Main

Obstructed defecation is one of the two main subtypes of chronic constipation (the other being represented by the slow transit variety),1 and it is estimated that it occurs in about 7% of the adult population.2 Even though it appears now quite clear that this is a heterogeneous entity,3, 4 traditionally obstructed defecation has been considered as due to rectoanal dysfunction, including failure to relax or paradoxical contraction of the pelvic floor while attempting to defecate,5, 6, 7 lack of rectal motor activity,8 and abnormal rectal sensitivity.9, 10, 11

However, there is also evidence that pathophysiological abnormalities in obstructed defecation may be not be confined to the very distal colonic area. Data from radiologic and scintigraphic studies during and after evacuation have shown that the act of defecation can empty a large part of the large bowel,12, 13 and manometric recordings reported that defecation follows a peculiar pattern of propagated high-amplitude colonic contractions14, 15 according to a complex sequence that starts up to 1 h before stools expulsion.16 Studies carried out by these manometric techniques have then demonstrated that in some patients with obstructed defecation motor abnormalities are also present higher in the colon, in both the basal state17 and after chemical or mechanical stimulation.18 Thus, alterations of the neuroenteric circuitry have been postulated as a pathophysiological ground for these patients17, 18 although to date no study has demonstrated such abnormalities.

Purpose of the present study was to investigate the neuropathological aspects of the colon in a group of patients with obstructed defecation unresponsive to treatment, to see whether such patients may display abnormalities that could justify an altered motor behaviour of the large bowel.

Patients

Eleven patients (nine women, age range 39–66 years) fulfilling the criteria for obstructed defecation and undergoing surgery (colectomy with ileorectostomy) for constipation refractory to any therapeutic intervention (see below) entered the study.

Inclusion criteria were as follows: (1) severe constipation present for more than 12 months and unresponsive to standard medical treatment, including dietary fiber, laxatives (including polyethylene glycol), enemas and suppositories; (2) fulfilment of Rome II criteria for constipation,19 that is two or more of six symptoms present for at least 12 weeks of the preceding 12 months: straining, lumpy or hard stools, sensation of incomplete evacuation, sensation of anorectal obstruction/blockage, or manual manoeuvres to facilitate defecation on more than one fourth of bowel movements, or less that three evacuations per week; (3) paradoxical contraction or failure to relax pelvic floor muscles during attempts to defecate, as shown by anorectal manometry and defecography; (4) inability to defecate a 50-ml, water-filled balloon within 5 min on repeated attempts; (5) failure to respond to biofeedback treatment, which has been shown as effective in a high percentage of cases.7, 20 It is worth noting that according to these criteria the patients also met the recently published Rome III criteria for functional defecation disorder.21

Exclusion criteria were as follows: (1) slow transit constipation, defined by evacuation of less than 80% of radiopaque markers 5 days after ingestion; however, patients with transit delay (two, with 85 and 88% of markers still present within the viscus) were included if the markers were concentrated in the rectosigmoid area; (2) barium enema consistent with abnormally dilated rectum or colon; (3) colonoscopy showing structural abnormalities of the large bowel; (4) abnormal biochemistry or thyroid function tests.

Controls

Ten patients (nine women, one man, age range 43–75 years) undergoing left hemicolectomy for non-obstructing colorectal cancer were used as controls, since there is evidence that the distribution of interstitial cells of Cajal is relatively uniform throughout the human colon.22 No data are available on the regional density of the enteric neurons and glial cells in man, although in preliminary observations we did not detect significant regional differences between the various colonic segments, except in the rectum (G Bassotti and V Villanacci, personal observations). The control specimens were taken at least 5 cm from the resection margin in tumour free areas.

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 rules, following the recommendations of the Declaration of Helsinki (Edinburgh revision, 2000).

Methods

Tissue samples were processed as described previously.23, 24, 25, 26 After removal, the surgical specimens were immediately fixed in 10% neutral-buffered formalin for 24 h, then 12–20 full-thickness samples from the whole ablated colon were taken and transversal sections obtained. For conventional histology 5 μm paraffin sections were stained with Hematoxylin–Eosin, Pas and Trichrome stain.

