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Rahman AA, Ohkura T, Bhave S, Pan W, Ohishi K, Ott L, Han C, Leavitt A, Stavely R, Burns AJ, Goldstein AM, Hotta R. Enteric neural stem cell transplant restores gut motility in mice with Hirschsprung disease. JCI Insight 2024; 9:e179755. [PMID: 39042470 PMCID: PMC11385093 DOI: 10.1172/jci.insight.179755] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2024] [Accepted: 07/18/2024] [Indexed: 07/25/2024] Open
Abstract
The goal of this study was to determine if transplantation of enteric neural stem cells (ENSCs) can rescue the enteric nervous system, restore gut motility, reduce colonic inflammation, and improve survival in the Ednrb-KO mouse model of Hirschsprung disease (HSCR). ENSCs were isolated from mouse intestine, expanded to form neurospheres, and microinjected into the colons of recipient Ednrb-KO mice. Transplanted ENSCs were identified in recipient colons as cell clusters in "neo-ganglia." Immunohistochemical evaluation demonstrated extensive cell migration away from the sites of cell delivery and across the muscle layers. Electrical field stimulation and optogenetics showed significantly enhanced contractile activity of aganglionic colonic smooth muscle following ENSC transplantation and confirmed functional neuromuscular integration of the transplanted ENSC-derived neurons. ENSC injection also partially restored the colonic migrating motor complex. Histological examination revealed a significant reduction in inflammation in ENSC-transplanted aganglionic recipient colon compared with that of sham-operated mice. Interestingly, mice that received cell transplant also had prolonged survival compared with controls. This study demonstrates that ENSC transplantation can improve outcomes in HSCR by restoring gut motility and reducing the severity of Hirschsprung-associated enterocolitis, the leading cause of death in human HSCR.
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Affiliation(s)
- Ahmed A Rahman
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Takahiro Ohkura
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Sukhada Bhave
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Weikang Pan
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
- Department of Surgery, Boston Children's Hospital, Boston, Massachusetts, USA
| | - Kensuke Ohishi
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
- Drug Discovery Laboratory, Wakunaga Pharmaceutical Co. Ltd., Hiroshima, Japan
| | - Leah Ott
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Christopher Han
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Abigail Leavitt
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Rhian Stavely
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Alan J Burns
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
- Stem Cells and Regenerative Medicine, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
| | - Allan M Goldstein
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Ryo Hotta
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
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Woods C, Flockton AR, Belkind-Gerson J. Phosphatase and Tensin Homolog Inhibition in Proteolipid Protein 1-Expressing Cells Stimulates Neurogenesis and Gliogenesis in the Postnatal Enteric Nervous System. Biomolecules 2024; 14:346. [PMID: 38540765 PMCID: PMC10967813 DOI: 10.3390/biom14030346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Revised: 03/07/2024] [Accepted: 03/11/2024] [Indexed: 04/04/2024] Open
Abstract
Phosphatase and tensin homolog (Pten) is a key regulator of cell proliferation and a potential target to stimulate postnatal enteric neuro- and/or gliogenesis. To investigate this, we generated two tamoxifen-inducible Cre recombinase murine models in which Pten was conditionally ablated, (1) in glia (Plp1-expressing cells) and (2) in neurons (Calb2-expressing cells). Tamoxifen-treated adult (7-12 weeks of age; n = 4-15) mice were given DSS to induce colitis, EdU to monitor cell proliferation, and were evaluated at two timepoints: (1) early (3-4 days post-DSS) and (2) late (3-4 weeks post-DSS). We investigated gut motility and evaluated the enteric nervous system. Pten inhibition in Plp1-expressing cells elicited gliogenesis at baseline and post-DSS (early and late) in the colon, and neurogenesis post-DSS late in the proximal colon. They also exhibited an increased frequency of colonic migrating motor complexes (CMMC) and slower whole gut transit times. Pten inhibition in Calb2-expressing cells did not induce enteric neuro- or gliogenesis, and no alterations were detected in CMMC or whole gut transit times when compared to the control at baseline or post-DSS (early and late). Our results merit further research into Pten modulation where increased glia and/or slower intestinal transit times are desired (e.g., short-bowel syndrome and rapid-transit disorders).
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Affiliation(s)
- Crystal Woods
- Department of Pediatrics, Section of Gastroenterology, Hepatology and Nutrition, University of Colorado, Aurora, CO 80045, USA; (C.W.); (A.R.F.)
| | - Amanda R. Flockton
- Department of Pediatrics, Section of Gastroenterology, Hepatology and Nutrition, University of Colorado, Aurora, CO 80045, USA; (C.W.); (A.R.F.)
| | - Jaime Belkind-Gerson
- Department of Pediatrics, Section of Gastroenterology, Hepatology and Nutrition, University of Colorado, Aurora, CO 80045, USA; (C.W.); (A.R.F.)
- Neurogastroenterology and Motility Program, Digestive Health Institute, Children’s Hospital Colorado, Aurora, CO 80045, USA
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Woods C, Flockton AR, Wallace LE, Keenan CM, Macklin WB, Sharkey KA, Belkind-Gerson J. Proteolipid protein 1 is involved in the regulation of intestinal motility and barrier function in the mouse. Am J Physiol Gastrointest Liver Physiol 2023; 324:G115-G130. [PMID: 36511517 DOI: 10.1152/ajpgi.00171.2022] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Proteolipid protein 1 (Plp1) is highly expressed in enteric glia, labeling cells throughout the mucosa, muscularis, and the extrinsic innervation. Plp1 is a major constituent of myelin in the central and peripheral nervous systems, but the absence of myelin in the enteric nervous system (ENS) suggests another role for Plp1 in the gut. Although the functions of enteric glia are still being established, there is strong evidence that they regulate intestinal motility and permeability. To interrogate the role of Plp1 in enteric glia, we investigated gut motility, secretomotor function and permeability, and evaluated the ENS in mice lacking Plp1. We studied two time points: ∼3 mo (young) and >1 yr (old). Old Plp1 null mice exhibited increased fecal output, decreased fecal water content, faster whole gut transit times, reduced intestinal permeability, and faster colonic migrating motor complexes. Interestingly, in both young and old mice, the ENS exhibited normal glial and neuronal numbers as well as glial arborization density in the absence of Plp1. As Plp1-associated functions involve mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (Mapk/Erk1/2) signaling and Mapk/Erk1/2 are reported to have a regulatory role in intestinal motility, we measured protein expression of Erk1/2 and its active form in the small intestine. Old Plp1 null mice had reduced levels of phosphorylated-Erk1/2. Although Plp1 is not required for the normal appearance of enteric glial cells, it has a regulatory role in intestinal motility and barrier function. Our results suggest that functional changes mediated by Plp1-expressing enteric glia may involve Erk1/2 activation.NEW & NOTEWORTHY Here, we describe that Plp1 regulates gut motility and barrier function. The functional effects of Plp1 eradication are only seen in old mice, not young. The effects of Plp1 appear to be mediated through the Erk1/2 pathway.
