Mucosal Nerve Fibers Research Articles

Oxidative stress–mediated proapoptosis signaling: A novel theory on the mechanism underlying the pathogenesis of burning mouth syndrome

BackgroundBurning mouth syndrome (BMS) is a chronic oral pain disorder characterized by a generalized burning sensation in the oral mucosa without apparent medical or dental causes. Despite various hypotheses proposed to explain BMS pathogenesis, a clear understanding of the cellular-level events and associated histologic and molecular findings is lacking. Advancing our understanding of BMS pathogenesis could facilitate the development of more targeted therapeutic interventions. Types of Studies ReviewedThe authors conducted an extensive literature search and review of cellular mechanisms, focusing on evidence-based data that support a comprehensive hypothesis for BMS pathogenesis. The authors explored novel and detailed mechanisms that may account for the characteristic features of BMS. ResultsThe authors proposed that BMS symptoms arise from the uncontrolled activation of proapoptotic transmembrane calcium permeable channels expressed in intraoral mucosal nerve fibers. Elevated levels of reactive oxygen species or dysfunctional antiapoptosis pathways may lead to uncontrolled oxidative stress–mediated apoptosis signaling, resulting in upregulation of transmembrane transient receptor potential vanilloid type 1 and P2X 3 calcium channels in nociceptive fibers. Activation of these channels can cause nerve terminal depolarization, leading to generation of action potentials that are centrally interpreted as pain. Conclusions and Practical ImplicationsThe authors present a novel hypothesis for BMS pathogenesis, highlighting the role of proapoptotic transmembrane calcium permeable channels and oxidative stress–mediated apoptosis signaling in the development of BMS symptoms. Understanding these underlying mechanisms could provide new insights into the development of targeted therapeutic interventions for BMS. Additional research is warranted to validate this hypothesis and explore potential avenues for effective management of BMS.

Associations of Mucosal Nerve Fiber Innervation Density with Hirschsprung-Associated Enterocolitis: A Retrospective Three-Center Cohort Study.

Hirschsprung's disease (HSCR) is a congenital intestinal neurodevelopmental disorder characterized by the absence of enteric ganglion cells in the distal colon. Although Hirschsprung-associated enterocolitis (HAEC) is the most frequent life-threatening complication in HSCR, to date reliable biomarkers predicting the likelihood of HAEC are yet to be established. We established a three-center retrospective study including 104 HSCR patients surgically treated between 1998 and 2019. Patient-derived cryopreserved or paraffin-preserved colonic tissue at surgery was analyzed via βIII-tubulin immunohistochemistry. We subsequently determined extrinsic mucosal nerve fiber density in resected rectosigmoid specimens and classified HSCR patients accordingly into nerve fiber-high or fiber-low groups. We compared the distribution of clinical parameters obtained from medical records between the fiber-high (n = 36) and fiber-low (n = 68) patient groups. We assessed the association between fiber phenotype and enterocolitis using univariate and multivariate logistic regression adjusted for age at operation. Enterocolitis was more prevalent in patients with sparse mucosal nerve fiber innervation (fiber-low phenotype, 87%) compared with the fiber-high phenotype (13%; p = 0.002). In addition, patients developing enterocolitis had a younger age at surgery (3 vs. 7 months; p = 0.016). In the univariate analysis, the odds for enterocolitis development in the fiber-low phenotype was 5.26 (95% confidence interval [CI], 1.67-16.59; p = 0.005) and 4.01 (95% CI, 1.22-13.17; p = 0.022) when adjusted for age. Here, we showed that HSCR patients with a low mucosal nerve fiber innervation grade in the distal aganglionic colon have a higher risk of developing HAEC. Consequently, histopathologic analysis of the nerve fiber innervation grade could serve as a novel sensitive prognostic marker associated with the development of enterocolitis in HSCR patients.

