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Background & Aims: Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning., Methods: Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged., Results: Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm 2 , and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-μm 2  × 500-μm 2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel., Conclusions: Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Following colonic inflammation, the uninjured bladder afferent neurons are also activated. The mechanisms and pathways underlying this sensory neuron cross-activation (from injured neurons to uninjured neurons) are not fully understood. Colonic and bladder afferent neurons reside in the same spinal segments and are separated by satellite glial cells (SGCs) and extracellular matrix in dorsal root ganglia (DRG). SGCs communicate with sensory neurons in a bidirectional fashion. This review summarizes the differentially regulated genes/proteins in the injured and uninjured DRG neurons and explores the role of SGCs in regulation of sensory neuron crosstalk in visceral cross-organ sensitization. The review also highlights the paracrine pathways in mediating neuron-SGC and SGC-neuron coupling with an emphasis on the neurotrophins and purinergic systems. Finally, I discuss the results from recent RNAseq profiling of SGCs to reveal useful molecular markers for characterization, functional study, and therapeutic targets of SGCs. SIGNIFICANCE STATEMENT: Satellite glial cells (SGCs) are the largest glial subtypes in sensory ganglia and play a critical role in mediating sensory neuron crosstalk, an underlying mechanism in colon-bladder cross-sensitization. Identification of novel and unique molecular markers of SGCs can advance the discovery of therapeutic targets in treatment of chronic pain including visceral pain comorbidity., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Here, we present our protocol to culture enteric glial cells from the submucosal and myenteric plexus of neonatal and juvenile pig colons. We describe steps for colon isolation, microdissection, and enzymatic and mechanical dissociation. We include procedures for passaging and analyzing cell yield, freeze/thaw efficiency, and purity. This protocol allows for the generation of primary cultures of enteric glial cells from single-cell suspensions of microdissected layers of the colon wall and can be used to culture enteric glia from human colon specimens. For complete details on the use and execution of this protocol, please refer to Ziegler et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Understanding how the gut communicates with the brain, via sensory nerves, is of significant interest to medical science. Enteroendocrine cells (EEC) that line the mucosa of the gastrointestinal tract release neurochemicals, including the largest quantity of 5-hydroxytryptamine (5-HT). How the release of substances, like 5-HT, from enterochromaffin (EC) cells activates vagal afferent nerve endings is unresolved. We performed anterograde labelling from nodose ganglia in vivo and identified vagal afferent axons and nerve endings in the mucosa of whole-mount full-length preparations of mouse colon. We then determined the spatial relationship between mucosal-projecting vagal afferent nerve endings and EC cells in situ using 3D imaging. The mean distances between vagal afferent nerve endings in the mucosa, or nearest varicosities along vagal afferent axon branches, and the nearest EC cell were 29.6 ± 19.2 μm (n = 107, N = 6) and 25.7 ± 15.2 μm (n = 119, N = 6), respectively. No vagal afferent endings made close contacts with EC cells. The distances between EC cells and vagal afferent endings are many hundreds of times greater than known distances between pre- and post-synaptic membranes (typically 10-20 nm) that underlie synaptic transmission in vertebrates. The absence of any close physical contacts between 5-HT-containing EC cells and vagal afferent nerve endings in the mucosa leads to the inescapable conclusion that the mechanism by which 5-HT release from ECs in the colonic mucosa occurs in a paracrine fashion, to activate vagal afferents., (© 2024. The Author(s).)

Abdominal pain is a cardinal symptom of inflammatory bowel disease (IBD). Transient receptor potential (TRP) channels contribute to abdominal pain in preclinical models of IBD, and TRP melastatin 3 (TRPM3) has recently been implicated in inflammatory bladder and joint pain in rodents. We hypothesized that TRPM3 is involved in colonic sensation and is sensitized during colitis. We used immunohistochemistry, ratiometric Ca 2+ imaging, and colonic afferent nerve recordings in mice to evaluate TRPM3 protein expression in colon-projecting dorsal root ganglion (DRG) neurons, as well as functional activity in DRG neurons and colonic afferent nerves. Colitis was induced using dextran sulfate sodium (DSS) in drinking water. TRPM3 protein expression was observed in 76% of colon-projecting DRG neurons and was often colocalized with calcitonin gene-related peptide. The magnitudes of intracellular Ca 2+ transients in DRG neurons in response to the TRPM3 agonists CIM-0216 and pregnenolone sulfate sodium were significantly greater in neurons from mice with colitis compared with controls. In addition, the percentage of DRG neurons from mice with colitis that responded to CIM-0216 was significantly increased. CIM-0216 also increased the firing rate of colonic afferent nerves from control and mice with colitis. The TRPM3 inhibitor isosakuranetin inhibited the mechanosensitive response to distension of wide dynamic range afferent nerve units from mice with colitis but had no effect in control mice. Thus, TRPM3 contributes to colonic sensory transduction and may be a potential target for treating pain in IBD. NEW & NOTEWORTHY This is the first study to characterize TRPM3 protein expression and function in colon-projecting DRG neurons. A TRPM3 agonist excited DRG neurons and colonic afferent nerves from healthy mice. TRPM3 agonist responses in DRG neurons were elevated during colitis. Inhibiting TRPM3 reduced the firing of wide dynamic range afferent nerves from mice with colitis but had no effect in control mice.

Background & Aims: Intestinal inflammation is associated with loss of enteric cholinergic neurons. Given the systemic anti-inflammatory role of cholinergic innervation, we hypothesized that enteric cholinergic neurons similarly possess anti-inflammatory properties and may represent a novel target to treat inflammatory bowel disease., Methods: Mice were fed 2.5% dextran sodium sulfate (DSS) for 7 days to induce colitis. Cholinergic enteric neurons, which express choline acetyltransferase (ChAT), were focally ablated in the midcolon of ChAT::Cre;R26-iDTR mice by local injection of diphtheria toxin before colitis induction. Activation of enteric cholinergic neurons was achieved using ChAT::Cre;R26-ChR2 mice, in which ChAT+ neurons express channelrhodopsin-2, with daily blue light stimulation delivered via an intracolonic probe during the 7 days of DSS treatment. Colitis severity, ENS structure, and smooth muscle contractility were assessed by histology, immunohistochemistry, quantitative polymerase chain reaction, organ bath, and electromyography. In vitro studies assessed the anti-inflammatory role of enteric cholinergic neurons on cultured muscularis macrophages., Results: Ablation of ChAT+ neurons in DSS-treated mice exacerbated colitis, as measured by weight loss, colon shortening, histologic inflammation, and CD45 + cell infiltration, and led to colonic dysmotility. Conversely, optogenetic activation of enteric cholinergic neurons improved colitis, preserved smooth muscle contractility, protected against loss of cholinergic neurons, and reduced proinflammatory cytokine production. Both acetylcholine and optogenetic cholinergic neuron activation in vitro reduced proinflammatory cytokine expression in lipopolysaccharide-stimulated muscularis macrophages., Conclusions: These findings show that enteric cholinergic neurons have an anti-inflammatory role in the colon and should be explored as a potential inflammatory bowel disease treatment., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction:  "Tuft" cells, also known as brush or caveolated cells, are characteristically fusiform shaped, with a distinct apical "tuft" of microvilli extending into the lumen. Double cortin-like kinase 1 (DCLK1) is a microtubule kinase and is a specific marker of intestinal tuft cells. DCLK1-positive tuft cells have been shown to play a key role in gastrointestinal chemosensation, inflammation, and neurotransmission. DCLK1 and Choline acetyltransferase (ChAT), the enzymes responsible for acetylcholine production, are reported to be coexpressed within the gastrointestinal tract. We designed this study to investigate the hypothesis that DCLK1 gene expression is altered in Hirschsprung's disease (HSCR).Materials and Methods:  HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was undertaken to quantify DCLK1 gene expression, and immunolabeling of DCLK1-positive tuft cells was visualized using confocal microscopy.Results:  qRT-PCR analysis revealed significant downregulation of the DCLK1 gene in both aganglionic and ganglionic HSCR specimens compared with controls (p < 0.05). Confocal microscopy revealed DCLK1-positive tuft cell expression within the colonic mucosa, with a reduction in expression in both aganglionic and ganglionic HSCR colon compared with controls.Conclusion:  DCLK1 is significantly downregulated in HSCR colon, suggesting a role for tuft cells in cholinergic neurotransmission of the distal colon. The marked decrease in DCLK1 expression within ganglionic specimens highlights the physiologically abnormal nature of this segment in HSCR patients. [ABSTRACT FROM AUTHOR]

