Three patients with intestinal pseudo-obstruction were studied. In two patients, the small intestinal wall was available and in the third case, a sample of the large intestine was studied. Case 1 was a 41 year old woman with an eight year history of abdominal pain, in whom small bowel dysmotility had been diagnosed after manometry studies, and who had previously undergone right hemicolectomy for a caecal volvulus. Case 2 was a 28 year old woman with a clinical diagnosis of small intestinal dysmotility, who underwent laparoscopy and full thickness ileal biopsy. Case 3 was a 54 year old woman with a history of chronic constipation since adolescence and a clinical diagnosis of pseudo-obstruction who underwent subtotal colectomy. No mechanical cause for bowel obstruction was identified within the resection specimens.
Control material was sourced from archival resections from 15 patients undergoing right hemicolectomy for caecal or proximal colonic carcinoma. Once suboptimally orientated sections had been excluded, a total of 15 small intestine samples and 11 large intestine samples served as controls.
In all cases and controls, histological examination revealed morphologically normal submucosal and myenteric nerve plexuses and muscularis propria smooth muscle. In particular, the muscularis propria smooth muscle showed no evidence of muscle layer loss, inflammation, fibrosis, or inclusion bodies using haematoxylin and eosin and diastase–periodic acid Schiff staining.
Formalin fixed and paraffin wax embedded sections of tissues were cut at 4 μm, dewaxed, and rehydrated in xylene and ethanol before antigen retrieval. Using the standard avidin–biotin–peroxidase complex staining technique, immunohistochemical staining was performed on all of the cases using the following primary antibodies: monoclonal anti-ASMA (clone 1A4; Sigma, Poole, Dorset, UK) at a 1/200 dilution, monoclonal anti-SMMHC (Dako, High Wycombe, UK) at a 1/200 dilution, and monoclonal anti-desmin (ICN Biomedicals, Basingstoke, UK) at a 1/30 dilution. For each sample, an adjacent tissue section was included without primary antibody as a negative control.
Endogenous peroxidase was inhibited using 0.5% hydrogen peroxide in methanol. The sections were then rinsed in buffer at pH 7.6. For ASMA and SMMHC, antigen retrieval was performed by placing the sections in a citrate buffer (0.01M citric acid with 80 g of sodium hydroxide/litre, adjusted to pH 6.0). Sections to be stained for ASMA were heated in a microwave oven (Panasonic NN-5452, 800 Watts, seven minutes at high power and seven minutes at medium power) and those for SMMHC were pressure cooked (Tefal CLIP50 X–PRESS at 13 lb pressure for two minutes). Sections to be stained with desmin were not subjected to antigen retrieval but were treated with 1% bovine serum albumin for 30 minutes. The sections were incubated with the primary antibody: at room temperature for 30 minutes in the case of ASMA and desmin, and for 24 hours at 4°C for SMMHC. After rinsing the sections in buffer, immunoreactivity was demonstrated using the LSAB kit (Dako), followed by 3-3′ diaminobenzidine-hydrogen peroxide solution. The slides were counterstained with haematoxylin, dehydrated, and mounted.
Each section was assessed by comparing the intensity of immunohistochemical labelling within the circular and longitudinal muscle layers of the muscularis propria, for each of the primary antibodies. Immunoreactivity within the muscularis mucosa and vessel walls acted as positive internal controls. Each section was scored independently by two consultant gastrointestinal pathologists (ACB and NJC).