5-HT7 receptor-dependent intestinal neurite outgrowth contributes to visceral hypersensitivity in irritable bowel syndrome

AbstractIrritable bowel syndrome (IBS) is characterized by visceral hypersensitivity (VH) associated with abnormal serotonin/5-hydroxytryptamine (5-HT) metabolism and neurotrophin-dependent mucosal neurite outgrowth. The underlying mechanisms of VH remain poorly understood. We investigated the role of 5-HT7 receptor in mucosal innervation and intestinal hyperalgesia. A high density of mucosal nerve fibres stained for 5-HT7 was observed in colonoscopic biopsy specimens from IBS patients compared with those from healthy controls. Staining of 5-HT3 and 5-HT4 receptors was observed mainly in colonic epithelia with comparable levels between IBS and controls. Visceromotor responses to colorectal distension were evaluated in two mouse models, one postinfectious with Giardia and subjected to water avoidance stress (GW) and the other postinflammatory with trinitrobenzene sulfonic acid-induced colitis (PT). Increased VH was associated with higher mucosal density of 5-HT7-expressing nerve fibres and elevated neurotrophin and neurotrophin receptor levels in the GW and PT mice. The increased VH was inhibited by intraperitoneal injection of SB-269970 (a selective 5-HT7 antagonist). Peroral multiple doses of CYY1005 (a novel 5-HT7 ligand) decreased VH and reduced mucosal density of 5-HT7-expressing nerve fibres in mouse colon. Human neuroblastoma SH-SY5Y cells incubated with bacteria-free mouse colonic supernatant, 5-HT, nerve growth factor, or brain-derived neurotrophic factor exhibited nerve fibre elongation, which was inhibited by 5-HT7 antagonists. Gene silencing of HTR7 also reduced the nerve fibre length. Activation of 5-HT7 upregulated NGF and BDNF gene expression, while stimulation with neurotrophins increased the levels of tryptophan hydroxylase 2 and 5-HT7 in neurons. A positive-feedback loop was observed between serotonin and neurotrophin pathways via 5-HT7 activation to aggravate fibre elongation, whereby 5-HT3 and 5-HT4 had no roles. In conclusion, 5-HT7-dependent mucosal neurite outgrowth contributed to VH. A novel 5-HT7 antagonist could be used as peroral analgesics for IBS-related pain.

Characterization of the Transition Zone in Short Segment Hirschsprung Disease Using Calretinin Immunostaining.

Introduction: The detailed expression pattern of calretinin immunohistochemistry in the transition zone (TZ) of Hirschsprung disease (HSCR) has not yet been reported. This study aims to examine the value of calretinin immunohistochemistry for more accurately determining the distal and proximal border of the TZ in short segment HSCR. Methods: Specimens of pull-through surgery from 51 patients with short form of HSCR were analyzed on two longitudinal strips using hematoxylin and eosin (H&E) staining and calretinin immunohistochemistry. Results: In all but two patients, the first appearance of calretinin expression was seen on mucosal nerve fibers before the appearance of any ganglion cells, indicating the distal border of the TZ. The maximum distance between the distal border of the TZ and the proximal border of the TZ, defined by ganglion cells in a normal density on H&E stained sections, a strong calretinin expression on mucosal nerve fibers and in >80% of submucosal and myenteric ganglion cells, with no nerve hypertrophy and absence of ganglionitis was 60mm. Conclusion: The distal border of the TZ is characterized by calretinin positive intramucosal neurites in nearly all of short form of HSCR and not by calretinin expression on ganglion cells.

Lack of Mucosal Cholinergic Innervation Is Associated With Increased Risk of Enterocolitis in Hirschsprung’s Disease

Background & AimsHirschsprung’s disease (HSCR) is a congenital intestinal motility disorder defined by the absence of enteric neuronal cells (ganglia) in the distal gut. The development of HSCR-associated enterocolitis remains a life-threatening complication. Absence of enteric ganglia implicates innervation of acetylcholine-secreting (cholinergic) nerve fibers. Cholinergic signals have been reported to control excessive inflammation, but the impact on HSCR-associated enterocolitis is unknown.MethodsWe enrolled 44 HSCR patients in a prospective multicenter study and grouped them according to their degree of colonic mucosal acetylcholinesterase-positive innervation into low-fiber and high-fiber patient groups. The fiber phenotype was correlated with the tissue cytokine profile as well as immune cell frequencies using Luminex analysis and fluorescence-activated cell sorting analysis of colonic tissue and immune cells. Using confocal immunofluorescence microscopy, macrophages were identified in close proximity to nerve fibers and characterized by RNA-seq analysis. Microbial dysbiosis was analyzed in colonic tissue using 16S-rDNA gene sequencing. Finally, the fiber phenotype was correlated with postoperative enterocolitis manifestation.ResultsThe presence of mucosal nerve fiber innervation correlated with reduced T-helper 17 cytokines and cell frequencies. In high-fiber tissue, macrophages co-localized with nerve fibers and expressed significantly less interleukin 23 than macrophages from low-fiber tissue. HSCR patients lacking mucosal nerve fibers showed microbial dysbiosis and had a higher incidence of postoperative enterocolitis.ConclusionsThe mucosal fiber phenotype might serve as a prognostic marker for enterocolitis development in HSCR patients and may offer an approach to personalized patient care and new therapeutic options.

