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Enteric neuropathology of the terminal ileum in patients with intractable slow-transit constipation
Human Pathology (2006) 37, 1252 – 1258
www.elsevier.com/locate/humpath
Original contribution
Enteric neuropathology of the terminal ileum in patients with intractable slow-transit constipation Gabrio Bassotti MD, PhDa,*, Vincenzo Villanacci MDb, Gieri Cathomas MDe, Christoph A. Maurer MDf, Simona Fisogni MDb, Morris Cadei MDb, Luigi Baron MDd, Antonio Morelli MDa, Eleonora Valloncini MDc, Bruno Salerni MDc a
Department of Clinical and Experimental Medicine, University of Perugia, 06100 Perugia, Italy 2nd Department of Pathology, Spedali Civili, 25100 Brescia, Italy c Department of Surgery, University of Brescia, 25100 Brescia, Italy d Pathology Unit, San Leonardo Hospital, Castellammare di Stabia, 80100 Naples, Italy e Cantonal Institute of Pathology, Liestal Hospital, CH-4410 Liestal, Switzerland f Department of Surgery, Liestal Hospital, CH-4410 Liestal, Switzerland b
Received 10 March 2006; revised 14 April 2006; accepted 26 April 2006
Keywords: Apoptosis; Enteric neurons; Glial cells; Interstitial cells of Cajal; Slow-transit constipation; Terminal ileum
Summary Slow-transit constipation is usually considered a colonic motor disorder. However, there is some evidence that abnormalities may be present in locations other than the colon. In particular, several studies have reported abnormal motor activity of the small bowel in these patients. We evaluated the neuropathological aspects of the terminal ileum in patients with slow-transit constipation to see whether abnormalities are present that may explain an abnormal motility of the small intestine. Specimens of the terminal ileum were obtained from 16 female patients (age range, 42-76 years) with slow-transit constipation undergoing surgery for intractable symptoms. Fifteen age- and sex-matched controls were used for comparison. Histologic and immunohistochemical evaluation of the myenteric plexus and the smooth muscle of the proximal ileal resection margin was carried out by means of hematoxylin and eosin, trichrome and periodic acid–Schiff stain, neuron-specific enolase, S-100, CD117, CD34, anti–a-actin, desmin, and vimentin antibodies. The patient group displayed a significantly reduced number of glial cells, compared with controls, in both the submucosal and the myenteric plexus. Only 1 of the 3 populations of interstitial cells of Cajal (that associated with the deep muscular plexus) was decreased in patients. No differences were found between patients and controls concerning ganglia neurons, fibroblast-like cells, enteric neurons, apoptotic phenomena, and smooth muscle. Patients with slow-transit constipation display neuropathological abnormalities of the terminal ileum to a lesser extent than those we previously found in the colon, which might explain the abnormal motor aspects sometimes found in these patients. D 2006 Elsevier Inc. All rights reserved.
1. Introduction * Corresponding author. Clinica di Gastroenterologia ed Epatologia, Via Enrico Dal Pozzo, Padiglione W, 06100 Perugia, Italy. E-mail address: [email protected] (G. Bassotti). 0046-8177/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2006.04.027
Among constipated patients, those with slow-transit constipation (STC) represent the subgroup that has the
Ileal neuropathology in slow-transit constipation most severe symptoms [1] and that also tends to be less influenced by medical therapeutic measures [2]. These patients’ symptoms are frequently labeled as intractable [3], and the most severe cases are often considered for surgery [4,5]. In patients with STC, several qualitative and quantitative changes in the enteric nervous system of the colon may be displayed. These include abnormal enteric neurochemistry [6,7], decreased argyrophilic neurons [8], decreased intraganglionic neurofilaments [9], hypoganglionosis of the myenteric plexus [10], and a decrease in colonic interstitial cells of Cajal (ICC) [11-13]. More recently, we have shown that patients with STC requiring surgery for intractable constipation, in addition to decreased ICC and enteric glial cells, have increased apoptotic phenomena of the enteric neurons [14]. These findings might represent a possible pathophysiological mechanism accounting for colonic neural loss and motor disturbances often described in these patients [4,15,16]. However, patients with STC may complain of upper gut symptoms, which often persist after subtotal colectomy [17]. Several studies have shown motor abnormalities related to functional disorders of the esophagus [18,19], the stomach [20,21], and the gall bladder [22]. Moreover, attention has been focused on abnormal motility of the small intestine, which may be present in these patients [23-25], even in relation to the outcome after colectomy [26]. These motor disturbances of the small bowel invariably display neuropathic features. Because abnormal motor activity may also be documented in the terminal ileum of patients with severe idiopathic constipation [27], the aim of the present study was to assess the number of ganglia neurons and glial cells within the submucosal and myenteric plexus and in the smooth muscle of the terminal ileum in patients undergoing surgery for intractable STC. Our working hypothesis was that these patients display alterations that can give origin to an abnormal motility of the small bowel.
