Annotated translation of Georg Meissner's first description of the submucosal plexus.

BACKGROUND: The discovery and detailed descriptions of the enteric nervous system dates back only ˂200 years. The 19th century was a golden age of histological, morphological, and physiological breakthroughs propelled by technological advances in microscopy, electricity, and scientific methodology. As a matter of fact, German-speaking scientists were highly successful during this period as can still be appreciated by the sheer number of German eponyms in anatomy. Therefore, the main language in scientific literature of this field was German at the time, thus, limiting the accessibility to the publications and scientific discussions from back then for the broader English-speaking scientific community today. PURPOSE: Here, an annotated translation of Meissner's first description of the submucosal plexus is provided along with a short biography of Georg Meissner and a review of the scientific literature and controversy surrounding his discovery.

Role of PD-L1 Expression during the Progression of Submucosal Gastric Cancer.

INTRODUCTION: Programmed death-ligand 1 (PD-L1) expression is a prognostic marker for gastric cancer that correlates with tumor diameter and depth of penetration. But the role of PD-L1 and mechanism(s) employed in the initial phase of invasion in early gastric cancer is yet to be understood. OBJECTIVE: This study aims to elucidate the role of PD-L1 during the progression of gastric cancer, specifically invading the submucosa beyond the lamina muscularis mucosa. METHODS: Using 107 patients with pathological submucosal gastric cancer, we determined the expression of PD-L1 based on the staining of the cell membrane or cytoplasm of tumor cells in the central and invasive front of the tumor. Samples were categorized into 3 groups based on the intensity of PD-L1 expression. CD8+ lymphocytes expressing PD-1 and CD163+ macrophages were used to determine the number of cell nuclei at the invasive front, similar to PD-L1. CMTM6 levels were determined and used to stratify samples into 3 groups. RESULTS: PD-L1 expression was higher in the invasive front (26.2%) than in the central portion of the tumors (7.4%; p < 0.001). Moreover, lymphatic and vascular invasion were more frequently observed in samples with high levels of PD-L1 (lymphatic invasion: 60.7 vs. 35.4%, p = 0.0026, and vascular invasion: 39.3 vs. 16.5%, p = 0.0018). There was no correlation between PD-L1 expression and the levels of PD-1, CD8, CD163, and CMTM6. CONCLUSIONS: PD-L1-expressing cancer cells at the invasive front of gastric cancer influence the initial stages of tumor invasion and lymphovascular permeation in early-stage gastric cancers. Immune checkpoint signaling may be the driving force in the invasive front during the invasion of the submucosa beyond the lamina muscularis mucosa.

The Endocrine Disruptor Bisphenol A (BPA) Affects the Enteric Neurons Immunoreactive to Neuregulin 1 (NRG1) in the Enteric Nervous System of the Porcine Large Intestine.

The enteric nervous system (ENS), located in the wall of the gastrointestinal (GI) tract, is characterized by complex organization and a high degree of neurochemical diversity of neurons. One of the less known active neuronal substances found in the enteric neurons is neuregulin 1 (NRG1), a factor known to be involved in the assurance of normal development of the nervous system. During the study, made up using the double immunofluorescence technique, the presence of NRG1 in the ENS of the selected segment of porcine large intestine (caecum, ascending and descending colon) was observed in physiological conditions, as well as under the impact of low and high doses of bisphenol A (BPA) which is commonly used in the production of plastics. In control animals in all types of the enteric plexuses, the percentage of NRG1-positive neurons oscillated around 20% of all neurons. The administration of BPA caused an increase in the number of NRG1-positive neurons in all types of the enteric plexuses and in all segments of the large intestine studied. The most visible changes were noted in the inner submucous plexus of the ascending colon, where in animals treated with high doses of BPA, the percentage of NRG1-positive neurons amounted to above 45% of all neuronal cells. The mechanisms of observed changes are not entirely clear, but probably result from neurotoxic, neurodegenerative and/or proinflammatory activity of BPA and are protective and adaptive in nature.

Robust, 3-Dimensional Visualization of Human Colon Enteric Nervous System Without Tissue Sectioning.

