An assessment of enteric nervous system and estroprogestinic ...

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Nov 22, 2011 - intussusception and/or anterior rectocele on dynamic defecogra- phy were present in all patients. A validated constipation score for defecation ...
Neurogastroenterology & Motility Neurogastroenterol Motil (2012) 24, e155–e161

doi: 10.1111/j.1365-2982.2011.01850.x

An assessment of enteric nervous system and estroprogestinic receptors in obstructed defecation associated with rectal intussusception GABRIO BASSOTTI ,* VINCENZO VILLANACCI ,  ALBERTO BELLOMI ,à ROSSELLA FANTE ,à MORIS CADEI ,  LUCA VICENZI ,§ FRANCESCO TONELLI ,– GABRIELLA NESI **

&

CORRADO R ASTERIA §

*Gastroenterology and Hepatology Section, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy  2nd Pathology Section, Spedali Civili, Brescia, Brescia, Italy àDepartment of Human Pathology, Hospital ‘‘Carlo Poma’’, Mantova, Italy §General Surgery Unit Asola, Department of Surgery and Orthopaedics, Hospital ‘‘Carlo Poma’’, Mantova, Italy –Surgical Unit, Department of Clinical Pathophysiology, University of Firenze, Firenze, Italy **Division of Pathologic Anatomy, University of Firenze, Firenze, Italy

Abstract Background The pathophysiological basis of obstructed defecation (OD) is still incompletely understood. In particular, few or no data are available concerning the enteric nervous system (ENS) in this condition. We investigated ENS abnormalities in patients with OD, undergoing surgery, together with the presence of estrogen (a and b) and progesterone receptors, and compare the results with those obtained in controls. Methods Full-thickness rectal samples were obtained from 17 patients undergoing stapled transanal rectal resection for OD associated with rectal intussusception. Samples were analyzed by immunohistochemistry for enteric neurons, enteric glial cells, interstitial cells of Cajal (ICC), and for estrogen and progesterone receptors. Data were compared with those obtained in 10 controls. Key Results No differences between patients and controls were found for enteric neurons, whereas (compared with controls) OD patients displayed a significant decrease of enteric glial cells in both the submucous (P = 0.0006) and the myenteric (P < 0.0001) plexus. ICC were significantly increased in patients in the submucosal surface (P < 0.0001) and the myenteric

area (P < 0.0001). Concerning estroprogestinic receptors, both were present on ICC in patients and controls. Estrogen receptors a and progesterone receptors were absent on enteric neurons and enteric glial cells in patients and controls, whereas estrogen receptors b were present in all controls and in 69% of patients’ enteric neurons (P = 0.18) and in 12% of patients’ glial cells (P = 0.0001). Conclusions & Inferences Patients with OD associated to rectal intussusception display abnormalities of the ENS and of estrogen receptors b. Keywords constipation, enteric plexus, estroprogestinic receptors, neuropathology, obstructed defecation.

INTRODUCTION Chronic constipation may broadly be divided into two main subtypes, slow-transit constipation and obstructed defecation (OD),1 the latter occurring in about 7% of the adult population2 and representing approximately 50% of constipated patients referred to tertiary care centers.3 Although OD is considered as a heterogeneous entity,4–6 most authors consider it basically related to rectoanal dysfunction, including failure to relax or paradoxical contraction of the pelvic floor while attempting to defecate,7–9 lack of rectal motor activity,10 and abnormal rectal sensitivity.11–13 However, in addition to functional causes, OD may also be due to anatomic causes,14 in particular rectocele and rectal intussusceptions,15 and these may require surgical correction.16,17 For this purpose, a variety of surgical techniques, including abdominal,

Address for Correspondence Professor Gabrio Bassotti, Clinica di Gastroenterologia ed Epatologia, Ospedale Santa Maria della Misericordia, Piazzale Menghini, 1, 06156 San Sisto (Perugia), Italy. Tel: +39 075 578 4423; fax: +39 075 5847570; e-mail: [email protected] Received: 23 May 2011 Accepted for publication: 22 November 2011

