Thilo Wedel

The London Classification of gastrointestinal neuromuscular pathology: report on behalf of the Gastro 2009 International Working Group

Objective Guidelines on histopathological techniques and reporting for adult and paediatric gastrointestinal neuromuscular pathology have been produced recently by an international working group (IWG). These addressed the important but relatively neglected areas of histopathological practice of the general pathologist, including suction rectal biopsy and full-thickness intestinal tissue. Recommendations were presented for the indications, safe acquisition of tissue, histological techniques, reporting and referral of such histological material. Design Consensual processes undertaken by the IWG and following established guideline decision group methodologies. Results and conclusion This report presents a contemporary and structured classification of gastrointestinal neuromuscular pathology based on defined histopathological criteria derived from the existing guidelines. In recognition of its origins and first presentation in London at the World Congress of Gastroenterology 2009, this has been named ‘The London Classification’. The implementation of this classification should allow some diagnostic standardisation, but should necessarily be viewed as a starting point for future modification as new data become available.

Gastrointestinal neuromuscular pathology: guidelines for histological techniques and reporting on behalf of the Gastro 2009 International Working Group

The term gastrointestinal neuromuscular disease describes a clinically heterogeneous group of disorders of children and adults in which symptoms are presumed or proven to arise as a result of neuromuscular, including interstitial cell of Cajal, dysfunction. Such disorders commonly have impaired motor activity, i.e. slowed or obstructed transit with radiological evidence of transient or persistent visceral dilatation. Whilst sensorimotor abnormalities have been demonstrated by a variety of methods in these conditions, standards for histopathological reporting remain relatively neglected. Significant differences in methodologies and expertise continue to confound the reliable delineation of normality and specificity of particular pathological changes for disease. Such issues require urgent clarification to standardize acquisition and handling of tissue specimens, interpretation of findings and make informed decisions on risk-benefit of full-thickness tissue biopsy of bowel or other diagnostic procedures. Such information will also allow increased certainty of diagnosis, facilitating factual discussion between patients and caregivers, as well as giving prognostic and therapeutic information. The following report, produced by an international working group, using established consensus methodology, presents proposed guidelines on histological techniques and reporting for adult and paediatric gastrointestinal neuromuscular pathology. The report addresses the main areas of histopathological practice as confronted by the pathologist, including suction rectal biopsy and full-thickness tissue obtained with diagnostic or therapeutic intent. For each, indications, safe acquisition of tissue, histological techniques, reporting and referral recommendations are presented.

Organization of the enteric nervous system in the human colon demonstrated by wholemount immunohistochemistry with special reference to the submucous plexus

To demonstrate the normal topography and structure of the enteric nervous system (ENS) in the human colon, the colonic wall of patients (n = 10, mean age 66.3 years), who underwent abdominal surgery unrelated to intestinal motility disorders, was submitted to wholemount immunohistochemistry. The specimens were stretched out and separated into the tunica muscularis, the outer and inner portion of the tela submucosa and the tunica mucosa. Prior to the application of the neuronal marker Protein Gene Product (PGP) 9.5, the laminar preparations were pretreated with the maceration agent KOH. The plexus myentericus was composed of prominent ganglia and interconnecting nerve fiber strands (NFS) forming a polygonal network, which was denser in the descending than in the ascending colon. Nerve cells were observed within the ganglia as well as in primary, secondary and tertiary NFS. The latter ramified into the adjacent smooth muscle layers, which contained the aganglionated plexus muscularis longitudinalis and circularis. The submucous plexus comprised three nerve networks of different topography and architecture: the delicate plexus submucosus extremus consisted of parallel orientated NFS with isolated nerve cells and small ganglia and was located at the outermost border of the tela submucosa adjacent to the circular muscle layer. The plexus submucosus externus was closely associated with the plexus submucosus extremus and composed of larger ganglia and thicker NFS. The plexus submucosus internus was situated adjacent to the lamina muscularis mucosae and formed a network with denser meshes but smaller ganglia and NFS than the plexus submucosus externus. The NFS of the aganglionated plexus muscularis mucosae followed the course of the smooth muscle cells of the lamina muscularis mucosae. The honeycomb-like network of the plexus mucosus was located within the lamina propria mucosae and divided into a subglandular and a periglandular portion. Single and accumulated nerve cells were observed within the plexus mucosus as a regular feature. The findings confirm the complex structural organisation of the ENS encountered in larger mammals, in particular the subdivision of the submucous plexus into three different compartments. PGP 9.5-immunohistochemistry applied to wholemount preparations comprehensively visualized the architecture of the intramural nerve plexus in human colonic specimens. In addition to conventional cross-sections, this technique allows a subtle assessment and classification of structural alterations of the ENS in patients with colorectal motor disorders.