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Affiliation(s)
- Crystal Woods
- Section of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, University of Colorado, Aurora, Colorado
| | - Amanda R Flockton
- Section of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, University of Colorado, Aurora, Colorado
| | - Laurie E Wallace
- Department of Physiology and Pharmacology, Cumming School of Medicine, Hotchkiss Brain Institute and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada
| | - Catherine M Keenan
- Department of Physiology and Pharmacology, Cumming School of Medicine, Hotchkiss Brain Institute and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada
| | - Wendy B Macklin
- Department of Cell & Developmental Biology, University of Colorado, Aurora, Colorado
| | - Keith A Sharkey
- Department of Physiology and Pharmacology, Cumming School of Medicine, Hotchkiss Brain Institute and Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada
| | - Jaime Belkind-Gerson
- Section of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, University of Colorado, Aurora, Colorado.,Neurogastroenterology and Motility Program, Digestive Health Institute, Children's Hospital Colorado, Aurora, Colorado
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Costa M, Wiklendt L, Hibberd T, Dinning P, Spencer NJ, Brookes S. Analysis of Intestinal Movements with Spatiotemporal Maps: Beyond Anatomy and Physiology. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2022; 1383:271-294. [PMID: 36587166 DOI: 10.1007/978-3-031-05843-1_26] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Over 150 years ago, methods for quantitative analysis of gastrointestinal motor patterns first appeared. Graphic representations of physiological variables were recorded with the kymograph after the mid-1800s. Changes in force or length of intestinal muscles could be quantified, however most recordings were limited to a single point along the digestive tract.In parallel, photography and cinematography with X-Rays visualised changes in intestinal shape, but were hard to quantify. More recently, the ability to record physiological events at many sites along the gut in combination with computer processing allowed construction of spatiotemporal maps. These included diameter maps (DMaps), constructed from video recordings of intestinal movements and pressure maps (PMaps), constructed using data from high-resolution manometry catheters. Combining different kinds of spatiotemporal maps revealed additional details about gut wall status, including compliance, which relates forces to changes in length. Plotting compliance values along the intestine enabled combined DPMaps to be constructed, which can distinguish active contractions and relaxations from passive changes. From combinations of spatiotemporal maps, it is possible to deduce the role of enteric circuits and pacemaker cells in the generation of complex motor patterns. Development and application of spatiotemporal methods to normal and abnormal motor patterns in animals and humans is ongoing, with further technical improvements arising from their combination with impedance manometry, magnetic resonance imaging, electrophysiology, and ultrasonography.
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Affiliation(s)
- Marcello Costa
- College of Medicine and Public Health, Department of Human Physiology, Flinders University, Bedford Park, SA, Australia.
| | - Luke Wiklendt
- Department of Gastroenterology and Surgery, Flinders Medical Centre, Bedford Park, SA, Australia
| | - Tim Hibberd
- College of Medicine and Public Health, Department of Human Physiology, Flinders University, Bedford Park, SA, Australia
| | - Phil Dinning
- Department of Gastroenterology and Surgery, Flinders Medical Centre, Bedford Park, SA, Australia
| | - Nick J Spencer
- College of Medicine and Public Health, Department of Human Physiology, Flinders University, Bedford Park, SA, Australia
| | - Simon Brookes
- College of Medicine and Public Health, Department of Human Physiology, Flinders University, Bedford Park, SA, Australia
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Primary Cilia Structure Is Prolonged in Enteric Neurons of 5xFAD Alzheimer's Disease Model Mice. Int J Mol Sci 2021; 22:ijms222413564. [PMID: 34948356 PMCID: PMC8707868 DOI: 10.3390/ijms222413564] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2021] [Revised: 12/09/2021] [Accepted: 12/15/2021] [Indexed: 02/06/2023] Open
Abstract
Neurodegenerative diseases such as Alzheimer’s disease (AD) have long been acknowledged as mere disorders of the central nervous system (CNS). However, in recent years the gut with its autonomous nervous system and the multitude of microbial commensals has come into focus. Changes in gut properties have been described in patients and animal disease models such as altered enzyme secretion or architecture of the enteric nervous system. The underlying cellular mechanisms have so far only been poorly investigated. An important organelle for integrating potentially toxic signals such as the AD characteristic A-beta peptide is the primary cilium. This microtubule-based signaling organelle regulates numerous cellular processes. Even though the role of primary cilia in a variety of developmental and disease processes has recently been recognized, the contribution of defective ciliary signaling to neurodegenerative diseases such as AD, however, has not been investigated in detail so far. The AD mouse model 5xFAD was used to analyze possible changes in gut functionality by organ bath measurement of peristalsis movement. Subsequently, we cultured primary enteric neurons from mutant mice and wild type littermate controls and assessed for cellular pathomechanisms. Neurite mass was quantified within transwell culturing experiments. Using a combination of different markers for the primary cilium, cilia number and length were determined using fluorescence microscopy. 5xFAD mice showed altered gut anatomy, motility, and neurite mass of enteric neurons. Moreover, primary cilia could be demonstrated on the surface of enteric neurons and exhibited an elongated phenotype in 5xFAD mice. In parallel, we observed reduced β-Catenin expression, a key signaling molecule that regulates Wnt signaling, which is regulated in part via ciliary associated mechanisms. Both results could be recapitulated via in vitro treatments of enteric neurons from wild type mice with A-beta. So far, only a few reports on the probable role of primary cilia in AD can be found. Here, we reveal for the first time an architectural altered phenotype of primary cilia in the enteric nervous system of AD model mice, elicited potentially by neurotoxic A-beta. Potential changes on the sub-organelle level—also in CNS-derived neurons—require further investigations.
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6
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Konen JR, Haag MM, Guseva D, Hurd M, Linton AA, Lavoie B, Kerrigan CB, Joyce E, Bischoff SC, Swann S, Griffin L, Matsukawa J, Falk MD, Gibson TS, Hennig GW, Wykosky J, Mawe GM. Prokinetic actions of luminally acting 5-HT 4 receptor agonists. Neurogastroenterol Motil 2021; 33:e14026. [PMID: 33185015 PMCID: PMC7990683 DOI: 10.1111/nmo.14026] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/09/2020] [Accepted: 10/15/2020] [Indexed: 12/12/2022]
Abstract
BACKGROUND 5-HT4 receptor (5-HT4 R) agonists exert prokinetic actions in the GI tract, but non-selective actions and potential for stimulation of non-target 5-HT4 Rs have limited their use. Since 5-HT4 Rs are expressed in the colonic epithelium and their stimulation accelerates colonic propulsion in vitro, we tested whether luminally acting 5-HT4 R agonists promote intestinal motility. METHODS Non-absorbed 5-HT4 R agonists, based on prucalopride and naronapride, were assessed for potency at the 5-HT4 R in vitro, and for tissue and serum distribution in vivo in mice. In vivo assessment of prokinetic potential included whole gut transit, colonic motility, fecal output, and fecal water content. Colonic motility was also studied ex vivo in mice treated in vivo. Immunofluorescence was used to evaluate receptor distribution in human intestinal mucosa. KEY RESULTS Pharmacological screening demonstrated selectivity and potency of test agonists for 5-HT4 R. Bioavailability studies showed negligible serum detection. Gavage of agonists caused faster whole gut transit and colonic motility, increased fecal output, and elevated fecal water content. Prokinetic actions were blocked by a 5-HT4 R antagonist and were not detected in 5-HT4 R knockout mice. Agonist administration promoted motility in models of constipation. Evaluation of motility patterns ex vivo revealed enhanced contractility in the middle and distal colon. Immunoreactivity for 5-HT4 R is present in the epithelial layer of the human small and large intestines. CONCLUSIONS AND INFERENCES These findings demonstrated that stimulation of epithelial 5-HT4 Rs can potentiate propulsive motility and support the concept that mucosal 5-HT4 Rs could represent a safe and effective therapeutic target for the treatment of constipation.
