, Biogenex).

12
  • 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

13
  • NSE and S-100 immunostaining was carried out
    using a peroxidase-based visualization kit (Dako
    LSAB),following the manufacturers
    recommendations.
  • Diaminobenzidine tetrahydrochloride was used as
    chromogen.
  • The slides were then counterstained with Mayers
    hematoxylin for 5 seconds, dehydrated and mounted
    in Clarion (Biomeda).

14
  • 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. .

15
  • Sections were then subjected to heat-induced
    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

16
  • Kit immunostaining was carried out using a
    peroxidasebased visualization kit (Dako
    EnVision), following the manufacturers
    recommendations

17
Antisingle-stranded DNAImmunohistochemistry
  • 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

18
  • After cooling, endogenous peroxidase activity was
    suppressed by incubation with 3 solution of H2O2
    for 5 minutes.
  • Normal serum diluted 150 was applied for 10
    minutes to room temperature, followed by anti-DNA
    MAb for 30 minutes, according to the
    manufacturers 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

19
  • a 5-minute reaction in the dark with
    diaminobenzidine (Bio-Optica) was carried out,
    and the sections were then counterstained with
    Mayers hematoxylin for 5 seconds, dehydrated,
    and mounted in Clarion (Biomeda).
  • Positivity was observed under the microscope as
    an intense brown reaction.

20
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)

21
  • 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 well-oriented
    fields at _20 magnification
  • Not only nucleated cells but also Kit-positive
    labeled elongated structures were considered for
    analysis

22
STATISTICAL ANALYSIS
  • Nonparametric tests were employed to analyze the
    data. The Kruskall-Wallis test, the Wilcoxons
    signed rank test, and the w2 test were employed,
    where appropriate.
  • Values of Plt0.05 were chosen for rejection of the
    null hypothesis.
  • Data are presented as median (95 CI).

23
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.

24
RESULTSConventional 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

25
  • and in the colonic smooth muscle
  • 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

26
  • 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).

27
(C), compared to that of a chagasic patient (D),
showing no enteric neurons (hematoxylin and
eosin, original magnifications C, 20 D, 40).
28
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. 2AF).

29
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30
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31
  • 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

32
  • and their decrease in a chagasic patient
  • ICC-IM in a control

33
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34
(C) and in a chagasic patient (D) showing an
increase in the latter (A-D CD 117, original
magnification 40).
35
  • 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).
36
  • 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).
37
  • 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 (P0.13)

38
  • 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.

39
Presence of lymphocytes (arrows) within a colonic
myenteric ganglion of a chagasic patient (CD3
C, original magnification 40 D, 100).
40
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

41
  • 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.

42
  • Compared to controls, we found a significant
    reduction of enteric neurons
  • to a lesser extent, of ICC (ICC-SM and ICC-MY) 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

43
  • 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

44
  • 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

45
  • 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

46
  • 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

47
  • 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
  • cholecystokinins failure in stimulating
    rectosigmoid motility in patients with megacolon
    due to Chagas disease.

48
  • 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

49
  • 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

50
  • 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.

51
  • 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

52
  • 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

53
  • 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.

54
  • Important abnormalities of the enteric nervous
    system are present in patients with chagasic and
    idiopathic megacolon.

55
Thanks for attention
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