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Background Purpose The incidence of constipation increases among the elderly (>65 years), while abdominal pain decreases. Causes include changes in lifestyle (e.g., diet and reduced exercise), disease and medications affecting gastrointestinal functions. Degenerative changes may also occur within the colo‐rectum. However, most evidence is from rodents, animals with relatively high rates of metabolism and accelerated aging, with considerable variation in time course. In humans, cellular and non‐cellular changes in the aging intestine are poorly investigated.To examine all available studies which reported the effects of aging on cellular and tissue functions of human isolated colon, noting the region studied, sex and age of tissue donors and study size. The focus on human colon reflects the ability to access full‐thickness tissue over a wide age range, compared with other gastrointestinal regions. Details are important because of natural human variability.We found age‐related changes within the muscle, in the enteric and nociceptor innervation, and in the submucosa. Some involve all regions of colon, but the ascending colon appears more vulnerable. Changes can be cell‐ and sublayer‐dependent. Mechanisms are unclear but may include development of “senescent‐like” and associated inflammaging, perhaps associated with increased mucosal permeability to harmful luminal contents.In summary, reduced nociceptor innervation can explain diminished abdominal pain among the elderly. Degenerative changes within the colon wall may have little impact on symptoms and colonic functions, because of high “functional reserve,” but are likely to facilitate the development of constipation during age‐related challenges (e.g., lifestyle, disease, and medications), now operating against a reduced functional reserve. [ABSTRACT FROM AUTHOR]

Background: Gastrointestinal (GI) motility disorders affect millions of people worldwide, yet they remain poorly treated in part due to insufficient knowledge of the molecular networks controlling GI motility. Interstitial cells of Cajal (ICC) are critical GI pacemaker cells, and abnormalities in ICC are implicated in GI motility disorders. Two cell surface proteins, KIT and ANO1, are used for identifying ICC. However, difficulties accessing human tissue and the low frequency of ICC in GI tissues have meant human ICC are insufficiently characterized. Here, a range of characterization assays including single‐cell RNA sequencing (scRNA‐seq) was performed using KIT+CD45−CD11B− primary human gastric ICC to better understand networks controlling human ICC biology. Methods: Excess sleeve gastrectomy tissues were dissected; ICC were analyzed by immunofluorescence, fluorescence‐activated cell sorting (FACSorting), real‐time PCR, mass spectrometry, and scRNA‐seq. Key Results: Immunofluorescence identified ANO1+/KIT+ cells throughout the gastric muscle. Compared to the FACSorted negative cells, PCR showed the KIT+CD45−CD11B− ICC were enriched 28‐fold in ANO1 expression (p < 0.01). scRNA‐seq analysis of the KIT−CD45+CD11B+ and KIT+CD45−CD11B− ICC revealed separate clusters of immune cells and ICC (respectively); cells in the ICC cluster expressed critical GI motility genes (eg, CAV1 and PRKG1). The scRNA‐seq data for these two cell clusters predicted protein interaction networks consistent with immune cell and ICC biology, respectively. Conclusions & Inferences: The single‐cell transcriptome of purified KIT+CD45−CD11B− human gastric ICC presented here provides new molecular insights and hypotheses into evolving models of GI motility. This knowledge will provide an improved framework to investigate targeted therapies for GI motility disorders. [ABSTRACT FROM AUTHOR]

Background: Aging is a complex biological process and associated with a progressive decline in functions of most organs including the gastrointestinal (GI) tract. Age‐related GI motor disorders/dysfunctions include esophageal reflux, dysphagia, constipation, fecal incontinence, reduced compliance, and accommodation. Although the incidence and severity of these diseases and conditions increase with age, they are often underestimated due in part to nonspecific and variable symptoms and lack of sufficient medical attention. They negatively affect quality of life and predispose the elderly to other diseases, sarcopenia, and frailty. The mechanisms underlying aging‐associated GI dysfunctions remain unclear, and there is limited data examining the effect of aging on GI motor functions. Many studies on aging‐associated changes to cells within the tunica muscularis including enteric neurons, smooth muscles, and interstitial cells have proposed that cell loss and/or molecular changes may be involved in the pathogenesis of age‐related GI motor disorders/dysfunctions. There is also evidence that the aging contributes to phenotypic changes in innate immune cells, which are physically and functionally linked to other cells in the tunica muscularis and can alter GI (patho) physiology. However, various patterns of changes have been reported, some of which are contradictory, indicating a need for additional work in this area. Purpose: Although GI infection due to intestinal bacterial overgrowth, bleeding, and cancers are also important and common problems in the elderly patients, this mini‐review focuses on data obtained from enteric neuromuscular aging research with the goal of better understanding the cellular and molecular mechanisms of enteric neuromuscular aging to enhance future therapy. [ABSTRACT FROM AUTHOR]

Background: Gastrointestinal (GI) motility disorders affect a large proportion of the population with limited treatment options. The aims of this study were to investigate the potential of a non‐invasive method of auricular vagal nerve stimulation (aVNS) for treating GI dysmotility and to explore possible mechanisms involving slow waves and interstitial cells of Cajal (ICC). Methods: Normal rats were treated daily with loperamide for 1 week and then treated, while still on daily loperamide, with aVNS/Sham‐aVNS for another 1 week. Gastric emptying (GE), small intestine transit (SIT), and GI slow waves were measured. The plasma level of pancreatic polypeptide (PP) and noradrenaline (NE) was assessed by ELISA. ICC in the gastric antrum were detected by immunohistochemistry. Key results: (a) aVNS significantly increased the percentage of normal GI slow waves (p < 0.05 for both fasting and postprandial states, vs. Sham‐aVNS) and accelerated GE (p < 0.05, vs. Sham‐aVNS) and SIT (p < 0.05, vs. Sham‐aVNS) impaired by loperamide. (b) aVNS increased plasma PP (p < 0.01) and decreased plasma NE (p < 0.01), compared with Sham‐aVNS. (c) Gastric ICC was decreased by loperamide (p < 0.01) but increased after aVNS (p < 0.01, vs. Sham aVNS). Conclusions & Inferences: Loperamide induces upper GI dysmotility. aVNS accelerates upper GI transit and improving pace‐making activity mediated via the ICC. Non‐invasive aVNS may have a therapeutic potential for upper GI dysmotility. [ABSTRACT FROM AUTHOR]

Background: Normal gastrointestinal motility depends on electrical slow‐wave activity generated by interstitial cells of Cajal (ICC) in the tunica muscularis of the gastrointestinal tract. A requirement for HCO3− in extracellular solutions used to record slow waves indicates a role for HCO3− transport in ICC pacemaking. The Slc4a4 gene transcript encoding the electrogenic Na+/HCO3− cotransporter, NBCe1, is enriched in mouse small intestinal myenteric region ICC (ICC‐MY) that generate slow waves. This study aimed to determine how extracellular HCO3− concentrations affect electrical activity in mouse small intestine and to determine the contribution of NBCe1 activity to these effects. Methods: Immunohistochemistry and sharp electrode electrical recordings were used. Key Results: The NBCe1 immunoreactivity was localized to ICC‐MY of the tunica muscularis. In sharp electrode electrical recordings, removal of HCO3‐ from extracellular solutions caused significant, reversible, depolarization of the smooth muscle and a reduction in slow‐wave amplitude and frequency. In 100 mM HCO3−, the muscle hyperpolarized and slow wave amplitude and frequency increased. The effects of replacing extracellular Na+ with Li+, an ion that does not support NBCe1 activity, were similar to, but larger than, the effects of removing HCO3‐. There were no additional changes to electrical activity when HCO3‐ was removed from Li+ containing solutions. The Na+/HCO3− cotransport inhibitor, S‐0859 (30µM) significantly reduced the effect of removing HCO3− on electrical activity. Conclusions & Inferences: These studies demonstrate a major role for Na+/HCO3− cotransport by NBCe1 in electrical activity of mouse small intestine and indicated that regulation of intracellular acid:base homeostasis contributes to generation of normal pacemaker activity in the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