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vaginal, transanal, and perineal approaches, have been devised.18,19 More recently, a new surgical approach, known as ‘stapled transanal rectal resection’ (STARR), has been proposed, and involves a double-stapling technique to produce a transanal full-thickness rectal resection with the goal of correcting structural abnormalities associated with OD.20–22 The enteric nervous system (ENS) is organized in a complex structure that controls motility, blood flow, uptake of nutrients, secretion, immunologic, and inflammatory processes in the gut,23 and there is now discrete evidence that abnormalities within this network may contribute to the pathophysiologic basis of some forms of ‘idiopathic’ constipation.24–28 It is, however, worth noting that, notwithstanding numerous studies present in literature on the various pathophysiologic aspects of OD, almost nil is known concerning its neuropathologic aspects, particularly the ENS. Although alterations of the neuroenteric circuitry have been postulated as a pathophysiologic ground for these patients,29,30 only one study so far showed that a subgroup of patients with OD and severe, intractable symptoms display abnormalities of the ENS,31 even though the analysis was done in segments proximal to the rectum. The purpose of the present study was to evaluate some aspects of the ENS in patients with OD undergoing STARR for rectal intussusception, to assess whether such patients display abnormalities that may be responsible for some symptoms. In addition, as there is evidence in both humans and experimental animal models that sex hormones (especially progesterone) are involved in colorectal motility and some subtypes of constipation by means of inhibitory effects on smooth muscle,32,33 we investigated the localization of receptors for estrogens (ER) and progesterone (PR) in the ENS of OD patients.

was carried out according to the method described by Boccasanta and colleagues.37 Tissue samples were processed as previously described.28,31,38,39 After removal, the surgical specimens were immediately fixed in 10% neutral-buffered formalin for 24 h and transversal sections obtained. For conventional histology 5-lm paraffin sections were stained with Hematoxylin-Eosin, Pas, and Trichrome stain. At least 10 slides for each patient were processed for immunohistochemistry (IHC). To evaluate markers of the ENS, 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. For the interstitial cells of Cajal (ICC) an anti-Kit antibody (rabbit polyclonal antibody, IgG, dilution 1 : 50, Dako) was used. The 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 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. Expression of Kit: consecutive formalin-fixed, paraffin sections were dewaxed and rehydrated through decreasing alcohol series up to distilled water. Sections were then subjected to heatinduced epitope retrieval by immersion in a heat-resistant container filled with citrate buffer solution (pH 6.0) placed in a pressure cooker and microwaved for 20 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; Dako, Carpinteria, CA, USA), following the manufacturer’s recommendations. Kit-positive mast cells served as internal control. Concerning the estrogen–progesterone receptors, a panel of three monoclonal antibodies was used: rabbit monoclonal antihuman ER (clone SP1; Ventana Medical Systems, Inc., Tucson, AZ, USA), directed against the estrogen receptor alpha (ERa) molecule, mouse antihuman directed against the estrogen receptor beta (ERb, clone EMR02; Novocastra) and rabbit monoclonal antihuman progestinic receptor (PR, clone 1E2; Ventana, that recognizes the A and B forms of PR receptor). Immunostaining for ERa and PR was performed on 4-lm thick paraffin sections using the BenchMark XT automated staining system (Ventana). For antigen retrieval, slides were heated with Cell Conditioning Solution 1 (CC1) for 30 min. Endogenous biotin was blocked with the aid of the appropriate kit. Primary prediluted antibodies against ERa and PR were applied for 32 min at 37 C in the BenchMark XT autostainer and highlighted with the iVIEW DAB detection kit, yielding a brown reaction product. The signal was enhanced with the Ventana Amplification Kit. After the staining run had been completed, the slides were removed from the autostainer, counterstained with hematoxylin, dehydrated, and mounted in permanent mounting medium. A human breast tumor section, strongly positive for ERa and PR, was used as positive control, whereas the negative control was performed by substituting the primary antibody with a Ventana dispenser filled with non-immune serum at the same concentration as the primary antibody. For ERb immunohistochemical analysis, the sections were deparaffinized in Bio-Clear (Bio-Optica, Milan, Italy) and hydrated with grade ethanol concentrations until distilled water. Antigen retrieval was performed by immersing the slides in a thermostatic

