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Introduction: Esophageal pain is mediated by sensory nerves, most importantly by the activation of the transient receptor potential vanilloid 1 (TRPV1) capsaicin receptor. TRPV1 is activated and sensitized by a broad range of pungent compounds, as well as inflammatory mediators and tissue irritants. Luminal stressors are suggested to impair the barrier function, which results in consequent activation of these sensory nerve terminals and pain. In this study, we investigated the effect of the perfusion of capsaicin, a TRPV1 agonist, on mucosal impedance and pain in asymptomatic volunteers., Methods: Thirteen asymptomatic volunteers completed a single-blind, saline-controlled, randomized crossover study. Capsaicin or saline was perfused for 30 minutes in the distal esophagus. Visual analog scale pain intensity scores and intraluminal impedance indicating mucosal integrity were determined. Distal and proximal biopsies were obtained 10 minutes later to measure TRPV1 messenger RNA and TRPV1 immunopositivity, as well as the intercellular space area., Results: Capsaicin perfusion resulted in significantly greater pain intensity (P = 0.047) and impaired recovery of the mucosal impedance compared with saline-treated controls (P = 0.027). Pain response was significantly associated with decreased mucosal impedance. Similar dynamics were seen in the proximal esophagus, but mucosal impedance recovered entirely to the preinfusion values there. There was a significant association between mucosal impedance and intercellular space width in the distal esophagus. TRPV1 transcription and expression were not significantly altered within this observation period., Discussion: Esophageal capsaicin perfusion results in pain, which is likely to be explained by impaired mucosal impedance and defective restoration capacity in the distal esophagus., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Neutrophil responses against pathogens must be balanced between protection and immunopathology. Factors that determine these outcomes are not well-understood. In a mouse model of genital herpes simplex virus-2 (HSV-2) infection, which results in severe genital inflammation, antibody-mediated neutrophil depletion reduced disease. Comparative single-cell RNA-sequencing analysis of vaginal cells against a model of genital HSV-1 infection, which results in mild inflammation, demonstrated sustained expression of interferon-stimulated genes (ISGs) only after HSV-2 infection primarily within the neutrophil population. Both therapeutic blockade of IFNα/β receptor 1 (IFNAR1) and genetic deletion of IFNAR1 in neutrophils concomitantly decreased HSV-2 genital disease severity and vaginal IL-18 levels. Therapeutic neutralization of IL-18 also diminished genital inflammation, indicating an important role for this cytokine in promoting neutrophil-dependent immunopathology. Our study reveals that sustained type I interferon (IFN) signaling is a driver of pathogenic neutrophil responses and identifies IL-18 as a novel component of disease during genital HSV-2 infection., Competing Interests: TL, YL, AC, PA, XJ, JS, MF, AO, CP, RC, MA, MD, HS No competing interests declared, (© 2021, Lebratti et al.)

Bladder afferents play a pivotal role in bladder function such as urine storage and micturition as well as conscious sensations such as urgency and pain. Endocannabinoids are ligands of cannabinoid 1 and 2 (CB1 and CB2) receptors but can influence the activity of a variety of G protein-coupled receptors as well as ligand-gated and voltage-gated channels. It is still not known which classes of bladder afferents are influenced by CB1 and CB2 receptor agonists. This study aimed to determine the role of CB2 receptors in two major classes of afferents in the guinea pig bladder: mucosal and muscular-mucosal. The mechanosensitivity of these two classes was determined by an ex vivo extracellular electrophysiological recording technique. A stable analog of endocannabinoid anandamide, methanandamide (mAEA), potentiated the mechanosensitivity of mucosal bladder afferents in response to stroking. In the presence of a transient receptor potential vanilloid 1 antagonist (capsazepine), the effect of mAEA switched from excitatory to inhibitory. A selective CB2 receptor agonist, 4-quinolone-3-carboxyamide (4Q3C), significantly inhibited the mechanosensitivity of mucosal bladder afferents to stroking. In the presence of a CB2 receptor antagonist, the inhibitory effect of 4Q3C was lost. mAEA and 4Q3C did not affect responses to stretch and/or mucosal stroking of muscular-mucosal afferents. Our findings revealed that agonists of CB2 receptors selectively inhibited the mechanosensitivity of capsaicin-sensitive mucosal bladder afferents but not muscular-mucosal afferents. This may have important implications for understanding of the role of endocannabinoids in modulating bladder function and sensation in health and diseases. NEW & NOTEWORTHY This article describes, for the first time, to our knowledge, the direct inhibitory effect of cannabinoid 2 receptor agonists on guinea pig mucosal bladder afferents. The cannabinoid 2 receptor is involved in pain and inflammation, suggesting that this may be a viable target for treatment of bladder disorders such as cystitis.

Mucosal Schwann cell hamartoma (MSCH) is an uncommon neural lesion characterized by an ill-defined proliferation of S100-positive Schwann cells in the lamina propria, with reported cases exclusively occurring in the colorectum. Here we describe the first series of MSCHs arising in the gastroesophageal junction (GEJ) and discuss their clinicopathologic features in comparison with their colorectal counterparts. We searched the UCLA pathology database from 01/2014 to 12/2018 to identify cases carrying the diagnosis of MSCH. A total of 48 cases (45 in-house, 3 consults) of colorectal MSCHs and 6 cases (1 in-house, 5 consults) of GEJ MSCHs were identified. For GEJ MSCHs, there were 4 males and 2 females with an average age of 70.2 years (range: 57-76 years). Clinical indications for endoscopy included history of gastroesophageal reflux disease (n = 2), heartburn (n = 2), dysphagia (n = 1), and iron deficiency anemia (n = 1). Endoscopic findings at the GEJ were available for 5 patients including irregular Z-line (n = 3), mild nodular carditis (n = 1), and normal (n = 1). None of them showed a polyp or nodule. The mean size of the lesion was 2.8 mm (range: 2-4 mm) microscopically. None of the colorectal or GEJ MSCH cases had an association with inherited syndromes. In conclusion, MSCH of the gastrointestinal tract is predominantly seen in the colorectum, but also infrequently seen in the GEJ. GEJ MSCH shares histologic and immunohistochemical features with its colorectal counterpart, but is usually an incidental finding with no endoscopically visible lesion. As there is no syndromic association with MSCH, additional treatment, work-up and follow-up are unnecessary., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The fate of macromolecules of biological or pharmacological interest that enter the mucus barrier is a current field of investigation. Studies of the interaction between the main constituent of mucus, mucins, and molecules involved in topical transmucoidal drug or gene delivery is a prerequisite for nanomedicine design. We studied the interaction of mucin with the bio-inspired arginine-derived amphoteric polymer d,l-ARGO7 by applying complementary techniques. Small angle X-ray scattering in bulk unveiled the formation of hundreds of nanometer-sized clusters, phase separated from the mucin mesh. Quartz microbalance with dissipation and neutron reflectometry measurements on thin mucin layers deposited on silica supports highlighted the occurrence of polymer interaction with mucin on the molecular scale. Rinsing procedures on both experimental set ups showed that interaction induces alteration of the deposited hydrogel. We succeeded in building up a new significant model for epithelial tissues covered by mucus, obtaining the deposition of a mucin layer 20 Å thick on the top of a glycolipid enriched phospholipid single membrane, suitable to be investigated by neutron reflectometry. The model is applicable to unveil the cross structural details of mucus-covered epithelia in interaction with macromolecules within the Å discreteness., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Experimental models of malaria have shown that infection with specific Plasmodium species in certain mouse strains can transiently modulate gut microbiota and cause intestinal shortening, indicating a disruption of gut homeostasis. Importantly, changes in gut homeostasis have not been characterized in the context of mild versus severe malaria. We show that severe Plasmodium infection in mice disrupts homeostasis along the gut-liver axis in multiple ways compared to mild infection. High parasite burden results in a larger influx of immune cells in the lamina propria and mice with high parasitemia display specific metabolomic profiles in the ceca and plasma during infection compared to mice with mild parasitemia. Liver damage was also more pronounced and longer lasting during severe infection, with concomitant changes in bile acids in the gut. Finally, severe Plasmodium infection changes the functional capacity of the microbiota, enhancing bacterial motility and amino acid metabolism in mice with high parasite burden compared to a mild infection. Taken together, Plasmodium infections have diverse effects on host gut homeostasis relative to the severity of infection that may contribute to enteric bacteremia that is associated with malaria.