BACKGROUND & AIMS: Small, 2-dimensional sections routinely used for human pathology analysis provide limited information about bowel innervation. We developed a technique to image human enteric nervous system (ENS) and other intramural cells in 3 dimensions. METHODS: Using mouse and human colon tissues, we developed a method that combines tissue clearing, immunohistochemistry, confocal microscopy, and quantitative analysis of full-thickness bowel without sectioning to quantify ENS and other intramural cells in 3 dimensions. RESULTS: We provided 280 adult human colon confocal Z-stacks from persons without known bowel motility disorders. Most of our images were of myenteric ganglia, captured using a 20× objective lens. Full-thickness colon images, viewed with a 10× objective lens, were as large as 4 × 5 mm2. Colon from 2 pediatric patients with Hirschsprung disease was used to show distal colon without enteric ganglia, as well as a transition zone and proximal pull-through resection margin where ENS was present. After testing a panel of antibodies with our method, we identified 16 antibodies that bind to molecules in neurons, glia, interstitial cells of Cajal, and muscularis macrophages. Quantitative analyses demonstrated myenteric plexus in 24.5% ± 2.4% of flattened colon Z-stack area. Myenteric ganglia occupied 34% ± 4% of myenteric plexus. Single myenteric ganglion volume averaged 3,527,678 ± 573,832 mm3 with 38,706 ± 5763 neuron/mm3 and 129,321 ± 25,356 glia/mm3. Images of large areas provided insight into why published values of ENS density vary up to 150-fold-ENS density varies greatly, across millimeters, so analyses of small numbers of thin sections from the same bowel region can produce varying results. Neuron subtype analysis revealed that approximately 56% of myenteric neurons stained with neuronal nitric oxide synthase antibody and approximately 33% of neurons produce and store acetylcholine. Transition zone regions from colon tissues of patients with Hirschsprung disease had ganglia in multiple layers and thick nerve fiber bundles without neurons. Submucosal neuron distribution varied among imaged colon regions. CONCLUSIONS: We developed a 3-dimensional imaging method for colon that provides more information about ENS structure than tissue sectioning. This approach could improve diagnosis for human bowel motility disorders and may be useful for other bowel diseases as well.

Autologous Transplantation of Skin-Derived Precursor Cells in a Porcine Model.

BACKGROUND: Hirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. METHODS: Juvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. RESULTS: Prior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. CONCLUSIONS: Autologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. TYPE OF STUDY: Basic science.

Enteric neuron density correlates with clinical features of severe gut dysmotility.

Gastrointestinal (GI) symptoms can originate from severe dysmotility due to enteric neuropathies. Current methods used to demonstrate enteric neuropathies are based mainly on classic qualitative histopathological/immunohistochemical evaluation. This study was designed to identify an objective morphometric method for paraffin-embedded tissue samples to quantify the interganglionic distance between neighboring myenteric ganglia immunoreactive for neuron-specific enolase, as well as the number of myenteric and submucosal neuronal cell bodies/ganglion in jejunal specimens of patients with severe GI dysmotility. Jejunal full-thickness biopsies were collected from 32 patients (22 females; 16-77 yr) with well-characterized severe dysmotility and 8 controls (4 females; 47-73 yr). A symptom questionnaire was filled before surgery. Mann-Whitney U test, Kruskal-Wallis coupled with Dunn's posttest and nonparametric linear regression tests were used for analyzing morphometric data and clinical correlations, respectively. Compared with controls, patients with severe dysmotility exhibited a significant increase in myenteric interganglionic distance (P = 0.0005) along with a decrease in the number of myenteric (P < 0.00001) and submucosal (P < 0.0004) neurons. A 50% reduction in the number of submucosal and myenteric neurons correlated with an increased interganglionic distance and severity of dysmotility. Our study proposes a relatively simple tool that can be applied for quantitative evaluation of paraffin sections from patients with severe dysmotility. The finding of an increased interganglionic distance may aid diagnosis and limit the direct quantitative analysis of neurons per ganglion in patients with an interganglionic distance within the control range.NEW & NOTEWORTHY Enteric neuropathies are challenging conditions characterized by a severe impairment of gut physiology, including motility. An accurate, unambiguous assessment of enteric neurons provided by quantitative analysis of routine paraffin sections may help to define neuropathy-related gut dysmotility. We showed that patients with severe gut dysmotility exhibited an increased interganglionic distance associated with a decreased number of myenteric and submucosal neurons, which correlated with symptoms and clinical manifestations of deranged intestinal motility.

Shrinkage of the myenteric neurons of the small intestine in patients with multiple system atrophy.