Background: High‐resolution colonic manometry gives an unprecedented window into motor patterns of the human colon. Our objective was to characterize motor activities throughout the entire colon that possessed persistent rhythmicity and spanning at least 5 cm. Methods: High‐resolution colonic manometry using an 84‐channel water‐perfused catheter was performed in 19 healthy volunteers. Rhythmic activity was assessed during baseline, proximal balloon distention, meal, and bisacodyl administration. Key Results: Throughout the entire colon, a cyclic motor pattern occurred either in isolation or following a high‐amplitude propagating pressure wave (HAPW), consisting of clusters of pressure waves at a frequency centered on 11‐13 cycles/min, unrelated to breathing. The cluster duration was 1‐6 minutes; the pressure waves traveled for 8‐27 cm, lasting 5‐8 seconds. The clusters itself could be rhythmic at 0.5‐2 cpm. The propagation direction of the individual pressure waves was mixed with >50% occurring simultaneous. This high‐frequency cyclic motor pattern co‐existed with the well‐known low‐frequency cyclic motor pattern centered on 3‐4 cpm. In the rectum, the low‐frequency cyclic motor pattern dominated, propagating predominantly in retrograde direction. Proximal balloon distention, a meal and bisacodyl administration induced HAPWs followed by cyclic motor patterns. Conclusions and Inferences: Within cyclic motor patterns, retrograde propagating, low‐frequency pressure waves dominate in the rectum, likely keeping the rectum empty; and mixed propagation, high‐frequency pressure waves dominate in the colon, likely promoting absorption and storage, hence contributing to continence. Propagation and frequency characteristics are likely determined by network properties of the interstitial cells of Cajal. [ABSTRACT FROM AUTHOR]

Background: Neuromuscular transmission has been extensively studied in the circular layer of the mouse colon where a co‐transmission of purines acting on P2Y1 receptors and NO has been previously described. However, the corresponding mechanisms in the longitudinal layer are less known. Methods: Electrophysiological and myography techniques were used to evaluate spontaneous phasic contractions (SPC) and neural‐mediated responses in the proximal, mid, and distal colon devoid of CD1 mice. Immunohistochemistry against c‐kit and PDGFRα was performed in each colonic segment. Key Results: SPC were recorded in both muscle layers at a similar frequency being about four contractions per minute (c.p.m.) in the proximal and distal colon compared to the mid colon (2 c.p.m.). In non‐adrenergic, non‐cholinergic conditions, L‐NNA (1 mmol/L) increased contractility in the circular but not in the longitudinal layer. In the longitudinal muscle, both electrophysiological and mechanical neural‐mediated inhibitory responses were L‐NNA and ODQ (10 µmol/L) sensitive. NaNP (1 µmol/L) caused cessation of SPC and the response was blocked by ODQ. Neither ADPßS (10 µmol/L) nor CYPPA (10 µmol/L), which both targeted the purinergic pathway, altered longitudinal contractions. PDGFRα + cells were located in both muscle layers and were more numerous compared with cKit + cells, which both formed a heterologous cellular network. A decreasing gradient of the PDGFRα labeling was observed along the colon. Conclusion: An inhibitory neural tone was absent in the longitudinal layer and neuronal inhibitory responses were mainly nitrergic. Despite the presence of PDGFRα + cells, purinergic responses were absent. Post‐junctional pathways located in different cell types might be responsible for neurotransmitter transduction. [ABSTRACT FROM AUTHOR]

Background: The internal anal sphincter (IAS) exhibits slow waves (SWs) and tone that are dependent upon L‐type Ca2+ channels (CavL) suggesting that phasic events (ie, SWs) play a fundamental role in tone generation. The present study further examined phasic activity in the IAS by measuring the spatiotemporal properties of Ca2+ transients (CTs) in IAS smooth muscle cells (SMCs). Methods: Ca2+ transients were recorded with spinning disk confocal microscopy from the IAS of SM‐GCaMP mice. Muscles were pinned submucosal surface up at two different lengths. Drugs were applied by inclusion in the superfusate. Key Results: Ca2+ transients displayed ongoing rhythmic firings at both lengths and were abolished by nifedipine and the KATP channel activator pinacidil indicating their dependence upon CavL. Like SWs, CTs were greatest in frequency (average 70.6 cpm) and amplitude at the distal extremity and conducted proximally. Removal of the distal IAS reduced but did not abolish CTs. The time constant for clearing cytoplasmic Ca2+ averaged 0.46 seconds and basal Ca2+ levels were significantly elevated. Conclusions & Inferences: The similarities in spatiotemporal and pharmacological properties of CTs and SWs suggest that SW gives rise to CTs while muscle stretch is not required. Elevated relative basal Ca2+ in the IAS is likely due to the inability of cells to clear or sequester Ca2+ between rapid frequency voltage‐dependent Ca2+ entry events, that is, conditions that will lead to tone development. The conduction of CTs from distal to proximal IAS will lead to orally directed contractions and likely contribute to the maintenance of fecal continence. [ABSTRACT FROM AUTHOR]

Abstract: Background: Interstitial cells of Cajal (ICC) serve as intestinal pacemakers. Postoperative ileus (POI) is a gastrointestinal motility disorder that occurs following abdominal surgery, which is caused by inflammation‐induced dysfunction of smooth muscles and enteric neurons. However, the participation of ICC in POI is not well understood. In this study, we investigated the functional changes of ICC in a mouse model of POI. Methods: Intestinal manipulation (IM) was performed to induce POI. At 24 h or 48 h after IM, the field potential of the intestinal tunica muscularis was investigated. Tissues were also examined by immunohistochemistry and electron microscopic analysis. Key Results: Gastrointestinal transit was significantly decreased with intestinal tunica muscularis inflammation at 24 h after IM, which was ameliorated at 48 h after IM. The generation and propagation of pacemaker potentials were disrupted at 24 h after IM and recovered to the control level at 48 h after IM. ICC networks, detected by c‐Kit immunoreactivity, were remarkably disrupted at 24 h after IM. Electron microscopic analysis revealed abnormal vacuoles in the ICC cytoplasm. Interestingly, the ICC networks recovered at 48 h after IM. Administration of aminoguanidine, an inducible nitric oxide synthase inhibitor, suppressed the disruption of ICC networks. Ileal smooth muscle tissue cultured in the presence of nitric oxide donor, showed disrupted ICC networks. Conclusions and Inferences: The generation and propagation of pacemaker potentials by ICC are disrupted via nitric oxide after IM, and this disruption may contribute to POI. When inflammation is ameliorated, ICC can recover their pacemaker function. [ABSTRACT FROM AUTHOR]

Abstract: Background: Regulation of gastrointestinal motility involves excitatory and inhibitory neurotransmission. Nitric oxide (NO), the major inhibitory neurotransmitter, acts via its receptor NO‐sensitive guanylyl cyclase (NO‐GC). In the GI tract, NO‐GC is expressed in several cell types such as smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Using cell‐specific knockout mice, we have previously shown that NO‐GC modulates spontaneous contractions in colonic longitudinal smooth muscle. However, its detailed role in the colonic circular smooth muscle is still unclear. Methods: Myography was performed to evaluate spontaneous contractions in rings of proximal colon (2.5 mm) from global (GCKO) and cell‐specific knockout mice for NO‐GC. Immunohistochemistry and in situ hybridization were used to specify NO‐GC expression. Key results: Colonic circular smooth muscle showed three different contraction patterns: high‐frequency ripples, slow phasic contractions, and large contractions. Ripples formed independently of NO‐GC. Slow phasic contractions occurred intermittently in WT, SMC‐GCKO, and ICC‐GCKO tissue, whereas they were more prominent and prolonged in GCKO and SMC/ICC‐GCKO tissue. Tetrodotoxin and the NO‐GC inhibitor ODQ transformed slow phasic contractions of WT and single cell‐specific knockout into GCKO‐like contractions. ODQ increased the frequency of large contractions in WT and ICC‐GCKO colon but not in GCKO, SMC‐GCKO, and SMC/ICC‐GCKO preparations. Tetrodotoxin and hexamethonium abolished large contractions. Conclusions and Inferences: We conclude that short rings of murine colon can be effectively used to record spontaneous contractions. Although NO‐GC in SMC determines smooth muscle tone, concerted action of NO‐GC in both SMC and ICC modulates slow phasic contractions and large contractions. [ABSTRACT FROM AUTHOR]

Abstract: Background: Gastric motility is controlled by the autonomic and enteric nervous systems and by interstitial cells of Cajal (ICCs). Although histamine is known to be released from enterochromaffin‐like cells in the gastric mucosa, its regulatory roles in gastric motility are still controversial. Therefore, we investigated the functional roles of histamine in gastric motility. Methods: Stomach preparations from hamsters were used because the stomach of hamsters can be easily separated into the forestomach and the glandular stomach. A whole preparation of the stomach was mounted in a Magnus tube, and mechanical responses were recorded using a force transducer. Key Results: Exogenous application of histamine had little effect on contractile activity of the glandular stomach. In contrast, the monoamine evoked regular, periodic contractions in the forestomach. An H1 receptor agonist reproduced the contractile responses and an H1 receptor antagonist blocked histamine‐evoked contractions. Atropine and tetrodotoxin did not affect the histamine‐evoked contractions. Pretreatment with drugs that inhibit the activity of ICCs abolished the effects of histamine. Conclusion & Inferences: The findings suggest that histamine regulates gastric motility by acting on ICCs via H1 receptors in the hamster. The remarkable ability of histamine to induce rhythmic contractions would be useful for treatment of gastric dysmotility. [ABSTRACT FROM AUTHOR]