The aim of this study was to characterize the number, type and distribution of immunochemically identified nerves in epithelium and lamina propria of the female rat urethra. Urethras from female Sprague-Dawley rats (n = 12) were fixed, frozen and sectioned (8 μm). Standard immunohistochemical techniques were used to identify putative nerves using the following antibodies: calcitonin gene related peptide (cgrp), neuronal nitric oxide synthase (nNos), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (vacht). The number, distribution and characteristics of all immunoreactive (IR) structures adjacent to the urethral epithelium and in the lamina propria was assessed. In the bladder, few cgrp-IR and vacht-IR fibers were associated with the urothelium or suburothelium of the lateral wall. In contrast, large numbers of vacht-IR, nNos-IR and cgrp-IR fibers were found close to the epithelium and subepithelium of the bladder neck and throughout the urethra. The number of cgrp-IR fibers was significantly higher in the urethra in comparison with the bladder neck. A population of undescribed cgrp-IR cells associated with the bladder neck and proximal urethra has been characterized. Each of these cells appears to be associated with a nerve fiber. In the distal urethra, the number of peptidergic fibers penetrating the epithelium was significantly higher than the rest of the urethra. Clearly, this study has revealed a highly complex and heterogeneous network of putative afferent nerves fibers along the length of the urethra. These structural specializations need to be taken into account when probing the different functions of the urethra. Anat Rec, 302:201-214, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Purpose: Stress urinary incontinence (SUI) and pelvic organ prolapse (POP) are common medical problems, particularlyamong older women. In this study, we aim to explore the relationship between the neurotransmitter nNOS in the vaginal epithelium, and the occurrence of SUI and changes of nNOS levels according to menopausal status.Matherials and Methods: Fourty women were enrolled. The patients were divided into four groups according to menstruaiton status and SUI. The vagina specimens were taken during transobturator tape application. The specimens were examined pathologically in terms of n-NOS expression. nNOS expression was compared between SUI and control groups. The results were evaluated statistically., Result: Epithelial total nNOS score in group 1 and group 3 were 2.4 ± 0.5 and 1.4 ± 0.5 respectively (P = .003). Stromal total nNOS score was found 2.2 ± 0.4 in group 1 and 1.3 ± 0.5 in group 3 (P = .001). Epithelial total nNOS score in group 2 and group 4 were 4.4 ± 0.5 and 3.5 ± 0.5 respectively (P = .003). Stromal total nNOS score was found 4.4 ± 0.5 in group 2 and 3.6 ± 0.5 in group 4 ( P = .006)., Conclusion: Our results show that expression of nNOs in the anterior vaginal epithelium decreased significantly in the SUI group. Altough our findings indicate important results, well designed further studies are needed to comprehend the role of NOS pathways better in SUI pathophysiology.

Background: Provoked vestibulodynia manifests as allodynia of the vulvar vestibular mucosa. The exact mechanisms that result in altered pain sensation are unknown. Recently, we demonstrated the presence of secondary lymphoid tissue, which is the vestibule-associated lymphoid tissue in the vestibular mucosa, and showed that this tissue becomes activated in provoked vestibulodynia., Objective: The purpose of this study was to examine whether expression of intraepithelial nerve fibers and nerve growth factor are related to immune activation in provoked vestibulodynia., Study Design: Vestibular mucosal specimens were obtained from 27 patients with severe provoked vestibulodynia that was treated by vestibulectomy and from 15 control subjects. We used antibodies against the protein gene product 9.5, the neuron specific neurofilament, and nerve growth factor for immunohistochemistry to detect intraepithelial nerve fibers and nerve growth factor expressing immune cells in the vestibular mucosa. For intraepithelial nerve fibers, we determined their linear density (fiber counts per millimeter of the outer epithelial surface, protein gene product 9.5) or presence (neuron specific neurofilament). Nerve growth factor was analyzed by counting the staining-positive immune cells. Antibodies against CD20 (B lymphocytes) and CD3 (T lymphocytes) were used to identify and locate mucosal areas with increased density of lymphocytes and the presence of germinal centers (ie, signs of immune activation). B-cell activation index was used to describe the overall intensity of B-cell infiltration., Results: We found more protein gene product 9.5-positive intraepithelial fibers in vestibulodynia than in the control samples (6.3/mm [range, 0.0-15.8] vs 2.0/mm [range, 0.0-12.0]; P=.006). Neuron specific neurofilament -positive intraepithelial fibers were found in 17 of 27 vestibulodynia cases (63.0%) and in none of the control cases. Protein gene product 9.5-positive intraepithelial fibers were more common in samples with more pronounced immune activation. The density of these fibers was higher in samples with than without germinal centers (6.1/mm [range, 4.3-15.8] vs 3.0/mm [range, 0.0-13.4]; P=.020). A positive correlation between the fiber density and B-cell activation index score of the sample was found (Spearman's Rho, 0.400; P=.004; R 2 =0.128). No significant difference, however, was found in the density or presence of nerve fibers between samples with high and low T-cell densities. We identified areas of minor and major vestibular glands in 16 of the patient samples and in 1 control sample. Protein gene product 9.5-positive nerve fibers were found more often in glandular epithelium surrounded by B-cell infiltration than in glands without B cells (P=.013). Also, the presence of neuron specific neurofilament-positive fibers in glandular epithelium was associated with B-cell infiltrates (P=.053). Nerve growth factor-positive immune cells were more common in mucosal areas with than without B-cell infiltration and intraepithelial nerve fibers., Conclusion: Excessive epithelial nerve growth in provoked vestibulodynia is associated with increased B-cell infiltration and the presence of germinal centers. This supports the fundamental role of immune activation in provoked vestibulodynia., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In visceral organs of mammals, most noxious (painful) stimuli as well as innocuous stimuli are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRGs). One of the major unresolved questions is the location, morphology, and neurochemistry of the nerve endings of spinal afferents that actually detect these stimuli in the viscera. In the upper gastrointestinal (GI) tract, there have been many anterograde tracing studies of vagal afferent endings, but none on spinal afferent endings. Recently, we developed a technique that now provides selective labeling of only spinal afferents. We used this approach to identify spinal afferent nerve endings in the upper GI tract of mice. Animals were anesthetized, and injections of dextran-amine were made into thoracic DRGs (T8-T12). Seven days post surgery, mice were euthanized, and the stomach and esophagus were removed, fixed, and stained for calcitonin gene-related peptide (CGRP). Spinal afferent axons were identified that ramified extensively through many rows of myenteric ganglia and formed nerve endings in discrete anatomical layers. Most commonly, intraganglionic varicose endings (IGVEs) were identified in myenteric ganglia of the stomach and varicose simple-type endings in the circular muscle and mucosa. Less commonly, nerve endings were identified in internodal strands, blood vessels, submucosal ganglia, and longitudinal muscle. In the esophagus, only IGVEs were identified in myenteric ganglia. No intraganglionic lamellar endings (IGLEs) were identified in the stomach or esophagus. We present the first identification of spinal afferent endings in the upper GI tract. Eight distinct types of spinal afferent endings were identified in the stomach, and most of them were CGRP immunoreactive. J. Comp. Neurol. 524:3064-3083, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The internal face of the detrusor smooth muscle wall of the urinary bladder is covered by a mucosa, separating muscle from the hostile environment of urine. However, the mucosa is more than a very low permeability structure and offers a sensory function that monitors the extent of bladder filling and composition of the urine. The mucosa may be considered as a single functional structure and comprises a tight epithelial layer under which is a basement membrane and lamina propria. The latter region itself is a complex of afferent nerves, blood vessels, interstitial cells and in some species including human beings a muscularis mucosae. Stress on the bladder wall through physical or chemical stressors elicits release of chemicals, such as ATP, acetylcholine, prostaglandins and nitric oxide that modulate the activity of either afferent nerves or the muscular components of the bladder wall. The release and responses are graded so that the mucosa forms a dynamic sensory structure, and there is evidence that the gain of this system is increased in pathologies such as overactive bladder and bladder pain syndrome. This system therefore potentially provides a number of drug targets against these conditions, once a number of fundamental questions are answered. These include how is mediator release regulated; what are the intermediate roles of interstitial cells that surround afferent nerves and blood vessels; and what is the mode of communication between urothelium and muscle - by diffusion of mediators or by cell-to-cell communication?, (© 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