This study aimed to determine whether enteric neurons are involved in multiple system atrophy (MSA). Four-µm-thick slices of small intestine were prepared from 10%-formalin-fixed and paraffin-embedded materials obtained from autopsied cases. Enteric neurons were stained using an anti-peripherin antibody. Immunostaining of phosphorylated α-synuclein was also performed. Areas of the cytoplasm and nucleus that showed nucleoli were measured using computer software. Both areas of myenteric neurons were significantly smaller in MSA cases (n = 3) than in control subjects (n = 3) (P < 0.0001); however, no deposits of phosphorylated α-synuclein were observed. These findings suggest that myenteric neurons in MSA are affected independent of α-synuclein accumulation.

Disruption of the network between Onuf's nucleus and myenteric ganglia, and developing Hirschsprung-like disease following spinal subarachnoid haemorrhage: an experimental study.

Purpose/Aim of the study: Auerbach/Meissner network of lower abdominopelvic organs managed by parasympathetic nerve fibres of lumbosacral roots arising from Onuf's nucleus located in conus medullaris. Aim of this study is to evaluate if there is any relationship between Onuf's nucleus ischemia and Auerbach/Meissner network degeneration following spinal subarachnoid haemorrhage (SAH). Materials and Methods: Study was conducted on 24 male rabbits included control (Group I, n = 5), serum saline-SHAM (Group II, n = 5), and spinal SAH (Group III, n = 14) groups. Spinal SAH performed by injecting homologous blood into subarachnoid space at Th12-L4 level and followed three weeks. Live and degenerated neuron densities of Onuf's nucleus, Auerbach and Meissner ganglia (n/mm3) were determined by Stereological methods. Results: The mean degenerated neuron density of Onuf's nucleus was significantly higher in Group III than in Groups I-II (152 ± 26, 2 ± 1 and 5 ± 2/mm3 respectively, p < 0.005). The degenerated neuron density of Auerbach's ganglia was significantly higher in Group III than in Groups I-II (365 ± 112, 3 ± 1 and 9 ± 3/mm3 respectively, p < 0.005). The degenerated neuron density of Meissner's ganglia was significantly higher in Group III than in Groups I-II (413 ± 132, 2 ± 1 and 11 ± 4/mm3 respectively, p < 0.005). Conclusions: Onuf's nucleus pathologies should be considered as Auerbach/Meissner ganglia degeneration and also related Hirschsprung-like diseases in the future.

Can Sertraline and Nortriptyline Protect the Neurons in Submucosal and Myenteric Plexuses of Rat's Colon Against Stress?

BACKGROUND: The colon is partly controlled by myenteric and submucosal plexuses, which respond to stress and lead to some gastrointestinal disorders. These plexuses play roles in irritable bowel syndrome. Patients suffering from this syndrome can be treated with some antidepressants, including sertraline and nortriptyline. AIMS: The primary aim of study was to compare the effect of a sertraline and a nortriptyline on the structural changes of the enteric neurons after stress exposure in both sexes. The secondary objectives were to evaluate the effects of stress on the submucosal and myenteric plexuses. METHODS: Male and female Sprague-Dawley rats were assigned to four subgroups. The first subgroup received no stress. The other three subgroups received chronic variable stress (CVS) and were given phosphate buffer, sertraline (10 mg/kg/day), or nortriptyline (10 mg/kg/day). After 45 days, the neuron number in their colon plexuses was estimated using the stereologic method. RESULTS: The number of neurons increased by 40-51% in the submucosal plexus and by 57-69% in the myenteric plexus in the CVS group compared with the control group (p < 0.002) without any sex preference. The increment was significantly higher in the myenteric plexus than in the submucosal plexus (p < 0.05). Moreover, co-treatment of stressed rats with sertraline and nortriptyline could prevent the cellular hyperplasia of the plexuses, with more effective action for sertraline (p < 0.02). CONCLUSIONS: Stress exposure for 45 days induced hyperplasia of the colon's enteric plexuses in both sexes. However, these drugs could prevent the changes, with a more effective action for sertraline.

Acute Toxoplasma gondii infection alters the number of neurons and the proportion of enteric glial cells in the duodenum in Wistar rats.