Enteric glia promote intestinal mucosal healing via activation of focal adhesion kinase and release of proEGF

Wound healing of the gastrointestinal mucosa is essential for the maintenance of gut homeostasis and integrity. Enteric glial cells play a major role in regulating intestinal barrier function, but their role in mucosal barrier repair remains unknown. The impact of conditional ablation of enteric glia on dextran sodium sulfate (DSS)-induced mucosal damage and on healing of diclofenac-induced mucosal ulcerations was evaluated in vivo in GFAP-HSVtk transgenic mice. A mechanically induced model of intestinal wound healing was developed to study glial-induced epithelial restitution. Glial-epithelial signaling mechanisms were analyzed by using pharmacological inhibitors, neutralizing antibodies, and genetically engineered intestinal epithelial cells. Enteric glial cells were shown to be abundant in the gut mucosa, where they associate closely with intestinal epithelial cells as a distinct cell population from myofibroblasts. Conditional ablation of enteric glia worsened mucosal damage after DSS treatment and significantly delayed mucosal wound healing following diclofenac-induced small intestinal enteropathy in transgenic mice. Enteric glial cells enhanced epithelial restitution and cell spreading in vitro. These enhanced repair processes were reproduced by use of glial-conditioned media, and soluble proEGF was identified as a secreted glial mediator leading to consecutive activation of epidermal growth factor receptor and focal adhesion kinase signaling pathways in intestinal epithelial cells. Our study shows that enteric glia represent a functionally important cellular component of the intestinal epithelial barrier microenvironment and that the disruption of this cellular network attenuates the mucosal healing process.

Diverticular disease is associated with an enteric neuropathy as revealed by morphometric analysis

Background  The pathogenesis of diverticular disease (DD) is attributed to several aetiological factors (e.g. age, diet, connective tissue disorders) but also includes distinct intestinal motor abnormalities. Although the enteric nervous system (ENS) is the key‐regulator of intestinal motility, data on neuropathological alterations are limited. The study aimed to investigate the ENS by a systematic morphometric analysis.

Oligoneuronal hypoganglionosis in patients with idiopathic slow-transit constipation.

PURPOSE: Several alterations of the enteric nervous system have been described as an underlying neuropathologic correlate in patients with idiopathic slow-transit constipation. To obtain comprehensive data on the structural components of the intramural nerve plexus, the colonic enteric nervous system was investigated in patients with slow-transit constipation and compared with controls by means of a quantitative morphometric analysis. METHODS: Resected specimens were obtained from ten patients with slow-transit constipation and ten controls (nonobstructive neoplasias) and processed for immunohistochemistry with the neuronal marker Protein Gene Product 9.5. The morphometric analysis was performed separately for the myenteric plexus and submucous plexus compartments and included the quantification of ganglia, neurons, glial cells, and nerve fibers. RESULTS: In patients with slow-transit constipation, the total ganglionic area and neuronal number per intestinal length as well as the mean neuron count per ganglion were significantly decreased within the myenteric plexus and external submucous plexus. The ratio of glial cells to neurons was significantly increased in myenteric ganglia but not in submucous ganglia. On statistical analysis, the histopathologic criteria (submucous giant ganglia and hypertrophic nerve fibers) of intestinal neuronal dysplasia previously described in patients with slow-transit constipation were not completely fulfilled. CONCLUSION: The colonic motor dysfunction in slow-transit constipation is associated with quantitative alterations of the enteric nervous system. The underlying defect is characterized morphologically by oligoneuronal hypoganglionosis. Because the neuropathologic alterations primarily affect the myenteric plexus and external submucous plexus, superficial submucous biopsies are not suitable to detect these innervational disorders.

Cell-free supernatants of Escherichia coli Nissle 1917 modulate human colonic motility: evidence from an in vitro organ bath study

Abstract  Clinical studies have shown that probiotics influence gastrointestinal motility, e.g. Escherichia coli Nissle 1917 (EcN) (Mutaflor®) proved to be at least as efficacious as lactulose and more potent than placebo in constipated patients. As the underlying mechanisms are not clarified, the effects of EcN culture supernatants on human colonic motility were assessed in vitro. Human colonic circular smooth muscle strips (n = 94, 17 patients) were isometrically examined in an organ bath and exposed to different concentrations of EcN supernatants. Contractility responses were recorded under (i) native conditions, (ii) electrical field stimulation (EFS), (iii) non‐adrenergic non‐cholinergic conditions, and (iv) enteric nerve blockade by tetrodotoxin (TTX). As concentrations of acetic acid were increased in EcN supernatants, contractility responses to acetic acid were additionally tested. EcN supernatants significantly increased the maximal tension forces both at low and high concentrations. Neither blockade of both adrenergic and cholinergic nerves nor application of TTX abolished these effects. EFS‐induced contractility responses were not altered after exposure to EcN supernatants. Acetic acid elicited effects comparable to EcN supernatants only under TTX conditions. EcN supernatants modulate in vitro contractility of the human colon. As neither partial nor TTX blockade of enteric nerves abolished these effects, EcN supernatants appear to enhance colonic contractility by direct stimulation of smooth muscle cells. Active metabolites may include other substances than acetic acid, as acetic acid only partially resembled the effects elicited by EcN supernatants. The data provide a rationale for therapeutical application of probiotics in gastrointestinal motility disorders.