The fibrocartilaginous eustachian tube is part of a system of contiguous organs including the nose, palate, rhinopharynx, and middle ear cleft. The middle ear cleft consists of the tympanic cavity, which includes the bony eustachian tube (protympanum) and the mastoid gas cells system. The tympanic cavity and mastoid gas cells are interconnected and allow gaseous exchange and pressure regulation. The fibrocartilaginous eustachian tube is a complex organ consisting of a dynamic conduit with its mucosa, cartilage, surrounding soft tissue, peritubal muscles (ie, tensor and levator veli palatine, salpingopharyngeus and tensor tympani), and superior bony support (the sphenoid sulcus)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: We investigated the neurohumoral modulation of the contractility of bladder muscularis mucosae (mucosa) compared with that of detrusor smooth muscle., Materials and Methods: Changes in the contractility of mucosal and detrusor bundles from guinea pig bladders were measured using isometric tension recording. The morphological relationship between the muscularis mucosae and blood vessels, and their sensory innervation was examined by fluorescence immunohistochemistry., Results: Meshworks of muscularis mucosae with numerous branches and anastomosis preferentially ran parallel with suburothelial blood vessels. Although PTHrPRs (parathyroid hormone-related peptide receptors) were expressed in detrusor and mucosa, the endogenous detrusor relaxant PTHrP (parathyroid hormone-related peptide) (1 nM) suppressed spontaneous contractions in detrusor but not in mucosa. A higher concentration of PTHrP (10 nM) was required to inhibit mucosal contractility. Capsaicin (1 μM) abolished spontaneous contractions in mucosa but had an excitatory action on detrusor contractility. hCGRP (human calcitonin gene-related peptide) (1 nM) attenuated spontaneous mucosal contractions. Pretreatment with the CGRP (calcitonin gene-related peptide) antagonist hCGRP 8-37 (2 μM) inhibited CGRP or capsaicin induced suppression of spontaneous contractions. Consistently, CGRP immunoreactive primary afferent nerves were abundant in muscularis mucosae., Conclusions: Co-localization of muscularis mucosae with the suburothelial microvasculature suggests that spontaneous contractions of mucosa might function to prevent microvasculature stretching upon bladder wall distension during the storage phase. It is likely that PTHrP selectively suppresses spontaneous contractions in detrusor but not in mucosa. Thus, endogenous PTHrP may well increase bladder compliance without an associated distension induced deformation of mucosal elements. Excessive stimulations of sensory nerves may suppress mucosal contractility by releasing CGRP., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Multidirectional interactions between the nervous and immune systems have been documented in homeostasis and pathologies ranging from multiple sclerosis to autism, and from leukemia to acute and chronic inflammation. Recent studies have addressed this crosstalk using cell-specific targeting, novel sequencing, imaging, and analytical tools, shedding light on unappreciated mechanisms of neuro-immune regulation. This Review focuses on neuro-immune interactions at barrier surfaces-mostly the gut, but also including the skin and the airways, areas densely populated by neurons and immune cells that constantly sense and adapt to tissue-specific environmental challenges., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background and Aims: The GI tract is innervated by the autonomic enteric nervous system, mainly composed of submucosal Meissner's plexus and myenteric Auerbach's plexus, which is essential for motility, blood flow regulation, and secretory functions. In vivo visualization of the esophageal enteric nervous system (EENS) during endoscopy has not been possible without invasive mucosal resection. This study aimed to visualize the EENS without mucosal resection, in vivo by using the novel probe, needle-based confocal laser-induced endomicroscopy (nCLE) with a fluorescence neuronal probe, NeuroTrace, under EUS guidance and to evaluate the feasibility of ex vivo imaging of the neuronal network in submucosal biopsy samples acquired at endoscopy., Methods: Four Yorkshire pigs were anesthetized and examined. In vivo experiment: During endoscopy, NeuroTrace was injected into the submucosa and muscularis propria of the middle and distal esophagus under EUS guidance, and nCLE imaging was performed. Ex vivo experiment: Submucosal tissue biopsy specimens from the porcine esophagus were obtained for ex vivo evaluation by using a "through-the-needle" forceps technique. After incubation of the samples in NeuroTrace solution, pCLE was used to visualize the EENS elements in the tissue., Results: Imaging of the EENS network by using EUS-guided nCLE was successful, both within the submucosa and the muscularis propria, and clearly visualized neuronal cells, glial cells, nerve bundles, and nerve fibers provided distinctive image patterns with excellent imaging quality. The use of the "through-the-needle" forceps technique achieved ex vivo images similar to those acquired in vivo., Conclusions: EUS-guided in vivo imaging of the enteric nervous system is feasible without mucosal resection and provides a novel ex vivo imaging alternative for human application. These novel, minimally invasive imaging approaches could be of tremendous diagnostic value to better characterize and explore the EENS of the GI tract., (Copyright © 2015 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Sensing mechanisms for nutrients, in particular dietary fat, operate in the mouth, brain, and gastrointestinal tract and play a key role in regulating feeding behavior and energy balance. Critical to these regulatory mechanisms are the specialized receptors present on taste buds on the tongue, on neurons in specialized centers in the brain, and on epithelial and enteroendocrine cells in the intestinal mucosa. These receptors recognize nutrients and respond by inducing intracellular signals that trigger release of bioactive compounds that influence other organs and help coordinate the response to the meal. Components of dietary fat that are recognized by these receptors are the long-chain fatty acids that act as ligands for 2 G protein-coupled receptors, GPR40 and GPR120, and the fatty acid (FA) translocase/CD36. Recent evidence that emphasizes the important role of CD36 in orosensory, intestinal, and neuronal sensing of FAs under physiologic conditions is highlighted in the review. How this role intersects with that of GPR120 and GPR40 in the regulation of food preference and energy balance is briefly discussed., (© 2015 American Society for Nutrition.)

Conclusion: In the sublingual gland, the serous lobule usually carried a higher density of NSE-positive nerve elements than the mucous lobule, whereas the mucous acinus in the mucous lobule was larger than the serous acinus in the serous lobule., Objectives: To demonstrate quantitative differences in nerve elements between the mucous and serous lobules of sublingual glands., Methods: This study investigated using specimens from 14 donated cadavers (mean age = 78 years). Since immunohistochemistry for neuron-specific enolase (NSE) stains all nerves in addition to other mesenchymal cells possibly of nerve origin, the present quantitative evaluation was based on NSE-positive areas per visual field under a ×20 objective lens (0.6 × 0.45 mm when printed)., Results: In mucous lobules, the areas occupied by NSE-positive nerve elements ranged from 5798-16,541 μm(2) (mean ± SD = 9280 ± 2584 μm(2)). In contrast, the corresponding areas in serous lobules ranged from 7853-23,540 μm(2) (mean ± SD = 13,520 ± 4351 μm(2)). The difference in NSE-positive areas was statistically significant (p = 0.0022). However, the mucous acinus in the mucous lobule was 2-times larger than the serous acinus in the serous lobule (2474 ± 1477 μm(2) vs 1119 ± 632 μm(2)).

Little is known about the mucosal phenotype of the proximal human esophagus. There is evidence to suggest that the proximal esophagus is more sensitive to chemical and mechanical stimulation compared with the distal. This may have physiological relevance (e.g., in prevention of aspiration of gastroesophageal refluxate), but also pathological relevance (e.g., in reflux perception or dysphagia). Reasons for this increased sensitivity are unclear but may include impairment in mucosal barrier integrity or changes in sensory innervation. We assessed mucosal barrier integrity and afferent nerve distribution in the proximal and distal esophagus of healthy human volunteers. In 10 healthy volunteers baseline proximal and distal esophageal impedance was measured in vivo. Esophageal mucosal biopsies from the distal and proximal esophagus were taken, and baseline transepithelial electrical resistance (TER) was measured in Ussing chambers. Biopsies were examined immunohistochemically for presence and location of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers. In a further four healthy volunteers we investigated for colocalization of CGRP and protein gene product (PGP) 9.5 immunoreactivity in nerve fibers. Baseline impedance was higher in the proximal than in the distal esophagus [2,936 Ω (SD578) vs. 2,229 Ω (SD821); P = 0.03], however, baseline TER was not significantly different between them. Mucosal CGRP-immunoreactive nerves were found in the epithelium of both proximal and distal esophagus, but were located more superficially in the proximal mucosa compared with the distal [11.5 (SD7) vs. 21.7 (SD5) cell layers from lumen, P = 0.002] 19% of proximal, and 10% of distal mucosal PGP-immunoreactive fibers colocalized with CGRP. PGP-immunoreactive fibers were also significantly closer to the luminal surface in the proximal compared with the distal esophagus (P < 0.001). We conclude that mucosal barrier integrity is similar in proximal and distal esophagus, but proximal mucosal afferent nerves are in a more superficial location. The enhanced sensitivity to reflux-evoked symptoms of the proximal esophagus most likely has an anatomical basis., (Copyright © 2015 the American Physiological Society.)

The gastrointestinal (GI) tract is a highly specialized sensory organ that provides crucial negative feedback during a meal, partly via a gut-brain axis. More specifically, enteroendocrine cells located throughout the GI tract are able to sense and respond to specific nutrients, releasing gut peptides that act in a paracrine, autocrine or endocrine fashion to regulate energy balance, thus controlling both food intake and possibly energy expenditure. Furthermore, the gut microbiota has been shown to provide a substantial metabolic and physiological contribution to the host, and metabolic disease such as obesity has been associated with aberrant gut microbiota and microbiome. Interestingly, recent evidence suggests that the gut microbiota can impact the gut-brain axis controlling energy balance, at both the level of intestinal nutrient-sensing mechanisms, as well as potentially at the sites of integration in the central nervous system. A better understanding of the intricate relationship between the gut microbiota and host energy-regulating pathways is crucial for uncovering the mechanisms responsible for the development of metabolic diseases and for possible therapeutic strategies., (© 2014 John Wiley & Sons Ltd.)