2. Materials and methods 2.1. Patients Specimens from 5 to 7 cm of terminal ileum, taken at least 3 cm from the ileocecal valve, were obtained from a clinically homogeneous group of 16 female patients (age range, 42 to 76 years) fulfilling the Rome II criteria for constipation [28], undergoing colectomy with ileoproctostomy for intractable STC. The inclusion criteria were (1) long-standing history of constipation (N3 years; average, 15 years; range, 12-20 years); (2) 1 or fewer evacuations per week; (3) absence of frequent (N2 episodes per month) or chronic abdominal pain; (4) sensation of incomplete evacuation in more than 1 of 4 defecations; (5) negative history for (sub)obstructive episodes; and (6) unresponsiveness to appropriate and intensive medical treatment,
1253 including high-fiber diet, stimulant and osmotic laxatives, and enemas. Intestinal transit time, measured by means of radiopaque markers, was delayed in all patients (up to N240 hours). Causes of secondary constipation were excluded by drug history, physical examination, and laboratory screening (blood chemistry; thyroid hormones; and where appropriate, oral glucose tolerance test, sex hormone profiles, and antinuclear antibodies). To exclude organic diseases or mechanical causes of constipation and megacolon or megarectum, each patient underwent doublecontrast barium enema and/or colonoscopy. Absence of Hirschsprung’s disease was demonstrated by normal relaxation of the internal anal sphincter at anorectal manometry. No patient had evidence of obstructed defecation, as documented by anorectal manometry and/ or defecography. These patients were part of a study on colonic enteric nervous system abnormalities and were shown to have a significant decline in enteric nervous structures compared with controls [14]. Apart from sporadic dyspeptic symptoms, no patients had other upper gut complaints.
2.2. Methods After ileocolectomy, the surgical specimens were immediately fixed in 10% neutral-buffered formalin for 24 hours, then 4 to 6 full-thickness samples from the resected ileum were taken and transversal sections obtained. For conventional histology, 5-lm paraffin sections were stained with hematoxylin-eosin, periodic acid–Schiff, and trichrome.
2.3. Immunohistochemistry At least 30 slides for each patient were processed for immunohistochemistry. To evaluate markers of the enteric nervous system, monoclonal antibodies (MoAbs) toward neuron-specific enolase (NCL-NSE2; dilution, 1:50; Novocastra Laboratories, Newcastle upon Tyne, UK), acting as a marker of ganglion cells, and the glial marker protein S-100 (S-100; dilution, 1:50; Dako, Carpinteria, Calif) were used [29,30]. An anti-Kit antibody (rabbit polyclonal antibody; IgG; dilution, 1:50; Dako) was used to detect ICC, as previously reported [31]. To evaluate the population of fibroblast-like cells, which are intimately associated with the ICC [32], CD34 staining (CD34 Clone QBEnd/ 10; dilution, 1:30; Neo markers, Union City, Calif) was also used. Two methods were used as markers for apoptosis in the enteric nervous system: (a) the expression of Bcl-2 protein (BCL2 oncoprotein clone 124; dilution, 1:10; DBS, Pleasantown, Australia), a protooncogene responsible for specific suppression of apoptosis in several important situations and well-displayed in human enteric neurons [33], and (b) the MoAb to single-stranded DNA [34], using the formamide MoAb method (Mab F7-26 BMS 156; Bender MedSystem, Vienna, Austria), which detects apoptotic cells in tissue processed with routine histologic techniques and allows discrimination of apoptosis from
1254 necrosis [35]. The presence of lymphocytes was assessed by means of a monoclonal mouse antihuman CD3 antibody (Dako; dilution 1:40). The colonic smooth muscle was evaluated by means of anti–a-actin MoAb (dilution, 1:100; Biogenex, San Ramon, Calif), muscle-specific actin MoAb (clone HHF35, Dako), vimentin (mouse MoAb, Biogenex), and desmin (MoAb, Biogenex). Neuron-specific enolase, S-100, CD34, and Bcl-2 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, Foster City, Calif). 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. For Bcl-2, the expression in mucosal lymphoid cells served as internal control.
2.4. Expression of c-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 suppressed by incubation with 3% solution of H2O2 for 5 minutes. Kit immunostaining was carried out using a peroxidase-based visualization kit (Dako EnVision), following the manufacturer’s recommendations. Kit-positive mast cells served as internal control. Anti–single-stranded DNA immunohistochemistry. Sections 2 to 3 lm thick were warmed overnight at 608C 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 608C 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 MoAb 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 supersensitive nonbiotin detection system; Menarini, Firenze, Italy) for 30 minutes. Finally, a 5-minute reaction in the dark with diaminobenzidine (Bio-Optica; Milano, Italy) was carried out, and the sections were then counterstained with Mayer’s hematoxylin for 5 seconds, dehydrated, and mounted in
G. Bassotti et al. Clarion (Biomeda). Positivity was observed under the microscope as an intense brown reaction. For anti–a-actin, desmin, and vimentin, the immunoprecipitate was visualized by treatment with 3V3-diaminobenzidine (Lab Vision Corporation) and counterstaining with hematoxylin.
2.5. Data analysis All slides were coded and analyzed together by 2 pathologists (V. V. and S. F.), blinded with respect to patients and controls. For neuron-specific enolase, S-100, and CD3, as well as for Bcl-2 and formamide MoAb–positive cells, both the submucosal and the myenteric plexuses were taken into account by optical microscopy at 20 magnification (BX 40, Olympus, Tokyo, Japan). For each patient, the number of immunopositive cells was calculated and expressed as the mean of cells on 10 well-stained and well-oriented microscopic fields for each region of interest. To be considered as positive, the intensity of cell immunostaining had to be from moderate to strong, as described previously [14,36]. The density of ICC was graded, according to a previously described method [37,38], after the evaluation of 10 well-stained and well-oriented fields at 20 magnification. The 3 previously identified populations of ICC in the small bowel were taken into consideration [39-41]: ICC–deep muscular plexus (ICC-DMP), located between the internal thin layer and the thick outer layer of the circular muscle; ICC–myenteric plexus (ICC-MP), located between the circular and longitudinal muscle layers; and ICC– intramuscular, within the muscle fibers of the circular and longitudinal muscle layers. Not only nucleated cells but also Kit-positive, labeled elongated structures were considered for analysis [14,37]. For CD34, the strength of the immunostaining (graded as either present or severely depleted/absent, according to recently reported criteria [42]) was calculated around the myenteric plexus, between the elements of the plexus, within the longitudinal and circular muscle elements. Care was taken not to include vessels in the evaluation; however, the effectiveness of CD34 staining was indicated by the staining of capillaries in subjects with severe depletion/ absence in the other locations [42].