Background: Irritable bowel syndrome (IBS) is a common disease with intestinal dysmotility, whose mechanism remains elusive. TMEM16A is a calcium-activated chloride channel (CaCC) involved in intestinal slow-wave generation.Aims: To investigate whether TMEM16A is involved in colonic dysmotility in IBS.Methods: A rat model of IBS was established by chronic water avoidance stress (WAS). Colonic pathological alterations were evaluated histologically, and intestinal motility was assessed by intestinal transit time (ITT) and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response (VMR) to colorectal distension (CRD). TMEM16A expression was evaluated by RT-PCR, Western blot, and immunofluorescence. Colonic muscle strip contractility was measured by isometric transducers, and the effect of niflumic acid (NFA), a CaCC antagonist, on colonic motility was examined.Results: After 10 days of WAS exposure, ITT was decreased and FWC was elevated. Furthermore, VMR magnitude of WAS rats in response to CRD was significantly enhanced. Protein and mRNA levels of TMEM16A in colon were considerably increased after WAS. The percentage of TMEM16A-positive neurons in myenteric plexus (MP) of WAS rats was significantly higher than controls. Pharmacological blockade of TMEM16A activity by NFA considerably enhanced ITT, with concentration-dependent declines in FWC and VMR magnitude in NFA-treated rats. Further, spontaneous contraction of colonic strips of NFA-treated rats was significantly ameliorated in a concentration-dependent manner in vitro.Conclusions: Upregulation of TMEM16A in MP neurons may play an important role in chronic stress-induced colonic hypermotility, making CaCC-blocking drugs a putatively effective treatment method for colonic hypermotility in IBS. [ABSTRACT FROM AUTHOR]

Dr. Orvar Swenson is best remembered for developing the Swenson pull-through, a technique he developed to treat Hirschsprung's disease. After graduating from Harvard Medical School and beginning his residency at Peter Bent Brigham Hospital, Dr. Swenson observed that patients with Hirschsprung's disease and toxic megacolon resumed normal bowel function after placement of transverse colostomies. His observation led to studying the patency of his patients' colons using barium enema contrast studies. At the collapsed portion of the colon, he performed rectal biopsies leading to the discovery that the cause of Hirschsprung's disease is that the collapsed portion of the colon lacks the Auerbach plexus. The Swenson pull-through removes this aganglionic portion of the colon and cures the patient. His career grew from there as he traveled to academic institutions teaching his technique. He is remembered fondly for his contributions to pediatric surgery through the restructuring of pediatric surgery departments, pediatric surgery research, and writing and editing multiple volumes of Pediatric Surgery, the standard textbook for pediatric surgeons. He died peacefully in 2012 at the age of 103 years. [ABSTRACT FROM AUTHOR]

Propulsion of contents in the gastrointestinal tract requires coordinated functions of the extrinsic nerves to the gut from the brain and spinal cord, as well as the neuromuscular apparatus within the gut. The latter includes excitatory and inhibitory neurons, pacemaker cells such as the interstitial cells of Cajal and fibroblast-like cells, and smooth muscle cells. Coordination between these extrinsic and enteric neurons results in propulsive functions which include peristaltic reflexes, migrating motor complexes in the small intestine which serve as the housekeeper propelling to the colon the residual content after digestion, and mass movements in the colon which lead to defecation., Competing Interests: M.C. received funding for research in gastroparesis and stomach motility from Takeda and Vanda, and he serves as a consultant to Takeda with compensation to his employer, Mayo Clinic., (Thieme. All rights reserved.)

Purpose: Laminin, an extracellular matrix molecule, is essential for normal development of the nervous system. The alpha1 subunit of laminin-1 (LAMA1) has been reported to promote neurites and outgrowth and is expressed only during embryogenesis. Previously, we developed a Sox10 transgenic version of the Endothelin receptor-B (Ednrb) mouse to visualize Enteric neural crest-derived cell (ENCC)s with a green fluorescent protein, Venus. We designed this study to investigate the expression of LAMA1 using Sox10-VENUS mice gut.Methods: We harvested the gut on days 13.5 (E13.5) and 15.5 (E15.5) of gestation. Sox10-VENUS+/Ednrb -/- mice (n = 8) were compared with Sox10-VENUS+/Ednrb +/+ mice (n = 8) as controls. Gene expression of LAMA1 was analysed by real-time RT-PCR. Fluorescent immunohistochemistry was performed to assess protein distribution.Results: The relative mRNA expression levels of LAMA1 were significantly increased in HD in the proximal and distal colon on E15.5 compared to controls (p < 0.05), whereas there were no significant differences on E13.5. LAMA1 was expressed in the serosa, submucosa and basal lamina in the gut, and was markedly increased in the proximal and distal colon of HD on E15.5.Conclusions: Altered LAMA1 expression in the aganglionic region may contribute to impaired ENCC migration, resulting in HD. These data could help in understanding the pathophysiologic interactions between LAMA1 and ENCC migration. [ABSTRACT FROM AUTHOR]

Abdominal pain is one of the major symptoms in bowel obstruction (BO); its cellular mechanisms remain incompletely understood. We tested the hypothesis that mechanical stress in obstruction upregulates expression of nociception mediator nerve growth factor (NGF) in gut smooth muscle cells (SMCs), and NGF sensitizes primary sensory nerve to contribute to pain in BO. Partial colon obstruction was induced with a silicon band implanted in the distal bowel of Sprague-Dawley rats. Colon-projecting sensory neurons in the dorsal root ganglia (T13 to L2) were identified for patch-clamp and gene expression studies. Referred visceral sensitivity was assessed by measuring withdrawal response to stimulation by von Frey filaments in the lower abdomen. Membrane excitability of colon-projecting dorsal root ganglia neurons was significantly enhanced, and the withdrawal response to von Frey filament stimulation markedly increased in BO rats. The expression of NGF mRNA and protein was increased in a time-dependent manner (day 1-day 7) in colonic SMC but not in mucosa/submucosa of the obstructed colon. Mechanical stretch in vitro caused robust NGF mRNA and protein expression in colonic SMC. Treatment with anti-NGF antibody attenuated colon neuron hyperexcitability and referred hypersensitivity in BO rats. Obstruction led to significant increases of tetrodotoxin-resistant Na currents and mRNA expression of Nav1.8 but not Nav1.6 and Nav1.7 in colon neurons; these changes were abolished by anti-NGF treatment. In conclusion, mechanical stress-induced upregulation of NGF in colon SMC underlies the visceral hypersensitivity in BO through increased gene expression and activity of tetrodotoxin-resistant Na channels in sensory neurons. [ABSTRACT FROM AUTHOR]

The gut-brain axis has received increasing attention recently due to evidence that colonic microbes can affect brain function and behavior. However, little is known about the innervation of the colon by a major component of the gut-brain axis, vagal afferent neurons. Furthermore, it is currently unknown whether individual NG neurons or DRG neurons innervate both the proximal and distal colon. We aimed to quantify the number of vagal and spinal afferent neurons that innervate the colon; and determine whether these individual neurons simultaneously innervate the mouse proximal and distal colon. C57Bl/6 mice received injections of a combination of retrograde tracers that were either injected into the muscularis externa of the proximal or the distal colon: fast blue, DiI and DiO. Five to seven percent of lumbosacral and thoracolumbar spinal afferent neurons, and 25% of vagal afferent neurons were labelled by injections of DiI and DiO into the colon. We also found that approximately 8% of NG neurons innervate the distal colon. Ten percent of labeled thoracolumbar and 15% of labeled lumbosacral DRG neurons innervate both the distal and proximal colon. Eighteen percent of labeled NG neurons innervated both the distal and proximal colon. In conclusion, vagal afferent innervation of the distal colon is less extensive than the proximal colon, whereas a similar gradient was not observed for the spinal afferent innervation. Furthermore, overlap appears to exist between the receptive fields of vagal and spinal afferent neurons that innervate the proximal and distal colon., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The effective management of visceral pain is a significant unmet clinical need for those affected by gastrointestinal diseases, such as inflammatory bowel disease (IBD). The rational design of novel analgesics requires a greater understanding of the mediators and mechanisms underpinning visceral pain. Interleukin-13 (IL-13) production by immune cells residing in the gut is elevated in IBD, and IL-13 appears to be important in the development of experimental colitis. Furthermore, receptors for IL-13 are expressed by neurons innervating the colon, though it is not known whether IL-13 plays any role in visceral nociception per se. To resolve this, we used Ca 2+ imaging of cultured sensory neurons and ex vivo electrophysiological recording from the lumbar splanchnic nerve innervating the distal colon. Ca 2+ imaging revealed the stimulation of small-diameter, capsaicin-sensitive sensory neurons by IL-13, indicating that IL-13 likely stimulates nociceptors. IL-13-evoked Ca 2+ signals were attenuated by inhibition of Janus (JAK) and p38 kinases. In the lumbar splanchnic nerve, IL-13 did not elevate baseline firing, nor sensitize the response to capsaicin application, but did enhance the response to distention of the colon. In line with Ca 2+ imaging experiments, IL-13-mediated sensitization of the afferent response to colon distention was blocked by inhibition of either JAK or p38 kinase signaling. Together, these data highlight a potential role for IL-13 in visceral nociception and implicate JAK and p38 kinases in pronociceptive signaling downstream of IL-13.