Increased esophageal sensitivity and impaired mucosal integrity have both been described in patients with gastroesophageal reflux disease, but the relationship between hypersensitivity and mucosal integrity is unclear. The aim of the present study was to investigate acid sensitivity in patients with erosive and nonerosive reflux disease and control subjects to determine the relation with functional esophageal mucosal integrity changes as well as to investigate cellular mechanisms of impaired mucosal integrity in these patients. In this prospective experimental study, 12 patients with nonerosive reflux disease, 12 patients with esophagitis grade A or B, and 11 healthy control subjects underwent an acid perfusion test and upper endoscopy. Mucosal integrity was measured during endoscopy by electrical tissue impedance spectroscopy and biopsy specimens were analyzed in Ussing chambers for transepithelial electrical resistance, transepithelial permeability and gene expression of tight junction proteins and filaggrin. Patients with nonerosive reflux disease and esophagitis were more sensitive to acid perfusion compared with control subjects, having a shorter time to perception of heartburn and higher perceived intensity of heartburn. In reflux patients, enhanced acid sensitivity was associated with impairment of in vivo and vitro esophageal mucosal integrity. Mucosal integrity was significantly impaired in patients with esophagitis, displaying higher transepithelial permeability and lower extracellular impedance. Although no significant differences in the expression of tight junction proteins were found in biopsies among patient groups, mucosal integrity parameters in reflux patients correlated negatively with the expression of filaggrin. In conclusion, sensitivity to acid is enhanced in patients with gastroesophageal reflux disease, irrespective of the presence of erosions, and is associated with impaired esophageal mucosal integrity. Mucosal integrity of the esophagus is associated with the expression of filaggrin., (Copyright © 2014 the American Physiological Society.)

Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered peptide inducing strong anxiogenic-like effect. CART distribution and its role(s) at periphery are not well understood. Immunohistochemisty was utilized to investigate the distribution patterns of CART in the stomach of the pig and wild boar. Double immunohistochemisty was applied to elucidate whether CART-immunoreactive (IR) neuronal elements coexpress galanin, substance P (SP) and neuropeptide Y (NPY). In the pig stomach, different proportions of CART-IR myenteric neurons were found in the antrum (42.3 ± 3.5%), corpus (18.0 ± 1.9%) and pylorus (33.2 ± 3.0%). CART-IR myeneric neurons were also found in the antrum, corpus and pylorus of the wild boar stomach (41.7 ± 3.2, 36.0 ± 2.2 and 35.8 ± 3.5%; respectively). In both species, none of gastric submucous neurons were CART-IR; however, CART-IR nerve fibres encircled submucous perikarya. In all portions of the pig and wild boar stomach, CART-IR nerve fibres were frequently found in the smooth muscle layer as well as in the lamina muscularis mucosae. In all regions of the pig and wild boar stomach, the expression of galanin and SP was found in CART-IR myenteric neurons and smooth muscle-supplying nerve fibres. CART/NPY coexpression was not found in the porcine stomach; however, in different regions of the wild boar stomach, subpopulations of CART-IR/NPY-IR myenteric neurons were noted. In conclusion, in this study, the existence and distribution patterns of CART in discrete regions of the pig and wild boar stomach were described in details. Colocalization studies revealed that in both animal species, a functional cooperation of CART with several neuropeptides is likely., (© 2013 Blackwell Verlag GmbH.)

The bladder mucosa consists of the urothelium, basement membrane, and lamina propria (LP). Although the urothelium has been given much attention, it may be regarded as one part of a signaling system involving another equally important component of the bladder mucosa, namely, the LP. The LP lies between the basement membrane of the mucosa and the detrusor muscle and is composed of an extracellular matrix containing several types of cells, including fibroblasts, adipocytes, interstitial cells, and afferent and efferent nerve endings. In addition, the LP contains a rich vascular network, lymphatic vessels, elastic fibers, and smooth muscle fascicles (muscularis mucosae). The roles of the LP and its components in bladder function have not been definitively established, though it has been suggested to be the capacitance layer of the bladder, determining bladder compliance and enabling adaptive changes to increasing volumes. However, the bladder LP may also serve as a communication center, with an important integrative role in signal transduction to the central nervous system (nociception, mechanosensation). The LP may also, by means of its different components, make it possible for the urothelium to transmit information to other components of the bladder wall, contributing to activation of the detrusor muscle. In addition, the LP may serve as a source for production of factors influencing the growth of both the overlying urothelium and the underlying detrusor muscle., (© 2013 Wiley Periodicals, Inc.)

The expression of short-chain fatty acid receptors GPR41/FFAR3 and GPR43/ free fatty acid receptor 2 (FFAR2) was studied in the gastrointestinal tract of transgenic monomeric red fluorescent protein (mRFP) reporter mice. In the stomach free fatty acid receptor 3 (FFAR3)-mRFP was expressed in a subpopulation of ghrelin and gastrin cells. In contrast, strong expression of FFAR3-mRFP was observed in all cholecystokinin, glucose-dependent insulinotropic peptide (GIP), and secretin cells of the proximal small intestine and in all glucagon-like peptide-1 (GLP-1), peptide YY, and neurotensin cells of the distal small intestine. Throughout the colon and rectum, FFAR3-mRFP was strongly expressed in the large population of peptide YY and GLP-1 cells and in the neurotensin cells of the proximal colon. A gradient of expression of FFAR3-mRFP was observed in the somatostatin cells from less than 5% in the stomach to more than 95% in the rectum. Substance P-containing enterochromaffin cells displayed a similar gradient of FFAR3-mRFP expression throughout the small intestine. Surprisingly, FFAR3-mRFP was also expressed in the neuronal cells of the submucosal and myenteric ganglia. Quantitative PCR analysis of fluorescence-activated cell sorting (FACS) purified FFAR3-mRFP positive cells confirmed the coexpression with the various peptide hormones as well as key neuronal marker proteins. The FFAR2-mRFP reporter was strongly expressed in a large population of leukocytes in the lamina propria of in particular the small intestine but surprisingly only weakly in a subpopulation of enteroendocrine cells. Nevertheless, synthetic ligands specific for either FFAR3 or FFAR2 each released GLP-1 from colonic crypt cultures and the FFAR2 agonist mobilized intracellular Ca²⁺ in FFAR2 positive enteroendocrine cells. It is concluded that FFAR3-mRFP serves as a useful marker for the majority of enteroendocrine cells of the small and large intestine and that FFAR3 and FFAR2 both act as sensors for short-chain fatty acids in enteroendocrine cells, whereas FFAR3 apparently has this role alone in enteric neurons and FFAR2 in enteric leukocytes.

Several populations of interstitial cells of Cajal (ICC) exist in the bladder, associated with intramural nerves. Although ICC respond to exogenous agonists, there is currently no evidence of their functional innervation. The objective was to determine whether bladder ICC are functionally innervated. Guinea-pig bladder tissues, loaded with fluo-4AM were imaged with fluorescent microscopy and challenged with neurogenic electrical field stimulation (EFS). All subtypes of ICC and smooth muscle cells (SMC) displayed spontaneous Ca(2+)-oscillations. EFS (0.5 Hz, 2 Hz, 10 Hz) evoked tetrodotoxin (1 µM)-sensitive Ca(2+)-transients in lamina propria ICC (ICC-LP), detrusor ICC and perivascular ICC (PICC) associated with mucosal microvessels. EFS responses in ICC-LP were significantly reduced by atropine or suramin. SMC and vascular SMC (VSM) also responded to EFS. Spontaneous Ca(2+)-oscillations in individual ICC-LP within networks occurred asynchronously whereas EFS evoked coordinated Ca(2+)-transients in all ICC-LP within a field of view. Non-correlated Ca(2+)-oscillations in detrusor ICC and adjacent SMC pre-EFS, contrasted with simultaneous neurogenic Ca(2+) transients evoked by EFS. Spontaneous Ca(2+)-oscillations in PICC were little affected by EFS, whereas large Ca(2+)-transients were evoked in pre-EFS quiescent PICC. EFS also increased the frequency of VSM Ca(2+)-oscillations. In conclusion, ICC-LP, detrusor ICC and PICC are functionally innervated. Interestingly, Ca(2+)-activity within ICC-LP networks and between detrusor ICC and their adjacent SMC were synchronous under neural control. VSM and PICC Ca(2+)-activity was regulated by bladder nerves. These novel findings demonstrate functional neural control of bladder ICC. Similar studies should now be carried out on neurogenic bladder to elucidate the contribution of impaired nerve-ICC communication to bladder pathophysiology.