Vasoactive Intestinal Polypeptide (VIP) in the Intestinal Mucosal Nerve Fibers in Dogs with Inflammatory Bowel Disease.

Simple SummaryCanine inflammatory bowel disease (IBD)—a group of gastrointestinal disorders—is a serious problem in veterinary medicine. The etiology of IBD remains unknown, and its diagnosis and effective treatment are difficult. One of the less-known aspects of IBD pathology is the influence of this disease on the enteric nervous system, which is located in the intestinal wall and regulates most of the gastrointestinal functions. Therefore, the aim of the present study was to evaluate the influence of IBD on the intramucosal nerve fibers containing vasoactive intestinal polypeptide (VIP). VIP is one of the most important substances produced by the enteric nervous structures that is involved in many regulatory processes in the gastrointestinal tract. The obtained results show that IBD induces changes in the density of intramucosal VIP-positive nerve fibers in the canine gastrointestinal tract. It suggests that VIP is involved in the pathological processes occurring during this disease. Observed changes may be a result of neuroprotective and/or adaptive processes regulated by VIP, aimed at the homeostasis maintenance in the inflamed gastrointestinal (GI) tract and induced by proinflammatory factors.Canine inflammatory bowel disease (IBD) is a group of enteropathies with nonspecific chronic symptoms and poorly understood etiology. Many aspects connected with IBD are not understood. One of them is the participation of the intestinal nervous system in the development of pathological processes. Thus, this study aimed to demonstrate changes in the density of intramucosal nerve fibers containing vasoactive intestinal polypeptide (VIP)—one of the most important intestinal nervous factors caused by the various stages of IBD development. Mucosal biopsy specimens collected from the duodenum, jejunum and descending colon of healthy dogs and dogs with varied severity of IBD were included in the experiment. The density of VIP-like immunoreactive (VIP-LI) nerves was determined by a single immunofluorescence technique and a semi-quantitative method consisting in VIP-LI fiber counts in the field of view (0.1 mm2). The obtained results indicate that IBD induces changes in the density of mucosal VIP-LI nerve fibers in the canine gastrointestinal tract. The initial decrease is followed by an increase in VIP-like immunoreactivity in successive stages of the disease. These observations show that VIP is a neuronal factor that participates in the pathological processes connected with canine IBD. The observed changes probably result from the neuroprotective and/or adaptive properties of VIP. Protective and adaptive reactions induced by inflammation aim to protect the GI tract against damage by proinflammatory factors and ensure the homeostasis in the enteric nervous system (ENS) under the conditions changed by the disease process.

Transition Zone in Total Colonic Aganglionosis and Colorectal Hirschsprung's Disease Shows a Similar Trend of Mucosal Innervation: Image Processing and Analysis Study.

The aganglionic segment of bowel in Hirschsprung's disease (HD) varies in length. It is not clear whether total colonic aganglionosis (TCA) merely represents a long form of HD or a different phenotype of the disease. Animal model studies suggest that TCA may have a longer transition zone (TZ) than conventional colorectal HD. We compared mucosal innervation of TZ in 2 TCA cases and 10 conventional colorectal HD cases by quantifying calretinin-positive mucosal nerve fibers using image processing and analysis. One TCA was associated with esophageal atresia-tracheoesophageal fistula, the other with trisomy 21. The gradients of calretinin-stained pixel count increase per distance from the beginning of TZ (slope) for TCA were not significantly different from those for the conventional HD group. Given this observation, it is speculated that the length of TZ in TCA may fall within the range of and may not be much longer than conventional colorectal HD.

Esophageal mucosal innervation in functional heartburn: Closer to healthy asymptomatic subjects than to non-erosive reflux disease patients.