2.6. Controls Fifteen patients (all women; age range, 47-78 years) undergoing right hemicolectomy with ileocolonic anastomosis for nonobstructing colorectal cancer were used as controls. The control specimens were taken at least 3 cm from the resection margin in tumor-free areas.
2.7. Ethical considerations After explanation about the aims of the study, informed consent was obtained from both patients and controls, and the investigation was carried out according to local ethical
Ileal neuropathology in slow-transit constipation
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rules, following the recommendations of the Declaration of Helsinki (Edinburgh revision, 2000).
2.8. Statistical analysis Data were analyzed by means of nonparametric tests. The Wilcoxon signed rank test and the v 2 test were used, where appropriate. Values of P b .05 were chosen for rejection of the null hypothesis. Data are presented as median (95% confidence interval).
3. Results 3.1. Conventional histology In both groups, the mucosa, submucosa, smooth muscle, and nerve plexus architecture appeared normal on hematoxylin-eosin, trichrome and periodic acid–Schiff staining. In particular, no hypertrophy of the muscularis mucosa or the muscularis propria was found in patients. The trichrome staining did not highlight intra- or perimuscular fibrosis. No inflammatory cells (nor any intranuclear or viral inclusions) were observed in or around muscular or nervous structures. Moreover, no inclusion body myopathy was found in the smooth muscle [43].
Fig. 2 CD117 expression showing ICC-DMP (arrows) in a control (A) and in a patient (B). Note that in the patient, only 1 cell may be visualized. Original magnification 40.
3.2. Immunohistochemistry
Fig. 1 S-100 expression in the myenteric plexus of a control (A) and a patient (B); the latter displays a reduced number of glial cells. The arrows show enteric glial cells. Original magnification 40.
Neuron-specific enolase expression was not significantly different between patients and controls, in both the submucosal (25 [21-51] cells versus 39 [37-60] cells, P = .08) and the myenteric plexus (66.5 [39-78] cells versus 66 [50-89] cells, P = .40). Conversely, S-100–positive cells were significantly different between patients and controls in the submucosal (76 [57-87] cells versus 97 [73-126] cells, P = .05) and in the myenteric plexus (163 [132-210] cells versus 212 [194-240] cells, P = .034) (Fig. 1). As regards ICC, ICC-DMP was significantly decreased in patients with respect to controls (23 [13-35] cells versus 32 [24-41] cells, P = .04) (Fig. 2), whereas no significant differences between patients and controls were found for ICC-MP (208 [169-283] cells versus 273 [228-337] cells, P = .07) and ICC-intramuscular (68 [50-79] cells versus 70 [53-75] cells, P = .91). Scattered c-kit–positive mast cells were observed in the patients’ mucosa and were distributed as in the control tissue. The expression of CD34 was severely depleted in 6% of patients and 9% of controls ( P = .84) around the myenteric
1256 plexus, in 6% of patients and 7% of controls ( P = .58) between the elements of the plexus, and in none of the subjects of both groups within the longitudinal and circular muscle elements. The expression of Bcl-2 was not significantly different between patients and controls, in both the submucosal (88 [61-92] cells versus 84 [68-96] cells, P = .96) and the myenteric plexus (200 [163-282] cells versus 248 [199-291] cells, P = .20); no differences were also found between the 2 groups concerning the number of apoptotic neurons, in the submucosal (0 [0-0] cells versus 0 [0-0.4] cells, P = .72). and in the myenteric plexus (0 [0-2] cells versus 0 [0-1.4] cells, P = .69). No lymphocytic infiltration (assessed by CD3) was observed in either the submucosal or the myenteric plexus of the patients and controls. All patients and controls showed strong intensity for a-actin, muscle-specific actin, vimentin, and desmin immunostaining so that the ileal smooth muscle was judged to display normal characteristics.