Acetylcholine, and to a lesser extent ATP, mediates neurogenic contractions of bladder smooth muscle. Recently, the urothelium and lamina propria have also been shown to have contractile properties, but the neurotransmitters involved in mediating responses to nerve stimulation have not been investigated. Isolated strips of porcine urothelium with lamina propria were electrically field stimulated and contractions recorded. Drugs interfering with neurotransmission were then employed to identify which neurotransmitters mediated responses. Strips of urothelium/lamina propria developed spontaneous contractions with a frequency of 3.5±0.1 cycles min⁻¹ and amplitude of 0.84±0.06 g. Electrical field stimulation at 5, 10, and 20 Hz resulted in frequency-related contractions (1.13±0.36 g, 1.59±0.46 g and 2.20±0.53 g, respectively, n=13), and these were reduced in the presence of tetrodotoxin (1 μm) by 77±20% at 5 Hz, 79±7% at 10 Hz and 74±12% at 20 Hz (all P<0.01), indicating they were predominantly neurogenic in nature. Neither the muscarinic antagonist atropine (10 μm), the adrenergic neurone blocker guanethidine (10 μm) nor desensitization of the purinergic receptors with α,β-methylene ATP (10 μm) affected the contractile amplitude. Similarly, responses were not affected by the nitric oxide synthase inhibitor L-NNA (100 μm) or drugs that interfere with peptide neurotransmission (capsaicin, NK2 antagonist GR159897, protease inhibitors). In conclusion, electrical depolarization of the nerves present in the porcine urothelium/lamina propria results in frequency-dependent contractions, which are predominantly neurogenic in nature. These contractions are resistant to drugs that inhibit the adrenergic, cholinergic and purinergic systems. The neurotransmitter involved in the responses of this tissue is therefore unknown but does not appear to be a peptide., (© 2012 Blackwell Publishing Ltd.)

Unlabelled: Vestibulodynia, the most common type of chronic vulvovaginal pain, impairs the psychological, physical health of nearly 10% of women at some point in their lifetime. The aim of this investigation was to establish reliable standardized methodologies for assessment of pain sensitivity in vulvar mucosa and pelvic musculature. We enrolled 34 women with vestibulodynia and 21 pain-free controls. The participants underwent a nuanced exam that consisted of palpation of precisely located vulvar mucosal and pelvic muscle sites. These measurements remained highly stable when participants were reexamined after 2 weeks, with high within-examiner correlation. Vestibulodynia patients reported greater sensitivity than pain-free controls at the majority of examination sites, particularly at mucosal sites on the lower vestibule. The pain threshold measures at the lower mucosal sites were also associated with the participants' self-reported pain levels during intercourse. These mucosal pain threshold measurements were used to discriminate between vestibulodynia cases and controls with high sensitivity and specificity. This data supports the feasibility of contemporaneous assessment of vulvar mucosa and underlying musculature in the pelvic region, offering the hope of a more precise case definition for vestibulodynia and related disorders., Perspective: This study describes performance characteristics of novel methodologies for assessing pelvic muscle and mucosal sensitivity. These pain sensitivity measures were reproducible and associated with subjective pain reports and vestibulodynia case status and represent an important step toward a more precise case definition for vestibulodynia and related disorders., (Copyright © 2012 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Changes in the distribution of interstitial cells (IC) are reportedly associated with dysfunctional bladder. This study investigated whether spinal cord injury (SCI) resulted in changes to IC subpopulations (vimentin-positive with the ultrastructural profile of IC), smooth muscle and nerves within the bladder wall and correlated cellular remodelling with functional properties. Bladders from SCI (T8/9 transection) and sham-operated rats 5 weeks post-injury were used for ex vivo pressure-volume experiments or processed for morphological analysis with transmission electron microscopy (TEM) and light/confocal microscopy. Pressure-volume relationships revealed low-pressure, hypercompliance in SCI bladders indicative of decompensation. Extensive networks of vimentin-positive IC were typical in sham lamina propria and detrusor but were markedly reduced post-SCI; semi-quantitative analysis showed significant reduction. Nerves labelled with anti-neurofilament and anti-vAChT were notably decreased post-SCI. TEM revealed lamina propria IC and detrusor IC which formed close synaptic-like contacts with vesicle-containing nerve varicosities in shams. Lamina propria and detrusor IC were ultrastructurally damaged post-SCI with retracted/lost cell processes and were adjacent to areas of cellular debris and neuronal degradation. Smooth muscle hypertrophy was common to SCI tissues. In conclusion, IC populations in bladder wall were decreased 5 weeks post-SCI, accompanied with reduced innervation, smooth muscle hypertrophy and increased compliance. These novel findings indicate that bladder wall remodelling post-SCI affects the integrity of interactions between smooth muscle, nerves and IC, with compromised IC populations. Correlation between IC reduction and a hypercompliant phenotype suggests that disruption to bladder IC contribute to pathophysiological processes underpinning the dysfunctional SCI bladder., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Perineural involvement is a well-recognized clinicopathologic entity found in head and neck (H&N) cancers, including mucosal epithelial carcinomas and salivary gland malignancies. Perineural disease remains a diagnostic, prognostic and therapeutic challenge for the multidisciplinary H&N oncology team. Nerves are important routes of tumor spread in H&N malignancies, yet the biology and prognostic implications of perineural tumor growth are not fully understood. On balance, the available evidence suggests that it is associated with an increased risk of locoregional recurrence but the impact on survival remains uncertain. Perineural involvement has implications for locoregional disease diagnosis and management. MRI is the best imaging modality to detect tumor extent. Advanced radiotherapy technologies such as intensity-modulated radiation therapy and image-guided radiation therapy have the potential for more accurate targeting and treatment of anatomically complex patterns of disease spread. This review is limited to nondermatologic H&N cancers.

The purpose of this study was to explore the innervation of neuropeptide Y (NPY) in the anterior vaginal mucosa of menopausal patients suffering from pelvic organ prolapse (POP). To do this, we analyzed the distribution and expression of NPY and its correlation with the occurrence and development of POP. Changes in NPY abundance in the anterior vaginal mucosa were assessed by immunohistochemistry in tissue samples collected from 41 POP patients and 9 control subjects. We divided patients into 4 populations, designated POP IV-POP I, exhibiting decreasing levels of NPY innervation. Through multivariate regression analysis, the level of reduction in NPY innervation was shown to be associated with an increasing severity of POP disease in a statistically significant manner. In conclusion, our data reveal that as the symptoms of POP intensify, the expression of NPY in anterior vaginal mucosa decreases progressively. Menopause, in combination with stress urinary incontinence (SUI), results in a stronger effect on both nerve damage to the pelvic floor and NPY alteration, compared to other risk factors, such as parity and weight.

Unlabelled: With 30 figures, 3 histograms and 3 tables, Summary: The vomeronasal organ (VNO) is a chemosensory organ that detects environmental pheromones. The morphology of the 'non-sensory' epithelium (NSE) of the VNO and its lamina propria, as well as how it relates to ageing has received little attention. Histological, histochemical, morphometric and ultrastructural techniques were used to study the morphological structure of the rat NSE in five adult (3 months old) and five aged (2-2.5 years old) male albino rats. In adult rats, the NSE contained dark and light columnar cells with predominance of the latter. The surface of the epithelial cells was covered with microvilli and/or cilia. The lamina propria contained serous vomeronasal glands (VNGs), smooth muscles with numerous variable-sized mitochondria, vessels including lymphatic capillaries and nerve bundles. The following changes were detected in aged rats. The NSE exhibited an increase in number of dark columnar cells. Some cells revealed a prominent cell coat, dense aggregation of filaments in the luminal cytoplasm and appearance of multinucleated cells. Their surface revealed malformed configuration. Large mitochondria (2 μm), formed by fusion, were frequently observed in the smooth muscle cells of the lamina propria. Lipid droplets were frequently detected both in the VNGs acini and in the lymphatic endothelium. Ageing affected both the cells of the tissues and the extracellular matrix., (© 2011 Blackwell Verlag GmbH.)

Objectives: To investigate how hyaluronic acid (HA) affects nerve growth factor (NGF) production and bladder overactivity in a cyclophosphamide (CYP)-induced cystitis rat model., Methods: Female Sprague-Dawley rats received three intermittent intraperitoneal injections of CYP (75 mg/kg) or saline. Before or after CYP injection, HA was given intravesically and urine NGF was checked with creatinine correction. Bladder function was evaluated by cystometrograms under Zoletil anesthesia. Furthermore, the effect of HA was counteracted with hyaluronidase (HYAL). Bladder structural change was compared among groups with trichrome stain., Results: The intercontraction interval (ICI) significantly decreased in CYP-injected rats in comparison to the saline-injected controls. In the CYP-injected groups, bladder HA instillation significantly increased the ICI, but did not change the maximum voiding pressure in comparison to the saline instillation. NGF production significantly increased in CYP-injected rats, but decreased significantly with HA treatment. Treatment with HA had a more significant effect on urine NGF and the use of HYAL would eliminate this effect. Specific staining showed mucosa swelling after CYP treatment. Little HA coating on bladder mucosa could be found in HA-treated rats., Conclusions: Present findings raise the possibility that HA could be an effective treatment for CYP-related bladder overactivity through the involvement of NGF signaling., (© 2011 The Japanese Urological Association.)