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Affiliation(s)
- John R Konen
- Department of Neurological Sciences, University of Vermont, Burlington, VT, USA,Department of Surgery, University of Vermont Medical Center, Burlington, VT, USA
| | - Melody M Haag
- Department of Neurological Sciences, University of Vermont, Burlington, VT, USA
| | - Daria Guseva
- Department of Nutritional Medicine, University of Hohenheim, Stuttgart, Germany
| | - Molly Hurd
- Department of Neurological Sciences, University of Vermont, Burlington, VT, USA
| | - Alisha A. Linton
- Department of Neurological Sciences, University of Vermont, Burlington, VT, USA
| | - Brigitte Lavoie
- Department of Neurological Sciences, University of Vermont, Burlington, VT, USA
| | - Colleen B Kerrigan
- Department of Neurological Sciences, University of Vermont, Burlington, VT, USA,Department of Surgery, University of Vermont Medical Center, Burlington, VT, USA
| | - Emily Joyce
- Department of Neurological Sciences, University of Vermont, Burlington, VT, USA
| | - Stephan C Bischoff
- Department of Nutritional Medicine, University of Hohenheim, Stuttgart, Germany
| | - Steve Swann
- Takeda Pharmaceutical Company, San Diego, CA, USA
| | | | | | | | | | - Grant W Hennig
- Department of Pharmacology, University of Vermont, Burlington, VT, USA
| | - Jill Wykosky
- Takeda Pharmaceutical Company, San Diego, CA, USA
| | - Gary M Mawe
- Department of Neurological Sciences, University of Vermont, Burlington, VT, USA,Department of Pharmacology, University of Vermont, Burlington, VT, USA
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7
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Physical and nutrient stimuli differentially modulate gut motility patterns, gut transit rate, and transcriptome in an agastric fish, the ballan wrasse. PLoS One 2021; 16:e0247076. [PMID: 33571240 PMCID: PMC7877642 DOI: 10.1371/journal.pone.0247076] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Accepted: 01/31/2021] [Indexed: 12/14/2022] Open
Abstract
The effects of nutrient and mechanical sensing on gut motility and intestinal metabolism in lower vertebrates remains largely unknown. Here we present the transcriptome response to luminal stimulation by nutrients and an inert bolus on nutrient response pathways and also the response on gut motility in a stomachless fish with a short digestive tract; the ballan wrasse (Labrus berggylta). Using an in vitro model, we differentiate how signals initiated by physical stretch (cellulose and plastic beads) and nutrients (lipid and protein) modulate the gut evacuation rate, motility patterns and the transcriptome. Intestinal stretch generated by inert cellulose initiated a faster evacuation of digesta out of the anterior intestine compared to digestible protein and lipid. Stretch on the intestine upregulated genes associated with increased muscle activity, whereas nutrients stimulated increased expression of several neuropeptides and receptors which are directly involved in gut motility regulation. Although administration of protein and lipid resulted in similar bulbous evacuation times, differences in intestinal motility, transit between the segments and gene expression between the two were observed. Lipid induced increased frequency of ripples and standing contraction in the middle section of the intestine compared to the protein group. We suggest that this difference in motility was modulated by factors [prepronociceptin (pnoca), prodynorphin (pdyn) and neuromedin U (nmu), opioid neurotransmitters and peptides] that are known to inhibit gastrointestinal motility and were upregulated by protein and not lipid. Our findings show that physical pressure in the intestine initiate contractions propelling the bolus distally, directly towards the exit, whereas the stimuli from nutrients modulates the motility to prolong the residence time of digesta in the digestive tract for optimal digestion.
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8
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Bhave S, Arciero E, Baker C, Ho WL, Guyer RA, Hotta R, Goldstein AM. Pan-enteric neuropathy and dysmotility are present in a mouse model of short-segment Hirschsprung disease and may contribute to post-pullthrough morbidity. J Pediatr Surg 2021; 56:250-256. [PMID: 32414519 PMCID: PMC7572464 DOI: 10.1016/j.jpedsurg.2020.04.002] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/17/2020] [Revised: 03/14/2020] [Accepted: 04/06/2020] [Indexed: 12/13/2022]
Abstract
PURPOSE Hirschsprung disease (HSCR) is characterized by distal intestinal aganglionosis. While surgery is lifesaving, gastrointestinal (GI) motility disorders persist in many patients. Our objective was to determine whether enteric nervous system (ENS) abnormalities exist in the ganglionated portions of the GI tract far proximal to the aganglionic region and whether these are associated with GI dysmotility. METHODS Using Ednrb-null mice, a model of HSCR, immunohistochemical analysis was performed to evaluate quantitatively ENS structure in proximal colon, small intestine, and stomach. Gastric emptying and intestinal transit were measured in vivo and small and large bowel contractility was assessed by spatiotemporal mapping ex vivo. RESULTS Proximal colon of HSCR mice had smaller ganglia and decreased neuronal fiber density, along with a marked reduction in migrating motor complexes. The distal small intestine exhibited significantly fewer ganglia and decreased neuronal fiber density, and this was associated with delayed small intestinal transit time. Finally, in the stomach of HSCR mice, enteric neuronal packing density was increased and gastric emptying was faster. CONCLUSIONS ENS abnormalities and motility defects are present throughout the ganglionated portions of the GI tract in Ednrb-deficient mice. This may explain the GI morbidity that often occurs following pull-through surgery for HSCR.
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Affiliation(s)
- Sukhada Bhave
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA
| | - Emily Arciero
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA
| | - Corey Baker
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA
| | - Wing Lam Ho
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA
| | - Richard A Guyer
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA
| | - Ryo Hotta
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA
| | - Allan M Goldstein
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA.