Immunohistochemistry

At least 20 slides for each patient were processed for immunohistochemistry. To evaluate markers of the enteric nervous system monoclonal antibodies toward neuron-specific enolase (NSE, NCL-NSE2, dilution 1:50, Novocastra Laboratories, Newcastle upon Tyne, UK) acting as a marker of gangliar cells, and the glial marker protein S100 (S-100, dilution 1:50, Dako, Carpinteria, CA, USA) were used. As interstitial cells of Cajal express Kit,27 an anti-Kit antibody (rabbit polyclonal antibody, IgG, dilution 1:50, Dako, Carpinteria, CA, USA) was used to detect these cells, as reported previously.28 Moreover, CD34 staining (CD34 Clone QBEnd/10, dilution 1:30, Neo markers, Union City, CA, USA) was used to evaluate the population of fibroblast-like cells which are intimately associated with the interstitial cells of Cajal.29 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 proto-oncogene responsible for specific suppression of apoptosis in several important situations,30 and well displayed in human enteric neurons,31, 32 and (b) the monoclonal antibody to single-stranded DNA, using the formamide monoclonal antibody (formamide-MAb) method (Mab F7–26 BMS 156, Bender MedSystem, Vienna, Austria), which detects apoptotic cells in tissue processed with routine histological techniques and allows discrimination of apoptosis from necrosis.33 This method has also been proved to yield the best results to identify enteric neuronal apoptosis when compared to other methods.34

NSE, 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 s, dehydrated and mounted in Clarion (Biomeda, Foster City, CA, USA). To account for non-specific 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.

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 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 min. Endogenous peroxidase activity was suppressed by incubation with 3% solution of H2O2 for 5 min. 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 2–3 μm thick were warmed overnight at 60°C, then dewaxed and rehydrated through decreasing alcohol series up to distilled water. Thereafter, the sections were incubated for 5 min in PBS with the addition of 20% Tween 20, followed by a passage with proteinase K (Dako) for 20 min. The sections were then rinsed with distilled water and heated in 50% formamide prewarmed to 60°C for 20 min. After cooling, endogenous peroxidase activity was suppressed by incubation with 3% solution of H2O2 for 5 min. Normal serum diluted 1:50 was applied for 10 min to room temperature, followed by anti-DNA MAb for 30 min, according to the manufacturer's recommendations. After that, the sections were incubated at room temperature with secondary polymeric antibody for 20 min and ABC (Kit super sensitive non biotin detection system, Menarini, Firenze, Italy) for 30 min. Finally, a 5 min 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 s, dehydrated and mounted in Clarion (Biomeda). Positivity was observed under the microscope as an intense brown reaction.

The presence of lymphocytes was assessed by means of a monoclonal mouse anti-human CD 3 antibody (Dako Cytomation, dilution 1:40).

The colonic smooth muscle was evaluated by means of an anti alfa-actin monoclonal antibody (dilution 1:100, Biogenex, CA, USA).

Data analysis

All slides were coded and analysed blind by two pathologists. For NSE, S100, and CD3, as well as for Bcl-2 and formamide-MAb positive cells, both the submucosal and the myenteric plexuses were taken into account by optical microscopy at × 20 magnification (Olympus BX 40). 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.35 The density of interstitial cells of Cajal was graded, according to a previously described method,23, 24, 25, 26, 36 after the evaluation of 10 well-stained and well-oriented fields at × 20 magnification. The three previously identified populations of interstitial cells of Cajal were taken into consideration:37, 38 IC-SM, along the submucosal surface of the circular muscle bundle, IC-MY, within the intermuscular space between circular and longitudinal muscle layers (myenteric region, which displays the highest yield of interstitial cells of Cajal in normal tissue22, 35, 39), and IC-IM, within the muscle fibers of the circular and longitudinal muscle layers. Not only nucleated cells but also Kit positive labelled elongated structures were considered for analysis.23, 24, 25, 26

For CD34, the strength of the immunostaining (graded as either present or severely depleted/absent, according to recently reported criteria40) 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.40

Statistical analysis

Data were analysed by means of nonparametric tests. The Wilcoxon's signed rank test and the χ2 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).

Results

Conventional Histology

In both patients and controls the mucosa, submucosa, smooth muscle and nerve plexus architecture appeared normal at Hematoxylin–Eosin, Trichrome and Pas staining. No inflammatory cells (nor any intranuclear or viral inclusions) were observed in or around muscular or nervous structures.

No patient exhibited hyperplastic changes (eg giant ganglia) of the submucosal plexus, thereby excluding a diagnosis of intestinal neuronal dysplasia.41

Immunohistochemistry

Table 1 summarizes the main pathological findings. No differences between groups were found concerning NSE expression in the myenteric plexus, whereas a significant decrease was found in the patients' submucosal plexus (Figure 1). S100 expression was significantly decreased in the patients' group, in both the myenteric (Figure 2c and d) and the submucosal plexus (Figure 2a and b). With respect to interstitial cells of Cajal, no significant differences between patients and controls were detected for the three populations.

Table 1 Immunohistochemical findings (number of positive cells) in controls and patients with obstructed defecation
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Figure 1
figure 1

NSE expression in the submucosal plexus of a control (a, b) and of a OD patients (c, d): the latter show reduced number of enteric neurons. Original magnifications: × 40 (a, d), × 100 (b), × 20 (c).