Objective: To investigate re-innervation in the neovaginal mucosa of patients underwent sigmoid colon vaginoplasty in treatment of Mayer-Rokitansky-Kistner-Hauser Syndrome (MRKHS)., Methods: Biopsies in the upper third of the posterior neovagina were taken in 20 patients treated by sigmoid colon vaginoplasty at 1, 2 and 3 years after surgery, respectively. Protein gene product 9.5 (PGP 9.5), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) were detected by immunohistochemical method and compared with those in intact sigmoid colon mucosa., Results: (1) Density of nerve fiber: abundant distribution of PGP 9.5 nerve fibers were observed in the mucosal muscle layer, submucosa, and smooth muscle layer of the neovagina. The nerve fibers of VIP and NPY immunoreactivity were mainly distributed around blood vessels and in the smooth muscles. In the neovagina, the density of nerve fibers of PGP 9.5 of 17 ± 6 were much more than VIP of 2.9 ± 1.0 and NPY of 2.5 ± 0.8 significantly (P < 0.05). (2) Expression of PGP 9.5 in neovagina: at 1 year after surgery, PGP 9.5 positive expression of 14 ± 4 was significantly lower in the neovagina than 28 ± 7 in the intact sigmoid colon (P < 0.05). However, after 2 to 3 years, its expression displayed an upgrade tendency in the neovagina and was significantly higher at the 3 year postoperatively than that at the 1 years postoperatively (22 ± 7 vs. 14 ± 4, P < 0.05). The changes were much more obvious in submucosa. (3) The expression of VIP and NPY in neovagina: at 1 year after surgery, VIP and NPY positive nerve fibers were also decreased in the neovagina when compared with those in the intact sigmoid colon (2.3 ± 0.7 vs. 5.3 ± 1.4, P < 0.05; 2.5 ± 1.1 vs. 5.5 ± 1.1, P < 0.05). At 2 to 3 years after surgery, the positive VIP fiber showed initially decreased and subsequently increased tendency. The density of VIP of 3.7 ± 0.7 in the neovagina at 3 years postoperatively was higher than 2.3 ± 0.7 at 1 years postoperatively (P < 0.05). No significant up-regulation was observed in NPY-positive expression in the neovagina within 3 years after operation., Conclusions: Distribution of sensory PGP 9.5, VIP and NPY immunoreactive nerve fibers was similar to the pattern observed within the intact sigmoid colon wall. The number of nerve fibers in the neovagina decreased after surgery and then increased subsequently within 3 years after surgery.

Aims: To better understand the anatomy of the region of the male membranous urethra in order to preserve continence during radical cystectomy and prostatectomy., Methods: Cadaveric dissections of 15 male specimens were undertaken to investigate the nerves to membranous urethra. The nerves were traced from both an intrapelvic approach and a perineal approach. The origin, course, and distribution of the branches to the membranous urethra region were investigated in detail., Results: The membranous urethra is innervated by branches of inferior hypogastric plexus (IHP) and intrapelvic and extrapelvic branches of pudendal nerve (PN). The pelvic nerve from IHP originated from the caudal most root of the pelvic splanchnic nerve, running along the surface of the levator ani muscle (LAM) to enter the membranous urethra at the 5 and 7 O'clock positions. In 40% of specimens we found that the intrapelvic branches were supplied by the PN. Before exiting the pudendal canal, PN gives off an intrapelvic branch that traverses the LAM to course with the pelvic nerve and innervate the membranous urethra; the distance between these intrapelvic branches and prostatic apex is 5.3 +/- 1.8 mm. The branches originating from the dorsal nerve of penis innervate the membranous urethra in 53.3% of specimens; these nerve branches are located 4.2 +/- 1.1 mm from the prostatic apex., Conclusions: Dissection of the seminal vesicles and the prostatic apex during radical cystectomy and prostatectomy likely injures the nerve responsible for continence., ((c) 2009 Wiley-Liss, Inc.)

Bradykinin acts as an inflammatory mediator in the gut. In the present study we characterized bradykinin-induced changes in the intracellular calcium concentration ([Ca(2+)](i)) in whole-mount submucosal preparations from rat distal colon and examined the bradykinin receptors and subsequent signalling cascades involved. Bradykinin (2.10(-10)-2.10(-7)mol/l) evoked a concentration-dependent increase in [Ca(2+)](i) in about 90% of the investigated neurones. This Ca(2+) response was abolished by the bradykinin B(2) receptor antagonist HOE 140. The B(2) receptor agonist [Hyp(3)]-bradykinin mimicked the kinin response. In contrast, the B(1) receptor antagonist [des-Arg(10)]-HOE 140 and the B(1) receptor agonist bradykinin fragment 1-8 were ineffective. Immunohistochemical experiments confirmed the presence of bradykinin B(2) receptors in submucosal neurones. The effect of bradykinin on [Ca(2+)](i) was not mediated by a release of prostaglandins, as it was resistant against the cyclooxygenase inhibitor indomethacin. Blocking of G(q/11) proteins with YM-254890 suppressed the action of bradykinin, revealing that neuronal bradykinin B(2) receptors are coupled to this G protein. However, the subsequent signalling cascade differed from the classical phospholipase C signalling pathway, as the bradykinin response was resistant against the phospholipase C inhibitor U-73221, the ryanodine receptor antagonist dehydroryanodine, and only marginally sensitive against the blocker of IP(3)-receptors xestospongin C. Vice versa, the effect of bradykinin was nearly completely dependent on the presence of external Ca(2+) and could be reduced by lanthanum, a blocker of voltage-operated Ca(2+) channels, suggesting that the bradykinin-induced Ca(2+) response is achieved by an influx from the extracellular space via voltage-operated Ca(2+) channels., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Organ failure secondary to fibrosis is the main cause of morbidity and death in patients with systemic sclerosis. Gastrointestinal tract dysmotility is a major visceral manifestation, clinically ranging from an asymptomatic form to severe paresis. Although the oesophagus is the most frequently affected part of the gastrointestinal tract, all other segments can be involved. The present study was undertaken to evaluate the histopathological changes of the gastric wall in a series of full-thickness biopsies from systemic sclerosis patients who underwent gastric surgery due to severe gastroesophageal involvement. Gastric biopsies were processed for light microscopy and transmission electron microscopy. The histological and ultrastructural observations revealed a generalized fibrosis affecting all the gastric wall layers. The most severe changes were observed in the muscularis mucosae and muscle layers. Wide areas of marked focal fibrosis with dense collagen bundles and elastic fibre deposition surrounding smooth muscle cells were found. Myofilaments and thickened dense bodies were severely disarranged or absent in most smooth muscle cells. Nerve fibres showed ultrastructural alterations, such as oedematous axoplasm and scarce cytoskeletal elements. Abundant elastic and collagen fibres enveloped nerve fibres, nerve endings and interstitial cells of Cajal, thereby separating them from smooth muscle cells and blood microvessels. This study provides evidence for a prominent fibrosis and severe ultrastructural alterations of smooth muscle cells and nerve fibres as the main histopathological hallmarks in the gastric wall of systemic sclerosis patients.

Since the discovery of two cyclooxygenase isoforms (COX-1, COX-2), efforts have been made to characterize the roles played by these enzymes in the regulation of physiological functions, as well as to explore their involvement in the pathophysiology of inflammatory disorders. In the digestive tract, the majority of evidence has been obtained at mucosal level, where both isoforms regulate various functions, and contribute to the development of inflammatory and neoplastic disorders. The role of COX isoforms in the gut neuromuscular compartment, where their expression has been detected in different species, is still unclear. However, the characterization of actions exerted by COX-derived prostanoids on gut motility has been under investigation for many years, and it is becoming increasingly appreciated that these mediators subserve complex regulatory patterns of COX on digestive motility. More recently, several studies have strengthened the concept that both COX-1 and COX-2 are involved in the modulation of gastrointestinal neuromuscular activity under normal conditions, and that changes in their regulatory activities occur in the presence of various digestive disorders, including inflammatory bowel diseases and postoperative ileus. Despite a large body of preclinical evidence, studies aimed at translating these findings into clinically relevant applications are needed, in an attempt to identify novel therapeutic approaches for treatment of gut disorders associated with motility alterations. This review illustrates and discusses current knowledge of the roles played by COX pathways in the regulation of gastrointestinal neuromuscular functions, both under normal conditions and in the presence of gut disorders.