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Kacmaz H, Alto A, Knutson K, Linden DR, Gibbons SJ, Farrugia G, Beyder A. A simple automated approach to measure mouse whole gut transit. Neurogastroenterol Motil 2021; 33:e13994. [PMID: 33000540 PMCID: PMC7899194 DOI: 10.1111/nmo.13994] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/26/2020] [Revised: 07/23/2020] [Accepted: 08/25/2020] [Indexed: 12/05/2022]
Abstract
BACKGROUND Gastrointestinal (GI) motility is a complex physiological process that is critical for normal GI function. Disruption of GI motility frequently occurs in GI diseases or as side effects of therapeutics. Whole gut transit measurements, like carmine red leading-edge transit, in mice form the cornerstone of in vivo preclinical GI motility studies. METHOD We have developed an easily achievable, labor-saving method to measure whole gut transit time in mice. This approach uses inexpensive, commercially available materials to monitor pellet production over time via high definition cameras capturing time-lapse video for offline analysis. KEY RESULT We describe the assembly of our automated gut transit setup and validate this approach by comparing the results with loperamide to delay transit and conventional transit measurements. We demonstrate that compared to the control group, the loperamide group had slowed transit, evidenced by a decrease in total pellet production and prolonged whole gut transit time. The control group had an extended transit time compared with the results reported in the literature. Whole gut transit rates accelerated to times comparable to the literature by disrupting cages every 10-15 min to imitate the conventional approach, suggesting that disruption affects the assay and supports the use of an automated approach. CONCLUSION & INFERENCES A novel automated, inexpensive, and easily assembled whole gut transit setup is labor-saving and allows minimal disruption to animal behavior compared with the conventional approach.
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Affiliation(s)
- Halil Kacmaz
- Enteric Neuroscience Program (ENSP), Mayo Clinic, Rochester, Minnesota, USA.,Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Alecia Alto
- Enteric Neuroscience Program (ENSP), Mayo Clinic, Rochester, Minnesota, USA.,Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Kaitlyn Knutson
- Enteric Neuroscience Program (ENSP), Mayo Clinic, Rochester, Minnesota, USA.,Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - David R. Linden
- Enteric Neuroscience Program (ENSP), Mayo Clinic, Rochester, Minnesota, USA.,Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, USA
| | - Simon J. Gibbons
- Enteric Neuroscience Program (ENSP), Mayo Clinic, Rochester, Minnesota, USA.,Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, USA
| | - Gianrico Farrugia
- Enteric Neuroscience Program (ENSP), Mayo Clinic, Rochester, Minnesota, USA.,Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA.,Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, USA
| | - Arthur Beyder
- Enteric Neuroscience Program (ENSP), Mayo Clinic, Rochester, Minnesota, USA.,Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA.,Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, USA.,Corresponding author: Arthur Beyder, MD, PhD, Enteric NeuroScience Program (ENSP), Division of Gastroenterology & Hepatology, Departments of Medicine and Physiology and Biomedical Engineering Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Telephone: (507) 284-2511, Fax: (507) 284-0266,
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10
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Influence of Acetylcholine Esterase Inhibitors and Memantine, Clinically Approved for Alzheimer's Dementia Treatment, on Intestinal Properties of the Mouse. Int J Mol Sci 2021; 22:ijms22031015. [PMID: 33498392 PMCID: PMC7864027 DOI: 10.3390/ijms22031015] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2020] [Revised: 01/11/2021] [Accepted: 01/19/2021] [Indexed: 02/07/2023] Open
Abstract
Four drugs are currently approved for the treatment of Alzheimer’s disease (AD) by the FDA. Three of these drugs—donepezil, rivastigmine, and galantamine—belong to the class of acetylcholine esterase inhibitors. Memantine, a NMDA receptor antagonist, represents the fourth and a combination of donepezil and memantine the fifth treatment option. Recently, the gut and its habitants, its microbiome, came into focus of AD research and added another important factor to therapeutic considerations. While the first data provide evidence that AD patients might carry an altered microbiome, the influence of administered drugs on gut properties and commensals have been largely ignored so far. However, the occurrence of digestive side effects with these drugs and the knowledge that cholinergic transmission is crucial for several gut functions enforces the question if, and how, this medication influences the gastrointestinal system and its microbial stocking. Here, we investigated aspects such as microbial viability, colonic propulsion, and properties of enteric neurons, affected by assumed intestinal concentration of the four drugs using the mouse as a model organism. All ex vivo administered drugs revealed no direct effect on fecal bacteria viability and only a high dosage of memantine resulted in reduced biofilm formation of E. coli. Memantine was additionally the only compound that elevated calcium influx in enteric neurons, while all acetylcholine esterase inhibitors significantly reduced esterase activity in colonic tissue specimen and prolonged propulsion time. Both, acetylcholine esterase inhibitors and memantine, had no effect on general viability and neurite outgrowth of enteric neurons. In sum, our findings indicate that all AD symptomatic drugs have the potential to affect distinct intestinal functions and with this—directly or indirectly—microbial commensals.
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11
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Bhave S, Arciero E, Baker C, Ho WL, Stavely R, Goldstein AM, Hotta R. Enteric neuronal cell therapy reverses architectural changes in a novel diphtheria toxin-mediated model of colonic aganglionosis. Sci Rep 2019; 9:18756. [PMID: 31822721 PMCID: PMC6904570 DOI: 10.1038/s41598-019-55128-4] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2019] [Accepted: 11/19/2019] [Indexed: 01/14/2023] Open
Abstract
Hirschsprung disease (HSCR) is characterized by absence of the enteric nervous system (ENS) in the distal bowel. Despite removal of the aganglionic segment, gastrointestinal (GI) problems persist. Cell therapy offers potential treatment but use of genetic models is limited by their poor survival. We have developed a novel model of aganglionosis in which enteric neural crest-derived cells (ENCDCs) express diphtheria toxin (DT) receptor. Local DT injection into the colon wall results in focal, specific, and sustained ENS ablation without altering GI transit or colonic contractility, allowing improved survival over other aganglionosis models. Focal ENS ablation leads to increased smooth muscle and mucosal thickness, and localized inflammation. Transplantation of ENCDCs into this region leads to engraftment, migration, and differentiation of enteric neurons and glial cells, with restoration of normal architecture of the colonic epithelium and muscle, reduction in inflammation, and improved survival.