Objectives: The Enteric Nervous System (ENS) present in the wall of the gut is currently being explored because of its influence on the gut and beyond. In this context, the morphology of developing ENS has not been completely understood in humans due to lack of adequate literature. The aim of the present study was to observe the morphology of the enteric neurons in the human fetal colon and compare the findings in ascending colon a midgut derivative and descending colon a hindgut derivative at various weeks of gestation (WG)., Material and Methods: Tissue samples from 15 aborted fetuses (11 WG to 2 months postnatal) were processed for Cresyl violet, H & E staining, and NADPH Diaphorase histochemistry. The morphometric analysis was done by calculating the neuronal number density and neuronal fractional area. The Student t-test; Mann-Whitney test and Wilcoxon signed-rank test were used to analyze the data., Results: The muscularis externa with two distinct layers was visible as early as 13 WG and the muscularis mucosae was first observed at 18 WG. The size of the myenteric neurons appeared to be larger with increasing weeks of gestation suggesting a process of neuronal maturation. The neuronal number density and neuronal fractional area seemed to be reduced with advancing fetal age. There was no marked difference between the ascending and sigmoid colon. At 23 and 26 WG, a mature pattern of nitrergic innervation was observed., Conclusion: This study is done on human fetal tissue samples unlike previous studies on animal samples to comprehend the morphology of developing ENS. It will aid in understanding the effect of ENS on various neurological disorders., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

The porcine gut is increasingly regarded as a useful translational model. The enteric nervous system in the colon coordinates diverse functions. However, knowledge of the molecular profiling of porcine enteric nerve system and its similarity to that of human is still lacking. We identified the distinct transcriptional programs associated with functional characteristics between inner submucosal and myenteric ganglia in porcine proximal and distal colon using bulk RNA and single-cell RNA sequencing. Comparative transcriptomics of myenteric ganglia in corresponding colonic regions of pig and human revealed highly conserved programs in porcine proximal and distal colon, which explained >96% of their transcriptomic responses to vagal nerve stimulation, suggesting that porcine proximal and distal colon could serve as predictors in translational studies. The conserved programs specific for inflammatory modulation were displayed in pigs with vagal nerve stimulation. This study provides a valuable transcriptomic resource for understanding of human colonic functions and neuromodulation using porcine model., (© 2023. The Author(s).)

The speed of pellet propulsion through the isolated guinea pig distal colon in vitro significantly exceeds in vivo measurements, suggesting a role for inhibitory mechanisms from sources outside the gut. The aim of this study was to investigate the effects of sympathetic nerve stimulation on three different neurogenic motor behaviors of the distal colon: transient neural events (TNEs), colonic motor complexes (CMCs), and pellet propulsion. To do this, segments of guinea pig distal colon with intact connections to the inferior mesenteric ganglion (IMG) were set up in organ baths allowing for simultaneous extracellular suction electrode recordings from smooth muscle, video recordings for diameter mapping, and intraluminal manometry. Electrical stimulation (1-20 Hz) of colonic nerves surrounding the inferior mesenteric artery caused a statistically significant, frequency-dependent inhibition of TNEs, as well as single pellet propulsion, from frequencies of 5 Hz and greater. Significant inhibition of CMCs required stimulation frequencies of 10 Hz and greater. Phentolamine (3.6 μM) abolished effects of colonic nerve stimulation, consistent with a sympathetic noradrenergic mechanism. Sympathetic inhibition was constrained to regions with intact extrinsic nerve pathways, allowing normal motor behaviors to continue without modulation in adjacent extrinsically denervated regions of the same colonic segments. The results demonstrate differential sensitivities to sympathetic input among distinct neurogenic motor behaviors of the colon. Together with findings indicating CMCs activate colo-colonic sympathetic reflexes through the IMG, these results raise the possibility that CMCs may paradoxically facilitate suppression of pellet movement in vivo, through peripheral sympathetic reflex circuits., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Background and Aims: Gut functions including motility, secretion, and blood flow are largely controlled by the enteric nervous system. Characterizing the different classes of enteric neurons in the human gut is an important step to understand how its circuitry is organized and how it is affected by disease., Methods: Using multiplexed immunohistochemistry, 12 discriminating antisera were applied to distinguish different classes of myenteric neurons in the human colon (2596 neurons, 12 patients) according to their chemical coding. All antisera were applied to every neuron, in multiple layers, separated by elutions., Results: A total of 164 combinations of immunohistochemical markers were present among the 2596 neurons, which could be divided into 20 classes, with statistical validation. Putative functions were ascribed for 4 classes of putative excitatory motor neurons (EMN1-4), 4 inhibitory motor neurons (IMN1-4), 3 ascending interneurons (AIN1-3), 6 descending interneurons (DIN1-6), 2 classes of multiaxonal sensory neurons (SN1-2), and a small, miscellaneous group (1.8% of total). Soma-dendritic morphology was analyzed, revealing 5 common shapes distributed differentially between the 20 classes. Distinctive baskets of axonal varicosities surrounded 45% of myenteric nerve cell bodies and were associated with close appositions, suggesting possible connectivity. Baskets of cholinergic terminals and several other types of baskets selectively targeted ascending interneurons and excitatory motor neurons but were significantly sparser around inhibitory motor neurons., Conclusions: Using a simple immunohistochemical method, human myenteric neurons were shown to comprise multiple classes based on chemical coding and morphology and dense clusters of axonal varicosities were selectively associated with some classes., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Isolectin B4-binding (IB4+) dorsal root ganglion (DRG) neurons are distinct from peptidergic DRG neurons in their terminal location in the spinal cord and respective contributions to various classes and modalities of nociception. In DRG neurons innervating the mouse colon (c-DRG neurons), the reported proportion of IB4+ population is inconsistent across studies, and little is known regarding their role in colorectal mechanonociception. To address these issues, in C57BL/6J mice, we quantified IB4+ binding after labeling c-DRG neurons with Fast Blue and examined functional consequences of ablating these neurons by IB4-conjugated saporin. Sixty-one percent of Fast Blue-labeled neurons in the L6 DRG were IB4+, and 95% of these IB4+ c-DRG neurons were peptidergic. Intrathecal administration of IB4-conjugated saporin reduced the proportion of IB4+ c-DRG neurons to 37%, which was due to the loss of c-DRG neurons showing strong to medium IB4+ intensity; c-DRG neurons with weak IB4+ intensity were spared. However, this loss altered neither nociceptive behaviors to colorectal distension nor the relative proportions of stretch-sensitive colorectal afferent classes characterized by single-fiber recordings. These findings demonstrate that more than 1 half of viscerosensory L6 c-DRG neurons in C57BL/6J mouse are IB4+ and suggest, in contrast to the reported roles of IB4+/nonpeptidergic neurons in cutaneous mechanonociception, c-DRG neurons with strong-to-medium IB4+ intensity do not play a significant role in colorectal mechanonociception. [ABSTRACT FROM AUTHOR]

Hirschsprung's disease (HSCR) is a severe congenital anomaly of the enteric nervous system (ENS) characterized by functional intestinal obstruction due to a lack of intrinsic innervation in the distal bowel. Distal innervation deficiency results from incomplete colonization of the bowel by enteric neural crest cells (eNCCs), the ENS precursors. Here, we report the generation of a mouse model for HSCR--named Holstein--that contains an untargeted transgenic insertion upstream of the collagen-6α4 (Col6a4) gene. This insertion induces eNCC-specific upregulation of Col6a4 expression that increases total collagen VI protein levels in the extracellular matrix (ECM) surrounding both the developing and the postnatal ENS. Increased collagen VI levels during development mainly result in slower migration of eNCCs. This appears to be due to the fact that collagen VI is a poor substratum for supporting eNCC migration and can even interfere with the migration-promoting effects of fibronectin. Importantly, for a majority of patients in a HSCR cohort, the myenteric ganglia from the ganglionated region are also specifically surrounded by abundant collagen VI microfibrils, an outcome accentuated by Down syndrome. Collectively, our data thus unveil a clinically relevant pathogenic mechanism for HSCR that involves cell-autonomous changes in ECM composition surrounding eNCCs. Moreover, as COL6A1 and COL6A2 are on human Chr.21q, this mechanism is highly relevant to the predisposition of patients with Down syndrome to HSCR. [ABSTRACT FROM AUTHOR]