To test whether transient receptor potential channel vanilloid subfamily member-1 (TRPV1) mediates acid-induced inflammation in the esophagus, a tubular segment of esophageal mucosa was tied at both ends, forming a sac. The sac was filled with 0.01 N HCl (or Krebs buffer for control) and kept in oxygenated Krebs buffer at 37 degrees C. The medium around the sac (supernatant) was collected after 3 h. Supernatant of the HCl-filled sac abolished contraction of esophageal circular muscle strips in response to electric field stimulation. Contraction was similarly abolished by supernatant of mucosal sac filled with the TRPV1 agonist capsaicin (10(-6) M). These effects were reversed by the selective TRPV1 antagonist 5'-iodoresiniferatoxin (IRTX) and by the platelet-activating factor (PAF) receptor antagonist CV9388. Substance P and CGRP levels in mucosa and in supernatant increased in response to HCl, and these increases were abolished by IRTX and by tetrodotoxin (TTX) but not affected by CV9388, indicating that substance P and CGRP are neurally released and PAF independent. In contrast, the increase in PAF was blocked by IRTX but not by TTX. Presence of TRPV1 receptor was confirmed by RT-PCR and by Western blot analysis in whole mucosa and in esophageal epithelial cells enzymatically isolated and sorted by flow cytometry or immunoprecipitated with cytokeratin antibodies. In epithelial cells PAF increased in response to HCl, and the increase was abolished by IRTX. We conclude that HCl-induced activation of TRPV1 receptors in esophageal mucosa causes release of substance P and CGRP from neurons and release of PAF from epithelial cells.

The aim of this study is to elucidate the innervation of human vagina by performing a microdissection and PGP immunohistochemistry study. Seven fresh Korean cadavers were used. In five cadavers, the site at which the nerve entered the vaginal wall was observed by microdissection. Two vaginas were stained with trichrome and protein gene product 9.5 (PGP) immunostain. Terminal nerve branches in the vaginal wall were most dense at the second 1/5 partition from the inferior anterior wall (1.49 +/- 0.51/cm(2)). Its density started decreasing (0.57 +/- 0.09/cm(2) -0.89 +/- 0.46/cm(2)) and became scarce at the fourth and fifth 1/5 partitions from the bottom (0.37 +/- 0.14/cm(2)/3-0/cm(2)). The mucosa of the anterior vaginal wall at the distal 1/3 point (designated 1) was 3.50 +/- 2.06 mm in thickness and was 1.18 +/- 0.20 mm at the proximal point (designated 2). It was 1.57 +/- 0.78 mm at the distal 1/3 point (designated 3) of the lateral wall and 1.43 +/- 0.39 mm at the proximal 1/3 point (designated 4) of the posterior wall. Point 1 was thicker than 2, 3, and 4. The vaginal muscle of 1 (5.59 +/- 2.74 mm) was thicker than 2 (3.19 +/- 0.62 mm), 3 (3.31 +/- 1.31 mm), and 4 (2.98 +/- 0.33 mm). Small nerve fibers were most dense at 1 (6.5 +/- 1.1/mm(2)) and scarce at 4 (3.8 +/- 1.5/mm(2)). Large nerve fibers were most dense at 1 (1.9 +/- 0.9/mm(2)) and scarce at 2 (0.7 +/- 0.7/mm(2)) and 4 (0.7 +/- 1.0/mm(2)). The density of small nerve fibers is 2 approximately 2.9/mm(2) in the muscle and 0.7 approximately 1.5/mm(2) in large nerve fibers. The second 1/5 partition of the distal anterior wall had significantly richer innervation than the surrounding areas. Therefore, we think this 1/5 partition of the distal anterior wall could be so called "G-spot."

We examined the modulation of intrinsic (i.e., spontaneous) detrusor contractions by the urothelium and the lamina propria through optical mapping approaches. Normal adult and spinal cord-transected (SCT) rat bladders were stained with Ca2+- and voltage-sensitive dyes, and optical activity generated from intrinsic contractions was mapped from the mucosal surface of whole bladder sheets. Both normal adult and SCT rat bladders displayed intrinsic contractions, where normal bladders showed low-amplitude, high-frequency contractions with disorganized patterns of activity. In contrast, in the SCT animals there were high-amplitude, low-frequency contractions that displayed an organized spread of membrane potential and intracellular Ca2+. The difference in contractile activity was mirrored in the Ca2+ and membrane potential maps of bladder sheets. Normal bladders showed multiple initiation sites across the mucosal surface, whereas SCT bladders showed only one or two fixed initiation sites localized to the dome. The magnitude of intrinsic contractions could be enhanced by stretch or low-dose arecaidine (50 nM), a muscarinic-specific agonist. Partial removal of the mucosa decreased the amplitude of the intrinsic contractions and decreased the response to stretch or arecaidine. Optical mapping of mucosa-denuded sheets, where enhanced spontaneous activity was abolished, or application of 1 microM nifedipine to remove smooth muscle signals, but not the mucosal signals, shows that intrinsic activity in pathological bladders is driven by the mucosal layer. In summary, we suggest an urotheliogenic origin for intrinsic activity, where structures within the mucosal layer organize and thereby enhance intrinsic detrusor contractions.

Purpose: We developed and tested the efficacy of an implantable bladder device which, when combined with the Neurometer, can be used to assess fiber specific afferent bladder sensation in the rat., Materials and Methods: We developed an implantable bladder device that applies selective nerve fiber stimuli (250 Hz for small myelinated Adelta fibers and 5 Hz for unmyelinated C fibers) to the bladder mucosa in the rat to determine bladder sensory perception threshold values. We performed 3 experiments in 55 female Sprague-Dawley rats to examine the effects of our device on voiding habits, assess the interobserver reliability of the sensory perception threshold and determine the effects of intravesical administration of resiniferatoxin (Sigma) and lidocaine on the sensory perception threshold., Results: Sensory perception threshold values obtained by 2 blinded, independent observers were not different from each other (p = 0.41). Sensory perception threshold values obtained at the 2 stimulation frequencies remained constant for at least 3 weeks after device implantation. A significant increase in sensory perception threshold values after resiniferatoxin instillation was noted at a stimulus frequency of 5 Hz (p = 0.02), whereas intravesical lidocaine led to an immediate increase in the sensory perception threshold at 250 and 5 Hz. Device implantation led to an early decreased voided volume and increased frequency of voids, although these parameters returned to normal after 4 days., Conclusions: Assessment of bladder afferent sensation with our newly developed device is feasible in rats. It provides sensory perception thresholds that appear to be fiber-type selective for autonomic bladder afferent nerves.

Sensory neurons represent an attractive target for pharmacological treatment of various bladder disorders. However the properties of major classes of mechano-sensory neurons projecting to the bladder have not been systematically established. An in vitro bladder preparation was used to examine the effects of a range of mechanical stimuli (stretch, von Frey hair stroking and focal compression of receptive fields) and chemical stimuli (1 mm alpha,beta-methylene ATP, hypertonic solutions (500 mm NaCl) and 3 microm capsaicin) during electrophysiological recordings from guinea pig bladder afferents. Four functionally distinct populations of bladder sensory neurons were distinguished by these stimuli. The first class, muscle mechanoreceptors, were activated by stretch but not by mucosal stroking with light (0.05-0.1 mN) von Frey hairs or by hypertonic saline, alpha,beta-methylene ATP or capsaicin. Removal of the urothelium did not affect their stretch-induced firing. The second class, muscle-mucosal mechanoreceptors, were activated by both stretch and mucosal stroking with light von Frey hairs or by hypertonic saline and by alpha,beta-methylene ATP, but not by capsaicin. Removal of the urothelium reduced their stretch- and stroking-induced firing. The third class, mucosal high-responding mechanoreceptors, were stretch-insensitive but could be activated by mucosal stroking with light von Frey hairs or by hypertonic saline, alpha,beta-methylene ATP and capsaicin. Stroking-induced firing was significantly reduced by removal of the urothelium. The fourth class, mucosal low-responding mechanoreceptors, were stretch insensitive but could be weakly activated by mucosal stroking with light von Frey hairs but not by hypertonic saline, alpha,beta-methylene ATP or capsaicin. Removal of the urothelium reduced mucosal stroking-induced firing. All four populations of afferents conducted in the C-fibre range and showed class-dependent differences in spike amplitude and duration. At least four functional classes of bladder mechanoreceptors can be readily distinguished by different mechanisms of activation and are likely to transmit different types of information to the central nervous system.