Interstitial cells of Cajal are involved in the afferent limb of the rectoanal inhibitory reflex

Background and aims: Interstitial cells of Cajal (ICC) have been shown to be involved in nitrergic neurotransmission of the lower oesophageal sphincter and pylorus. Here we studied the role of ICC and nitric oxide (NO) in the inhibitory neurotransmission of the murine internal anal sphincter (IAS). Methods: The rectoanal inhibitory reflex, rectal compliance, and relaxation of the isolated IAS to electrical stimulation were measured in controls, KIT W/KIT Wv mice, and neuronal NO synthase (nNOS) deficient mice. In addition, we evaluated the effect of blockade of nNOS using N-nitro-L-arginine methyl ester. Distribution of nNOS positive neurones and ICC in the IAS was assessed immunohistochemically. Results: KIT positive ICC were present in a dense network in the IAS of controls but not in KIT W/KITWv mice. Relaxation of IAS muscle strips induced by electrical stimulation was diminished in nNOS−/− mice but not in KIT W/KIT Wv mice. Blockade of NOS reduced the relaxation of IAS muscle strips in both mice. Relaxation of the IAS to rectal distension was significantly diminished in KIT W/KIT Wv mice and nNOS deficient mice. In concert, in vivo blockade of NOS attenuated the relaxation of the IAS in controls. No significant difference in compliance was found. Conclusion: The inhibitory innervation of the IAS and the rectoanal inhibitory reflex are mediated by NO and the rectoanal inhibitory reflex requires an intact network of ICC in the IAS. Thus both loss of nitrergic innervation and deficiency of ICC lead to impaired anal relaxation and may play an important role in rectal evacuation disorders.

Expression pattern and localization of alpha-synuclein in the human enteric nervous system

BACKGROUND Alpha-synuclein (α-syn) is abundantly expressed in the central nervous system and involved in the regulation of neurotransmission. Insoluble fibrils of phosphorylated α-synuclein (p-α-syn) have been implicated in several neurodegenerative diseases (e.g. Parkinsons disease, Alzheimers disease). The aim of the study was to determine the gene expression pattern and localization of α-syn/p-α-syn in the human enteric nervous system (ENS). METHODS Human colonic specimens (n=13, 15-83 years) were processed for α-syn and p-α-syn immunohistochemistry. Colocalization of α-syn was assessed by dual-labeling with pan-neuronal markers (PGP 9.5, HuC/D). For qPCR studies, tissue was obtained from full-thickness sections, tunica muscularis, submucosa, mucosa, and laser-microdissected (LMD) enteric ganglia. RESULTS Highest α-syn levels were detectable within the tunica muscularis and submucosa. Ganglia isolated by LMD showed high expression of α-syn mRNA. All myenteric and submucosal ganglia and nerve fibers were immunoreactive for α-syn. Dual-labeling revealed colocalization of α-syn with both pan-neuronal markers. p-α-syn immunoreactivity was consistently observed in specimens from adults with increasing age. CONCLUSIONS α-syn is abundantly expressed in all nerve plexus of the human ENS including both neuronal somata and processes. The presence of p-α-syn within the ENS is a regular finding in adults with increasing age and may not be regarded as pathological correlate. The data provide a basis to unravel the functions of α-syn and to evaluate altered α-syn in enteric neuropathies and α-synucleinopathies of the CNS with gastrointestinal manifestations.

Novel smooth muscle markers reveal abnormalities of the intestinal musculature in severe colorectal motility disorders.

Abstract  Histopathological studies of gastrointestinal motility disorders have mainly focused on enteric nerves and interstitial cells of Cajal, but rarely considered the enteric musculature. Here we used both classical and novel smooth muscle markers and transmission electron microscopy (TEM) to investigate muscular alterations in severe colorectal motility disorders. Full‐thickness specimens from Hirschsprungs disease, idiopathic megacolon, slow‐transit constipation and controls were stained with haematoxylin/eosin (HE) and Massons trichrome (MT), incubated with antibodies against smooth muscle α‐actin (α‐SMA), smooth muscle myosin heavy chain (SMMHC), smoothelin (SM) and histone deacetylase 8 (HDAC8) and processed for TEM. Control specimens exhibited homogeneous immunoreactivity for all antibodies. Diseased specimens showed normal smooth muscle morphology by HE and MT. While anti‐α‐SMA staining was generally normal, immunoreactivity for SMMHC, HDAC8 and/or SM was either absent or focally lacking in Hirschsprungs disease (80%), idiopathic megacolon (75%) and slow‐transit constipation (70%). Ultrastructurally, clusters of myocytes with noticeably decreased myofilaments were observed in all diseases. SMMHC and the novel smooth muscle markers SM and HDAC8 often display striking abnormalities linked to the smooth muscle contractile apparatus unnoticed by both routine stainings and α‐SMA, suggesting specific defects of smooth muscle cells involved in the pathogenesis of gastrointestinal motility disorders.