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Affiliation(s)
- Sukhada Bhave
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | - Emily Arciero
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | - Corey Baker
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | - Wing Lam Ho
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | - Rhian Stavely
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | - Allan M Goldstein
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | - Ryo Hotta
- Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
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12
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Holmes GM, Hubscher CH, Krassioukov A, Jakeman LB, Kleitman N. Recommendations for evaluation of bladder and bowel function in pre-clinical spinal cord injury research. J Spinal Cord Med 2019; 43:165-176. [PMID: 31556844 PMCID: PMC7054945 DOI: 10.1080/10790268.2019.1661697] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Objective: In order to encourage the inclusion of bladder and bowel outcome measures in preclinical spinal cord injury (SCI) research, this paper identifies and categorizes 1) fundamental, 2) recommended, 3) supplemental and 4) exploratory sets of outcome measures for pre-clinical assessment of bladder and bowel function with broad applicability to animal models of SCI.Methods: Drawing upon the collective research experience of autonomic physiologists and informed in consultation with clinical experts, a critical assessment of currently available bladder and bowel outcome measures (histological, biochemical, in vivo functional, ex vivo physiological and electrophysiological tests) was made to identify the strengths, deficiencies and ease of inclusion for future studies of experimental SCI.Results: Based upon pre-established criteria generated by the Neurogenic Bladder and Bowel Working Group that included history of use in experimental settings, citations in the literature by multiple independent groups, ease of general use, reproducibility and sensitivity to change, three fundamental measures each for bladder and bowel assessments were identified. Briefly defined, these assessments centered upon tissue morphology, voiding efficiency/volume and smooth muscle-mediated pressure studies. Additional assessment measures were categorized as recommended, supplemental or exploratory based upon the balance between technical requirements and potential mechanistic insights to be gained by the study.Conclusion: Several fundamental assessments share reasonable levels of technical and material investment, including some that could assess bladder and bowel function non-invasively and simultaneously. Such measures used more inclusively across SCI studies would advance progress in this high priority area. When complemented with a few additional investigator-selected study-relevant supplemental measures, they are highly recommended for research programs investigating the efficacy of therapeutic interventions in preclinical animal models of SCI that have a bladder and/or bowel focus.
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Affiliation(s)
- Gregory M. Holmes
- Neural and Behavioral Sciences, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA,Correspondence to: Gregory M. Holmes, Neural and Behavioral Sciences, Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17036, USA. ;
| | - Charles H. Hubscher
- Department of Anatomical Sciences and Neurobiology, University of Louisville, Louisville, Kentucky, USA,Kentucky Spinal Cord Injury Research Center, University of Louisville, Louisville, Kentucky, USA
| | - Andrei Krassioukov
- ICORD, University of British Columbia, GF Strong Rehabilitation Centre, Vancouver, Canada
| | - Lyn B. Jakeman
- National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA
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13
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Hamzah HH, Keattch O, Yeoman MS, Covill D, Patel BA. Three-Dimensional-Printed Electrochemical Sensor for Simultaneous Dual Monitoring of Serotonin Overflow and Circular Muscle Contraction. Anal Chem 2019; 91:12014-12020. [DOI: 10.1021/acs.analchem.9b02958] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
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14
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Walsh KT, Zemper AE. The Enteric Nervous System for Epithelial Researchers: Basic Anatomy, Techniques, and Interactions With the Epithelium. Cell Mol Gastroenterol Hepatol 2019; 8:369-378. [PMID: 31108231 PMCID: PMC6718943 DOI: 10.1016/j.jcmgh.2019.05.003] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/17/2018] [Revised: 05/02/2019] [Accepted: 05/03/2019] [Indexed: 02/08/2023]
Abstract
The intestinal epithelium does not function in isolation, but interacts with many components including the Enteric Nervous System (ENS). Understanding ENS and intestinal epithelium interactions requires multidisciplinary approaches to uncover cells involved, mechanisms used, and the ultimate influence on intestinal physiology. This review is intended to serve as a reference for epithelial biologists interested in studying these interactions. With this in mind, this review aims to summarize the basic anatomy of the epithelium and ENS, mechanisms by which they interact, and techniques used to study these interactions. We highlight in vitro, ex vivo and in vivo techniques. Additionally, ENS influence on epithelial proliferation and gene expression within stem and differentiated cells as well as gastrointestinal cancer are discussed.
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Key Words
- 5-ht, 5-hydroxytryptamine
- 5-ht3r, 5-hydroxytryptamine 3 receptor
- ach, acetylcholine
- aitc, allyl isothicyanate
- cpi, crypt proliferation index
- eec, enteroendocrine cell
- ens, enteric nervous system
- gi, gastrointestinal
- hio, human intestinal organoid
- isc, intestinal stem cell
- lgr5, leucine-rich repeat–containing g protein–coupled receptor
- ne, norepinephrine
- ngf, nerve growth factor
- si, small intestine
- ta, transit-amplifying
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Affiliation(s)
- Kathleen T. Walsh
- Institute of Molecular Biology, University of Oregon, Eugene, Oregon,Institute of Neuroscience, University of Oregon, Eugene, Oregon,Department of Biology, University of Oregon, Eugene, Oregon
| | - Anne E. Zemper
- Institute of Molecular Biology, University of Oregon, Eugene, Oregon,Department of Biology, University of Oregon, Eugene, Oregon,Correspondence Address correspondence to: Anne E. Zemper, PhD, University of Oregon, 218 Streisinger Hall, 1370 Franklin Boulevard, Eugene, Oregon 97401. fax: (541) 346–6056.
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15
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Kanemasa T, Koike K, Arai T, Ono H, Horita N, Chiba H, Nakamura A, Morioka Y, Kihara T, Hasegawa M. Pharmacologic effects of naldemedine, a peripherally acting μ-opioid receptor antagonist, in in vitro and in vivo models of opioid-induced constipation. Neurogastroenterol Motil 2019; 31:e13563. [PMID: 30821019 PMCID: PMC6850587 DOI: 10.1111/nmo.13563] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/06/2018] [Revised: 01/07/2019] [Accepted: 01/08/2019] [Indexed: 12/25/2022]
Abstract
BACKGROUND Naldemedine (S-297995) is a peripherally acting μ-opioid receptor antagonist developed as a once-daily oral drug for opioid-induced constipation (OIC) in adults with chronic noncancer or cancer pain. This study characterized the pharmacological effects of naldemedine in vitro and in vivo. METHODS The binding affinity and antagonist activity of naldemedine against recombinant human μ-, δ-, and κ-opioid receptors were assayed in vitro. Pharmacologic effects of naldemedine were investigated using animal models of morphine-induced inhibition of small and large intestinal transit, castor oil-induced diarrhea, antinociception, and morphine withdrawal. KEY RESULTS Naldemedine showed potent binding affinity and antagonist activities for recombinant human μ-, δ-, and κ-opioid receptors. Naldemedine significantly reduced opioid-induced inhibition of small intestinal transit (0.03-10 mg kg-1 ; P < 0.05) and large intestinal transit (0.3-1 μmol L-1 ; P < 0.05). Naldemedine (0.03-1 mg kg-1 ) pretreatment significantly reversed the inhibition of castor oil-induced diarrhea by subcutaneous morphine (P < 0.01). Naldemedine (1-30 mg kg-1 ) pretreatment (1 or 2 hours) did not alter the analgesic effects of morphine in a model measuring the latency of a rat to flick its tail following thermal stimulation. However, a significant delayed reduction of the analgesic effect of morphine was seen with higher doses of naldemedine (10-30 mg kg-1 ). Some centrally mediated and peripherally mediated withdrawal signs in morphine-dependent rats were seen with naldemedine doses ≥3 and ≥0.3 mg kg-1 , respectively. CONCLUSIONS & INFERENCES Naldemedine displayed potent binding affinity to, and antagonistic activity against, μ-, δ-, and κ-opioid receptors. Naldemedine tempered OIC in vivo without compromising opioid analgesia.