Mucosal innervation in non-erosive reflux disease (NERD; pathological esophageal acid exposure, normal macroscopic mucosa) is clearly distinct from that of healthy volunteers (HV) and from patients with esophagitis or Barrett's esophagus: The nerves in NERD are situated much closer to the luminal surface of the mucosa. Patients with functional heartburn (FH) have a similar symptom profile to patients with NERD and indistinguishable macroscopic appearances. However, they have physiological acid exposure and no reflux-symptom association. The aim of our study was to delineate the position of esophageal mucosal nerve fibers in patients with FH and compare it with that in NERD and HV. Distal esophageal biopsies from patients with FH were immunohistochemically stained for CGRP. CGRP-positive nerve fibers were identified, and their position relative to the lumen was determined. These results were compared to our previously published cohort of HV and NERD. Eleven patients were included in the FH group with a mean age of 46years (range 33-69); 7F:4M. Nine patients had visible nerve fibers. The location of the afferent nerve fibers in the distal esophageal mucosa (median of 22, range 10.4-28) was similar to the HV group (median 25.5) and significantly deeper than the superficial nerves seen in NERD (median 9.5). The mucosal innervation pattern in FH is more alike that of healthy individuals than that of NERD, with afferent nerves lying deep in the mucosa, away from the luminal surface. This supports the theory that heartburn in FH has a distinct nociceptive pathophysiology.

Degranulated Eosinophils Contain More Fine Nerve Fibers in the Duodenal Mucosa of Patients With Functional Dyspepsia.

Background/AimsFunctional dyspepsia (FD) is characterized as chronic recurrent upper gastrointestinal symptoms in the absence of any organic disorder. We hypothesized that duodenal low-grade inflammation activates superficial afferent nerve sprouting, thereby contributing to hypersensitivity in patients with FD.MethodsA prospective case-control study was conducted in a tertiary referral center. FD was defined using the Rome III criteria. Standardized endoscopic biopsies were performed in the stomach and duodenum. Hematoxylin and eosin staining and immunohistochemical staining for major basic proteins were performed to detect granulated eosinophil-derived granules, and S-100 staining was performed to detect fine nerve fibers.ResultsA total of 51 patients with FD (82% female; mean age 35.8 ± 13.4 years) and 35 controls were enrolled. Activated eosinophil counts in the duodenum were significantly higher in patients with FD than in controls (41.4% vs 17.1%, P = 0.005). Microscopic duodenitis was more frequently detected in patients with FD than in controls. Fine nerve fibers were more abundant in patients with FD than in controls (45.1% vs 11.4%, P = 0.029). The abundance of fine nerve fibers highly correlated with the degree of activated eosinophils.ConclusionDuodenal low-grade inflammation, such as mucosal eosinophilic accumulation with degranulation, promoted mucosal enteric nerve fiber density and sprouting in patients with FD.

Increased expression of brain-derived neurotrophic factor is correlated with visceral hypersensitivity in patients with diarrhea-predominant irritable bowel syndrome.