Experiencing early life stress or injury increases a woman's likelihood of developing vulvodynia and concomitant dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. To investigate the outcome of neonatal vaginal irritation (NVI), female mouse pups were administered intravaginal zymosan on postnatal days 8 and 10 and were assessed as adults for vaginal hypersensitivity by measuring the visceromotor response to vaginal balloon distension (VBD). Western blotting and calcium imaging were performed to measure transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) in the vagina and innervating primary sensory neurons. Serum corticosterone (CORT), mast cell degranulation, and corticotropin-releasing factor receptor 1 (CRF1) expression were measured as indicators of peripheral HPA axis activation. Colorectal and hind paw sensitivity were measured to determine cross-sensitization resulting from NVI. Adult NVI mice had significantly larger visceromotor response during VBD than naive mice. TRPA1 protein expression was significantly elevated in the vagina, and calcium transients evoked by mustard oil (TRPA1 ligand) or capsaicin (TRPV1 ligand) were significantly decreased in dorsal root ganglion from NVI mice, despite displaying increased depolarization-evoked calcium transients. Serum CORT, vaginal mast cell degranulation, and CRF1 protein expression were all significantly increased in NVI mice, as were colorectal and hind paw mechanical and thermal sensitivity. Neonatal treatment with a CRF1 antagonist, NBI 35965, immediately before zymosan administration largely attenuated many of the effects of NVI. These results suggest that NVI produces chronic hypersensitivity of the vagina, as well as of adjacent visceral and distant somatic structures, driven in part by increased HPA axis activation. [ABSTRACT FROM AUTHOR]

Background: Irritable bowel syndrome and bladder pain syndrome often overlap and are both characterized by visceral hypersensitivity. Since pelvic organs share common sensory pathways, it is likely that those syndromes involve a cross-sensitization of the bladder and the colon. The precise pathophysiology remains poorly understood., Aim: To develop a model of chronic bladder-colon cross-sensitization and to investigate the mech-anisms involved., Methods: Chronic cross-organ visceral sensitization was obtained in C57BL/6 mice using ultrasound-guided intravesical injections of acetic acid under brief isoflurane anesthesia. Colorectal sensitivity was assessed in conscious mice by measuring intracolonic pressure during isobaric colorectal distensions. Myeloperoxidase, used as a marker of colorectal inflammation, was measured in the colon, and colorectal permeability was measured using chambers. c-Fos protein expression, used as a marker of neuronal activation, was assessed in the spinal cord (L6-S1 level) using immunohistochemistry. Green fluorescent protein on the fractalkine receptor-positive mice were used to identify and count microglia cells in the L6-S1 dorsal horn of the spinal cord. The expression of NK1 receptors and MAPK-p38 were quantified in the spinal cord using western blot., Results: Visceral hypersensitivity to colorectal distension was observed after the intravesical injection of acetic acid vs saline ( P < 0.0001). This effect started 1 h post-injection and lasted up to 7 d post-injection. No increased permeability or inflammation was shown in the bladder or colon 7 d post-injection. Visceral hypersensitivity was associated with the increased expression of c-Fos protein in the spinal cord ( P < 0.0001). In green fluorescent protein on the fractalkine receptor-positive mice, intravesical acetic acid injection resulted in an increased number of microglia cells in the L6-S1 dorsal horn of the spinal cord ( P < 0.0001). NK1 receptor and MAPK-p38 levels were increased in the spinal cord up to 7 d after injection ( P = 0.007 and 0.023 respectively). Colorectal sensitization was prevented by intrathecal or intracerebroventricular injections of minocycline, a microglia inhibitor, by intracerebroventricular injection of CP-99994 dihydrochloride, a NK1 antagonist, and by intracerebroventricular injection of SB203580, a MAPK-p38 inhibitor., Conclusion: We describe a new model of cross-organ visceral sensitization between the bladder and the colon in mice. Intravesical injections of acetic acid induced a long-lasting colorectal hypersensitivity to distension, mediated by neuroglial interactions, MAPK-p38 phosphorylation and the NK1 receptor., Competing Interests: Conflict-of-interest statement: None of the authors have any conflicts of interest., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

Doublecortin-like kinase 1 protein (DCLK1) is a gastrointestinal tuft cell marker that has been proposed to identify quiescent and tumor growth-sustaining stem cells. DCLK1⁺ tuft cells are increased in inflammation-induced carcinogenesis; however, the role of these cells within the gastrointestinal epithelium and their potential as cancer-initiating cells are poorly understood. Here, using a BAC-CreERT-dependent genetic lineage-tracing strategy, we determined that a subpopulation of DCLK1⁺ cells is extremely long lived and possesses rare stem cell abilities. Moreover, genetic ablation of Dclk1 revealed that DCLK1⁺ tuft cells contribute to recovery following intestinal and colonic injury. Surprisingly, conditional knockdown of the Wnt regulator APC in DCLK1⁺ cells was not sufficient to drive colonic carcinogenesis under normal conditions; however, dextran sodium sulfate-induced (DSS-induced) colitis promoted the development of poorly differentiated colonic adenocarcinoma in mice lacking APC in DCLK1⁺ cells. Importantly, colonic tumor formation occurred even when colitis onset was delayed for up to 3 months after induced APC loss in DCLK1⁺ cells. Thus, our data define an intestinal DCLK1⁺ tuft cell population that is long lived, quiescent, and important for intestinal homeostasis and regeneration. Long-lived DCLK1⁺ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve to initiate colon cancer. [ABSTRACT FROM AUTHOR]

Background and Aims: Researchers have not clearly described the clinical and pathogenetic features of hypoganglionosis and adult-onset Hirschsprung's disease, which cause pseudo-obstruction or intractable constipation. We conducted this study to explore these features of hypoganglionosis and adult-onset Hirschsprung's disease in Korean patients.Methods: We enrolled 24 patients pathologically confirmed as having hypoganglionosis and 11 as having adult-onset Hirschsprung's disease. We recruited 26 subjects who had undergone operation for nonobstructive colon cancer and 45 healthy volunteers as controls. We described their clinical features, investigated ganglion cells and interstitial cells of Cajal (ICC), and analyzed RET, EDNRB, EDN3, and SOX10 genes.Results: We classified hypoganglionosis patients into two groups: type I (focal type, n = 13), with focally narrowed transition zone (TZ); and type II (diffuse type, n = 11), without transition zone. Hypoganglionosis patients had significantly fewer ganglion cells than the controls, and those cells were scarcer in the transition zone than in the proximal dilated area (P < 0.05). The ICC numbers in both diseases were significantly lower than in controls; however, they were similar between transition zone and the proximal dilated area in hypoganglionosis. In adult-onset Hirschsprung's disease, two significant intronic RET polymorphic variants, IVS14-24G>A and IVS19+47T>C, were significantly associated with adult-onset Hirschsprung's disease (P = 0.0122 and 0.0295, respectively), but not with hypoganglionosis.Conclusions: Hypoganglionosis and adult-onset Hirschsprung's disease have different pathophysiologic characteristics, although their clinical presentations are similar. We suggest that there are two subgroups of hypoganglionosis: those with or without a focally narrowed transition zone with a profoundly diminished number of ganglion cells. [ABSTRACT FROM AUTHOR]

Some patients with chronic constipation may undergo colectomy yielding tissue appropriate to diagnosis of underlying neuromuscular pathology. The analysis of such tissue has, over the past 40 years, fuelled research that has explored the presence of neuropathy, myopathy and more recently changes in interstitial cells of Cajal (ICC). In this chapter, the data from these studies have been critically reviewed in the context of the significant methodological and interpretative issues that beset the field of gastrointestinal neuromuscular pathology. On this basis, reductions in ICC appear to a consistent finding but one whose role as a primary cause of slow-transit constipation requires further evaluation. Findings indicative of significant neuropathy or myopathy are variable and in many studies subject to considerable methodological bias. Methods with practical diagnostic utility in the individual patient have rarely been employed and require further validation in respect of normative data. [Copyright &y& Elsevier]