BACKGROUND: Toxoplasma gondii infection can occur through the ingestion of raw meat that contains tissue cysts or food that contains oocysts. Through the ingestion of oocysts, the parasite crosses the intestinal barrier, where the enteric nervous system is located. The objective was to investigate the kinetics of neuronal and glial responses during acute T. gondii infection. METHODS: We used 45 Wistar rats that were divided into a control group and infected groups that were evaluated at 6, 12, 24, 48, 72 hours, 7 days, 10 days, and 15 days after infection. The rats received 5000 sporulated oocysts of the parasite orally. To detect neurons and enteric glia cells, the myenteric and submucosal plexuses of the duodenum underwent double-labeling immunohistochemical techniques to evaluate HuC/HuD and S100, HuC/HuD and ChAT, and HuC/HuD and nNOS. KEY RESULTS: We observed a reduction of the total neuron population in the submucosal plexus 72 hours after infection. Cholinergic neurons decreased in the submucosal plexus 15 days after infection, and nitrergic neurons decreased in the myenteric plexus 72 hours after infection. A decrease in the number of glial cells was observed 7 days after infection in the submucosal plexus, and an increase in the enteric glial cell (EGC)/neuron ratio was found in both plexuses 48 hours after infection. CONCLUSIONS AND INFERENCES: We found decrease of neurons and increase in the EGC/neuron ratio in both plexuses caused by acute T. gondii infection, with major alterations 72 hours after oral infection. The number of cholinergic neurons decreased in the submucosal plexus, and the number of nitrergic neurons decreased in the myenteric plexus. A decrease in the number of enteric glial cells was observed in the submucosal plexus, and an increase in the enteric glial cell/neuron ratio was observed in both ganglionate plexuses of the duodenum.

Selective bladder denervation for overactive bladder (OAB) syndrome: From concept to healing outcomes using the ovine model.

AIMS: We evaluated a Selective Bladder Denervation (SBD) device, which uses radiofrequency ablation, for the treatment of overactive bladder syndrome in terms of its nerve denervation, ablation characteristics, and post-treatment healing. METHODS: Using the SBD device, eight fresh extirpated ovine bladder trigones were treated (90°C set point for 60 s) and nitroblue tetrazolium viability stained to characterize the ablation. In addition, 12 trigones were treated in vivo with three adjacent ablations and divided into survival cohorts: Day 7, Day 30, and Day 90 to assess the ablations and their associated healing. RESULTS: The ex vivo single trigone ablations had a 7.9 ± 0.9 mm width and 5.7 ± 1.0 mm thickness that involved the submucosa, detrusor muscle, adventitia, and vagina. Microscopic viability staining confirmed complete nerve necrosis within the targeted tissue. The in vivo Day 7 trigones supported the ex vivo ablation characteristics and showed up to minimal inflammation, granulation tissue, and collagen fibrosis. Day 30 trigones had essentially absent inflammation and granulation tissue with evolving collagen fibrosis at the ablation's periphery. Day 90 trigones had essentially absent acute inflammation, minimal chronic inflammation, essentially absent granulation tissue, and up to mild collagen fibrosis. No ureteral/urethral alterations, vesico-vaginal fistulas, or other complications were identified. CONCLUSIONS: The SBD device provided a targeted trigone ablation with resultant denervation. The tissue healing timeline followed that expected for a hyperthermic ablation and was characterized by a fibroproliferative healing response with limited inflammation and granulation tissue. The ablations did not impact the overlying bladder mucosal surface.

A comparison of submucosal tunneling endoscopic resection and endoscopic full-thickness resection for gastric fundus submucosal tumors

Aim: Both submucosal tunneling endoscopic resection (STER) and endoscopic full-thickness resection (EFTR) are effective methods for gastric fundus submucosal tumors (SMTs). However, there is little data that compares the two methods. The aim of this study was to compare the safety and efficacy of STER and EFTR for the treatment of SMTs in the gastric fundus. Methods: Clinical data was retrospectively collected from patients with gastric fundus SMTs who underwent STER or EFTR at our hospital from April 2011 to May 2016. Epidemiological data (gender, age), tumor size, procedure-related parameters, complications, postoperative hospital stay, cost and follow-up data were compared. Results: A total of 43 patients were enrolled: 15 underwent STER and the remaining 28 cases underwent EFTR. There were no significant differences between the two groups with regard to gender, age, tumor size, en bloc resection rate, operation time, pathohistological results and cost (p > 0.05). However, patients who underwent EFTR had a longer suture time, required a larger number of clips for closure and a prolonged postoperative hospital stay (p < 0.05). No recurrence was noted in either the STER or the EFTR group during a mean follow-up of 12.1 and 22.8 months, respectively. Conclusions: The treatment efficacy of STER and EFTR for the treatment of gastric fundus SMTs was comparable. However, STER has some advantages over EFTR in terms of suture time, the number of clips required for closure and postoperative hospital stay (AU)

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Massive Submucosal Ganglia in Colonic Inertia.