BACKGROUNDVisceral hypersensitivity is considered to play a vital role in the pathogenesis of irritable bowel syndrome (IBS). Neurotrophins have drawn much attention in IBS recently. Brain-derived neurotrophic factor (BDNF) was found to mediate visceral hypersensitivity via facilitating sensory nerve growth in pre-clinical studies. We hypothesized that BDNF might play a role in the pathogenesis of diarrhea-predominant IBS (IBS-D).AIMTo investigate BDNF levels in IBS-D patients and its role in IBS-D pathophysiology.METHODSThirty-one IBS-D patients meeting the Rome IV diagnostic criteria and 20 age- and sex-matched healthy controls were recruited. Clinical and psychological assessments were first conducted using standardized questionnaires. Visceral sensitivity to rectal distension was tested using a high-resolution manometry system. Colonoscopic examination was performed and four mucosal pinch biopsies were taken from the rectosigmoid junction. Mucosal BDNF expression and nerve fiber density were analyzed using immunohistochemistry. Mucosal BDNF mRNA levels were quantified by quantitative real-time polymerase chain reaction. Correlations between these parameters were examined.RESULTSThe patients had a higher anxiety score [median (interquartile range), 6.0 (2.0-10.0) vs 3.0 (1.0-4.0), P = 0.003] and visceral sensitivity index score [54.0 (44.0-61.0) vs 21.0 (17.3-30.0), P < 0.001] than controls. The defecating sensation threshold [60.0 (44.0-80.0) vs 80.0 (61.0-100.0), P = 0.009], maximum tolerable threshold [103.0 (90.0-128.0) vs 182.0 (142.5-209.3), P < 0.001] and rectoanal inhibitory reflex threshold [30.0 (20.0-30.0) vs 30.0 (30.0-47.5), P = 0.032] were significantly lower in IBS-D patients. Intestinal mucosal BDNF protein [3.46E-2 (3.06E-2-4.44E-2) vs 3.07E-2 (2.91E-2-3.48E-2), P = 0.031] and mRNA [1.57 (1.31-2.61) vs 1.09 (0.74-1.42), P = 0.001] expression and nerve fiber density [4.12E-2 (3.07E-2-7.46E-2) vs 1.98E-2 (1.21E-2-4.25E-2), P = 0.002] were significantly elevated in the patients. Increased BDNF expression was positively correlated with abdominal pain and disease severity and negatively correlated with visceral sensitivity parameters.CONCLUSIONElevated mucosal BDNF may participate in the pathogenesis of IBS-D via facilitating mucosal nerve growth and increasing visceral sensitivity.

Glucagon-like peptide 1 receptor (GLP-1R) expression by nerve fibres in inflammatory bowel disease and functional effects in cultured neurons.

IntroductionGlucagon like-peptide 1 receptor (GLP-1R) agonists diminish appetite and may contribute to the weight loss in inflammatory bowel disease (IBD).ObjectivesThe aim of this study was to determine, for the first time, the expression of GLP-1R by colon nerve fibres in patients with IBD, and functional effects of its agonists in cultured rat and human sensory neurons.MethodsGLP-1R and other nerve markers were studied by immunohistochemistry in colon biopsies from patients with IBD (n = 16) and controls (n = 8), human dorsal root ganglia (DRG) tissue, and in GLP-1R transfected HEK293 cells. The morphological effects of incretin hormones oxyntomodulin, exendin-4 and glucagon were studied on neurite extension in cultured DRG neurons, and their functional effects on capsaicin and ATP signalling, using calcium imaging.ResultsSignificantly increased numbers of colonic mucosal nerve fibres were observed in IBD biopsies expressing GLP-1R (p = 0.0013), the pan-neuronal marker PGP9.5 (p = 0.0008), and sensory neuropeptide CGRP (p = 0.0014). An increase of GLP-1R positive nerve fibres in IBD colon was confirmed with a different antibody to GLP-1R (p = 0.016). GLP-1R immunostaining was intensely positive in small and medium-sized neurons in human DRG, and in human and rat DRG cultured neurons. Co-localization of GLP-1R expression with neuronal markers in colon and DRG confirmed the neural expression of GLP-1R, and antibody specificity was confirmed in HEK293 cells transfected with the GLP-1R. Treatment with oxyntomodulin, exendin-4 and GLP-1 increased neurite length in cultured neurons compared with controls, but did not stimulate calcium influx directly, or affect capsaicin responses. However, exendin-4 significantly enhanced ATP responses in human DRG neurons.ConclusionOur results show that increased GLP-1R innervation in IBD bowel could mediate enhanced visceral afferent signalling, and provide a peripheral target for therapeutic intervention. The differential effect of GLP-1R agonists on capsaicin and ATP responses in neurons suggest they may not affect pain mechanisms mediated by the capsaicin receptor TRPV1, but may enhance the effects of purinergic agonists.