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Affiliation(s)
- Toshiyuki Kanemasa
- Neuroscience, Drug Discovery & Disease Research LaboratoryShionogi & Co., Ltd.OsakaJapan
| | - Katsumi Koike
- Neuroscience, Drug Discovery & Disease Research LaboratoryShionogi & Co., Ltd.OsakaJapan
| | - Tohko Arai
- Drug Safety Evaluation, Research Laboratory for DevelopmentShionogi & Co., LtdOsakaJapan
| | - Hiroko Ono
- Neuroscience, Drug Discovery & Disease Research LaboratoryShionogi & Co., Ltd.OsakaJapan
| | - Narumi Horita
- Neuroscience, Drug Discovery & Disease Research LaboratoryShionogi & Co., Ltd.OsakaJapan
| | - Hiroki Chiba
- Neuroscience, Drug Discovery & Disease Research LaboratoryShionogi & Co., Ltd.OsakaJapan
| | - Atsushi Nakamura
- Neuroscience, Drug Discovery & Disease Research LaboratoryShionogi & Co., Ltd.OsakaJapan
| | - Yasuhide Morioka
- Neuroscience, Drug Discovery & Disease Research LaboratoryShionogi & Co., Ltd.OsakaJapan
| | - Tsuyoshi Kihara
- Neuroscience, Drug Discovery & Disease Research LaboratoryShionogi & Co., Ltd.OsakaJapan
| | - Minoru Hasegawa
- Neuroscience, Drug Discovery & Disease Research LaboratoryShionogi & Co., Ltd.OsakaJapan
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16
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Nyavor Y, Estill R, Edwards H, Ogden H, Heideman K, Starks K, Miller C, May G, Flesch L, McMillan J, Gericke M, Forney L, Balemba O. Intestinal nerve cell injury occurs prior to insulin resistance in female mice ingesting a high-fat diet. Cell Tissue Res 2019; 376:325-340. [PMID: 30778729 DOI: 10.1007/s00441-019-03002-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2018] [Accepted: 01/30/2019] [Indexed: 12/11/2022]
Abstract
Diabetic patients suffer from gastrointestinal disorders associated with dysmotility, enteric neuropathy and dysbiosis of gut microbiota; however, gender differences are not fully known. Previous studies have shown that a high-fat diet (HFD) causes type two diabetes (T2D) in male mice after 4-8 weeks but only does so in female mice after 16 weeks. This study seeks to determine whether sex influences the development of intestinal dysmotility, enteric neuropathy and dysbiosis in mice fed HFD. We fed 8-week-old C57BL6 male and female mice a standard chow diet (SCD) or a 72% kcal HFD for 8 weeks. We analyzed the associations between sex and intestinal dysmotility, neuropathy and dysbiosis using motility assays, immunohistochemistry and next-generation sequencing. HFD ingestion caused obesity, glucose intolerance and insulin resistance in male but not female mice. However, HFD ingestion slowed intestinal propulsive motility in both male and female mice. This was associated with decreased inhibitory neuromuscular transmission, loss of myenteric inhibitory motor neurons and axonal swelling and loss of cytoskeletal filaments. HFD induced dysbiosis and changed the abundance of specific bacteria, especially Allobaculum, Bifidobacterium and Lactobacillus, which correlated with dysmotility and neuropathy. Female mice had higher immunoreactivity and numbers of myenteric inhibitory motor neurons, matching larger amplitudes of inhibitory junction potentials. This study suggests that sex influences the development of HFD-induced metabolic syndrome but dysmotility, neuropathy and dysbiosis occur independent of sex and prior to T2D conditions. Gastrointestinal dysmotility, neuropathy and dysbiosis might play a crucial role in the pathophysiology of T2D in humans irrespective of sex.
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Affiliation(s)
- Yvonne Nyavor
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA
| | - Rachel Estill
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA
| | - Hannah Edwards
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA
| | - Hailey Ogden
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA
| | - Kaila Heideman
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA
| | - Kiefer Starks
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA
| | - Christopher Miller
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA
| | - George May
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA
| | - Lance Flesch
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA
| | - John McMillan
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA
| | - Martin Gericke
- Institute for Anatomy, University of Leipzig, Liebigstraße 13, 04103, Leipzig, Germany
| | - Larry Forney
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA
| | - Onesmo Balemba
- Department of Biological Sciences, University of Idaho, 875 Perimeter Drive, LSS 252, Moscow, ID, 83844, USA.
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17
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Opportunities and Challenges for Single-Unit Recordings from Enteric Neurons in Awake Animals. MICROMACHINES 2018; 9:mi9090428. [PMID: 30424361 PMCID: PMC6187697 DOI: 10.3390/mi9090428] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Revised: 08/17/2018] [Accepted: 08/23/2018] [Indexed: 12/18/2022]
Abstract
Advanced electrode designs have made single-unit neural recordings commonplace in modern neuroscience research. However, single-unit resolution remains out of reach for the intrinsic neurons of the gastrointestinal system. Single-unit recordings of the enteric (gut) nervous system have been conducted in anesthetized animal models and excised tissue, but there is a large physiological gap between awake and anesthetized animals, particularly for the enteric nervous system. Here, we describe the opportunity for advancing enteric neuroscience offered by single-unit recording capabilities in awake animals. We highlight the primary challenges to microelectrodes in the gastrointestinal system including structural, physiological, and signal quality challenges, and we provide design criteria recommendations for enteric microelectrodes.
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18
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White AR, Holmes GM. Anatomical and Functional Changes to the Colonic Neuromuscular Compartment after Experimental Spinal Cord Injury. J Neurotrauma 2018; 35:1079-1090. [PMID: 29205096 DOI: 10.1089/neu.2017.5369] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
A profound reduction in colorectal transit time accompanies spinal cord injury (SCI), yet the colonic alterations after SCI have yet to be understood fully. The loss of descending supraspinal input to lumbosacral neural circuits innervating the colon is recognized as one causal mechanism. Remodeling of the colonic enteric nervous system/smooth muscle junction in response to inflammation, however, is recognized as one factor leading to colonic dysmotility in other pathophysiological models. We investigated the alterations to the neuromuscular junction in rats with experimental high-thoracic (T3) SCI. One day to three weeks post-injury, both injured and age-matched controls underwent in vivo experimentation followed by tissue harvest for histological evaluation. Spontaneous colonic contractions were reduced significantly in the proximal and distal colon of T3-SCI rats. Histological evaluation of proximal and distal colon demonstrated significant reductions of colonic mucosal crypt depth and width. Markers of intestinal inflammation were assayed by qRT-PCR. Specifically, Icam1, Ccl2 (MCP-1), and Ccl3 (MIP-1α) mRNA was acutely elevated after T3-SCI. Smooth muscle thickness and collagen content of the colon were increased significantly in T3-SCI rats. Colonic cross sections immunohistochemically processed for the pan-neuronal marker HuC/D displayed a significant decrease in colonic enteric neuron density that became more pronounced at three weeks after injury. Our data suggest that post-SCI inflammation and remodeling of the enteric neuromuscular compartment accompanies SCI. These morphological changes may provoke the diminished colonic motility that occurs during this same period, possibly through the disruption of intrinsic neuromuscular control of the colon.