Abstract: Background: The molecular changes that occur in the stomach that are associated with idiopathic gastroparesis are poorly described. The aim of this study was to use quantitative analysis of mRNA expression to identify changes in mRNAs encoding proteins required for the normal motility functions of the stomach. Methods: Full‐thickness stomach biopsy samples were collected from non‐diabetic control subjects who exhibited no symptoms of gastroparesis and from patients with idiopathic gastroparesis. mRNA was isolated from the muscularis externa and mRNA expression levels were determined by quantitative reverse transcriptase (RT)‐PCR. Key Results: Smooth muscle tissue from idiopathic gastroparesis patients had decreased expression of mRNAs encoding several contractile proteins, such as MYH11 and MYLK1. Conversely, there was no significant change in mRNAs characteristic of interstitial cells of Cajal (ICCs) such as KIT or ANO1. There was also a significant decrease in mRNA‐encoding platelet‐derived growth factor receptor α (PDGFRα) and its ligand PDGFB and in Heme oxygenase 1 in idiopathic gastroparesis subjects. In contrast, there was a small increase in mRNA characteristic of neurons. Although there was not an overall change in KIT expression in gastroparesis patients, KIT expression showed a significant correlation with gastric emptying whereas changes in MYLK1, ANO1 and PDGFRα showed weak correlations to the fullness/satiety subscore of patient assessment of upper gastrointestinal disorder‐symptom severity index scores. Conclusions and Inferences:: Our findings suggest that idiopathic gastroparesis is associated with altered smooth muscle cell contractile protein expression and loss of PDGFRα+ cells without a significant change in ICCs. [ABSTRACT FROM AUTHOR]

Background Animal studies have increasingly highlighted the role of macrophages in the development of delayed gastric emptying. However, their role in the pathophysiology of human gastroparesis is unclear. Our aim was to determine changes in macrophages and other cell types in the gastric antrum muscularis propria of patients with diabetic and idiopathic gastroparesis. Methods Full thickness gastric antrum biopsies were obtained from patients enrolled in the Gastroparesis Clinical Research Consortium (11 diabetic, 6 idiopathic) and 5 controls. Immunolabeling and quantitative assessment was done for interstitial cells of Cajal ( ICC) (Kit), enteric nerves protein gene product 9.5, neuronal nitric oxide synthase, vasoactive intestinal peptide, substance P, tyrosine hydroxylase), overall immune cells ( CD45) and anti-inflammatory macrophages ( CD206). Gastric emptying was assessed using nuclear medicine scintigraphy and symptom severity using the Gastroparesis Cardinal Symptom Index. Results Both diabetic and idiopathic gastroparesis patients showed loss of ICC as compared to controls (Mean [standard error of mean]/hpf: diabetic, 2.28 [0.16]; idiopathic, 2.53 [0.47]; controls, 6.05 [0.62]; P=.004). Overall immune cell population ( CD45) was unchanged but there was a loss of anti-inflammatory macrophages ( CD206) in circular muscle (diabetic, 3.87 [0.32]; idiopathic, 4.16 [0.52]; controls, 6.59 [1.09]; P=.04) and myenteric plexus (diabetic, 3.83 [0.27]; idiopathic, 3.59 [0.68]; controls, 7.46 [0.51]; P=.004). There was correlation between the number of ICC and CD206-positive cells ( r=.55, P=.008). Enteric nerves ( PGP9.5) were unchanged: diabetic, 33.64 (3.45); idiopathic, 41.26 (6.40); controls, 46.80 (6.04). Conclusion Loss of antral CD206-positive anti-inflammatory macrophages is a key feature in human gastroparesis and it is associates with ICC loss. [ABSTRACT FROM AUTHOR]

Background Histopathology of muscularis externa in primary esophageal motility disorders has been characterized previously. We aimed to correlate the results of high-resolution manometry with those of histopathology. Methods During peroral endoscopic myotomy, peroral esophageal muscle biopsy was performed in patients with primary esophageal motility disorders. Immunohistochemical staining for c-kit was performed to assess the interstitial cells of Cajal ( ICCs). Hematoxylin Eosin and Azan-Mallory staining were used to detect muscle atrophy, inflammation, and fibrosis, respectively. Key Results Slides from 30 patients with the following motility disorders were analyzed: achalasia (type I: 14, type II: 5, type III: 3), one diffuse esophageal spasm ( DES), two outflow obstruction ( OO), four jackhammer esophagus ( JE), and one nutcracker esophagus ( NE). ICCs were preserved in high numbers in type III achalasia ( n=9.4±1.2 cells/high power field [ HPF]), compared to types I ( n=3.7±0.3 cells/ HPF) and II ( n=3.5±1.0 cells/ HPF). Moreover, severe fibrosis was only observed in type I achalasia and not in other types of achalasia, OO, or DES. Four of five patients with JE and NE had severe inflammation with eosinophilic infiltration of the esophageal muscle layer (73.8±50.3 eosinophils/ HPF) with no epithelial eosinophils. One patient with JE showed a visceral myopathy pattern. Conclusions & Inferences Compared to types I and II, type III achalasia showed preserved ICCs, with variable data regarding DES and OO. In disorders considered as primary esophageal motility disorders, a disease category exists, which shows eosinophilic infiltration in the esophageal muscle layer with no eosinophils in the epithelium. [ABSTRACT FROM AUTHOR]

Background Histological assessment of the interstitial cells of Cajal ( ICCs) in the bowel is important for diagnosing patients with gastrointestinal neuromuscular diseases ( GINMD). Although the International Working Group on GINMD proposed reporting a decrease in ICC number of more than 50%, quantitative methods used in literature are not practical for daily routine of the pathologist. Consequently, this study presents a straightforward semiquantitative estimation method for myenteric ICCs of the bowel. Methods Formalin-fixed paraffin-embedded sections from small bowel ( n = 87) and colon ( n = 159) were collected to create two control groups and four groups composed of patients with gastrointestinal motility disorders. The control groups included material of resection and autopsy origin, respectively. Samples were stained with CD117 (c-kit) antibody to estimate the myenteric ICC network. Scores of two observers were compared to analyze inter- and intraobserver agreement and reliability. Key Results Interobserver reliability was almost perfect for small bowel (intraclass correlation coefficient 0.847; 95% confidence interval [ CI]: 0.774-0.897) and substantial for colon (0.683; 95% CI: 0.591-0.758). Almost perfect intraobserver reliability was found (intraclass correlation coefficient 0.918; 95% CI: 0.874-0.947). The small bowel showed more myenteric ICCs than the colon. Neither significant differences between colonic regions were found nor were there any differences in the orientation of the sections. Conclusions & Inferences The proposed estimation method for the myenteric ICC network showed generally good agreement and reliability. As the method is semiquantitative, simple, and capable to differentiate between normal and diseased tissue, it can be used in routine diagnostics of gastrointestinal neuromuscular disorders. [ABSTRACT FROM AUTHOR]

Background The ICC- DMP have been proposed to generate stimulus-dependent pacemaker activity, rhythmic transient depolarizations, that take part in orchestrating segmentation and clustered propulsive motor patterns in the small intestine. However, little is known about the fundamental properties of ICC- DMP. Methods This study was undertaken to increase our understanding of intrinsic properties of the ICC- DMP through calcium imaging and intracellular electrical recordings. Key Results Without stimulation, most ICC- DMP were quiescent. In some preparations ICC- DMP generated rhythmic low-frequency calcium oscillations (<10 cpm) with or without high frequency activity superimposed (>35 cpm). Immunohistochemistry proved the existence of NK1R on the ICC- DMP and close contacts between ICC- DMP and substance P-positive nerves. Substance P (25 nM) induced low-frequency calcium oscillations that were synchronized across the ICC- DMP network. Substance P also induced low frequency rhythmic transient depolarizations (<10cpm) in circular muscle cells close to the ICC- DMP. An intracellular recording from a positively identified ICC- DMP showed rhythmic transient depolarizations with superimposed high frequency activity. To investigate if quiescent ICC- DMP were chronically inhibited by nitrergic activity, nNOS was inhibited, but without effect. Conclusions & Inferences Substance P changes non-synchronized high frequency flickering or quiescence in ICC- DMP into strong rhythmic calcium transients that are synchronized within the network; they are associated with rhythmic transient depolarizations within the same frequency range. We hypothesize that Substance P, released from nerves, can evoke rhythmicity in ICC- DMP, thereby providing it with potential pacemaker activity. [ABSTRACT FROM AUTHOR]