Image Processing and Analysis of Mucosal Calretinin Staining to Define the Transition Zone in Hirschsprung Disease: A Pilot Study

Quantification of calretinin-stained mucosal nerve fibers by image processing and analysis (IPA) may objectively define the transition zone (TZ) of Hirschsprung disease (HD). We tested the utility of IPA as an adjunctive tool in HD. Calretinin immunostain was performed on 15 HD pull-through specimens, and multiple images were captured from the proximal aganglionic zone, TZ, and probable normal zone (NZ). Pixel count (PC), defined as the percentage of brown-stained pixels in the mucosa, was quantified and plotted against distance from the rectal distal end. To validate the method, PCs from 45 images were compared with three-tiered visual scoring by five pathologists. Results were correlated against pertinent variables, which were retrieved from the clinical record. The PC gradually increased in the TZ toward the proximal resection margin in 10/13 (77%) cases. The PC variation in the probable NZ and around the circumference was substantial by the coefficient of variation. The mean PC of images with a visual score of 1 was less than scores of 2 and 3 by all five (100%) pathologists (p < 0.01). One patient had possible TZ pull-through that was clinically confirmed. While the mucosal calretinin staining gradually increases in the TZ, for now, the boundaries of the TZ cannot be accurately defined by mucosal biopsies given the substantial variation of staining around the circumference at the same distance and in the NZ. However, the IPA technique does provide a continuous variable and warrants further utility in HD studies.

Superficial Esophageal Mucosal Afferent Nerves May Contribute to Reflux Hypersensitivity in Nonerosive Reflux Disease

Little is known about the causes of heartburn in patients with gastro-esophageal reflux disease. Visible epithelial damage is seldom associated with symptom severity, evidenced by the significant symptom burden in patients with nonerosive reflux disease (NERD) compared with patients with erosive reflux disease (ERD) or Barrett's esophagus (BE). We studied the distribution of mucosal nerve fibers in patients with NERD, ERD, and BE, and compared the results with those of healthy subjects. We performed a prospective study of 13 patients with NERD, 11 patients with ERD, and 16 patients with BE undergoing endoscopic evaluation in the United Kingdom or Greece. Biopsies were obtained from the proximal and distal esophageal mucosa of patients with NERD, from the distal esophageal mucosa of patients with ERD, and the distal-most squamous epithelium of patients with BE. These were examined for the presence and location of nerve fibers that reacted with a labeled antibody against calcitonin gene-related peptide (CGRP), a marker of nociceptive sensory nerves. The results were compared with those from 10 healthy volunteers (controls). The distribution of CGRP-positive nerves did not differ significantly between the distal esophageal mucosa of controls (median, 25.5 cell layers to surface; interquartile range [IQR], 21.4-28.8) vs patients with ERD (median, 23 cell layers to surface; IQR, 16-27.5), or patients with BE (median, 21.5 cell layers to surface; IQR, 16.1-27.5). However, CGRP-positive nerves were significantly more superficial in mucosa from patients with NERD-both distal (median, 9.5 cell layers to surface; IQR, 1.5-13.3; P < .0001 vs ERD, BE, and controls) and proximal (median, 5.0 cell layers to surface; IQR, 2.5-9.3 vs median 10.4 cell layers to surface; IQR, 8.0-16.9; P= .0098 vs controls). Proximal and distal esophageal mucosa of patients with NERD have more superficial afferent nerves compared with controls or patients with ERD or BE. Acid hypersensitivity in patients with NERD might be partially explained by the increased proximity of their afferent nerves to the esophageal lumen, and therefore greater exposure to noxious substances in refluxate.

Substance P and the neurokinin-1 receptor expression in dog ileum with and without inflammation

In the gastrointestinal tract, the tachykinin Substance P (SP) is involved in motility, fluid and electrolyte secretion, and blood flow and regulation of immunoinflammatory response. SP exerts its biological activity on target cells by interacting mainly with the neurokinin-1 receptor (NK1R). The present study aims to quantify the percentage of SP-immunoreactive (SP-IR) enteric neurons and the density of SP-IR nerve fibers in the ileum of control dogs (CTRL-dogs; n=7) vs dogs with spontaneous ileal inflammation (INF-dogs; n=8). In addition, the percentage of enteric neurons bearing NK1R, and nitrergic neurons (nNOS-IR) expressing NK1R immunoreactivity were evaluated in both groups. The percentages of SP-IR neurons were similar in CTRL- and INF-dogs, in either the myenteric (MP) (15±8% vs. 16±7%, respectively) and submucosal plexus (SMP) (26±7% vs. 24±14%, respectively). In INF-dogs, the density of SP-IR mucosal nerve fibers showed a trend to decrease (P=0.07). Myenteric neurons of CTRL- and INF-dogs expressed similar percentages of NK1R-immunoreactivity (39±5% vs. 38±20%, respectively). Submucosal NK1R-IR neurons were occasionally observed in a CTRL-dog. MP nitrergic neurons bearing NK1R showed a trend to decrease in INF-dogs vs. CTRL- dogs (41±22% vs. 65±10%, respectively; P=0.11). In INF-dogs, muscle cells and immune cells overexpressed NK1R immunoreactivity. These findings should be taken as a warning for possible intestinal motility disorders, which might occur during administration of NK1R-antagonist drugs. Conversely, the strong expression of NK1R immunoreactivity observed in muscle and mucosal immune cells of inflamed tissues may provide a rationale for the use of NK1R antagonist drugs in the treatment of intestinal inflammation.