4. Discussion It is presently recognized that STC is a severe condition of gut dysmotility, which may result in surgical intervention because of lack of response to medical therapeutic approaches [1]. Although several sources of evidence point to a prevalent colonic dysfunction, evidence has been accumulating over years that the impaired colonic motility may be part of a more generalized motility defect [44]. Indeed, studies investigating viscera other than the colon revealed abnormal motility in STC [18-22], more consistently in the small bowel [23-26], including the terminal ileum [27]. However, only 2 studies are available that investigated the basic mechanisms of small intestine abnormalities in severe idiopathic constipation. The first (focused on the ileocecal valve area) showed normal features at light and electron microscopic examination concerning neurons, ICC, and smooth muscle cells, whereas immunohistochemistry revealed reduced neuronal density and vasoactive intestinal polypeptide–immunoreactive neurons in the 2 enteric plexuses [45]. The second, focused on nerve fibers, demonstrated a deficiency of tachykinins and enkephalins in 75% of circular ileal muscle of these patients [46]. Our study was carried out in a homogeneous and relatively large group of patients with severe and intractable STC, and showed that the terminal ileum displays abnormal features, confined to the myenteric plexus, whereas the smooth muscle maintains its integrity. Interestingly, no differences in enteric neuronal counts were found between the 2 groups in both the myenteric and the submucosal plexus, whereas significantly reduced enteric glial cells were found in STC patients compared with controls in both the myenteric and the submucosal plexus. To our knowledge, this is the first time that such an alteration of enteric glial cells is described in the
G. Bassotti et al. small bowel of these patients, and we feel that it might have some pathophysiological relevance. In fact, glial cells are essential regulators of the formation, maintenance, and function of synapses, the key functional units of the nervous system [47]. Because enteric glial cells are thought to act as intermediaries in enteric neurotransmission [48], their decrease might impair the delicate neuroenteric balance coordinating bowel motility, as recently shown in experimental animal model of glial disruption [49]. Alternatively, impaired glial functions could result in insufficient support of enteric neurons, even in absence of inflammation, as recently proposed [50]. We have presently no explanation for this decrease of glial elements in our patients, and there is no literature support, except the reduction of these cells found in aged rats [51]. However, no such reduction was detected in the similarly aged control group; it therefore appears unlikely that the decrease was due to aging, at least in the proportion we found. Of course, such impairment of enteric glial cell function would require confirmation by means of electrophysiologic studies in fresh tissue samples. Concerning ICC, we found that only the ICC-DMP was significantly reduced in patients compared with controls. The role of ICC as intestinal pacemaker has been studied in experimental animal models, which have shown that a lack of ICC networks leads to the absence of slow waves and is accompanied by delayed or absent intestinal motility [52,53]. However, the different subtypes of ICC may have different physiological functions. Thus, it has been suggested that ICC-DMP and ICC-MP are devoted to neural transmission and induction of slow waves in the ileum, respectively [54,55]. A main role for ICC-DMP, as involved in inhibitory neurotransmission, was also inferred from the observation that nitric oxide synthase–immunoreactive nerve endings are close to these cells in the ileum [56] and that there are numerous gap junctions between ICCDMP and outer circular smooth muscle cells [57]. Therefore, a reduction of ICC-DMP function might impair the ileal neurotransmission, causing an abnormal motor activity, because the distribution of ICC in the ileum is quite similar to that of the more proximal segments of the small intestine [58,59]. The population of fibroblast-like CD34-positive cells associated with the ICC was not differently expressed at any of the anatomical locations taken into consideration and especially within and around the myenteric plexus. The concomitant loss of these cells and of ICC has been reported in patients with severe constipation due to pseudoobstruction [41], an entity with somewhat different pathophysiological and clinical characteristics [60,61]. We did not find increased apoptotic phenomena in the patients’ neuronal cells, as previously documented in the colon, and this is consistent with the absence of neuronal depletion in the terminal ileum. This, added to a loss of ICC limited to ICC-DMP in this area, could probably justify why our patients had minimal or no symptoms. It could be therefore hypothesized that the decrease of glial cells and, to
Ileal neuropathology in slow-transit constipation a lesser extent, of ICC-DMP, might synergistically act toward a derangement of intestinal motility, which is, however, somewhat controlled by the preservation of the enteric ganglia neurons and of ICC-MP populations, which probably limits the extent of the damage. On the other hand, intestinal manometric findings are sometimes abnormal in these patients [23-27]. However, it must be taken into consideration that these findings are completely nonspecific [62] and may occur in several different pathologic conditions [63], and no firm causal relationship may usually be established with symptoms. This is probably because the small bowel, from a motility point of view, responds in a monotonous manner to all series of triggering factors, including response to different foodstuffs [64]. Moreover, the patients’ series included in the above reports may have been more heterogeneous than our study cohort, which was selected for surgery after a rigorous workout. In conclusion, we have shown that in intractable STC requiring colectomy for symptoms’ relief, some neuropathological abnormalities of the terminal ileum may be detected, mainly consisting in a decrease of enteric glial cells and ICC-DMP. Because of their consistency with the almost constant documentation of a neuropathic-type pattern on manometric recordings, these abnormalities could be responsible for the alterations in small bowel motility sometimes documented in these patients (although the gastrointestinal hormones may also play a role in this setting [65,66]) and help toward a better understanding of the pathophysiological mechanisms of this entity.
