Expression and regulation of reelin and its receptors in the enteric nervous system.

BACKGROUND & AIMS: In the central nervous system (CNS), reelin coordinates migration and lamination of neurons and regulates synaptic plasticity, whereas its role in the enteric nervous system (ENS) remains enigmatic. Thus we determined the expression pattern and localization of reelin in the human ENS and monitored the time course of mRNA expression of the reelin signaling system in the rat intestine as well as in GDNF treated ENS cultures. RESULTS: Reelin, its receptors and Dab1 were expressed in all intestinal layers as well as in isolated myenteric ganglia. Enteric ganglia and nerve fibers were immunoreactive for reelin which co-localized with PGP 9.5 and synaptophysin. In the rat small intestine, highest expression levels of reelin were detected at early postnatal stages. Enteric nerve cell cultures treated with GDNF showed marked up-regulation of reelin and its receptors. CONCLUSIONS: Reelin and its receptors are strongly expressed in the human ENS. Reelin is specifically localized in enteric neurons with highest expression levels during early postnatal life as well as in neuronal network forming enteric nerve cell cultures pointing to putative functions in the differentiation and maintenance of the ENS. EXPERIMENTAL METHODS: Gene expression of reelin, its receptors and Dab1 were analyzed in the human colon and isolated enteric ganglia. Co-localization of reelin with the pan-neuronal marker PGP 9.5 and the synaptic vesicle marker synaptophysin was studied by dual-label-immunocytochemistry. The time course of reelin expression was monitored in an ontogenetic study of rat intestines as well as in GDNF-treated cultures of enteric neurons.

The enteric serotonergic system is altered in patients with diverticular disease.

OBJECTIVE: Disturbances of the enteric serotonergic system have been implicated in several intestinal motility disorders. Patients with diverticular disease (DD) have been reported to exhibit abnormal intestinal motility and innervation patterns. Gene expression profiles of the serotonergic system and distribution of the serotonin type 4 receptor (5HT-4R) were thus studied in patients with DD. DESIGN: Colonic specimens from patients with DD and controls were subjected to quantitative PCR for serotonin receptors 2B, 3A, 4, serotonin transporter and synthesising enzyme tryptophan hydroxylase. Localisation of 5HT-4R was determined by dual-label immunocytochemistry using smooth muscle actin (α-SMA) and pan-neuronal markers (PGP 9.5) and quantitative analysis was carried out. Site-specific gene expression analysis of 5HT-4R was assessed within myenteric ganglia and muscle layers. Correlation of 5HT-4R with muscarinic receptors 2 and 3 (M2R, M3R) messenger RNA expression was determined. RESULTS: 5HT-4R mRNA expression was downregulated in the tunica muscularis and upregulated in the mucosa of patients with DD, whereas the other components of the serotonergic system remained unchanged. 5HT-4R was detected in ganglia and muscle layers, but was decreased in the circular muscle layer and myenteric ganglia of patients with DD. 5HT-4R mRNA expression correlated with M2R/M3R mRNA expression in controls, but not in patients with DD. CONCLUSIONS: The serotonergic system is compromised in DD. Altered expression of 5HT-4R at mRNA and protein levels may contribute to intestinal motor disturbances reported in patients with DD. The findings support the hypothesis that DD is associated and possibly promoted by an enteric neuromuscular pathology.

A novel endoscopic prototype device for gastric full-thickness biopsy for the histopathologic diagnosis of GI neuromuscular pathology: in vivo porcine long-term survival study (with videos).

BACKGROUND: Many GI motility disorders are associated with underlying GI neuromuscular pathology, which requires full-thickness biopsies (FTB) for histopathologic diagnosis. Currently, none of the endoscopy-based attempts to obtain FTB specimens have proven suitable for routine use. This study evaluated a novel endoscopic prototype device (ED) for this purpose. OBJECTIVE: To determine (1) the ability of the ED to obtain suitable FTB specimens, (2) associated complications, (3) feasibility of reliable defect closure, and (4) ability to evaluate intramural neuromuscular components. DESIGN: Preclinical proof-of-concept study in 30 pigs. SETTING: Animal laboratory. INTERVENTION: Gastric FTB specimens were obtained with a circular cutter and anchor. The defect was closed by over-the-scope clips/T-tags. The resection site was inspected via laparoscopy. After 2 to 4 weeks, necropsy was carried out to evaluate late complications. MAIN OUTCOME MEASUREMENTS: Feasibility, safety, and closure rate of the procedure. FTB specimens were assessed by histology/immunohistochemistry to visualize enteric neuromusculature. RESULTS: A total of 29 of 30 procedures were successfully performed; one hemorrhage required endoscopic treatment. A total of 29 of 30 FTB specimens (mean diameter 9.1 mm) were retrieved in 7.1 ± 0.4 minutes (range 3.0-12.5 minutes), displaying optimal tissue quality. Defect closure took 10.8 ± 0.9 minutes (range 7.2-32 minutes). Laparoscopy did not reveal damage to adjacent organs. Necropsy showed well-healed scars at the resection site and no complications, peritonitis, or abscess formation. Histology showed smooth muscle layers and submucosal and myenteric ganglia. LIMITATIONS: Survival animal pilot study, no patients. CONCLUSION: The novel ED enabled safe harvesting of well-preserved FTB specimens. Defect closure proved to be reliable. All neuromuscular structures relevant for histopathologic evaluation of GI neuromuscular pathology were demonstrated. Further studies are needed to verify the efficacy of this prototype device in the entire gut and in humans.