Background Depletion and ultrastructural changes of interstitial cells of Cajal ( ICC) in the gastric body and antrum have been observed in gastroparesis. This research was performed to investigate the ICC population in the muscularis propria and fibrosis of the muscular layer of the pylorus in gastroparesis. Methods Full thickness pyloric and antral biopsies were obtained from 17 gastroparetic and 6 non-gastroparetic controls. Biopsies were stained with C-Kit for ICC and Trichrome for collagen fibrosis. Interstitial cells of Cajal depletion in the antrum was defined as mean ICC count <10 per 20 high power fields ( HPF) based on established data. Key Results The average pyloric ICC count was ≥10/ HPF in the control patients. Twelve (70.5%) gastroparetic patients had pyloric ICC loss. Only five patients (29.4%) had ICC loss in the antrum. Gastric emptying ( GE) was not significantly different in patients with depleted vs normal pyloric ICC. However, GE at 2 h was slower in patients with antral ICC <10/ HPF compared to those with normal antral ICC populations. Collagen fibrosis was observed in the pylorus of 14 (82.3%) patients. Inclusion bodies in the muscularis propria of the pylorus were identified in four patients, all with diabetic gastroparesis. Conclusions & Inferences In gastroparetic patients, ICC loss in the pylorus is twice as common as in the antrum and fibrosis in the pyloric smooth muscle is nearly three times more common than the antrum. These findings can provide one explanation for pyloric dysfunction which is a contributing factor to the pathophysiology of gastroparesis. [ABSTRACT FROM AUTHOR]

Background Gastric contractions are coordinated by slow waves, generated by interstitial cells of Cajal ( ICC). Gastric surgery affects slow wave conduction, potentially contributing to postoperative gastric dysfunction. However, the impact of gastric cuts on slow waves has not been comprehensively evaluated. This study aimed to define consequences of surgical excisions on gastric slow waves by applying high-resolution ( HR) electrical mapping and in silico modeling. Methods Patients undergoing gastric stimulator implantation ( n = 10) underwent full-thickness stapled excisions (25 × 15 mm, distal corpus) for histological evaluation, enabling HR mapping (256 electrodes; 36 cm2) over and adjacent to excisions. A biophysically based in silico model of bidirectionally coupled ICC networks was developed and applied to investigate the underlying conduction mechanisms and importance of excision orientation. Key Results Normal gastric slow waves propagated aborally (3.0 ± 0.2 cpm). Excisions induced complete conduction block and wavelets that rotated around blocks, then propagated rapidly circumferentially distal to the blocks (8.5 ± 1.2 vs normal 3.6 ± 0.4 mm/s; p < 0.01). This 'conduction anisotropy' homeostatically restored antegrade propagating gastric wavefronts distal to excisions. Excisions were associated with complex dysrhythmias in five patients: retrograde propagation (3/10), ectopics (3/10), functional blocks (2/10), and collisions (1/10). Simulations demonstrated conduction anisotropy emerged from bidirectional coupling within ICC layers and showed transverse incision length and orientation correlated with the degree of conduction distortion. Conclusions & Inferences Orienting incisions in the longitudinal gastric axis causes least disruption to electrical conduction and motility. However, if transverse incisions are made, a homeostatic mechanism of gastric conduction anisotropy compensates by restoring aborally propagating wavefronts. Complex dysrhythmias accompanying excisions could modify postoperative recovery in susceptible patients. [ABSTRACT FROM AUTHOR]

Background Otilonium bromide ( OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are well-characterized, little is known about the effects of chronic administration of OB on enteric neurons, neuromuscular transmission, and interstitial cells of Cajal ( ICC), key regulators of the gut function. Methods Adult Sprague-Dawley rats were treated with OB in drinking water at a dose of 2 mg/kg for 30 days. The colons of OB-treated and age-matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity ( IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology. Key Results Using immunohistochemistry, chronic OB administration did not change total neuron number, assessed by anti-Hu IR, but resulted in a significant increase in NK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. Chronic OB administration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonic ICC, detected by Kit IR, were not altered nor were slow waves or smooth muscle membrane potential. Conclusions & Inferences Chronic treatment with OB caused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon. [ABSTRACT FROM AUTHOR]

Waxing and waning of slow waves amplitude has been recently associated with a segmentation motor pattern in the murine small intestine. The 'wax and wane' phenomenon in this area of the gastrointestinal tract seems to be the result of modulation of slow waves by a second pacemaker of a lower frequency displayed by the interstitial cells of Cajal near the deep muscular plexus ( ICC- DMP). In the rat colon, smooth muscle cyclic depolarizations causing low-frequency ( LF) contractions (0.9 ± 0.1 cpm) occur together with slow wave activity associated to high-frequency ( HF) contractions (14 ± 0.3 cpm; ripples). In the present manuscript, we demonstrate the presence of 'wax and wane' in rat colonic slow waves. Depolarization from the 'wax' to the 'wane' was 7.6 ± 1.2 mV, i.e., smooth muscle cells went from a resting membrane potential ( RMP) of −50.0 mV to a RMP of −42.4 mV. The amplitude of the slow wave decreased from 14.0 ± 2.2 mV to 3.4 ± 0.7 mV. The wax and wane phenomenon occurred at 0.9 ± 0.1 cpm, coinciding with the frequency of cyclic depolarizations. Therefore, we hypothesized that the 'wax and wane' of slow waves in the rat colon could be the result of their interaction with the LF pacemaker. We describe three different myogenic motor patterns that depend on the level of smooth muscle and ICC excitation: (i) LF propulsive contractions, (ii) regular slow waves causing ripples, and (iii) a wax and wane pattern that may lead to segmentation. Different intra- and extra-luminal inputs probably determine the dominating motor pattern in each area through the enteric nervous system. [ABSTRACT FROM AUTHOR]

Background There is increasing evidence for specific cellular changes in the stomach of patients with diabetic ( DG) and idiopathic ( IG) gastroparesis. The most significant findings are loss of interstitial cells of Cajal ( ICC), neuronal abnormalities, and an immune cellular infiltrate. Studies done in diabetic mice have shown a cytoprotective effect of CD206+ M2 macrophages. To quantify overall immune cellular infiltrate, identify macrophage populations, and quantify CD206+ and i NOS+ cells. To investigate associations between cellular phenotypes and ICC. Methods Full thickness gastric body biopsies were obtained from non-diabetic controls (C), diabetic controls ( DC), DG, and IG patients. Sections were labeled for CD45, CD206, Kit, i NOS, and putative human macrophage markers ( HAM56, CD68, and EMR1). Immunoreactive cells were quantified from the circular muscle layer. Key Results Significantly fewer ICC were detected in DG and IG tissues, but there were no differences in the numbers of cells immunoreactive for other markers between patient groups. There was a significant correlation between the number of CD206+ cells and ICC in DG and DC patients, but not in C and IG and a significant correlation between i NOS+ cells and ICC in the DC group, but not the other groups. CD68 and HAM56 reliably labeled the same cell populations, but EMR1 labeled other cell types. Conclusions & Inferences Depletion of ICC and correlation with changes in CD206+ cell numbers in DC and DG patients suggests that in humans, like mice, CD206+ macrophages may play a cytoprotective role in diabetes. These findings may lead to novel therapeutic options, targeting alternatively activated macrophages. [ABSTRACT FROM AUTHOR]

Background Nitric oxide (NO) is an important inhibitory neurotransmitter in the gastrointestinal (GI) tract. The majority of nitrergic effects are transduced by NO-sensitive guanylyl cyclase (NO-GC) as the receptor for NO, and, thus, mediated by cGMP-dependent mechanisms. Work carried out during the past years has demonstrated NO to be largely involved in GI smooth muscle relaxation and motility. However, detailed investigation of nitrergic signaling has turned out to be complicated as NO- GC was identified in several different GI cell types such as smooth muscle cells, interstitial cells of Cajal and fibroblast-like cells. With regards to nitrergic neurotransmission, special focus has been placed on the role of interstitial cells of Cajal using mutant mice with reduced populations of ICC. Recently, global and cell-specific knockout mice for enzymes participating in nitrergic signaling have been generated providing a suitable approach to further examine the role of NO-mediated signaling in GI smooth muscle. Purpose This review discusses the current knowledge on nitrergic mechanisms in gastrointestinal neuromuscular transmission with a focus on genetic models and outlines possible further investigations to gain better understanding on NO-mediated effects in the GI tract. [ABSTRACT FROM AUTHOR]

