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 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

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, 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

11. 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).

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 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).

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 manufacturer’s recommendations

17. Anti–single-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 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

19. 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.

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 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).

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. 2A–F).

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

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 (P=0.13)

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 • cholecystokinin’s 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.