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Affiliation(s)
- Amanda R White
- Department of Neural and Behavioral Sciences, Penn State University College of Medicine , Hershey, Pennsylvania
| | - Gregory M Holmes
- Department of Neural and Behavioral Sciences, Penn State University College of Medicine , Hershey, Pennsylvania
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19
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Wong KKL, Tang LCY, Zhou J, Ho V. Analysis of spatiotemporal pattern and quantification of gastrointestinal slow waves caused by anticholinergic drugs. Organogenesis 2017; 13:39-62. [PMID: 28277890 DOI: 10.1080/15476278.2017.1295904] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
Anticholinergic drugs are well-known to cause adverse effects, such as constipation, but their effects on baseline contractile activity in the gut driven by slow waves is not well established. In a video-based gastrointestinal motility monitoring (GIMM) system, a mouse's small intestine was placed in Krebs solution and recorded using a high definition camera. Untreated controls were recorded for each specimen, then treated with a therapeutic concentration of the drug, and finally, treated with a supratherapeutic dose of the drug. Next, the video clips showing gastrointestinal motility were processed, giving us the segmentation motions of the intestine, which were then converted via Fast Fourier Transform (FFT) into their respective frequency spectrums. These contraction quantifications were analyzed from the video recordings under standardised conditions to evaluate the effect of drugs. Six experimental trials were included with benztropine and promethazine treatments. Only the supratherapeutic dose of benztropine was shown to significantly decrease the amplitude of contractions; at therapeutic doses of both drugs, neither frequency nor amplitude was significantly affected. We have demonstrated that intestinal slow waves can be analyzed based on the colonic frequency or amplitude at a supratherapeutic dose of the anticholinergic medications. More research is required on the effects of anticholinergic drugs on these slow waves to ascertain the true role of ICC in neurologic control of gastrointestinal motility.
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Affiliation(s)
- Kelvin K L Wong
- a School of Medicine, Western Sydney University , Campbelltown , NSW , Australia
| | - Lauren C Y Tang
- a School of Medicine, Western Sydney University , Campbelltown , NSW , Australia
| | - Jerry Zhou
- a School of Medicine, Western Sydney University , Campbelltown , NSW , Australia
| | - Vincent Ho
- a School of Medicine, Western Sydney University , Campbelltown , NSW , Australia
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20
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Spohn SN, Bianco F, Scott RB, Keenan CM, Linton AA, O’Neill CH, Bonora E, Dicay M, Lavoie B, Wilcox RL, MacNaughton WK, De Giorgio R, Sharkey KA, Mawe GM. Protective Actions of Epithelial 5-Hydroxytryptamine 4 Receptors in Normal and Inflamed Colon. Gastroenterology 2016; 151:933-944.e3. [PMID: 27480173 PMCID: PMC5159265 DOI: 10.1053/j.gastro.2016.07.032] [Citation(s) in RCA: 88] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/12/2016] [Revised: 06/16/2016] [Accepted: 07/06/2016] [Indexed: 12/12/2022]
Abstract
BACKGROUND & AIMS The 5-hydroxytryptamine receptor 4 (5-HT4R or HTR4) is expressed in the colonic epithelium but little is known about its functions there. We examined whether activation of colonic epithelial 5-HT4R protects colons of mice from inflammation. METHODS The 5-HT4R agonist tegaserod (1 mg/kg), the 5-HT4R antagonist GR113808 (1 mg/kg), or vehicle (control) were delivered by enema to wild-type or 5-HT4R knockout mice at the onset of, or during, active colitis, induced by administration of dextran sodium sulfate or trinitrobenzene sulfonic acid. Inflammation was measured using the colitis disease activity index and by histologic analysis of intestinal tissues. Epithelial proliferation, wound healing, and resistance to oxidative stress-induced apoptosis were assessed, as was colonic motility. RESULTS Rectal administration of tegaserod reduced the severity of colitis compared with mice given vehicle, and accelerated recovery from active colitis. Rectal tegaserod did not improve colitis in 5-HT4R knockout mice, and intraperitoneally administered tegaserod did not protect wild-type mice from colitis. Tegaserod increased proliferation of crypt epithelial cells. Stimulation of 5-HT4R increased Caco-2 cell migration and reduced oxidative stress-induced apoptosis; these actions were blocked by co-administration of the 5-HT4R antagonist GR113808. In noninflamed colons of wild-type mice not receiving tegaserod, inhibition of 5-HT4Rs resulted in signs of colitis within 3 days. In these mice, epithelial proliferation decreased and bacterial translocation to the liver and spleen was detected. Daily administration of tegaserod increased motility in inflamed colons of guinea pigs and mice, whereas administration of GR113808 disrupted motility in animals without colitis. CONCLUSIONS 5-HT4R activation maintains motility in healthy colons of mice and guinea pigs, and reduces inflammation in colons of mice with colitis. Agonists might be developed as treatments for patients with inflammatory bowel diseases.
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Affiliation(s)
| | - Francesca Bianco
- Department of Veterinary Medical Sciences,Department of Medical and Surgical Sciences, University of Bologna, Italy
| | | | - Catherine M. Keenan
- Hotchkiss Brain Institute, Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada,Snyder Institute for Chronic Diseases, Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada
| | | | | | - Elena Bonora
- Department of Medical and Surgical Sciences, University of Bologna, Italy
| | - Michael Dicay
- Hotchkiss Brain Institute, Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada
| | - Brigitte Lavoie
- Neurological Sciences, University of Vermont, Burlington, VT
| | | | - Wallace K. MacNaughton
- Hotchkiss Brain Institute, Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada
| | - Roberto De Giorgio
- Department of Medical and Surgical Sciences, University of Bologna, Italy
| | - Keith A. Sharkey
- Hotchkiss Brain Institute, Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada,Snyder Institute for Chronic Diseases, Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada
| | - Gary M. Mawe
- Neurological Sciences, University of Vermont, Burlington, VT
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21
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Electrochemical fecal pellet sensor for simultaneous real-time ex vivo detection of colonic serotonin signalling and motility. Sci Rep 2016; 6:23442. [PMID: 27000971 PMCID: PMC4802304 DOI: 10.1038/srep23442] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2015] [Accepted: 03/07/2016] [Indexed: 12/21/2022] Open
Abstract
Various investigations have focused on understanding the relationship between mucosal serotonin (5-HT) and colonic motility, however contradictory studies have questioned the importance of this intestinal transmitter. Here we described the fabrication and use of a fecal pellet electrochemical sensor that can be used to simultaneously detect the release of luminal 5-HT and colonic motility. Fecal pellet sensor devices were fabricated using carbon nanotube composite electrodes that were housed in 3D printed components in order to generate a device that had shape and size that mimicked a natural fecal pellet. Devices were fabricated where varying regions of the pellet contained the electrode. Devices showed that they were stable and sensitive for ex vivo detection of 5-HT, and no differences in the fecal pellet velocity was observed when compared to natural fecal pellets. The onset of mucosal 5-HT was observed prior to the movement of the fecal pellet. The release of mucosal 5-HT occurred oral to the fecal pellet and was linked to the contraction of the bowel wall that drove pellet propulsion. Taken, together these findings provide new insights into the role of mucosal 5-HT and suggest that the transmitter acts as a key initiator of fecal pellet propulsion.