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G. Bassotti et al. [50] Ruhl A. Glial cells in the gut. Neurogastroenterol Mot 2005;17: 777 - 90. [51] Phillips RJ, Kieffer EJ, Powley TL. Loss of glia and neurons in the myenteric plexus of the aged Fischer 344 rat. Anat Embryol (Berl) 2004;209:19 - 30. [52] Ward SM, Burns AJ, Torihashi S, Sanders KM. Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J Physiol (Lond) 1994; 480:91 - 7. [53] Huizinga JD, Thuneberg L, Kluppel M, Malysz J, Mikkelsen HB, Bernstein A. W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature 1995;373:347 - 9. [54] Ward SM, Burns AJ, Torihashi S, Harney SC, Sanders KM. Impaired development of interstitial cells and intestinal electrical rhythmicity in steel mutants. Am J Physiol 1995;269:C1577 - 85. [55] Takeuchi T, Fujinami K, Fujita A, Okishio Y, Takewari T, Hata F. Essential role of the interstitial cells of Cajal in nitric-oxide–mediated relaxation of longitudinal muscle of the mouse ileum. J Pharmacol Sci 2004;95:71 - 80. [56] Matini P, Faussone-Pellegrini MS. Ultrastructural localization of neuronal nitric oxide synthase-immunoreactivity in the rat ileum. Neurosci Lett 1997;229:45 - 8. [57] Ward SM, Sanders KM. Physiology and pathophysiology of the interstitial cells of Cajal: from bench to bedside. I. Functional development and plasticity of interstitial cells of Cajal networks. Am J Physiol 2001;281:G602. [58] Romert P, Mikkelsen HB. C-kit immunoreactive interstitial cells of Cajal in the human small and large intestine. Histochem Cell Biol 1998;109:195 - 202. [59] Vanderwinden JM, Rumessen JJ. Interstitial cells of Cajal in human gut and gastrointestinal disease. Microsc Res Tech 1999; 47:344 - 60. [60] Mann SD, Debinski HS, Kamm MA. Clinical characteristics of chronic idiopathic intestinal pseudo-obstruction in adults. Gut 1997;41:675 - 81. [61] Stanghellini V, Cogliandro RF, De Giorgio R, et al. Natural history of chronic idiopathic intestinal pseudo-obstruction in adults: a single center study. Clin Gastroenterol Hepatol 2005;3:449 - 58. [62] Hansen MB. Small bowel manometry. Physiol Res 2002;51: 541 - 56. [63] Malagelada JR, Camilleri M, Stanghellini V. Manometric diagnosis of gastrointestinal motility disorders. New York7 Thieme Inc; 1986. [64] Bassotti G, Bertotto A, Spinozzi F. Heretical thoughts about food hypersensitivity: small bowel manometry as an objective way to document gut reactions. Eur J Clin Nutr 1997;51:567 - 72. [65] van der Sijp JR, Kamm MA, Nightingale JM, et al. Circulating gastrointestinal hormone abnormalities in patients with severe idiopathic constipation. Am J Gastroenterol 1999;93:1351 - 6. [66] Hopman WP, Mollen RM, Kuijpers JH, Jansen JB. Peptide YY release after colectomy in slow transit constipation. Scand J Gastroenterol 2004;39:727 - 30.
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Original contribution
Enteric neuropathology of the terminal ileum in patients with intractable slow-transit constipation Gabrio Bassotti MD, PhDa,*, Vincenzo Villanacci MDb, Gieri Cathomas MDe, Christoph A. Maurer MDf, Simona Fisogni MDb, Morris Cadei MDb, Luigi Baron MDd, Antonio Morelli MDa, Eleonora Valloncini MDc, Bruno Salerni MDc a
Department of Clinical and Experimental Medicine, University of Perugia, 06100 Perugia, Italy 2nd Department of Pathology, Spedali Civili, 25100 Brescia, Italy c Department of Surgery, University of Brescia, 25100 Brescia, Italy d Pathology Unit, San Leonardo Hospital, Castellammare di Stabia, 80100 Naples, Italy e Cantonal Institute of Pathology, Liestal Hospital, CH-4410 Liestal, Switzerland f Department of Surgery, Liestal Hospital, CH-4410 Liestal, Switzerland b
Received 10 March 2006; revised 14 April 2006; accepted 26 April 2006
Keywords: Apoptosis; Enteric neurons; Glial cells; Interstitial cells of Cajal; Slow-transit constipation; Terminal ileum
Summary Slow-transit constipation is usually considered a colonic motor disorder. However, there is some evidence that abnormalities may be present in locations other than the colon. In particular, several studies have reported abnormal motor activity of the small bowel in these patients. We evaluated the neuropathological aspects of the terminal ileum in patients with slow-transit constipation to see whether abnormalities are present that may explain an abnormal motility of the small intestine. Specimens of the terminal ileum were obtained from 16 female patients (age range, 42-76 years) with slow-transit constipation undergoing surgery for intractable symptoms. Fifteen age- and sex-matched controls were used for comparison. Histologic and immunohistochemical evaluation of the myenteric plexus and the smooth muscle of the proximal ileal resection margin was carried out by means of hematoxylin and eosin, trichrome and periodic acid–Schiff stain, neuron-specific enolase, S-100, CD117, CD34, anti–a-actin, desmin, and vimentin antibodies. The patient group displayed a significantly reduced number of glial cells, compared with controls, in both the submucosal and the myenteric plexus. Only 1 of the 3 populations of interstitial cells of Cajal (that associated with the deep muscular plexus) was decreased in patients. No differences were found between patients and controls concerning ganglia neurons, fibroblast-like cells, enteric neurons, apoptotic phenomena, and smooth muscle. Patients with slow-transit constipation display neuropathological abnormalities of the terminal ileum to a lesser extent than those we previously found in the colon, which might explain the abnormal motor aspects sometimes found in these patients. D 2006 Elsevier Inc. All rights reserved.
1. Introduction * Corresponding author. Clinica di Gastroenterologia ed Epatologia, Via Enrico Dal Pozzo, Padiglione W, 06100 Perugia, Italy. E-mail address: [email protected] (G. Bassotti). 0046-8177/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2006.04.027
Among constipated patients, those with slow-transit constipation (STC) represent the subgroup that has the
Ileal neuropathology in slow-transit constipation most severe symptoms [1] and that also tends to be less influenced by medical therapeutic measures [2]. These patients’ symptoms are frequently labeled as intractable [3], and the most severe cases are often considered for surgery [4,5]. In patients with STC, several qualitative and quantitative changes in the enteric nervous system of the colon may be displayed. These include abnormal enteric neurochemistry [6,7], decreased argyrophilic neurons [8], decreased intraganglionic neurofilaments [9], hypoganglionosis of the myenteric plexus [10], and a decrease in colonic interstitial cells of Cajal (ICC) [11-13]. More recently, we have shown that patients with STC requiring surgery for intractable constipation, in addition to decreased ICC and enteric glial cells, have increased apoptotic phenomena of the enteric neurons [14]. These findings might represent a possible pathophysiological mechanism accounting for colonic neural loss and motor disturbances often described in these patients [4,15,16]. However, patients with STC may complain of upper gut symptoms, which often persist after subtotal colectomy [17]. Several studies have shown motor abnormalities related to functional disorders of the esophagus [18,19], the stomach [20,21], and the gall bladder [22]. Moreover, attention has been focused on abnormal motility of the small intestine, which may be present in these patients [23-25], even in relation to the outcome after colectomy [26]. These motor disturbances of the small bowel invariably display neuropathic features. Because abnormal motor activity may also be documented in the terminal ileum of patients with severe idiopathic constipation [27], the aim of the present study was to assess the number of ganglia neurons and glial cells within the submucosal and myenteric plexus and in the smooth muscle of the terminal ileum in patients undergoing surgery for intractable STC. Our working hypothesis was that these patients display alterations that can give origin to an abnormal motility of the small bowel.