Purpose: Hirschsprung's disease is characterised by the congenital absence of ganglion cells beginning in the distal rectum and extending proximally for varying distances. 'Zonal aganglionosis' is a phenomenon involving a zone of aganglionosis occurring within normally innervated intestine. 'Skip segment' Hirschsprung's disease (SSHD) involves a 'skip area' of normally ganglionated intestine, surrounded proximally and distally by aganglionosis. While Hirschsprung's disease is believed to be the result of incomplete craniocaudal migration of neural crest-derived cells, the occurrence of SSHD has no clear embryological explanation. The aim of this study was to perform a systematic review of SSHD, reported in the literature between 1954 and 2009, in order to determine the clinical characteristics of this rare entity and its significance.Methods: The first reported case of SSHD was published in 1954. A systematic review of SSHD cases in the literature, from 1954 to 2009, was carried out using the electronic database 'Pubmed'. Detailed information was recorded regarding the age, gender, presenting symptoms and location of the skip segment in each patient.Results: 24 cases of SSHD have been reported in the literature to date. 18/24 (75%) of these cases were males and 6/24 (25%) were females. Of these, 22/24 (92%) were cases of total colonic aganglionosis (TCA), and 2/24 (8%) were rectosigmoid Hirschsprung's disease. Of the 22 TCA cases, 9 (41%) had a skip segment in the transverse colon, 6 (27%) in the ascending colon, 2 (9%) in the caecum and 5 (23%) had multiple skip segments. In both rectosigmoid Hirschsprung's disease cases, the skip segment was in the sigmoid colon. Overall, the length of the skip segment was variable, with the entire transverse colon ganglionated in some cases.Conclusion: SSHD occurs predominantly in patients with TCA. The existence of a skip area of normally innervated colon in TCA may influence surgical management, enabling surgeons to preserve and use the ganglionated skip area during pull-through operations. [ABSTRACT FROM AUTHOR]

Purpose: The definitive surgical management of Hirschsprung's disease (HD) depends on an unambiguous histological diagnosis of bowel aganglionosis, as well as an accurate assessment of its proximal extent from the rectum or 'leveling'. This study aimed to evaluate and compare the transition zone in rectosigmoid and long segment colonic forms of HD.Methods: One hundred and fifteen cases of rectosigmoid Hirschsprung's disease (RSHD) and 20 cases of long segment colonic Hirschsprung's disease (LScHD) managed from 1998 to 2008 with a consensus prospective protocol were included in this clinicopathological analysis.Results: In RSHD, the radiologic transition zone (rTZ)/gross appearance at laparotomy correlated with the histological 'leveling' in the majority (96%); the histologic transition zone was also short and predictable. In contrast, the rTZ in LScHD was conclusive in only 53%; overall the radiologic and histological transition zones concurred in only 26%. The histologic transition zone extended proximally for an unpredictable length before normal circumferential innervation was noted.Conclusions: A stringent intraoperative histological evaluation of the colon as outlined is more helpful than radiology to establish the distal level of normally innervated bowel in LScHD and achieve an accurate leveling during a colostomy/pull through. [ABSTRACT FROM AUTHOR]

Background: The performance characteristics of colonic sensation and motility measurements are unclear.Aim: To compare left colon compliance, tone, and sensation in males and females and to evaluate inter and intra-individual coefficients of variation (COV) in these measurements.Methods: Data were acquired using standard barostat methods, by one technologist, in 72 human volunteers (38 males, 18-65 years). We measured compliance, fasting tone, and sensation during baseline and post-placebo; postprandial (PP) tone was measured only post-placebo. Compliance and thresholds for first sensation, gas, and pain were measured using ascending method of limits; sensory ratings (0-100 mm VAS) using random phasic distensions at 8-36 mmHg above baseline operating pressure. Change in PP tone was measured by barostat balloon volume for the first 30 min after a 1000-kcal meal. Inter-COV was calculated as 100 (SD/mean), and intra-COV as (100 x SD delta/overall mean).Results: There were no statistically significant associations with gender for most sensory or motor data at baseline. A modest association of fasting colonic tone and gender was observed. COV are lower (20-35%) for compliance, fasting tone, pain threshold, and sensation ratings than for PP tone and threshold for first or gas sensation (>45%). COV data are similar in males and females; sensation COVs appear smaller in females than in males.Conclusions: Testing of compliance, tone and pain, and gas sensation in left colon performs adequately to assess these functions in humans. Lower COV for sensation tests among females is relevant to plan studies of drugs intended for functional GI disorders. [ABSTRACT FROM AUTHOR]

Introduction: The enteric nervous system is an intrinsic network of nerve cells and glia within the gastrointestinal wall, which originates in the vagal and sacral neural tube. The vagal neural tube is known to supply the colorectum with the majority of its nerve cells, and its ablation during early development produces a hypoganglionic colorectum. We hypothesized that the cholinergic nerve activity in the chick embryo hypoganglionic colorectum is decreased similar to the human situation and, therefore, this study is designed to investigate cholinergic innervations in the chick embryo hypoganglionic colorectum.Materials and Methods: Chicken eggs were incubated until embryos reached the 10-12 somite stage. The vagal neural tube was microsurgically ablated and eggs were returned to the incubator until embryos reached E12 and E14. Whole embryos were fixed and embedded in paraffin wax. Transverse sections were cut and immunohistochemistry was performed using a neural crest cell antibody, human natural killer-1 (HNK-1), and a choline acetyltransferase antibody (ChAT).Results: The results showed that in normal embryos, the colorectum contained many nerve cells (HNK-1) and ChAT-positive nerve cells and fibres, while in embryos with a hypoganglionic colorectum, the number of nerve cells (HNK-1) and ChAT-positive nerve cells and fibres was decreased.Conclusion: Cholinergic nerve activity is decreased as a result of a reduction in nerve cell numbers in the chick embryo colorectum. These results suggest that the cholinergic activity in the hypoganglionic chick model resembles that of human hypoganglionosis. [ABSTRACT FROM AUTHOR]

Purpose: During pull-through for Hirschsprung's disease (HSCR), the assessment of innervation is mainly based on the presence of ganglion cells when conventional Hematoxylin and Eosin (HE) staining is used. In hypoganglionosis, the evaluation is difficult. We adapted a standardized methodology for the examination of resected bowel after HSCR surgery, using the technique described by Moolenbeek on rodent intestine and later by Meier-Ruge in children. We have analysed the entire innervation of surgically resected bowels and compared the results with the follow up of patients.Methods: Three longitudinal strips of colon were harvested from the mesenteric, anti-mesenteric and intermediate part in the whole length of resected colon of six patients with HSCR. Each strip was divided into two parts. One of the contiguous strips was assessed with HE and Hematoxylin-Phloxin-Safran, and the other one with acetylcholinesterase (AChE) histochemistry. We analyzed the distribution of ganglion cells and nerve arrangement along the strips with both techniques and compared the results obtained in the three different regions of the bowel.Results: There was no significant difference in the pattern of innervation circumferentially. There was a correlation between a progressive increase of AChE activity and nerve hypertrophy and a decrease of ganglion cells from the proximal to the distal part of the resected colon in the submucosa and the myenteric plexus. Nerve hypertrophy and AChE-positive reaction in the mucosa were found at the resection border in patients who presented postoperative complications.Conclusions: Simultaneous assessment of nerve cells, nerve fibers and AChE activity is important in the evaluation of the innervation of the bowel segment proximal to the aganglionic zone. The method described is feasible and can be adapted to older children and adults with larger bowels. These results point out the importance of assessing nerve fibers in intraoperative biopsies during pull-through procedures to prevent uncomplete surgical bowel resection. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effect of the purified extract of the Ginkgo biloba (EGb 761) plant on the myenteric plexus in the proximal and distal colon of Wistar rats for a period of 120 days. The experimental rats were divided into two age groups: a young group, sacrificed at age 90 days, and an adult group, sacrificed at age 210 days. We observed a significant reduction in the number of neurons in the myenteric plexus of the adult group compared to the young group in both of the segments studied (P < 0.01). The adult group treated with Ginkgo biloba showed a significant increase in neuronal profile area in both the segments studied (P < 0.001). It can be concluded from these results that treatment with the purified Ginkgo biloba (EGb 761) plant extract at a dose of 50 mg/kg body weight has neurotrophic effect on the myenteric plexus in the proximal and distal colon of rats after 120 days of treatment. [ABSTRACT FROM AUTHOR]