Background Gastrointestinal manifestations of diabetes are common and a source of significant discomfort and disability. Diabetes affects almost every part of gastrointestinal tract from the esophagus to the rectum and causes a variety of symptoms including heartburn, nausea, vomiting, abdominal pain, diarrhea and constipation. Understanding the underlying mechanisms of diabetic gastroenteropathy is important to guide development of therapies for this common problem. Over recent years, the data regarding the pathophysiology of diabetic gastroenteropathy is expanding. In addition to autonomic neuropathy causing gastrointestinal disturbances the role of enteric nervous system is becoming more evident. Purpose In this review, we summarize the reported alterations in enteric nervous system including enteric neurons, interstitial cells of Cajal and neurotransmission in diabetic animal models and patients. We also review the possible underlying mechanisms of these alterations, with focus on oxidative stress, growth factors and diabetes induced changes in gastrointestinal smooth muscle. Finally, we will discuss recent advances and potential areas for future research related to diabetes and the ENS such as gut microbiota, micro- RNAs and changes in the microvasculature and endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Background The roles of M2 and M3 muscarinic receptor subtypes in the regulation of gut motor activity were investigated. Methods We simultaneously recorded changes in the intraluminal pressure ( IP) and longitudinal tension ( LT) in small intestinal segments from M2 or M3 receptor knockout ( KO) and wild-type ( WT) mice. Key Results In the WT preparations, luminal distension induced a continuous rhythmic contractile activity that was characterized by synchronous rises in IP and LT, occurring periodically at a constant interval. Tetrodotoxin completely abolished the response, whereas atropine either abolished or attenuated it. In the majority of the M2 KO preparations, however, no rhythmic activity was observed in response to the luminal distention, even though networks of enteric neurons and interstitial cells of Cajal ( ICC) seemed to be intact. Where rhythmic activity did occur in M2 KO preparations, it was atropine resistant. In the M3 KO preparations, the IP and LT were synchronously changed by the luminal distention, but the changes occurred at irregular intervals. The W/Wv mutant preparations, which lack ICC in the myenteric plexus ( ICC- MY), showed results similar to those of the M3 KO preparations. In some of the M2/M3 double- KO preparations, rhythmic activity was not observed, but in the others, an atropine-resistant rhythmicity appeared. Conclusions & Inferences These results suggest that M2 and M3 muscarinic receptors differentially regulate the intestinal motor activity: M2 receptors play an essential role in the generation of rhythmic motor activity, and M3 receptors have a modulatory role in controlling the periodicity of the rhythmic activity together with the ICC- MY. [ABSTRACT FROM AUTHOR]

Background Loss or disruption of Kit+-interstitial cells of Cajal ( ICC) capable of generating pacemaker activity has been implicated in the development of numerous gastrointestinal motility disorders. We sought to develop a model where ICC could be allotransplanted into intestines naturally devoid of these cells. Methods Enzymatically dispersed cells from the intestinal tunica muscularis of Kit+/copGFP and KitV558Δ/+ gain-of-function mice were allotransplanted into myenteric plexus regions of W/WV mutant intestines that lack ICC at the level of the myenteric plexus ( ICC- MY) and pacemaker activity. Immunohistochemical analysis fate mapped the development of ICC- MY networks and intracellular microelectrode recordings provided evidence for the development of functional pacemaker activity. Key Results Kit+- ICC developed into distinct networks at the level of the myenteric plexus in organotypic cultures over 28 days and displayed robust rhythmic pacemaker activity. Conclusions & Inferences This study demonstrates the feasibility of allotransplantation of ICC into the myenteric region of the small intestine and the establishment of functional pacemaker activity into tissues normally devoid of ICC- MY and slow waves, thus providing a possible basis for the therapeutic treatment of patients where ICC networks have been disrupted due to a variety of pathophysiological conditions. [ABSTRACT FROM AUTHOR]

Background Slow-waves modulate the pattern of small intestine contractions. However, the large-scale spatial organization of intestinal slow-wave pacesetting remains uncertain because most previous studies have had limited resolution. This study applied high-resolution ( HR) mapping to evaluate intestinal pacesetting mechanisms and propagation patterns in vivo. Methods HR serosal mapping was performed in anesthetized pigs using flexible arrays (256 electrodes; 32 × 8; 4 mm spacing), applied along the jejunum. Slow-wave propagation patterns, frequencies, and velocities were calculated. Slow-wave initiation sources were identified and analyzed by animation and isochronal activation mapping. Key Results Analysis comprised 32 recordings from nine pigs (mean duration 5.1 ± 3.9 min). Slow-wave propagation was analyzed, and a total of 26 sources of slow-wave initiation were observed and classified as focal pacemakers (31%), sites of functional re-entry (23%) and circumferential re-entry (35%), or indeterminate sources (11%). The mean frequencies of circumferential and functional re-entry were similar (17.0 ± 0.3 vs 17.2 ± 0.4 cycle min−1; P = 0.5), and greater than that of focal pacemakers (12.7 ± 0.8 cycle min−1; P < 0.001). Velocity was anisotropic (12.9 ± 0.7 mm s−1 circumferential vs 9.0 ± 0.7 mm s−1 longitudinal; P < 0.05), contributing to the onset and maintenance of re-entry. Conclusions & Inferences This study has shown multiple patterns of slow-wave initiation in the jejunum of anesthetized pigs. These results constitute the first description and analysis of circumferential re-entry in the gastrointestinal tract and functional re-entry in the in vivo small intestine. Re-entry can control the direction, pattern, and frequency of slow-wave propagation, and its occurrence and functional significance merit further investigation. [ABSTRACT FROM AUTHOR]

Background The colon undergoes distension-induced changes in motor activity as luminal contents or feces increase wall pressure. Input from enteric motor neurons regulates this motility. Here we examined stretch-dependent responses in circular muscle strips of murine colon. Methods Length ramps (6-31 μm s−1) were applied in the axis of the circular muscle layer in a controlled manner until 5 mN isometric force was reached. Key Results Length ramps produced transient membrane potential hyperpolarizations and attenuation of action potential (AP) complexes. Responses were reproducible when ramps were applied every 30 s. Stretch-dependent hyperpolarization was blocked by TTX, suggesting AP-dependent release of inhibitory neurotransmitter(s). Atropine did not potentiate stretch-induced hyperpolarizations, but increased compliance of the circular layer. Nω-nitro- l-arginine ( l-NNA) inhibited stretch-dependent hyperpolarization and decreased muscle compliance, suggesting release of NO mediates stretch-dependent inhibition. Control membrane potential was restored by the NO donor sodium nitorprusside. Stretch-dependent hyperpolarizations were blocked by l-methionine, an inhibitor of stretch-dependent K+ (SDK) channels in colonic muscles. Loss of interstitial cells of Cajal, elicited by Kit neutralizing antibody, also inhibited responses to stretch. In presence of l-NNA and apamin, stretch responses became excitatory and were characterized by membrane depolarization and increased AP firing. A neurokinin-1 receptor antagonist inhibited this stretch-dependent increase in excitability. Conclusions and Inferences Our data show that stretch-dependent responses in colonic muscles require tonic firing of enteric inhibitory neurons, but reflex activation of neurons does not appear to be necessary. NO causes activation of SDK channels, and stretch of muscles further activates these channels, explaining the inhibitory response to stretch in colonic muscle strips. [ABSTRACT FROM AUTHOR]

Background The gastric sling muscle has not been investigated for possible sensory innervation, in spite of the key roles the structure plays in lower esophageal sphincter (LES) function and gastric physiology. Thus, the present experiment used tracing techniques to label vagal afferents and survey their projections in the lesser curvature. Methods Sprague-Dawley rats received injections of dextran biotin into the nodose ganglia. Fourteen days postinjection, animals were euthanized and their stomachs were processed to visualize the vagal afferent innervation. In different cases, neurons, muscle cells, or interstitial cells of Cajal (ICC) were counterstained. Key Results The sling muscle is innervated throughout its length by vagal afferent intramuscular arrays (IMAs) associated with ICC. In addition, the distal antral attachment site of the sling muscle is innervated by a novel vagal afferent terminal specialization, an antral web ending. The muscle wall of the distal antrum is also innervated by conventional IMAs and intraganglionic laminar endings, the two types of mechanoreceptors found throughout stomach smooth muscle. Conclusions & Inferences The innervation of sling muscle by IMAs, putative stretch receptors, suggests that sling sensory feedback may generate vago-vagal or other reflexes with vagal afferent limbs. The restricted distribution of afferent web endings near the antral attachments of sling fibers suggests the possibility of specialized mechanoreceptor functions linking antral and pyloric activity to the operation of the LES. Dysfunctional sling afferents could generate LES motor disturbances, or normative compensatory sensory feedback from the muscle could compromise therapies targeting only effectors. [ABSTRACT FROM AUTHOR]