CONTEXT: - Colonic inertia is a debilitating form of primary chronic constipation with unknown etiology and diagnostic criteria, often requiring pancolectomy. We have occasionally observed massively enlarged submucosal ganglia containing at least 20 perikarya, in addition to previously described giant ganglia with greater than 8 perikarya, in cases of colonic inertia. These massively enlarged ganglia have yet to be formally recognized. OBJECTIVE: - To determine whether such "massive submucosal ganglia," defined as ganglia harboring at least 20 perikarya, characterize colonic inertia. DESIGN: - We retrospectively reviewed specimens from colectomies of patients with colonic inertia and compared the prevalence of massive submucosal ganglia occurring in this setting to the prevalence of massive submucosal ganglia occurring in a set of control specimens from patients lacking chronic constipation. RESULTS: - Seven of 8 specimens affected by colonic inertia harbored 1 to 4 massive ganglia, for a total of 11 massive ganglia. One specimen lacked massive ganglia but had limited sampling and nearly massive ganglia. Massive ganglia occupied both superficial and deep submucosal plexus. The patient with 4 massive ganglia also had 1 mitotically active giant ganglion. Only 1 massive ganglion occupied the entire set of 10 specimens from patients lacking chronic constipation. CONCLUSIONS: - We performed the first, albeit distinctly small, study of massive submucosal ganglia and showed that massive ganglia may be linked to colonic inertia. Further, larger studies are necessary to determine whether massive ganglia are pathogenetic or secondary phenomena, and whether massive ganglia or mitotically active ganglia distinguish colonic inertia from other types of chronic constipation.

Postnatal human enteric neuronal progenitors can migrate, differentiate, and proliferate in embryonic and postnatal aganglionic gut environments.

BACKGROUND: Enteric neural stem/progenitor cells (ENSCs) offer an innovative approach to treating Hirschsprung disease (HSCR) and other enteric neuropathies. However, postnatal-derived human ENSCs have not been thoroughly characterized and their behavior in the embryonic and postnatal intestinal environment is unknown. METHODS: ENSCs were isolated from the intestines of 25 patients undergoing bowel resection, including 7 children with HSCR. Neuronal differentiation and proliferation of ENSCs from submucosal and myenteric plexuses from patients with and without HSCR were characterized. ENSC migration and differentiation were studied following transplantation into embryonic chick neural crest, embryonic chick hindgut, and postnatal mouse aganglionic colon. RESULTS: The proliferative and neurogenic potential of ENSCs from HSCR intestine is equivalent to that of non-HSCR controls. Similarly, no difference was observed between myenteric- and submucosal-derived ENSCs. Postnatal ENSCs transplanted to embryonic neural crest pathways and to aneural hindgut migrate normally and differentiate into appropriate neural crest-derived cell types. ENSCs in postnatal mouse aganglionic colon differentiate into neurons and glia both ex vivo and in vivo. CONCLUSIONS: ENSCs isolated from the postnatal intestine of patients with and without HSCR can behave like embryonic neural crest-derived cells. These results support the feasibility of cell-based therapy for future treatment of neurointestinal disease.

Submucosal Plexitis as a Predictive Factor for Postoperative Endoscopic Recurrence in Patients with Crohn's Disease Undergoing a Resection with Ileocolonic Anastomosis: Results from a Prospective Single-centre Study.