Alterations of Substance-P Expression in Gastrointestinal Mucosal Nerve Fibres of Dogs with Inflammatory Bowel Disease

Alterations of Substance-P Expression in Gastrointestinal Mucosal Nerve Fibres of Dogs with Inflammatory Bowel Disease

Tu1745 The TRPM8 Ion Channel Is Expressed in Colonic Mucosal Peptidergic Nerve Fibres, Which Increase During Inflammation and Associate With Dendritic Cells

G A A b st ra ct s in inflamed colons compared to WT + 2% DSS mice suggesting greater degree of vasculogenesis under inflammatory conditions. Conclusion: These results show that lymphatic dysregulation through genetic ablation of FOXC2 significantly exacerbates the onset, progression and severity of experimental IBD. These data suggest that alterations in lymphatic patterning and valve formation (under FOXC2 control) may contribute to IBD progression. Although abnormal lymphatic patterning is not clearly linked with IBD development, therapies which preserve postnatal valve integrity may help to resist gut injury or recover gut integrity in IBD associated gut injury. With lymphatics playing such an important potential role in the pathophysiology of IBD, research focusing on factors interfering with lymphatic damage and therapy to restore lymphatic functioning may open new doors for treatment modalities targeting this area. This work supported by Department of Defense Grant ‘Lymphatic Vascular Based Therapy in IBD' (W81XWH-11-1-0577)

414 Reversal of Opioid-Induced Constipation by Lubiprostone (Amitiza®) in Guinea Pig Ileum

Background & Aims: Brain-derived neurotrophic factor (BDNF) may play a critical role in gut motility. We aimed to investigate BDNF's physiologic effects on gut motility in slowtransit constipation (STC) and to explore the underlying molecular mechanisms. Methods: BDNF expression and alterations of colonic nerve fiber density in STC patients were first investigated. BDNF's effects on gastrointestinal motility of both BDNF+/− mice and loperamide-induced constipation mice were then examined in vivo and in vitro. Smooth muscle α-actin (α-SMA) expression, and nerve fiber, neuromuscular junction (NMJ), and smooth muscle cell (SMC) alterations were investigated. Finally, the effects of BDNF-induced TrkBphospholipase C/inositol trisphosphate (TrkB-PLC/IP3) pathway activation on gut motility were investigated. Results: In STC patients, BDNF expression and nerve fiber density were decreased, and mucosal nerve fiber ultrastructural degenerations were demonstrated. Gut motility was decreased in vivo and in vitro in BDNF+/− and constipation mice, with BDNF dose-dependently increasing gut motility. In BDNF+/− mice, α-SMA expression and nerve fiber density were decreased, and nerve fiber, NMJ, and SMC ultrastructural degenerations were observed. Finally, TrkB-PLC/IP3 pathway agonists dramatically attenuated BDNF's excitatory effect on gut motility, and exogenous BDNF induced an obvious increase in IP3 expression. Conclusions: BDNF plays an important regulatory role in gut motility in STC. It was mediated by altering the intestinal innervation structure, as well as smooth muscle secondary degeneration through a mechanism involving TrkB-PLC/IP3 pathway activation.