Background Morphological studies have revealed a close anatomical relationship between enteric nerve terminals and intramuscular ICC (ICC-IM) which supports a role for ICC-IM as intermediaries in enteric motor neurotransmission. Recently, a second type of interstitial cell previously described as 'fibroblast-like' but can now be identified by platelet-derived growth factor receptor-α expression, has also been implicated in enteric neurotransmission in rodents. The present study was performed to determine if enteric nerve fibers form close anatomical relationships with ICC and PDGFRα+ cells throughout the primate GI tract. Methods Immunohistochemical experiments and confocal microscopy were performed to examine the relationship between excitatory and inhibitory motor neurons, ICC and PDGFRα+ cells throughout the monkey GI tract. Key Results The pan neuronal marker. Protein gene product 9.5 (PGP9.5) was used to label all enteric neurons and substance-P (sub-P) and neuronal nitric oxide synthase (nNOS) to label excitatory and inhibitory neurons, respectively. Double labeling with Kit revealed that both classes of nerve fibers were closely apposed with ICC-IM in the stomach, small intestine and colon (taenia and inter-taenia regions), but not with ICC at the level of the myenteric plexus (ICC-MY). Varicose enteric nerve fibers were closely associated with ICC-IM for distances up to 250 μm. Both excitatory and inhibitory nerve fibers were also closely apposed to PDGFRα+ cells throughout the primate GI tract. Conclusions & Inferences The close anatomical relationship between enteric nerve fibers and ICC-IM and PDGFRα+ cells throughout the GI tract of the Cynomolgus monkey provides morphological evidence that these two classes of interstitial cells may provide a similar physiological function in primates as has been attributed in rodent animal models. [ABSTRACT FROM AUTHOR]

Background Emerging evidence suggests that 'fibroblast-like cells' (FLC) may play a role in the regulation of gastrointestinal (GI) motor function. FLC are ultrastructurally distinct from other interstitial cells, including interstitial cells of Cajal (ICC), and express small-conductance Ca2+-activated K+ channels (SK3). In mice, platelet-derived growth factor receptor α (PDGFR α) antibody has also been shown to label FLC. The aims of this study were to determine the morphology and distribution of PDGFR α-immunoreactive (ir) FLC in human gastric muscle and to determine if FLC are altered in gastroparesis, where ICC are reduced. Methods Full thickness gastric body biopsies from five healthy subjects, 10 diabetic, and 10 idiopathic gastroparesis patients were immunolabeled using SK3 and PDGFR α staining for FLC and Kit staining for ICC. Intramuscular FLC and ICC were quantified. Key Results Intramuscular PDGFR α-ir cells had slender cell bodies and long, thin processes and were more abundant in the longitudinal compared with the circular muscle. In the region of myenteric plexus, FLC had smaller, rounder cell bodies with 3-4 processes and formed networks, often around ganglia. All SK3-ir cell structures showed complete overlap with PDGFR α-ir. FLC were in close proximity to ICC, but their cell bodies did not overlap. No differences were seen in the distribution, morphology, or overall numbers of FLC in gastroparesis patients. Conclusions & Inferences In conclusion, PDGFR α identifies FLC in human gastric smooth muscle. FLC were not altered in distribution or overall numbers in gastroparesis. Additional studies are required to determine their role in human GI function. [ABSTRACT FROM AUTHOR]

The tunica muscularis of the gastrointestinal (GI) tract contains two layers of smooth muscle cells (SMC) oriented perpendicular to each other. SMC express a variety of voltage-dependent and voltage-independent ionic conductance(s) that develop membrane potential and control excitability. Resting membrane potentials (RMP) vary through the GI tract but generally are within the range of −80 to −40 mV. RMP sets the 'gain' of smooth muscle and regulates openings of voltage-dependent Ca2+ channels. A variety of K+ channels contribute to setting RMP of SMC. In most regions, RMP is considerably less negative than the K+ equilibrium potential, due to a finely tuned balance between background K+ channels and non-selective cation channels (NSCC). Variations in expression patterns and openings of K+ channels and NSCC account for differences of the RMP in different regions of the GI tract. Smooth muscle excitability is also regulated by interstitial cells (interstitial cells of Cajal (ICC) and PDGFRα+ cells) that express additional conductances and are electrically coupled to SMC. Thus, 'myogenic' activity results from the integrated behavior of the SMC/ICC/PDGFRα+ cell (SIP) syncytium. Inputs from excitatory and inhibitory motor neurons are required to produce the complex motor patterns of the gut. Motor neurons innervate three cell types in the SIP, and receptors, second messenger pathways, and ion channels in these cells mediate postjunctional responses. Studies of isolated SIP cells have begun to unravel the mechanisms responsible for neural responses. This review discusses ion channels that set and regulate RMP of SIP cells and how neurotransmitters regulate membrane potential. [ABSTRACT FROM AUTHOR]

Gastroparesis is often divided into subsets based on etiology and pathophysiology; however, the utility of these subsets in the diagnosis and treatment of gastro-paresis is not well defined. The objectives are to consider the subsets of gastroparesis from the perspectives of etiology and pathogenesis, pathophysiology, histopathology, and clinical associations, with particular focus on similarities and differences between diabetic and idiopathic gastroparesis and consideration of the potential subset of painful gastroparesis. We conclude that idiopathic and diabetic gastroparesis has similar initial presentations and manifestations, except that idiopathic gastroparesis tends to be associated more frequently with pain. Myopathic disorders are uncommon. Extrinsic denervation was considered the most common etiology; however, with the decline in surgery for peptic ulceration and in-depth study of full-thickness gastric biopsies, the most common intrinsic defects are being recognized in the interstitial cells of Cajal (ICC-opathy) and with immune infiltration and neuronal changes (intrinsic neuropathic gastroparesis). Histomorphological differences at the microscopic level between diabetic and idiopathic gastroparesis are still of unclear significance. Two gastroparesis subsets worthy of special mention, because they are potentially reversible with identification of the cause, are postviral gastroparesis, which has a generally good prognosis, and iatrogenic gastroparesis, especially in patients with non-surgical gastroparesis, such as diabetics exposed to incretins such as pramlintide and exenatide. [ABSTRACT FROM AUTHOR]

Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to lack of experimental models. Previously we established the progeric klotho mouse as a model of aging-associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in vivo function and cellular make-up of the small intestinal and colonic neuromuscular apparatus. Klotho expression was studied by RT-PCR and immunohistochemistry. Motility was assessed by dye transit and bead expulsion. Smooth muscle and neuron-specific gene expression was studied by Western immunoblotting. Interstitial cells of Cajal (ICC) and precursors were analyzed by flow cytometry, confocal microscopy, and three-dimensional reconstruction. HuC/D myenteric neurons were enumerated by fluorescent microscopy. Klotho protein was detected in neurons, smooth muscle cells, and some ICC classes. Small intestinal transit was slower but whole-gut transit of klotho mice was accelerated due to faster colonic transit and shorter intestinal lengths, apparent only after weaning. Fecal water content remained normal despite reduced output. Smooth muscle myosin expression was reduced. ICC, ICC precursors, as well as nitrergic and cholinergic neurons maintained their normal proportions in the shorter intestines. Progeric klotho mice express less contractile proteins and develop generalized intestinal neuromuscular hypoplasia mainly arising from stunted postweaning growth. As reduced fecal output in these mice occurs in the presence of accelerated colonic and whole-gut transit, it likely reflects reduced food intake rather than intestinal dysmotility. [ABSTRACT FROM AUTHOR]