Interstitial cells of Cajal (ICC) act as pacemaker in gastrointestinal smooth muscle. In animals, small bowel dilatation produces a reduction in ICC numbers and in pacemaker function. With resolution of dilatation, ICC numbers and pacemaking function are partially restored. In human colonic disease states, dilatation is associated with dysmotility. The effect of dilatation on ICC distribution has not previously been examined in the human colon. Tissues from a neonate with colonic atresia and a 17-year-old adolescent with acquired megasigmoid were fixed, sectioned and incubated with anti cKit antibodies followed by fluorescent secondary antibodies. Distended and non-distended segments of colon were examined for ICC distribution using immunohistochemistry to c-Kit. Images were obtained with confocal microscopy. In both patients, there was a marked reduction in cKit-immunoreactive cells in the circular muscle and the myenteric plexus of the distended colon compared to the distal non-distended colon. Dilatation of the human colon is associated with a marked reduction in ICC. A resulting loss of pacemaker function could contribute to dysmotility associated with distension. Further studies assessing pacemaking function in human subjects and investigating reversibility of ICC disruption may allow new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Conventional acetylcholinesterase (AChE) histochemistry is both time consuming and complicated and requires the mixing of reagents that are toxic to the human body. We developed a rapid technique for performing AChE histochemistry, which has already been published, and now present a kit for performing AChE histochemistry that is a further improvement. Rectal suction biopsy specimens taken from 20 constipated patients and three full thickness biopsy specimens taken from 4 Hirschsprung's disease (HD) patients during pull-through surgery from aganglionic, transitional, and ganglionic bowel segments were tested using our rapid technique and the new kit. Each specimen was incubated for only 6 min. All ganglion cells stained clearly for AchE in just 6 min using both techniques. However, the kit was able to stain AchE positive nerve fibers more clearly and did not detect endogenous peroxidase-containing histiocytes, as did the earlier rapid technique. The kit could also detect AchE positive nerve fibers in the circular and longitudinal muscle layers, unlike the earlier rapid technique. The kit allows AChE histochemistry to be performed rapidly with complete accuracy, without any risk for toxicity. Moreover, the kit provides more focused information on AchE distribution in the bowel itself without any extraneous staining and can be used for diagnosing HD and allied disorders as well as establishing the exact level of innervation for pull-through resection. [ABSTRACT FROM AUTHOR]

Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-kappaB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR2). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR2 antagonist and were absent in PAR2-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR2. [ABSTRACT FROM AUTHOR]

Intestinal neuronal dysplasia is a controversial form of dysganglionosis that has been described both as an isolated disorder and in association with Hirschsprung’s disease. It has been blamed for the bad outcome of bowel function in patients operated on for Hirschsprung’s disease. According to various authors, intestinal neuronal dysplasia could be a primary disorder or secondary to other diseases of the bowel. The aim of this study was to assess the plasticity of the enteric nervous system in patients with Hirschsprung’s disease-associated intestinal neuronal dysplasia and its ability to evolve spontaneously to normal innervation patterns. Since we prospectively introduced the assessment of the enteric nervous system of the ganglionated bowel in patients operated on for Hirschsprung’s disease, 31 patients have been operated on for Hirschsprung’s disease in our institution between 1995 and 2002. Among these patients, nine suffered postoperatively from severe constipation and five from bouts of entocolitis. IND was found in eight of them. We studied the evolution of the innervation in three of these patients by repeated serial full-thickness biopsies of the colon. All three patients underwent a colostomy before or after the pull-through procedure. Histopathological assessment of the enteric nervous system was made with conventional acetylcholinesterase histochemistry, rapid acetylcholinesterase histochemistry and immunohistochemistry for the Protein Gene Product 9.5 and the antigen CD56. This evolution was compared with the clinical outcome of bowel function. In our three patients with Hirschsprung’s disease-associated intestinal neuronal dysplasia, this form of dysganglionosis evolved to normal innervation patterns within a period ranging from 9 to 18 months. This evolution was accompanied by an improvement of bowel function in all. We conclude that Hirschsprung’s disease-associated intestinal neuronal dysplasia can evolve to a normal innervation, at least under certain circumstances such as a colostomy. Specific histopathological techniques are required to assess accurately the enteric nervous system. [ABSTRACT FROM AUTHOR]

We evaluated the neurotrophic effect of the neurokinin SP in the induced megacolon and the cell proliferation of the colonic epithelium after treatment. Colon segments of Wistar rats were chemically denervated by topical application of 2 mM benzalconium chloride and animals were daily injected intraperitoneally with SP (70 microg/kg body wt) for 15 days. Control rats received either SP or were denervated and treated with saline. Neuronal profiles of the myenteric plexus were studied by immunohistochemistry to motor protein myosin V and cell proliferation by PCNA immunolabeling. Denervation induced a significant reduction in the number of neurons and an enlargement of the surviving perikarya (from 263.7 microm2 in the control-saline group to 468.9 microm2 in the denervated groups). The total area occupied by neurons was maintained in the denarvated SP group but was significantly smaller in the denervated-saline group. The proliferative index was significantly higher in the denervated groups, of which the SP-treated group showed the highest index. These results suggest that SP may have a neurotrophic effect for the neurons of the myenteric plexus chemically denervated and that this denervation stimulates cell proliferation, especially after SP administration. [ABSTRACT FROM AUTHOR]

Focuses on the relationship of constipation and lack of colonic interstitial cells of Cajal. Discussion of a case; Case history; Treatment options.

Slow transit constipation (STC) is a severe motility disorder, which in the majority of cases is of unknown etiology. In some, symptoms arise de novo in childhood, but a proportion of patients present in later life, including after pelvic surgery or childbirth. Our aims were: (1) to describe our current knowledge of the anatomy and function of the pelvic autonomic nerves with respect to colonic motility (experimental and observational studies); (2) to discuss evidence for pelvic nerve injury in STC arising after pelvic surgery or childbirth; and (3), on the basis that such patients are clinically indistinguishable from patients with chronic idiopathic STC, to evaluate whether there is evidence that pelvic autonomic neuropathy has an etiologic role in patients with chronic idiopathic STC. The outcome was as follows: (1) The clear importance of the pelvic autonomic nerves in colonic motor function is documented. (2) While there is an association between pelvic surgery and childbirth, and the onset of STC, there is little direct anatomical evidence that pelvic denervation occurs in these patients. However the phenotype of these patients is similar to results of experimental and observational studies. (3) Clinical, physiological, and histological similarities exist between patients whose symptoms arose following pelvic intervention and those whose symptoms arise de novo (idiopathic). We further present evidence for possible pathogenetic mechanisms underlying pelvic autonomic neuropathy in chronic idiopathic STC. [ABSTRACT FROM AUTHOR]

Infants affected by Hirschsprung disease (HSCR), a neurodevelopmental congenital disorder, lack ganglia of the intrinsic enteric nervous system (aganglionosis) in a variable length of the colon, and are prone to developing severe Hirschsprung-associated enterocolitis (HAEC). HSCR patients typically show abnormal dense innervation of extrinsic cholinergic nerve fibers throughout the aganglionic rectosigmoid. Cholinergic signaling has been reported to reduce inflammatory response. Consequently, a sparse extrinsic cholinergic innervation in the mucosa of the rectosigmoid correlates with increased inflammatory immune cell frequencies and higher incidence of HAEC in HSCR patients. However, whether cholinergic signals influence the pro-inflammatory immune response of intestinal epithelial cells (IEC) is unknown. Here, we analyzed colonic IEC isolated from 43 HSCR patients with either a low or high mucosal cholinergic innervation density (fiber-low versus fiber-high) as well as from control tissue. Compared to fiber-high samples, IEC purified from fiber-low rectosigmoid expressed significantly higher levels of IL-8 but not TNF-α, IL-10, TGF-β1, Muc-2 or tight junction proteins. IEC from fiber-low rectosigmoid showed higher IL-8 protein concentrations in cell lysates as well as prominent IL-8 immunoreactivity compared to IEC from fiber-high tissue. Using the human colonic IEC cell line SW480 we demonstrated that cholinergic signals suppress lipopolysaccharide-induced IL-8 secretion via the alpha 7 nicotinic acetylcholine receptor (a7nAChR). In conclusion, we showed for the first time that the presence of a dense mucosal cholinergic innervation is associated with decreased secretion of IEC-derived pro-inflammatory IL-8 in the rectosigmoid of HSCR patients likely dependent on a7nAChR activation. Owing to the association between IL-8 and enterocolitis-prone, fiber-low HSCR patients, targeted therapies against IL-8 might be a promising immunotherapy candidate for HAEC treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Müller, Kym, Galati, Tharakan, Subotic, Krebs, Stathopoulos, Schmittenbecher, Cholewa, Romero, Reingruber, NIGStudy Group, Holland-Cunz and Keck.)