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. Parkinson's disease, Alzheimer's 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.

Alterations of the enteric smooth musculature in diverticular disease.

BACKGROUND: The pathogenesis of diverticular disease (DD) is considered to be multifactorial and involves intestinal motor disturbances and an underlying enteric neuromuscular pathology. While an enteric neuropathy has been well documented, actual studies on concomitant alterations of the enteric musculature are limited. This study is aimed at reassessing the smooth muscle tissue by histological, ultrastructural and molecular-biological approaches. METHODS: Full-thickness sigmoid specimens were obtained from patients with DD (n = 20) and controls (n = 19). Morphometric analysis was performed to evaluate the thickness and connective tissue index of the circular and longitudinal muscle layers as well as the myenteric plexus. Structural alterations were determined by light and transmission electron microscopy. mRNA profiles of components of the contractile smooth muscle apparatus including smooth muscle α-actin, smoothelin, histone deacetylase 8, and smooth muscle myosin heavy chain (SMMHC) were assessed by qPCR. Altered gene expression levels were confirmed at protein level by immunohistochemistry. RESULTS: Compared to controls, patients with DD showed (1) increased thickness of the circular and longitudinal muscle layers, (2) architectural alterations of smooth muscle cells, (3) increased connective tissue index of the longitudinal muscle layer, (4) focally reduced density of myofilaments at ultrastructural level, (5) specific down-regulation of SMMHC mRNA levels, (6) decreased immunoreactivity of SMMHC, (7) oligo-neuronal hypoganglionosis. CONCLUSIONS: DD is associated with distinct structural and functional alterations of the enteric musculature. The enteric myopathy is characterized by disturbed muscular architecture, connective tissue replacement and loss of specific myofilaments and thus may contribute to the pathogenesis and progression of DD.

The GDNF System Is Altered in Diverticular Disease - Implications for Pathogenesis.

BACKGROUND & AIMS: Absence of glial cell line-derived neurotrophic factor (GDNF) leads to intestinal aganglionosis. We recently demonstrated that patients with diverticular disease (DD) exhibit hypoganglionosis suggesting neurotrophic factor deprivation. Thus, we screened mRNA expression pattern of the GDNF system in DD and examined the effects of GDNF on cultured enteric neurons. METHODS: Colonic specimens obtained from patients with DD (n = 21) and controls (n = 20) were assessed for mRNA expression levels of the GDNF system (GDNF, GDNF receptors GFRα1 and RET). To identify the tissue source of GDNF and its receptors, laser-microdissected (LMD) samples of human myenteric ganglia and intestinal muscle layers were analyzed separately by qPCR. Furthermore, the effects of GDNF treatment on cultured enteric neurons (receptor expression, neuronal differentiation and plasticity) were monitored. RESULTS: mRNA expression of GDNF and its receptors was significantly down-regulated in the muscularis propria of patients with DD. LMD samples revealed high expression of GDNF in circular and longitudinal muscle layers, whereas GDNF receptors were also expressed in myenteric ganglia. GDNF treatment of cultured enteric neurons increased mRNA expression of its receptors and promoted neuronal differentiation and plasticity revealed by synaptophysin mRNA and protein expression. CONCLUSIONS: Our results suggest that the GDNF system is compromised in DD. In vitro studies demonstrate that GDNF enhances expression of its receptors and promotes enteric neuronal differentiation and plasticity. Since patients with DD exhibit hypoganglionosis, we propose that the observed enteric neuronal loss in DD may be due to lacking neurotrophic support mediated by the GDNF system.