Background Chloride channels are proposed to play a central role in the electrical pacemaking mechanism of interstitial cells of Cajal (ICC). A key unknown factor in the consideration of this role is the chloride equilibrium potential ( ECl), as determined by the relative concentrations of intra- ([Cl−]i) and extracellular ([Cl−]o) chloride ions. Methods To calculate the ECl of ICC, [Cl−]i was measured with the fluorescent chloride indicator N-(6-methoxyquinolyl) acetoethyl ester (MQAE). Pacemaker ICC in explant cultures or in situ, i.e. ICC associated with the myenteric plexus of the small intestine, were loaded with MQAE and fluorescence was measured by laser scanning confocal microscopy. The dye fluorescence was then calibrated against known [Cl−]i by treating the explants or in situ preparations with chloride ionophore and varying bath chloride concentrations. Key Results In explants, ICC [Cl−]i was measured as 13 mmol L−1 with [Cl−]o of 100 mmol L−1, giving an ECl of −23 mV. With [Cl−]o at 166 mmol L−1, [Cl−]i was 26 mmol L−1, giving an ECl of −21 mV. In situ, ICC [Cl−]i was measured as 26 mmol L−1 with [Cl−]o of 130 mmol L−1, giving an ECl of −18 mV. Importantly ICC compensate for changes in extracellular chloride by changing [Cl−]i and thus maintain ECl. In ICC explant clusters, [Cl−]i was seen to fluctuate, possibly evoked by rhythmic changes in intracellular calcium. Conclusions & Inferences The intracellular chloride concentration in ICC fluctuates to keep its equilibrium potential constant. The identification of ECl as positive to the resting membrane potential of ICC indicates that opening of chloride channels will depolarize ICC. [ABSTRACT FROM AUTHOR]

Background Gastrointestinal tract is one of the most susceptible organ systems to ischaemia. Not only mucosal injury but also alterations of the intestinal motility and loss of interstitial cells of Cajal (ICC) have been reported in response to ischaemia and reperfusion (I/R). However, there are few reports on the changes in the gastric motility after gastric I/R. The present study was designed to investigate the alterations in gastric emptying, the ICC and enteric nerves that regulate smooth muscle function in response to gastric I/R. Methods Seven-week-old male Wistar rats were exposed to gastric I/R, and the gastric emptying rates at 12 and 48 h after I/R were evaluated by the phenol red method. Expressions of gene product of c-kit receptor tyrosine kinase (c-Kit), a marker of ICC, and of neuronal proteins were also examined. Key Results Gastric emptying was transiently delayed at 12 h after I/R, but returned to normal by 48 h. Expression of c-Kit protein as assessed by Western blotting and immunofluorescent staining of the smooth muscle layer, as well as expression of the mRNA of stem cell factor, the ligand for c-Kit, were reduced at both 12 and 48 h after I/R. The expression of neuronal nitric oxide synthase (nNOS) protein as assessed by Western blotting and immunofluorescent staining was also decreased at 12 h after I/R, but was restored to normal by 48 h. Conclusions & Inferences Gastric I/R evokes transient gastroparesis with delayed gastric emptying, associated with disruption of the ICC network and nNOS-positive neurons. [ABSTRACT FROM AUTHOR]

Our goal was to investigate associations between the status of interstitial cells of Cajal (ICC) and electrogastrogram (EGG) parameters, gastric emptying and symptoms in a large cohort of patients with gastroparesis. Forty-one patients with refractory gastroparesis who were referred for gastric electrical stimulation (GES) underwent full thickness gastric (antrum) biopsy during the surgery to place the GES device. The biopsy samples were stained with c-kit and scored for the presence of ICC based on criteria obtained from 10 controls. All patients underwent EGG recordings, a 4-h standardized scintigraphic gastric emptying study and symptom assessment prior to the surgery. Based on antral biopsy, 15 patients (36%) had almost no ICC (ICC− group) and 26 patients had adequate cell numbers (ICC+ group). EGG recordings in the ICC− group displayed significantly less normal slow waves than in the ICC+ group both in the fasting and fed states. Tachygastria in the ICC− group was significantly more than in the ICC+ group both in the fasting (32 ± 8% vs 11 ± 2%) and fed states (27 ± 9% vs 12 ± 2%). There was no statistical difference in gastric emptying, symptom severity of gastroparesis, aetiology, age and gender between the two groups. Severely depleted ICC occurs in up to 36% of gastroparetic patients and significantly correlates with an abnormal EGG. Severely depleted ICC does not correlate with the severity of gastroparesis as assessed by gastric emptying or symptom status but did result in a poorer symptomatic response to GES. These data suggest that the EGG may have a role for predicting ICC status during clinical evaluation of gastroparetic patients. [ABSTRACT FROM AUTHOR]

Interstitial cells of Cajal (ICC) associated with Auerbach’s plexus in the small intestine, provide pacemaker activity to orchestrate peristalsis and mixing. Despite the close apposition between ICC and enteric nerves, little is known about the neural regulation of pacemaker activity. The present study pursues the hypothesis that substance P can affect pacemaker activity through action on non-selective cation channels. Cell-attached and inside-out patch clamp studies were performed on isolated ICC in short-term cultures that provided evidence that substance P increases open probability or initiates activity in non-selective cation channels in ICC. The single-channel conductance is ∼25 pS and in the on-cell configuration the activity can occur in a rhythmic fashion. Patches contained 1–10 channels and were most often accompanied by a ∼12 pS chloride channel that was also activated by substance P. In a recently developed preparation that allows patch clamping in ICC in their natural environment within tissue, i.e. in situ, the presence of the channel and substance P activation was confirmed. The non-selective cation channel is one of the channels that initiate intestinal pacemaker activity and the present study provides further single-channel data on this critical channel. Because of the close proximity of enteric motor and sensory nerves to ICC, these data provide a potential mechanism underlying neural regulation of pacemaker activity. The data also indicate that neurokinergic pharmacology is a promising avenue for excitation of the intestinal pacemaker system. [ABSTRACT FROM AUTHOR]

Mitochondrial neurogastrointestinal enceph-alomyopathy (MNGIE) is a rare autosomal-recessive multisystemic disorder with predominant gastrointestinal involvement, presenting with variable degrees of gut dysmotility up to frank chronic intestinal pseudoobstruction. Despite major advances in understanding its basic molecular pathogenesis in recent years, the distinct mechanisms and pathoanatomical substrate underlying MNGIE-associated gastrointestinal dysmotility are still widely unknown. As yet, though their critical role in proper gastrointestinal transit in terms of spontaneous pacemaker activity and enteric neurotransmission is well established, the population of the interstitial cells of Cajal (ICC) has not been investigated in MNGIE. Therefore, we examined small bowel samples of a well-characterized MNGIE patient by using conventional histology and immunohistochemistry techniques. The ICC network was studied by immunohistochemistry for the tyrosine kinase Kit (CD117), known to reliably detect ICCs, while mucosal mast cells served as an internal and normal small bowel specimen as external controls. At a light microscopic level, no gross structural alteration of the bowel wall composition and its neuromuscular elements was noted. However, a complete absence of Kit immunoreactive cells could be demonstrated in regions where ICCs are normally abundant, while internal and external controls retained strong Kit positivity. In conclusion, our preliminary results provide a first evidence for an alteration of the ICC network in MNGIE, and support the notion that ICC loss might be an early pathogenetic event in MNGIE-associated gut motor dysfunction before significant myopathic and/or neuropathic structural changes occur. [ABSTRACT FROM AUTHOR]

In the development of mouse gut, longitudinal smooth muscle cells (LMC) and interstitial cells of Cajal (ICC) originate from common precursor cells expressing c-Kit. Recently, some gastrointestinal stromal tumours, which develop from smooth muscle layers of the gut and have gain-of-function mutations of c-kit, have been reported to have gain-of-function mutations of platelet-derived growth factor (PDGF) receptor α gene. These data raise the possibility that PDGF signalling might be involved in the development of LMC. Therefore, we examined the expression pattern of the PDGF signal family of embryonic gut by immunohistochemistry and in situ hybridization, and investigated the role of PDGF signals in the development of smooth muscle layers in mouse gut using a new organ culture system. During embryonic development, the circular muscle layer expressed PDGF-A, enteric neurons expressed PDGF-B and common precursor cells of LMC and ICC expressed both PDGF receptor α and β. The selective PDGF receptor inhibitor AG1295 suppressed the differentiation of LMC in gut explants. We conclude that PDGF signals play critical roles in the differentiation of LMC in mouse embryonic gut. [ABSTRACT FROM AUTHOR]

Abnormalities of enteric nerves, interstitial cells of Cajal (ICC) and smooth muscle are often associated with severe gastrointestinal motility disorders. In this context, full-thickness biopsy of the gut may provide important diagnostic and prognostic clues as well as some possible therapeutic implications. Nonetheless, the unavoidable risk to further worsen prognosis evoked by laparotomy, and the unclear yield of histopathological analysis has hampered full-thickness gut sampling in patients with severe dysmotility. However, recent advances in minimally invasive surgery have refuelled enthusiasm in gastrointestinal neuromuscular pathology. In this issue of Neurogastroenterology and Motility, Miller et al. provide novel and exciting evidence that the appendix might be used as a surrogate tissue to analyse changes to enteric nerves, ICC and smooth muscle cells in patients with diabetic gastroenteropathy. The objective of this short review was to place this very important work in the context of current understanding of enteric neuromuscular dysfunction. [ABSTRACT FROM AUTHOR]

This section provides abstracts of poster sessions presented at the 21st International Symposium on Neurogastroenterology and Motility held in Jeju, South Korea in September 2007, including sessions on the sensitivity of guinea-pig small intestinal spinal afferents, telemetric recording of electrogastrogram, and changes in serum gastrin levels.