PATIENTS AND METHODS We retrospectively analyzed full-thickness rectal specimens from a series of patients with OD undergoing STARR for symptoms unresponsive to conventional measures (including lifestyle changes, dietary manipulation, laxatives, and biofeedback)34 undergoing surgery in the period September 2007–September 2009. All patients fulfilled Rome II criteria for constipation35 (i.e., 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 maneuvers to facilitate defecation on more than one-fourth of bowel movements, or less than three evacuations per week), colonoscopy was normal, and adequate external sphincter function on rectal examination and evidence of rectal intussusception and/or anterior rectocele on dynamic defecography were present in all patients. A validated constipation score for defecation disorders36 was also available. The surgical procedure

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Neuropathology of obstructed defecation

RESULTS

bath containing preheated EDTA Buffer, pH 8.0 for 30 min at 98 C and cooling down at room temperature for 20 min. To block endogenous peroxidase activity, the slides were treated with 3.0% hydrogen peroxidase in distilled water for 10 min. The sections were then incubated with monoclonal mouse antihuman ERb, at 1 : 50 dilution in Antibody Diluent (Ventana) for 1 h at room temperature. Staining was achieved employing the Ultravision Detection System antipolyvalent HRP (LabVision, Fremont, CA, USA). Bound antibody was visualized using 3,3¢diaminobenzidine (Peroxidase/DAB; Dako, Glostrup, Denmark) as the chromogen. Nuclei were slightly counterstained with Mayer’s hematoxylin. A skin section was used as positive control, whereas the negative control was performed by substituting the primary antibody with a non-immune mouse serum. The control sections were treated in parallel with the samples in the same run.

Overall, in the study period 17 patients with OD [16 women, 1 man, aged 60 (52–68) years] underwent the surgical procedure and full-thickness specimens were available for the entire group. Symptoms’ duration was more than 10 years in six (35%) patients, 5 years in seven (41%) patients, and 3 years in four (24%) patients. No patients had associated diseases (apart from mild hypertension in five patients), but six patients (35%) complained of recurrent urinary infections. Preoperative constipation score was 13 (8–16), and significantly decreased to four (2–9, P < 0.0001) 1 month after surgery. Conventional histology did not show specific abnormalities; in particular, no inflammatory cells were found in the muscle or infiltrating the submucous or myenteric plexus in both patients and controls, nor an increased amount of fibrosis was detected in patients compared with controls. Smooth muscle was also histologically normal in both groups. Table 1 shows the IHC results in controls and patients. Concerning ICC, patients displayed a significant increase of ICC-SM and ICC-MY, whereas no difference was found for ICC-IM. No differences were also found in the number of enteric neurons in both the submucous and the myenteric plexus, whereas EGC were found to be significantly decreased in both plexuses of OD patients. Representative images are shown in Fig. 1. Concerning estroprogestinic receptors, no differences between patients and controls were found for ERa and PR (expressed only on ICC, but not on enteric neurons or on EGC), whereas ERb was expressed on ICC in both groups, on 69% of enteric neurons in OD patients and 100% controls (v2 = 1.8, P = 0.18), and on 12% of EGC in OD vs 100% controls (v2 = 14.7, P = 0.0001). Representative images are shown in Fig. 2.

Controls Rectal tissue from ten patients [eight women, two men, aged 61 (54–70) years], undergoing rectal resection for cancer, was obtained. Sections were taken at least 3 cm from the neoplasms, and the margins were ascertained to be tumor-free.

Data analysis All slides were coded and analyzed blind by two pathologists. For NSE and S100 both the submucosal and the myenteric plexuses were taken into account by optical microscopy at ·20 magnification (Olympus BX 40; Olympus Corporation, Tokyo, Japan). For each patient, the number of immunopositive cells was calculated and expressed as the mean of cells on 10 well-stained and welloriented 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 previously described.28,31 The density of ICC was graded, according to a previously described method,28,31 after the evaluation of 10 well-stained and welloriented fields at ·20 magnification. The three previously identified populations of ICC were taken into consideration40,41: 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 ICC in normal tissue42–44), and IC-IM, 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.28,31,38 Concerning ER and PR, we evaluated the number of subjects in whom they were expressed and where their localization was in the ENS.

Table 1 Immunohistochemical results (ICC, enteric neurons, EGC) in the ENS of OD patients and controls

Statistical analysis Data from controls and patients were compared by non-parametric methods, using the Wilcoxon test and the test for difference of proportions. Values of P < 0.05 were chosen for rejection of the null hypothesis. Data are expressed as median (95% CI).

Controls

Patients

P

28.5 204 40 50 62 115 204

77 206 122 60 70 82 65