2. Materials and methods 2.1. Patients Specimens from 5 to 7 cm of terminal ileum, taken at least 3 cm from the ileocecal valve, were obtained from a clinically homogeneous group of 16 female patients (age range, 42 to 76 years) fulfilling the Rome II criteria for constipation [28], undergoing colectomy with ileoproctostomy for intractable STC. The inclusion criteria were (1) long-standing history of constipation (N3 years; average, 15 years; range, 12-20 years); (2) 1 or fewer evacuations per week; (3) absence of frequent (N2 episodes per month) or chronic abdominal pain; (4) sensation of incomplete evacuation in more than 1 of 4 defecations; (5) negative history for (sub)obstructive episodes; and (6) unresponsiveness to appropriate and intensive medical treatment,
1253 including high-fiber diet, stimulant and osmotic laxatives, and enemas. Intestinal transit time, measured by means of radiopaque markers, was delayed in all patients (up to N240 hours). Causes of secondary constipation were excluded by drug history, physical examination, and laboratory screening (blood chemistry; thyroid hormones; and where appropriate, oral glucose tolerance test, sex hormone profiles, and antinuclear antibodies). To exclude organic diseases or mechanical causes of constipation and megacolon or megarectum, each patient underwent doublecontrast barium enema and/or colonoscopy. Absence of Hirschsprung’s disease was demonstrated by normal relaxation of the internal anal sphincter at anorectal manometry. No patient had evidence of obstructed defecation, as documented by anorectal manometry and/ or defecography. These patients were part of a study on colonic enteric nervous system abnormalities and were shown to have a significant decline in enteric nervous structures compared with controls [14]. Apart from sporadic dyspeptic symptoms, no patients had other upper gut complaints.
2.2. Methods After ileocolectomy, the surgical specimens were immediately fixed in 10% neutral-buffered formalin for 24 hours, then 4 to 6 full-thickness samples from the resected ileum were taken and transversal sections obtained. For conventional histology, 5-lm paraffin sections were stained with hematoxylin-eosin, periodic acid–Schiff, and trichrome.
2.3. Immunohistochemistry At least 30 slides for each patient were processed for immunohistochemistry. To evaluate markers of the enteric nervous system, monoclonal antibodies (MoAbs) toward neuron-specific enolase (NCL-NSE2; dilution, 1:50; Novocastra Laboratories, Newcastle upon Tyne, UK), acting as a marker of ganglion cells, and the glial marker protein S-100 (S-100; dilution, 1:50; Dako, Carpinteria, Calif) were used [29,30]. An anti-Kit antibody (rabbit polyclonal antibody; IgG; dilution, 1:50; Dako) was used to detect ICC, as previously reported [31]. To evaluate the population of fibroblast-like cells, which are intimately associated with the ICC [32], CD34 staining (CD34 Clone QBEnd/ 10; dilution, 1:30; Neo markers, Union City, Calif) was also used. Two methods were used as markers for apoptosis in the enteric nervous system: (a) the expression of Bcl-2 protein (BCL2 oncoprotein clone 124; dilution, 1:10; DBS, Pleasantown, Australia), a protooncogene responsible for specific suppression of apoptosis in several important situations and well-displayed in human enteric neurons [33], and (b) the MoAb to single-stranded DNA [34], using the formamide MoAb method (Mab F7-26 BMS 156; Bender MedSystem, Vienna, Austria), which detects apoptotic cells in tissue processed with routine histologic techniques and allows discrimination of apoptosis from
1254 necrosis [35]. The presence of lymphocytes was assessed by means of a monoclonal mouse antihuman CD3 antibody (Dako; dilution 1:40). The colonic smooth muscle was evaluated by means of anti–a-actin MoAb (dilution, 1:100; Biogenex, San Ramon, Calif), muscle-specific actin MoAb (clone HHF35, Dako), vimentin (mouse MoAb, Biogenex), and desmin (MoAb, Biogenex). Neuron-specific enolase, S-100, CD34, and Bcl-2 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, Foster City, Calif). 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. For Bcl-2, the expression in mucosal lymphoid cells served as internal control.
2.4. Expression of c-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 suppressed by incubation with 3% solution of H2O2 for 5 minutes. Kit immunostaining was carried out using a peroxidase-based visualization kit (Dako EnVision), following the manufacturer’s recommendations. Kit-positive mast cells served as internal control. Anti–single-stranded DNA immunohistochemistry. Sections 2 to 3 lm thick were warmed overnight at 608C 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 608C 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 MoAb 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 supersensitive nonbiotin detection system; Menarini, Firenze, Italy) for 30 minutes. Finally, a 5-minute reaction in the dark with diaminobenzidine (Bio-Optica; Milano, Italy) was carried out, and the sections were then counterstained with Mayer’s hematoxylin for 5 seconds, dehydrated, and mounted in
G. Bassotti et al. Clarion (Biomeda). Positivity was observed under the microscope as an intense brown reaction. For anti–a-actin, desmin, and vimentin, the immunoprecipitate was visualized by treatment with 3V3-diaminobenzidine (Lab Vision Corporation) and counterstaining with hematoxylin.