This section provides abstracts of oral presentations given at the 21st International Symposium on Neurogastroenterology and Motility held in Jeju, South Korea, in September 2007, including presentations on the localization of chloride channels in myenteric neurons, gene expression of MLC20, and the mechanism and spread of pacemaker activity.

Nitric oxide (NO) has emerged as a major modulator of cellular function in health and disease. In addition to its well-known role as a mediator of smooth muscle relaxation, a rapidly developing body of research suggests, paradoxically, that NO can have both cytotoxic and cytoprotective effects. In this issue of Neurogastroenterology and Motility, Choi et al. provide evidence that supports NO has a prosurvival effect on interstitial cells of Cajal in the mouse stomach. The objective of this short review is to place this interesting report in the context of the current literature. [ABSTRACT FROM AUTHOR]

The distribution of interstitial cells of Cajal (ICC) and neurotransmission were investigated in lower oesophageal sphincter (LES) circular muscle strips from Sprague–Dawley (SD) rats, Ws/Ws mutant rats and their wild-type (+/+) siblings. Intramuscular c-Kit-positive cells, confirmed to be ICC-IM by electron microscopy, were observed throughout both muscle layers from SD and +/+ rats. In contrast, c-Kit-positive, ultrastructurally typical ICC-IM were absent in Ws/Ws. LES strips from Ws/Ws rats showed increased spontaneous contractile activity. Strips from SD and +/+ rats, responded to electrical neuronal stimulation with a relaxation that was in part L-NNA and in part apamin sensitive, followed by a contraction which was decreased by atropine. In Ws/Ws rats, similar to +/+ rats, neurally mediated relaxation was L-NNA and apamin sensitive and the contraction was decreased by atropine. We conclude that in the rat LES, relaxation is mediated by NO and an apamin-sensitive mediator, and contraction primarily by acetylcholine. Despite the absence of c-Kit-positive ICC, nerve–muscle interaction can be accomplished likely by diffusion of neurotransmitters to the smooth muscle cells. The lack of c-Kit-positive ICC is related to an increase in the basal tone and spontaneous contractile activity. The presence of fibroblast-like ICC in Ws/Ws rats might represent immature ICC whose possible functions need further investigation. [ABSTRACT FROM AUTHOR]

Crohn's disease associated dysmotility has been attributed to fibrosis and damage to enteric nerves but injury to interstitial cells of Cajal (ICC) could also be involved. We assessed ICC in specimens obtained from patients with Crohn's disease and determined the relation between ICC and the inflammatory infiltrate, particularly mast cells (MC) using quantitative immunohistochemistry and electron microscopy. Ultrastructural injury to ICC was patchy in all ICC subtypes but ICC-Auerbach's plexus (AP) showed damage more frequently, i.e. swelling of mitochondria, decreased electron density, autophagosomes and partial depletion of the cytoplasm. Light microscopy confirmed a significant decrease in c-kit immunoreactivity for ICC-AP and an increased number of MC in the muscularis externa. Electron microscopy showed MC exhibiting piecemeal degranulation and making frequent and selective membrane-to-membrane contact with all types of injured ICC which suggests chronic release of granule content to affect ICC. Extent of ICC injury was not associated with duration of the disease. In conclusion, ultrastructural injury and loss of ICC-AP is evident in Crohn's disease. Epidemiological and morphological data suggest that ICC have the capacity to regenerate in spite of the chronic insult. The muscularis hosts a marked number of MC that exhibit piecemeal degranulation associated with ICC and may facilitate ICC maintenance. [ABSTRACT FROM AUTHOR]

The rhythmic motility of the intestine is regulated by the interstitial cells of Cajal (ICC) and the enteric nervous system. Rhythmic motility is considered to occur after the differentiation of mesenchymal progenitor cells into ICC during the late embryonic period. In this study, we successfully reconstructed a gut-like tissue demonstrating rhythmic contractions by culturing dispersed cells enzymatically isolated from the mouse intestine during the mid-embryonic period. These intestinal cells were reconstituted into a collagen gel at high density, made to proliferate considerably, and grew into a gut-like tissue after 1 week of culturing. The reconstituted tissue showed rhythmic contractions and stained positive for the specific marker proteins of neurones and ICC, PGP9.5 and c-Kit. The tissue also demonstrated network formation by developing nerve cells and ICC. Moreover, in the presence of nifedipine, c-Kit-immunopositive cells showed spontaneous Ca2+ oscillation, which is considered to be coupled to the electrical activity that corresponds to slow waves. Therefore, this culture system may be of use in elucidating the developmental mechanisms of gastrointestinal motility. [ABSTRACT FROM AUTHOR]

Interstitial cells of Cajal (ICC) within the gastrointestinal (GI) tract play a critical role in the generation of electrical slow waves and as mediators of enteric motor neurotransmission. Kit immunohistochemistry has proven to be a reliable method to identify the location of these cells within the tunica muscularis and to provide information on how the distribution and density of these cells change in a variety of GI motility disorders. Because of the labile nature of Kit or its detection, ultrastructural immunocytochemistry using conventional chemical fixation methods has been difficult. We describe a novel in vivo technique to label ICC within GI tissues. Using antibodies directed against the extracellular domain of the Kit receptor, we have been able to live-label the stomach with Kit while the animal is under anaesthesia and the organ is still receiving normal blood supply. This approach provided optimum maintenance of ultrastructural features with significant binding of antibody to the Kit receptor. The loss of ICC in many human motility disorders suggests exciting new hypotheses for their aetiology. This method will prove useful to investigate the ultrastructural changes that occur in ICC networks in animal models of motility disorders that are associated with the loss of these cells. [ABSTRACT FROM AUTHOR]

We examined the time-dependent changes in the immunoreactivity of the smooth muscle region and the accompanying motility disorder in a hapten-induced rat model of colitis. Histological analysis and myeloperoxidase (MPO) activity indicated that inflammatory cells infiltrated into the muscle layer at 2 days after 2,4,6-trinitrobenzenesulphonic acid (TNBS) treatment. The infiltrated immune cells then gradually decreased in number, but were still present at 14 days. The expression of proinflammatory cytokine mRNAs (TNF- α, IL-1 β and IL-6) and proteins in the muscle layer was increased at 2 days, then began to decrease, returning to control levels at 14 days. The frequency of spontaneous rhythmicity was suppressed at 2 and 7 days, and returned to control levels at 14 days. Consistent with these observations, the immunoreactivity of the interstitial cells of Cajal network was disrupted at 2 and 7 days, which then gradually reformed at 14 days. On the other hand, the myogenic contractions induced by high K+ and carbachol were decreased at 2 days, and were still inhibited at 14 days. These results suggest that spontaneous rhythmicity dysfunction may improve more rapidly than myogenic contractility dysfunction in a hapten-induced rat model of colitis. [ABSTRACT FROM AUTHOR]

Achalasia is dominated by injury to inhibitory nerves. As intramuscular interstitial cells of Cajal (ICC-IM) are proposed to form functional units with nitrergic nerves, their fate in achalasia may be critically important. We studied the relationship between loss of nitrergic nerves and injury to ICC-IM in patients with achalasia and determined associations between ICC-IM and mast cells (MC), using quantitative immunohistochemistry and electron microscopy. Loss of neuronal nitric oxide synthase (nNOS) immunoreactivity was completed within 3 years of acquiring achalasia. Thereafter, progressive ultrastructural injury to remaining nerve structures was evident. Within the first 2 years, the number of ICC-IM did not decline although ultrastructural injury was already present. Thereafter, loss of ICC-IM occurred unrelated to duration of disease. Damage to ICC-IM appeared unrelated to nerve injury. A significant MC infiltration was observed in the musculature; the number of MC was positively related to the persistent number of ICC-IM. Mast cell formed close contacts with ICC-IM and piecemeal-degranulation occurred towards ICC-IM. In conclusion, injury to ICC-IM in achalasia is variable, but not related to duration of disease and injury to nitrergic nerves. MC are prominent and form close functional contact with ICC-IM which may be responsible for their relatively long survival. [ABSTRACT FROM AUTHOR]