Irritable bowel syndrome afflicts 10-20% of the global population, causing visceral pain with increased sensitivity to colorectal distension and normal bowel movements. Understanding and predicting these biomechanics will further advance our understanding of visceral pain and complement the existing literature on visceral neurophysiology. We recently performed a series of experiments at three longitudinal segments (colonic, intermediate, and rectal) of the distal 30 mm of colorectums of mice. We also established and fitted constitutive models addressing mechanical heterogeneity in both the through-thickness and longitudinal directions of the colorectum. Afferent nerve endings, strategically located within the submucosa, are likely nociceptors that detect concentrations of mechanical stresses to evoke the perception of pain from the viscera. In this study, we aim to: (1) establish and validate a method for incorporating residual stresses into models of colorectums, (2) predict the effects of residual stresses on the intratissue mechanics within the colorectum, and (3) establish intratissue distributions of stretches and stresses within the colorectum in vivo. To these ends we developed two-layered, composite finite element models of the colorectum based on our experimental evidence and validated our approaches against independent experimental data. We included layer- and segment-specific residual stretches/stresses in our simulations via the prestrain algorithm built into the finite element software febio. Our models and modeling approaches allow researchers to predict both organ and intratissue biomechanics of the colorectum and may facilitate better understanding of the underlying mechanical mechanisms of visceral pain., (Copyright © 2022 by ASME.)

Disruption in the nerve-tumor interaction is now considered as a possible anticancer strategy for treating various cancer types, particularly colorectal cancer. However, the underlying mechanisms are not still fully understood. Therefore, the present study aimed to evaluate the effects of sympathetic and parasympathetic denervation on the inhibition of colorectal cancer progression in early and late phases and assess the involvement of nerve growth factor in denervation mediated anticancer effects. One-hundred and fifty male Wistar rats were assigned into 15 groups. Seven groups comprising the control group, 1,2-dimethylhydrazine (DMH) group, sympathetic denervation group (celiac-mesenteric ganglionectomy and guanethidine sulphate administration), parasympathetic denervation group (vagotomy and atropine administration), and combination group were used in the early-stage protocol. For the late-stage protocol, eight groups comprising the control, DMH, surgical and pharmacological sympathetic and parasympathetic denervation groups, combination group, and 5-flourouracil group were considered. After 8 weeks, sympathetic and parasympathetic denervation significantly reduced ACF numbers in rats receiving DMH. On the other hand, in the late stages, parasympathetic but not sympathetic denervation resulted in significant reductions in tumor incidence, tumor volume and weight, cell proliferation (indicated by reduced immunostaining of PCNA and ki-67), and angiogenesis (indicated by reduced immunostaining of CD31 and VEGF expression levels), and downregulated NGF, β2 adrenergic, and M3 receptors. It can be concluded that parasympathetic denervation may be of high importance in colon carcinogenesis and suggested as a possible therapeutic modality in late stages of colorectal cancer., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Glucagon-like peptide-1 (GLP-1), a well-known insulin secretagogue, is released from enteroendocrine L cells both luminally and basolaterally to exert different effects. Basolaterally released GLP-1 increases epithelial ion transport by activating CGRP-containing enteric afferent neurons. Although bath-applied GLP-1 reduced the contractility of colonic segments, GLP-1-induced stimulation of afferent neurons could also accelerate peristaltic contractions. Here, the roles of endogenous GLP-1 in regulating colonic peristalsis were investigated using isolated colonic segments. Isolated segments of rat proximal colon were placed in an organ bath, serosally perfused with oxygenated physiological salt solution, and luminally perfused with degassed 0.9% saline. Colonic wall motion was recorded using a video camera and converted into spatiotemporal maps. Intraluminal administration of GLP-1 (100 nM) stimulating the secretion of GLP-1 from L cells increased the frequency of oro-aboral propagating peristaltic contractions. The acceleratory effect of GLP-1 was blocked by luminally applied exendin-3 (9-39) (100 nM), a GLP-1 receptor antagonist. GLP-1-induced acceleration of peristaltic contractions was also prevented by bath-applied BIBN4069 (1 μM), a CGRP receptor antagonist. In colonic segments that had been exposed to bath-applied capsaicin (100 nM) that desensitizes extrinsic afferents, GLP-1 was still capable of exerting its prokinetic effect. Stimulation of endogenous GLP-1 secretion with a luminally applied cocktail of short-chain fatty acids (1 mM) increased the frequency of peristaltic waves in an exendin-3 (9-39)-sensitive manner. Thus, GLP-1 activates CGRP-expressing intrinsic afferents to accelerate peristalsis in the proximal colon. Short-chain fatty acids appear to stimulate endogenous GLP-1 secretion from L cells resulting in the acceleration of colonic peristalsis. NEW & NOTEWORTHY Glucagon-like peptide-1 (GLP-1) activates CGRP-containing intrinsic afferent neurons resulting in the acceleration of colonic peristalsis. Short-chain fatty acids stimulate the secretion of endogenous GLP-1 from L cells that accelerates colonic peristalsis. Thus, besides the well-known humoral insulinotropic action, GLP-1 exerts a local action via the activation of the enteric nervous system to accelerate colonic motility. Such a prokinetic action of GLP-1 could underlie the mechanisms causing diarrhea in patients with type-2 diabetes treated with GLP-1 analogs.

Background: Gastrointestinal (GI) function is critically dependent on the control of the enteric nervous system (ENS), which is situated within the gut wall and organized into two ganglionated nerve plexuses: the submucosal and myenteric plexus. The ENS is optimally positioned and together with the intestinal epithelium, is well-equipped to monitor the luminal contents such as microbial metabolites and to coordinate appropriate responses accordingly. Despite the heightened interest in the gut microbiota and its influence on intestinal physiology and pathophysiology, how they interact with the host ENS remains unclear., Methods: Using full-thickness proximal colon preparations from transgenic Villin-CreERT2;R26R-GCaMP3 and Wnt1-Cre;R26R-GCaMP3 mice, which express a fluorescent Ca 2+ indicator in their intestinal epithelium or in their ENS, respectively, we examined the effects of key luminal microbial metabolites (SCFAs and 5-HT) on the mucosa and underlying enteric neurons., Key Results: We show that the SCFAs acetate, propionate, and butyrate, as well as 5-HT can, to varying extents, acutely elicit epithelial and neuronal Ca 2+ responses. Furthermore, SCFAs exert differential effects on submucosal and myenteric neurons. Additionally, we found that submucosal ganglia are predominantly aligned along the striations of the transverse mucosal folds in the proximal colon., Conclusions & Inferences: Taken together, our study demonstrates that different microbial metabolites, including SCFAs and 5-HT, can acutely stimulate Ca 2+ signaling in the mucosal epithelium and in enteric neurons., (© 2021 John Wiley & Sons Ltd.)

The aim of this study was to determine whether stimulation of sacral spinal nerve roots can induce defecation in cats. In anesthetized cats, bipolar hook electrodes were placed on the S1-S3 dorsal and/or ventral roots. Stimulus pulses (1-50 Hz, 0.2 ms) were applied to an individual S1-S3 root to induce proximal/distal colon contractions and defecation. Balloon catheters were inserted into the proximal and distal colon to measure contraction pressure. Glass marbles were inserted into the rectum to demonstrate defecation by videotaping the elimination of marbles. Stimulation of the S2 ventral root at 7 Hz induced significantly ( P < 0.05) larger contractions (32 ± 9 cmH 2 O) in both proximal and distal colon than stimulation of the S1 or S3 ventral root. Intermittent (5 times) stimulation (1 min on and 1 min off) of both dorsal and ventral S2 roots at 7 Hz produced reproducible colon contractions without fatigue, whereas continuous stimulation of 5-min duration caused significant fatigue in colon contractions. Stimulation (7 Hz) of both dorsal and ventral S2 roots together successfully induced defecation that eliminated 1 or 2 marbles from the rectum. This study indicates the possibility to develop a novel neuromodulation device to restore defecation function after spinal cord injury using a minimally invasive surgical approach to insert a lead electrode via the sacral foramen to stimulate a sacral spinal root. NEW & NOTEWORTHY This study in cats determined the optimal stimulation parameters and the spinal segment for sacral spinal root stimulation to induce colon contraction. The results have significant implications for design of a novel neuromodulation device to restore defecation function after spinal cord injury (SCI) and for optimizing sacral neuromodulation parameters to treat non-SCI people with chronic constipation.