AIM: Ileocolonoscopy allows early detection of recurrence after surgical resection for Crohn's disease [CD]. Plexitis, defined as presence of inflammatory cells in or around enteric ganglia or nerve bundles, in the proximal surgical margin has been associated with an increased overall recurrence risk. We investigated prospectively whether plexitis can predict endoscopic recurrence [ER] in a consecutive cohort of CD patients undergoing ileocolonic resection. METHODS: All CD patients undergoing ileocolonic resection in our institution between October 2009 and December 2012 were eligible for this study. Clinical data were obtained prospectively from the patients' files, and biopsies from the proximal surgical margins were analysed immunohistochemically for inflammation at the myenteric and submucosal plexus [lymphocytes, mast cells, eosinophils]. The degree of plexitis was correlated with the presence of ER at 6 months, defined as a modified Rutgeerts' score of ≥ i2b. Multivariate models were developed and tested to predict posterior probability of ER. RESULTS: A total of 74 patients were included. Six months after ileocolonic resection, 50% showed ER. Known risk factors such as penetrating disease, previous resections, and active smoking, showed no relation with ER. On the other hand, submucosal lymphocytic plexitis was associated with ER [p = 0.020]. The predictive value of lymphocytic cell count increased with more extensive biopsy sampling and with application of immunohistochemistry. CONCLUSIONS: Submucosal lymphocytic plexitis in the proximal surgical margin was significantly related with a higher risk for ER after ileocolonic resection. These data support development of a postoperative prevention trial with vedolizumab, which may block lymphocytic trafficking in the postoperative bowel.

Resección endoscópica mediante banda elástica de tumor rectal de células granulares / Endoscopic resection of rectal granular-cell tumor using elastic band ligation

El tumor de células granulares (TCG) es un tumor raro que se localiza en la capa submucosa del tubo digestivo. Presentamos a continuación a un varón con antecedentes de neoplasia de colon extirpada mediante cirugía en el que durante una colonoscopia de control se identificó un pólipo en el recto que extirpamos endoscópicamente realizando una mucosectomia asistida con banda elástica, resultando ser un TCG. Estamos ante lo que puede ser la segunda publicación de un TCG colorrectal tratado con éxito mediante banda elástica y el primer caso de TCG extirpado en el recto con esta técnica (AU)

Granular cell tumor (GCT) is a rare neoplasm that develops in the gut’s submucosal layer. We report the case of a male with a history of surgically excised colon neoplasm where a rectal polyp was identified during a follow-up endoscopy. The lesion, eventually identified as a GCT, was endoscopically removed by band ligation-assisted mucosectomy. This may be the second report of a colorectal GCT successfully managed using band ligation, and the first one on a rectal GCT excised with this technique (AU)

Myenteric plexuses atrophy in the vicinity of colorectal cancer tissue is not caused by apoptosis or necrosis.

INTRODUCTION: The previously performed studies showed that the presence of colorectal cancer (CRC) tumor is associated with the atrophy of myenteric plexuses in the vicinity of cancer invasion; however, the possible mechanisms of this phenomenon are not known. The aim of the present study was to determine whether the atrophic changes of the enteric nervous system (ENS) within an intestine wall of the CRC patients were caused by apoptosis or necrosis and whether they were associated with changes in the number of galanin-immunore-active (GAL-Ir) neurons. MATERIAL AND METHODS: Samples of the large intestine wall located close to the CRC invasion and control, distally-located part of the colon, were collected from 9 CRC patients. The size of ENS plexuses and the number of neurons were compared. Triple immunofluorescent staining was used to visualize the co-expression of caspase 3 (CASP3) or caspase 8 (CASP8) with GAL and protein gene-product 9.5 (PGP 9.5, panneuronal marker) in the submucosal and myenteric ENS plexuses. The cells expressing myeloperoxidase (MPO, marker of neutrophils) and CD68 (marker of macrophages) were detected by immunohistochemistry around/in myenteric plexuses (MPs) and in the muscularis externa of the colon wall in the vicinity of tumor invasion. RESULTS: Myenteric plexuses in the vicinity of the CRC tissue were significantly smaller and had lower number of neurons per plexus than distantly located plexuses. The number of CASP8- and CASP3-Ir neurons in the ENS plexuses was similar in the colon wall both close to and distally from tumor invasion. The number of CASP8-Ir neurons within MPs located close to CRC invasion was higher than of CASP3-Ir neurons. The percentage of neurons co-expressing CASP8 and GAL in myenteric plexuses close and distantly from tumor was three-fold lower than of those co-expressing CASP3 and GAL. The mean number of neutrophils and macrophages inside and around myenteric plexuses located close to tumor invasion was higher or similar, respectively, as compared with adjacent muscularis externa. CONCLUSIONS: The atrophy of myenteric plexuses in the vicinity of CRC invasion is not caused by apoptosis or necrosis. The differences in the proportions of neurons expressing galanin and the studied caspases suggest as yet unknown role of this neuropeptide in the mechanisms of neuron's atrophy in MPs located close to CRC tumor.

Decreased expression of NEDL2 in Hirschsprung's disease.