2.5. Data analysis All slides were coded and analyzed together by 2 pathologists (V. V. and S. F.), blinded with respect to patients and controls. For neuron-specific enolase, S-100, and CD3, as well as for Bcl-2 and formamide MoAb–positive cells, both the submucosal and the myenteric plexuses were taken into account by optical microscopy at 20 magnification (BX 40, Olympus, Tokyo, Japan). For each patient, the number of immunopositive cells was calculated and expressed as the mean of cells on 10 well-stained and well-oriented microscopic fields for each region of interest. To be considered as positive, the intensity of cell immunostaining had to be from moderate to strong, as described previously [14,36]. The density of ICC was graded, according to a previously described method [37,38], after the evaluation of 10 well-stained and well-oriented fields at 20 magnification. The 3 previously identified populations of ICC in the small bowel were taken into consideration [39-41]: ICC–deep muscular plexus (ICC-DMP), located between the internal thin layer and the thick outer layer of the circular muscle; ICC–myenteric plexus (ICC-MP), located between the circular and longitudinal muscle layers; and ICC– intramuscular, within the muscle fibers of the circular and longitudinal muscle layers. Not only nucleated cells but also Kit-positive, labeled elongated structures were considered for analysis [14,37]. For CD34, the strength of the immunostaining (graded as either present or severely depleted/absent, according to recently reported criteria [42]) was calculated around the myenteric plexus, between the elements of the plexus, within the longitudinal and circular muscle elements. Care was taken not to include vessels in the evaluation; however, the effectiveness of CD34 staining was indicated by the staining of capillaries in subjects with severe depletion/ absence in the other locations [42].
2.6. Controls Fifteen patients (all women; age range, 47-78 years) undergoing right hemicolectomy with ileocolonic anastomosis for nonobstructing colorectal cancer were used as controls. The control specimens were taken at least 3 cm from the resection margin in tumor-free areas.
2.7. Ethical considerations After explanation about the aims of the study, informed consent was obtained from both patients and controls, and the investigation was carried out according to local ethical
Ileal neuropathology in slow-transit constipation
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rules, following the recommendations of the Declaration of Helsinki (Edinburgh revision, 2000).
2.8. Statistical analysis Data were analyzed by means of nonparametric tests. The Wilcoxon signed rank test and the v 2 test were used, where appropriate. Values of P b .05 were chosen for rejection of the null hypothesis. Data are presented as median (95% confidence interval).
3. Results 3.1. Conventional histology In both groups, the mucosa, submucosa, smooth muscle, and nerve plexus architecture appeared normal on hematoxylin-eosin, trichrome and periodic acid–Schiff staining. In particular, no hypertrophy of the muscularis mucosa or the muscularis propria was found in patients. The trichrome staining did not highlight intra- or perimuscular fibrosis. No inflammatory cells (nor any intranuclear or viral inclusions) were observed in or around muscular or nervous structures. Moreover, no inclusion body myopathy was found in the smooth muscle [43].
Fig. 2 CD117 expression showing ICC-DMP (arrows) in a control (A) and in a patient (B). Note that in the patient, only 1 cell may be visualized. Original magnification 40.
3.2. Immunohistochemistry
Fig. 1 S-100 expression in the myenteric plexus of a control (A) and a patient (B); the latter displays a reduced number of glial cells. The arrows show enteric glial cells. Original magnification 40.
Neuron-specific enolase expression was not significantly different between patients and controls, in both the submucosal (25 [21-51] cells versus 39 [37-60] cells, P = .08) and the myenteric plexus (66.5 [39-78] cells versus 66 [50-89] cells, P = .40). Conversely, S-100–positive cells were significantly different between patients and controls in the submucosal (76 [57-87] cells versus 97 [73-126] cells, P = .05) and in the myenteric plexus (163 [132-210] cells versus 212 [194-240] cells, P = .034) (Fig. 1). As regards ICC, ICC-DMP was significantly decreased in patients with respect to controls (23 [13-35] cells versus 32 [24-41] cells, P = .04) (Fig. 2), whereas no significant differences between patients and controls were found for ICC-MP (208 [169-283] cells versus 273 [228-337] cells, P = .07) and ICC-intramuscular (68 [50-79] cells versus 70 [53-75] cells, P = .91). Scattered c-kit–positive mast cells were observed in the patients’ mucosa and were distributed as in the control tissue. The expression of CD34 was severely depleted in 6% of patients and 9% of controls ( P = .84) around the myenteric
1256 plexus, in 6% of patients and 7% of controls ( P = .58) between the elements of the plexus, and in none of the subjects of both groups within the longitudinal and circular muscle elements. The expression of Bcl-2 was not significantly different between patients and controls, in both the submucosal (88 [61-92] cells versus 84 [68-96] cells, P = .96) and the myenteric plexus (200 [163-282] cells versus 248 [199-291] cells, P = .20); no differences were also found between the 2 groups concerning the number of apoptotic neurons, in the submucosal (0 [0-0] cells versus 0 [0-0.4] cells, P = .72). and in the myenteric plexus (0 [0-2] cells versus 0 [0-1.4] cells, P = .69). No lymphocytic infiltration (assessed by CD3) was observed in either the submucosal or the myenteric plexus of the patients and controls. All patients and controls showed strong intensity for a-actin, muscle-specific actin, vimentin, and desmin immunostaining so that the ileal smooth muscle was judged to display normal characteristics.