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Figure 2
figure 2

S100 expression in the submucosal plexus in a control (a) and a patient with obstructed defecation (b), and in the myenteric plexus in a control (c) and a patient with OD (d). Note the decrease of enteric glial cells in patients. Original magnification, × 100.

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No significant differences between patients and controls were also found in the expression of Bcl-2 in the myenteric plexus, whereas this resulted significantly decreased in the submucosal plexus in the patients' group.

The expression of CD34 around the myenteric plexus, between the elements of the plexus, within the longitudinal and circular muscle elements was depleted/absent in three patients and two controls (χ2 test P=0.89).

The number of apoptotic enteric neurons was not significantly different in patients and controls, in both the myenteric and the submucosal plexus.

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 alfa-actin immunostaining, so that colonic smooth muscle was judged to display normal characteristics.

Discussion

This study gives evidence, for the first time, that in some patients with obstructed defecation and intractable symptoms the colonic enteric nervous system may be abnormal. It is interesting to note that these abnormalities are quite different from those reported in patients with severe slow transit constipation, requiring surgery for symptoms' relief.24 In fact, in patients with obstructed defecation the more striking alterations consisted in a significant loss of the overall population of enteric glial cells, decreased in both the myenteric and the submucosal plexus compared to controls, whereas a reduction of enteric neurons was observed only in the submucosal plexus, and no difference in the interstitial cells of Cajal subpopulations was found between patients and controls.

We found particularly intriguing that the population of enteric glial cells was globally reduced in obstructed defecation patients, together with the decrease of the enteric neurons only in the submucosal plexus and no involvement of the interstitial cells of Cajal. Some pathophysiological meanings can be inferred from these findings, and will be discussed below.

It is worth noting that enteric glial cells represent the most abundant cell population in the enteric nervous system,42, 43 and although they have always traditionally been considered as mere support structures for enteric ganglia, evidence is being accumulated that this cell population may have more active functions in the economy of the enteric neurophysiology.44 In fact, experimental human and animal studies have shown that enteric glial cells may also be involved in intestinal inflammation,45, 46 enteric neurotransmission47, 48, 49 and, importantly, are essential for the homeostasis of enteric neurons.50 With respect to this last aspect, a recent study showed that enteric glia disruption may alter the neurochemical coding of enteric neurons in an experimental animal model.51 All together, these data strongly indicate that enteric glial cells can orchestrate gastrointestinal motor activity interacting with several enteric functions, and especially controlling neurochemical phenotypes in the enteric nervous system.52

The enteric neuronal loss (not due to an increase of apoptotic phenomena) in patients with obstructed defecation was limited to the submucosal plexus; since this plexus in humans mainly regulates mucosal functions53 (although some neurons of the outer submucosal plexus also innervate the circular muscle54) it is likely that, together with the integrity of the interstitial cells of Cajal circuitry, the maintenance of the enteric neuronal populations within the myenteric plexus may account for the preservation of a discrete colonic motor activity, more effective than that usually recorded in patients with slow transit constipation.

These findings are interesting, and consistent with the recent hypothesis, based on abnormal colonic manometric findings, that at least one subpopulation of patients with obstructed defecation might result from defective colonic, rather than anorectal, function.17 Of practical importance, our results could give an explanation for the lack of response to treatments, especially to biofeedback, in these patients. Biofeedback is to one of the most effective forms of treatment for obstructed defecation7, 20 as also shown by controlled studies,55 and it is thought that it acts by a learned mechanism of relaxation of pelvic floor muscles.56, 57, 58 This would imply that most of the pathophysiological mechanisms are concentrated in the pelvic floor area; however, since patients with obstructed defecation represent an heterogeneous entity, it is likely that in a subgroup the abnormalities are also present higher up in the colon, accounting for refractoriness to medical and behavioural therapeutic measures.

We are at present unable to explain whether these abnormalities are primary or secondary to longstanding constipation or treatments. We favour the first hypothesis, since severe idiopathic constipation is relatively frequent also in children,59 and there is no firm evidence of treatment damage of the enteric nervous system.25, 60

Of course, the results of this study are limited to a very selected and homogeneous group of patients with obstructed defecation, and no rectal tissue was available to see whether this area was also involved. Therefore, more investigations are needed in different subgroups of these patients to ascertain whether these abnormalities are confined to particular subjects and to the segments above the anorectal area. Finally, we feel that these findings may have some importance to establish whether alternative treatment approaches (for instance, it could be hypothesized the development of molecular targets for therapeutic interventions apt to restore or replace glial functions and restart appropriate neurochemical phenotypes in order to ultimately restore a proper function of the enteric nervous system52) may be pursued in future.