PURPOSE: NEDD4-like ubiquitin protein ligase 2 (NEDL2) plays an important role in many physiological and pathological processes. NEDL2 is a positive regulator of GDNF/Ret signaling during enteric neurogenesis. Mice lacking NEDL2 exhibit decreased numbers of enteric neurons, progressive bowel dysmotility and intestinal hypoganglionosis. We designed this study to investigate the expression of NEDL2 in the normal human colon and in HSCR. METHODS: HSCR tissue specimens (n=10) were collected at the time of pull-through surgery and divided into aganglionic and ganglionic segments. Colonic control samples (n=10) were obtained from patients with imperforate anus at the time of colostomy closure. Immunolabeling of NEDL2 was visualized using confocal microscopy to assess protein distribution, while Western blot analysis was undertaken to quantify NEDL2 protein expression. RESULTS: Confocal microscopy revealed that NEDL2-immunoreactivity colocalized with ICCs and neurons within the submucosa, myenteric plexus and smooth muscle in controls and ganglionic specimens, with markedly reduced NEDL2-immunoreactivity in aganglionic specimens. Western blotting revealed high levels of the NEDL2 protein in normal controls and the ganglionic region of HSCR, while there was a marked decrease in NEDL2 protein expression in the aganglionic region of HSCR. CONCLUSION: We report, for the first time, the expression of NEDL2 in the human colon. The decreased expression of NEDL2 in the aganglionic colon suggests that NEDL2 may play a role in the pathophysiology of HSCR.

Effects of Oxaliplatin Treatment on the Enteric Glial Cells and Neurons in the Mouse Ileum.

Oxaliplatin, currently used for treatment of colorectal and other cancers, causes severe gastrointestinal side effects, including nausea, vomiting, diarrhea, and constipation that are attributed to mucosal damage. However, delayed onset and long-term persistence of these side effects suggest that damage to the enteric nervous system (ENS) regulating physiological function of the gastrointestinal tract may also occur. The ENS comprises myenteric and submucosal neurons and enteric glial cells (EGCs). This study aimed to investigate the effects of oxaliplatin treatment on enteric neurons and EGCs within the mouse ileum. BALB/c mice received repeated intraperitoneal injections of oxaliplatin (3 mg/kg, 3 injections/week). Tissues were collected 3, 7, 14, and 21 days from the commencement of treatment. Decreases in glial fibrillary acidic protein-immunoreactive (IR) EGCs and protein gene product 9.5/ß-Tubulin III-IR neurons as well as increase in s100ß-IR EGCs after chronic oxaliplatin administration were observed in both the myenteric and submucosal plexi. Changes in EGCs were further observed in cross-sections of the ileum at day 14 and confirmed by Western blotting. Alterations in EGCs correlated with loss of myenteric and submucosal neurons in the ileum from oxaliplatin-treated mice. These changes to the ENS may contribute to the mechanisms underlying gastrointestinal side effects associated with oxaliplatin treatment.

Post-translational modifications of α-synuclein contribute to neurodegeneration in the colon of elderly individuals.

Synucleinopathies and abnormalities in the nerves of the enteric nervous system are hypothesized to be involved in age-associated motility disorders. The aim of the present study was to investigate the expression of various antigens, including α­synuclein (Syn) and its post­translational modified forms, in the human colon at various ages. In addition, the study aimed to correlate the expression of Syn with neurodegeneration. Immunohistochemistry was used to detect the expression of neurofilament (NF), Syn, as well as its nitrated (N) form in the healthy colonic tissue of 12 young (34.08±5.12 years), 10 middle­aged (51.80±3.52 years), and 11 elderly (75.82±7.70 years) individuals. To the best of our knowledge, the current study is the first to demonstrate the presence of N­Syn in the colonic tissue. N­Syn was identified in the upper layer of the mucosa and submucosa layer. Furthermore, Syn (wild­type) was present in the mucosa and submucosa. The number of NF­positive neurons in the submucosal layer declined significantly with age (P<0.01). In addition, Syn and N­Syn significantly increased during aging (P<0.01). Furthermore, a negative correlation was identified between neuron number and synucleinopathies, indicating the abnormal accumulation of both wild-type Syn and N­Syn in the mucosa, submucosa, muscle layer and myenteric plexus. The present study demonstrates that the Syn pathology may be linked to colic neuronal degeneration during normal aging, and this link may cause functional deficits.