413 Brain-Derived Neurotrophic Factor Accelerates Gut Motility in Slow-Transit Constipation

Background & Aims: Brain-derived neurotrophic factor (BDNF) may play a critical role in gut motility. We aimed to investigate BDNF's physiologic effects on gut motility in slowtransit constipation (STC) and to explore the underlying molecular mechanisms. Methods: BDNF expression and alterations of colonic nerve fiber density in STC patients were first investigated. BDNF's effects on gastrointestinal motility of both BDNF+/− mice and loperamide-induced constipation mice were then examined in vivo and in vitro. Smooth muscle α-actin (α-SMA) expression, and nerve fiber, neuromuscular junction (NMJ), and smooth muscle cell (SMC) alterations were investigated. Finally, the effects of BDNF-induced TrkBphospholipase C/inositol trisphosphate (TrkB-PLC/IP3) pathway activation on gut motility were investigated. Results: In STC patients, BDNF expression and nerve fiber density were decreased, and mucosal nerve fiber ultrastructural degenerations were demonstrated. Gut motility was decreased in vivo and in vitro in BDNF+/− and constipation mice, with BDNF dose-dependently increasing gut motility. In BDNF+/− mice, α-SMA expression and nerve fiber density were decreased, and nerve fiber, NMJ, and SMC ultrastructural degenerations were observed. Finally, TrkB-PLC/IP3 pathway agonists dramatically attenuated BDNF's excitatory effect on gut motility, and exogenous BDNF induced an obvious increase in IP3 expression. Conclusions: BDNF plays an important regulatory role in gut motility in STC. It was mediated by altering the intestinal innervation structure, as well as smooth muscle secondary degeneration through a mechanism involving TrkB-PLC/IP3 pathway activation.

CGRP, a neurotransmitter of enteric sensory neurons, contributes to the development of food allergy due to the augmentation of microtubule reorganization in mucosal mast cells.

Neuro-immune interaction in the gut is substantially involved in the maintenance of intestinal immune homeostasis and the pathology of intestinal immune diseases. We have previously demonstrated that mucosal mast cells and nerve fibers containing CGRP, a neurotransmitter of intrinsic enteric sensory neurons, are markedly increased and exist in close proximity to each other in the colon of food allergy (FA) mice. In the present study, a CGRP-receptor antagonist BIBN4096BS significantly alleviated allergic symptoms in the murine FA model. In addition, the elevated numbers of mucosal mast cells in the proximal colon of FA mice were significantly decreased in that of BIBN4096BS-treated FA mice. Thus, we investigated the effects of CGRP on calcium-independent process in degranulation of mucosal mast cells since CGRP increases intracellular cAMP levels, but not Ca(2+) concentration. CGRP did not alter a calcium ionophore A23187-increased cytosolic Ca(2+) concentration in mucosal-type bone marrow-derived mast cells (mBMMCs), but did augment microtubule reorganization in resting and A23187-activated mBMMCs. Furthermore, CGRP alone failed to cause the degranulation of mBMMCs, but CGRP significantly enhanced the degranulation of mBMMCs induced by A23187. Together, these data indicate that CGRP- enhanced microtubule reorganization augments IgE-independent/non-antigenic stimuli-induced mucosal mast cell degranulation, thereby contributing to the development of FA.

Accumulation of α-synuclein in the bowel of patients in the pre-clinical phase of Parkinson’s disease

Parkinson's disease primarily affects the central nervous system, but autopsy and small patient studies have revealed autonomic nervous system pathology in most cases. We looked for α-synuclein pathology in routinely acquired biopsies from patients and matched controls. Immunocytochemistry was performed and assessed blind to the clinical diagnoses. One hundred and seventeen gastrointestinal tissue samples from 62 patients, and 161 samples from 161 controls, were examined. Twelve biopsies from seven patients showed accumulation of α-synuclein within mucosal and submucosal nerve fibres, and ganglia, which was more extensive with an antibody to phosphorylated, than with an antibody to non-phosphorylated, α-synuclein. These included gastric, duodenal and colonic biopsies, and were taken up to 8years prior to the onset of motor symptoms. All patients with positive biopsies had early autonomic symptoms and all controls were negative. This large scale study demonstrates that accumulation of α-synuclein in the gastrointestinal tract is a highly specific finding that could be used to confirm a clinical diagnosis of Parkinson's disease. We have shown that α-synuclein accumulation occurs prior to the onset of motor symptoms in the upper, as well as the lower gastrointestinal tract, remains present in serial biopsies until the onset of motor symptoms and is predominantly composed of phosphorylated α-synuclein. Accumulation of α-synuclein in the bowel therefore offers an accessible biomarker which allows further study of the early stages of the disease and could be of value in the assessment of disease modifying treatments.