Emerging evidence indicates a nociceptive role of vagal afferents. A distinct oesophageal innervation in the rat, with muscular and mucosal afferents travelling predominantly in the recurrent (RLN) and superior laryngeal nerve (SLN), respectively, enabled characterization of mucosal afferents with nociceptive properties, using novel isolated oesophagus-nerve preparations. SLN and RLN single-fibre recordings identified 55 and 14 units, respectively, with none conducting faster than 8.7 m s(-1). Mucosal response characteristics in the SLN distinguished mechanosensors (n = 13), mechanosensors with heat sensitivity (18) from those with cold sensitivity (19) and a mechanoinsensitive group (5). The mechanosensitive fibres, all slowly adapting, showed a unimodal distribution of mechanical thresholds (1.4-128 mN, peak approximately 5.7 mN). No difference in response characteristics of C and Adelta fibres was encountered. Mucosal proton stimulation (pH 5.4 for 3 min), mimicking gastro-oesophageal reflux disease (GORD), revealed in 31% of units a desensitizing response that peaked around 20 s and faded within 60 s. Cold stimulation (15 degrees C) was proportionally encoded but the response showed slow adaptation. In contrast, the noxious heat (48 degrees C) response showed no obvious adaptation with discharge rates reflecting the temperature's time course. Polymodal (69%) mucosal units, > 30% proton sensitive, were found in each fibre category and were considered nociceptors; they are tentatively attributed to vagal nerve endings type I, IV and V, previously morphologically described. All receptive fields were mapped and the distribution indicates that the posterior upper oesophagus may serve as a 'cutbank', detecting noxious matters, ingested or regurgitated, and triggering nocifensive reflexes such as bronchoconstriction in GORD.

Recent reports have proposed that the urothelium can sense mechanical stretch and communicate this information to sensory afferent neurons by the release of ATP into the vicinity of P2X-containing neurons. This report investigates the bidirectional release of ATP by in vitro rabbit urothelium. ATP was measured using the luciferin-luciferase assay. Immediately after washing of both sides of the epithelium, there was a linear increase in ATP content in the mucosal compartment with a rate of 23 +/- 6.5 fmol x min(-1) x cm(-2) (n = 18). Serosal ATP content increased as a saturating exponential function, suggesting a constant rate of release and degradation of ATP by ectonucleotidases/exonucleotidases. The presence of a serosal ectonucleotidase/exonucleotidases was demonstrated by the time-dependent decrease in exogenously added ATP. The maximum rate of hydrolysis was 11 pmol x min(-1) x cm(-2) with a K(m) of 0.49 microM. The time course of serosal ATP release was modeled as a constant rate of release (d: mol x min(-1) x cm(-2)) and rate constant of hydrolysis (k(h): min(-)). In control conditions d was 18 fmol x min(-1) x cm(-2) and k(h) of 0.056 +/- 0.01 min(-) (n = 18). Steady-state serosal chamber content is 370 +/- 90 fmol/cm(2), and concentration is 50 +/- 1.2 x 10(-12) M. Stretching the tissue resulted in a transient fivefold increase in the rate of mucosal ATP release and a transient sixfold increase in serosal ATP release. Half-osmotic strength solutions increased mucosal release by 10-fold and serosal release by 5-fold. Tissue damage resulted in a step-increase in mucosal chamber ATP content by 6.6 +/- 1 pmol/cm(2) and serosal chamber ATP by 0.1 +/- 0.06 pmol/cm(2) (n = 5).

Background/aim: Non-erosive reflux disease (NERD) is a common and heterogeneous disorder. We hypothesized that changes in peripheral innervation may lead to hyperalgesia and contribute to the development of the disorder., Methods: Patients referred for evaluation of reflux symptoms with wireless pH monitoring were asked to provide demographic and clinical data and complete a survey related to severity of reflux symptoms. Endoscopies were performed to rule out macroscopic abnormalities of the esophageal mucosa. Biopsies obtained 2 cm above the gastroesophageal junction were stained for protein gene product 9.5 (PGP 9.5; general neuronal marker) and TRPV1 (capsaicin receptor) immunoreactivity. The density of immunoreactive fibers in the esophageal mucosa was determined morphometrically., Results: A total of 39 patients without evidence of Barrett's metaplasia, erosive or ulcerative esophagitis were enrolled. Most patients had daily symptoms. The total esophageal acid exposure time was 5.6+/-0.6%, with 16 patients (41%) having increased acid reflux. Immunoreactivity for PGP 9.5 or TRPV1 was detected in papillary structures as well as within the epithelium (free intra-epithelial endings). Total acid-exposure time, but not symptom score or duration correlated significantly with density of PGP 9.5 immunoreactivity and TRPV1 positive fibers., Conclusion: Even in the absence of macroscopic injury, esophageal acid exposure is associated with changes in mucosal innervation of the esophagus, thus potentially further enhancing symptoms in patients with gastroesophageal reflux.

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Background: Gastro-oesophageal reflux disease (GORD) patients commonly describe symptoms of heartburn and chest pain. The capsaicin receptor vanilloid receptor 1 (TRPV1) (VR1) is a cation channel expressed by sensory neurones and activated by heat, acid pH and ethanol, which may trigger burning pain., Aim: To study the distribution of TRPV1-expressing nerve fibres in oesophageal mucosal biopsies from patients with symptomatic oesophagitis and in control subjects., Methods: Biopsies were taken at gastroscopy from the distal oesophagus of seven symptomatic oesophagitis patients and seven asymptomatic patients undergoing investigation for iron-deficiency anaemia. These biopsies were studied by immunohistochemistry using affinity-purified antibodies to TRPV1 and to the neuronal marker peripherin. The density of oesophageal epithelial TRPV1 innervation was assessed by calculating the proportion of papillae in each oesophageal epithelium biopsy specimen containing TRPV1-immunoreactive fibres., Results: TRPV1-immunoreactive nerves were distributed within the lamina propria in healthy subjects and in oesophagitis patients. The percentage of papillae positive for TRPV1 was elevated in oesophagitis patients compared with controls. Peripherin fibre density was not significantly different between the groups., Conclusions: TRPV1-immunoreactive sensory nerve fibres are expressed in human oesophageal mucosa both in health and in disease. Increased TRPV1 expression in the inflamed oesophagus may mediate the heartburn in oesophagitis, and TRPV1 blockers may provide novel treatment.

Objective: The aim of our study was to investigate the histological changes of vaginal mucosa innervation in stress urinary incontinence (SUI), to determine the alteration that contributed to them., Methods: Biopsy specimens of anterior vaginal wall were obtained from 56 subjects during operation, which belonged to one of the 3 groups: SUI (15 cases), POP (23 cases) and asymptomatic control (15 cases). Using immunohistochemical method we could semiquantitatively determine the expression of neurofilament (NF) in vaginal mucosa as the distribution of the nerve terminals., Results: There were brown-stained nerve fibers in the lamina propria. According to the expression staging (-, +, ++, +++), the number of patients was 6, 5, 4, 0 cases in SUI group, 17, 5, 1, 0 cases in POP group, and 3, 2, 4, 6 cases in control group, respectively. There was significant difference between SUI group and control group in the expression of NF (P < 0.05) and the expression of nerve fiber had no correlation with the clinical severity and leak point pressure (LPP) in SUI group (P > 0.05). The expression of NF in POP group was also weaker than control., Conclusion: The SUI group had a significant less total innervation of vaginal mucosa than asymptomatic controls, which may contribute to the pathogenesis of SUI.

Objective: To evaluate human bladder mucosal sensory function by neuroselective Current Perception Threshold (CPT) measures from healthy and neuropathic bladders., Methods: Eight healthy volunteers and 38 patients with urinary symptoms underwent conventional urodynamic tests including water-filling cystometry and ice water test. Standardized neuroselective CPT measures were obtained from the left index finger and the mucosa of the posterior bladder wall. Three different CPTs were obtained from each test site using a constant alternating current sinusoid waveform electrical stimulus presented at 2000Hz, 250Hz and 5Hz stimulation frequencies, which could selectively reflect the functions of the large myelinated fibers (A-beta-fiber), the small myelinated fibers (A-delta-fiber), and the unmyelinated fibers (C-fiber), respectively., Results: As the determination of CPT values on the finger skin, the CPT values in the bladder could be determined using the neuroselective measures in all patients but three who had no sensory response (absence of sensation) caused by complete spinal injury. In the 8 patients with detrusor hyperreflexia due to incomplete spinal cord injury (supra-sacral lesion), the bladder CPT value (4.0+/-1.9) at 5Hz was significantly lower (p<0.01) than that in the controls (26.2+/-17.7). In the neurogenic bladders determined to be underactive (n=11, including post pelvic surgery, post infra-sacral level spinal cord injury and diabetes patients), the higher CPT values of bladder mucosal sensory functions were found at 5Hz (p<0.05), 250Hz (p=0.07), and 2000Hz (p<0.05) compared to the controls., Conclusions: Quantitative neuroselective measurement of CPT values in the human bladder mucosal function was feasible. Hypersensitivity or hyposensitivity of the urinary sensory function could be determined using the CPT values in comparison to control. The quantitative neuroselective estimation of the bladder sensory functions in different types of sensory peripheral nerve fibers may contribute to the appropriate selection of therapeutic strategy in patients with urinary sensory dysfunction.