4. Discussion It is presently recognized that STC is a severe condition of gut dysmotility, which may result in surgical intervention because of lack of response to medical therapeutic approaches [1]. Although several sources of evidence point to a prevalent colonic dysfunction, evidence has been accumulating over years that the impaired colonic motility may be part of a more generalized motility defect [44]. Indeed, studies investigating viscera other than the colon revealed abnormal motility in STC [18-22], more consistently in the small bowel [23-26], including the terminal ileum [27]. However, only 2 studies are available that investigated the basic mechanisms of small intestine abnormalities in severe idiopathic constipation. The first (focused on the ileocecal valve area) showed normal features at light and electron microscopic examination concerning neurons, ICC, and smooth muscle cells, whereas immunohistochemistry revealed reduced neuronal density and vasoactive intestinal polypeptide–immunoreactive neurons in the 2 enteric plexuses [45]. The second, focused on nerve fibers, demonstrated a deficiency of tachykinins and enkephalins in 75% of circular ileal muscle of these patients [46]. Our study was carried out in a homogeneous and relatively large group of patients with severe and intractable STC, and showed that the terminal ileum displays abnormal features, confined to the myenteric plexus, whereas the smooth muscle maintains its integrity. Interestingly, no differences in enteric neuronal counts were found between the 2 groups in both the myenteric and the submucosal plexus, whereas significantly reduced enteric glial cells were found in STC patients compared with controls in both the myenteric and the submucosal plexus. To our knowledge, this is the first time that such an alteration of enteric glial cells is described in the
G. Bassotti et al. small bowel of these patients, and we feel that it might have some pathophysiological relevance. In fact, glial cells are essential regulators of the formation, maintenance, and function of synapses, the key functional units of the nervous system [47]. Because enteric glial cells are thought to act as intermediaries in enteric neurotransmission [48], their decrease might impair the delicate neuroenteric balance coordinating bowel motility, as recently shown in experimental animal model of glial disruption [49]. Alternatively, impaired glial functions could result in insufficient support of enteric neurons, even in absence of inflammation, as recently proposed [50]. We have presently no explanation for this decrease of glial elements in our patients, and there is no literature support, except the reduction of these cells found in aged rats [51]. However, no such reduction was detected in the similarly aged control group; it therefore appears unlikely that the decrease was due to aging, at least in the proportion we found. Of course, such impairment of enteric glial cell function would require confirmation by means of electrophysiologic studies in fresh tissue samples. Concerning ICC, we found that only the ICC-DMP was significantly reduced in patients compared with controls. The role of ICC as intestinal pacemaker has been studied in experimental animal models, which have shown that a lack of ICC networks leads to the absence of slow waves and is accompanied by delayed or absent intestinal motility [52,53]. However, the different subtypes of ICC may have different physiological functions. Thus, it has been suggested that ICC-DMP and ICC-MP are devoted to neural transmission and induction of slow waves in the ileum, respectively [54,55]. A main role for ICC-DMP, as involved in inhibitory neurotransmission, was also inferred from the observation that nitric oxide synthase–immunoreactive nerve endings are close to these cells in the ileum [56] and that there are numerous gap junctions between ICCDMP and outer circular smooth muscle cells [57]. Therefore, a reduction of ICC-DMP function might impair the ileal neurotransmission, causing an abnormal motor activity, because the distribution of ICC in the ileum is quite similar to that of the more proximal segments of the small intestine [58,59]. The population of fibroblast-like CD34-positive cells associated with the ICC was not differently expressed at any of the anatomical locations taken into consideration and especially within and around the myenteric plexus. The concomitant loss of these cells and of ICC has been reported in patients with severe constipation due to pseudoobstruction [41], an entity with somewhat different pathophysiological and clinical characteristics [60,61]. We did not find increased apoptotic phenomena in the patients’ neuronal cells, as previously documented in the colon, and this is consistent with the absence of neuronal depletion in the terminal ileum. This, added to a loss of ICC limited to ICC-DMP in this area, could probably justify why our patients had minimal or no symptoms. It could be therefore hypothesized that the decrease of glial cells and, to
Ileal neuropathology in slow-transit constipation a lesser extent, of ICC-DMP, might synergistically act toward a derangement of intestinal motility, which is, however, somewhat controlled by the preservation of the enteric ganglia neurons and of ICC-MP populations, which probably limits the extent of the damage. On the other hand, intestinal manometric findings are sometimes abnormal in these patients [23-27]. However, it must be taken into consideration that these findings are completely nonspecific [62] and may occur in several different pathologic conditions [63], and no firm causal relationship may usually be established with symptoms. This is probably because the small bowel, from a motility point of view, responds in a monotonous manner to all series of triggering factors, including response to different foodstuffs [64]. Moreover, the patients’ series included in the above reports may have been more heterogeneous than our study cohort, which was selected for surgery after a rigorous workout. In conclusion, we have shown that in intractable STC requiring colectomy for symptoms’ relief, some neuropathological abnormalities of the terminal ileum may be detected, mainly consisting in a decrease of enteric glial cells and ICC-DMP. Because of their consistency with the almost constant documentation of a neuropathic-type pattern on manometric recordings, these abnormalities could be responsible for the alterations in small bowel motility sometimes documented in these patients (although the gastrointestinal hormones may also play a role in this setting [65,66]) and help toward a better understanding of the pathophysiological mechanisms of this entity.
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