Background: Postoperative paralytic ileus prolongs hospitalization duration, increases medical expenses, and is even associated with postoperative mortality; however, effective prevention of postoperative paralytic ileus is not yet available. This trial aimed to assess the preventative effectiveness of transcutaneous electrical acupoint stimulation applied in the lower limbs on postoperative paralytic ileus incidence after colorectal surgery., Methods: After ethics approval and written informed consent, 610 patients from 10 hospitals who were scheduled for colorectal surgery between May 2018 and September 2019 were enrolled. Patients were randomly allocated into the transcutaneous electrical acupoint stimulation (stimulated on bilateral Zusanli, Shangjuxu, and Sanyinjiao acupoints in lower limbs for 30 minutes each time, total 4 times) or sham (without currents delivered) group with 1:1 ratio. The primary outcome was postoperative paralytic ileus incidence, defined as no flatus for >72 hours after surgery., Results: Compared to the sham treatment, transcutaneous electrical acupoint stimulation lowered the postoperative paralytic ileus incidence by 8.7% (32.3% vs 41.0%, P = .026) and decreased the risk of postoperative paralytic ileus by 32% (OR, 0.68; P = .029). Transcutaneous electrical acupoint stimulation also shortened the recovery time to flatus, defecation, normal diet, and bowel sounds. Transcutaneous electrical acupoint stimulation treatment significantly increased median serum acetylcholine by 55% (P = .007) and interleukin-10 by 88% (P < .001), but decreased interleukin-6 by 47% (P < .001) and inducible nitric oxide synthase by 42% (P = .002) at 72 hours postoperatively., Conclusion: Transcutaneous electrical acupoint stimulation attenuated the postoperative paralytic ileus incidence and enhanced gastrointestinal functional recovery, which may be associated with increasing parasympathetic nerve tone and its anti-inflammatory actions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Sigma-1 receptors (σ 1 R) have been implicated in several pain pathways. We assessed the implication of σ 1 Rs in the development of intestinal inflammation and inflammation-associated referred hypersensitivity in a model of colitis in σ 1 R knockout (KO) mice. Colitis was induced with dextran sulfate sodium (DSS) in wild type (WT) and σ 1 R KO mice. The development of referred mechanical hypersensitivity (von Frey test) was assessed. Colonic and spinal changes in expression of immune- and sensory-related markers were also investigated (RT-qPCR/Western blot). Absence of σ 1 Rs had little impact in colitis generation and progression, although during the chronic phase a reduction in edema and a down-regulation of iNOS gene expression was observed. In σ 1 R KO mice, inflammation-associated hypersensitivity was significantly attenuated (paw) or completely prevented (abdomen). During colitis, in WT mice, changes in the colonic expression of nociceptive markers were observed during the acute and chronic phases of inflammation. Although σ 1 R KO mice showed similar regulation in the acute phase, an attenuated response was observed during the chronic phase of colitis. These differences were especially relevant for CB2 and TRPV1 receptors, which could play an important role in σ 1 -mediated regulation of sensitivity. No changes were detected on ERK phosphorylation at the level of the lumbosacral spinal cord. In summary, intestinal inflammation-associated referred hyperalgesia was reduced (paw) or absent (abdomen) in σ 1 R KO mice, thus confirming an important role for σ 1 R in the development of colitis-associated hypersensitivity. These results identify σ 1 Rs as a possible therapeutic target for the treatment of hypersensitivity associated to intestinal inflammation., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The act of defaecation, although a ubiquitous human experience, requires the coordinated actions of the anorectum and colon, pelvic floor musculature, and the enteric, peripheral and central nervous systems. Defaecation is best appreciated through the description of four phases, which are, temporally and physiologically, reasonably discrete. However, given the complexity of this process, it is unsurprising that disorders of defaecation are both common and problematic; almost everyone will experience constipation at some time in their life and many will develop faecal incontinence. A detailed understanding of the normal physiology of defaecation and continence is critical to inform management of disorders of defaecation. During the past decade, there have been major advances in the investigative tools used to assess colonic and anorectal function. This Review details the current understanding of defaecation and continence. This includes an overview of the relevant anatomy and physiology, a description of the four phases of defaecation, and factors influencing defaecation (demographics, stool frequency/consistency, psychobehavioural factors, posture, circadian rhythm, dietary intake and medications). A summary of the known pathophysiology of defaecation disorders including constipation, faecal incontinence and irritable bowel syndrome is also included, as well as considerations for further research in this field., (© 2021. Springer Nature Limited.)

Our understanding of human colonic motility, and autonomic reflexes that generate motor patterns, has increased markedly through high-resolution manometry. Details of the motor patterns are emerging related to frequency and propagation characteristics that allow linkage to interstitial cells of Cajal (ICC) networks. In studies on colonic motor dysfunction requiring surgery, ICC are almost always abnormal or significantly reduced. However, there are still gaps in our knowledge about the role of ICC in the control of colonic motility and there is little understanding of a mechanistic link between ICC abnormalities and colonic motor dysfunction. This review will outline the various ICC networks in the human colon and their proven and likely associations with the enteric and extrinsic autonomic nervous systems. Based on our extensive knowledge of the role of ICC in the control of gastrointestinal motility of animal models and the human stomach and small intestine, we propose how ICC networks are underlying the motor patterns of the human colon. The role of ICC will be reviewed in the autonomic neural reflexes that evoke essential motor patterns for transit and defecation. Mechanisms underlying ICC injury, maintenance, and repair will be discussed. Hypotheses are formulated as to how ICC dysfunction can lead to motor abnormalities in slow transit constipation, chronic idiopathic pseudo-obstruction, Hirschsprung's disease, fecal incontinence, diverticular disease, and inflammatory conditions. Recent studies on ICC repair after injury hold promise for future therapies.

The cross talk between external genitalia and urinary bladder could be used as part of management to certain pathological conditions affecting urinary bladder. Since urinary bladder function is also affected by pathologies of other organs (e.g., colon and esophagus), the effect of genitalia stimuli on parameters of bladder function in normal or under different pathological conditions needs to be characterized. Cystometry recordings in male rats were used to examine the effect of low-threshold (LT) and high-threshold (HT) stimulation of the scrotum and penis on urinary bladder function. These effects were studied in intact, colon irritation (CI), and esophagus irritation (EI) groups. Although HT penile stimulation had a significant inhibitory effect on micturition reflex in all groups, CI hypersensitized the penile-bladder inhibitory reflex. In addition, LT penile stimulation had a significant inhibitory effect on micturition, which was significant in CI group only. On the other hand, HT penile stimulation in CI group significantly increased the timing parameters of cystometry. Whereas LT and HT penile stimuli in EI group had a significantly increasing effect on all pressure parameters of cystometry. The scrotal stimuli had minimal effect on bladder function in all groups except for HT scrotal stimulation in the CI group, where it had a significant inhibitory effect on micturition reflex and significantly increased the maximum pressure and pressure amplitude of micturition cycles. These results show that CI and EI exacerbate the effects of genitalia stimuli, especially penile stimuli, on urinary bladder function.

Chronic Overlapping Pain Conditions, including irritable bowel syndrome (IBS) and temporomandibular disorder (TMD), represent a group of idiopathic pain conditions that likely have peripheral and central mechanisms contributing to their pathology, but are poorly understood. These conditions are exacerbated by stress and have a female predominance. The presence of one condition predicts the presence or development of additional conditions, making this a significant pain management problem. The current study was designed to determine if the duration and magnitude of peripheral sensitization and spinal central sensitization differs between restraint stress-induced visceral hypersensitivity (SIH) and chronic comorbid pain hypersensitivity (CPH; stress during pre-existing orofacial pain). SIH in female rats, as determined by the visceromotor response, persisted at least four but resolved by seven weeks. In contrast, CPH persisted at least seven weeks. Surprisingly, colonic afferents in both SIH and CPH rats were sensitized at seven weeks. CPH rats also had referred pain through seven weeks, but locally anesthetizing the colon only attenuated the referred pain through four weeks, suggesting a transition to colonic afferent independent central sensitization. Different phenotypes of dorsal horn neurons were sensitized in the CPH rats seven weeks post stress compared to four weeks or SIH rats. The current study suggests differential processing of colonic afferent input to the lumbosacral spinal cord contributes to visceral hypersensitivity during comorbid chronic pain conditions. PERSPECTIVE: Chronic Overlapping Pain Conditions represent a unique challenge in pain management. The diverse nature of peripheral organs hinders a clear understanding of underlying mechanisms accounting for the comorbidity. This study highlights a mismatch between the condition-dependent behavior and peripheral and spinal mechanisms that contribute to visceral pain hypersensitivity., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)