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22
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Swaminathan M, Hill-Yardin E, Ellis M, Zygorodimos M, Johnston LA, Gwynne RM, Bornstein JC. Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice. J Vis Exp 2016:53828. [PMID: 26862815 DOI: 10.3791/53828] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3(R451C) mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.
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Affiliation(s)
| | | | - Melina Ellis
- Department of Physiology, The University of Melbourne
| | | | - Leigh A Johnston
- Department of Electrical and Electronic Engineering, The University of Melbourne
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23
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Kendig DM, Hurst NR, Grider JR. Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines. J Vis Exp 2016:e53263. [PMID: 26863156 PMCID: PMC4781693 DOI: 10.3791/53263] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.
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Affiliation(s)
- Derek M Kendig
- Department of Physiology and Biophysics, Virginia Commonwealth University; Department of Biology, Loyola University Maryland;
| | - Norm R Hurst
- Department of Physiology and Biophysics, Virginia Commonwealth University
| | - John R Grider
- Department of Physiology and Biophysics, Virginia Commonwealth University
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The Intrinsic Reflex Circuitry of the Inflamed Colon. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016; 891:153-7. [DOI: 10.1007/978-3-319-27592-5_15] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
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Mawe GM. Colitis-induced neuroplasticity disrupts motility in the inflamed and post-inflamed colon. J Clin Invest 2015; 125:949-55. [PMID: 25729851 DOI: 10.1172/jci76306] [Citation(s) in RCA: 73] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Effective colonic motility involves an intricate pattern of excitatory and inhibitory neuromuscular signals that arise from the enteric neural circuitry of the colon. Recent investigations have demonstrated that inflammation leads to a variety of changes in the physiological properties of the neurons in this circuitry, including hyperexcitability of neurons at the afferent end of the peristaltic reflex, synaptic facilitation, and attenuated inhibitory neuromuscular transmission. Furthermore, links have been established between these changes and disrupted motor activity in the colon, and we now know that some of these changes persist long after recovery from inflammation. It is highly likely that inflammation-induced neuroplasticity, which is not detectable by clinical diagnostics, contributes to disrupted motility in active and quiescent inflammatory bowel disease and in functional gastrointestinal disorders.
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Hurst NR, Kendig DM, Murthy KS, Grider JR. The short chain fatty acids, butyrate and propionate, have differential effects on the motility of the guinea pig colon. Neurogastroenterol Motil 2014; 26:1586-96. [PMID: 25223619 PMCID: PMC4438679 DOI: 10.1111/nmo.12425] [Citation(s) in RCA: 82] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/27/2014] [Accepted: 08/12/2014] [Indexed: 12/13/2022]
Abstract
BACKGROUND Colonic microbiota digest resistant starches producing short chain fatty acids (SCFAs). The main SCFAs produced are acetate, propionate, and butyrate. Both excitatory and inhibitory effects of SCFAs on motility have been reported. We hypothesized that the effect of SCFAs on colonic motility varies with chain length and aimed to determine the effects of SCFAs on propagating and non-propagating contractions of guinea pig proximal and distal colon. METHODS In isolated proximal colonic segments, Krebs solution alone or containing 10-100 mM acetate, propionate, or butyrate was injected into the lumen, motility was videorecorded over 10 min, and spatiotemporal maps created. In distal colon, the lumen was perfused with the same solutions of SCFAs at 0.1 mL/min, the movement of artificial fecal pellets videorecorded, and velocity of propulsion calculated. KEY RESULTS In proximal colon, butyrate increased the frequency of full-length propagations, decreased short propagations, and had a biphasic effect on non-propagating contractions. Propionate blocked full and short propagations and had a biphasic effect on non-propagating contractions. Acetate decreased short and total propagations. In distal colon, butyrate increased and propionate decreased velocity of propulsion. CONCLUSIONS & INFERENCES The data suggest that luminal SCFAs have differing effects on proximal and distal colonic motility depending on chain length. Thus, the net effect of SCFAs on colonic motility would depend on the balance of SCFAs produced by microbial digestion of resistant starches.
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Affiliation(s)
- Norm R Hurst
- Department of Physiology and Biophysics, VCU Program in Enteric Neuromuscular Science (VPENS), Virginia Commonwealth University, Richmond, VA, USA
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Roberts JA, Durnin L, Sharkey KA, Mutafova-Yambolieva VN, Mawe GM. Oxidative stress disrupts purinergic neuromuscular transmission in the inflamed colon. J Physiol 2013; 591:3725-37. [PMID: 23732648 DOI: 10.1113/jphysiol.2013.254136] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Colitis, induced by trinitrobenzene sulfonic acid (TNBS) in guinea pig, leads to decreased purinergic neuromuscular transmission resulting in a reduction in inhibitory junction potentials (IJPs) in colonic circular muscle. We explored possible mechanisms responsible for this inflammation-induced neurotransmitter plasticity. Previous studies have suggested that the deficit in inflamed tissue involves decreased ATP release. We therefore hypothesized that decreased purinergic transmission results from inflammation-induced free radical damage to mitochondria, leading to decreased purine synthesis and release. Stimulus-induced release of purines was measured using high-performance liquid chromatography, and quantities of all purines measured were significantly reduced in the inflamed colons as compared to controls. To test whether decreased mitochondrial function affects the IJP, colonic muscularis preparations were treated with the mitochondrial ATP synthase inhibitors oligomycin or dicyclohexylcarbodiimide, which resulted in a significant reduction of IJP amplitude. Induction of oxidative stress in vitro, by addition of H2O2 to the preparation, also significantly reduced IJP amplitude. Purinergic neuromuscular transmission was significantly restored in TNBS-inflamed guinea pigs, and in dextran sodium sulfate-inflamed mice, treated with a free radical scavenger. Furthermore, propulsive motility in the distal colons of guinea pigs with TNBS colitis was improved by in vivo treatment with the free radical scavenger. We conclude that oxidative stress contributes to the reduction in purinergic neuromuscular transmission measured in animal models of colitis, and that these changes can be prevented by treatment with a free radical scavenger, resulting in improved motility.
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Affiliation(s)
- Jane A Roberts
- Department of Neurological Sciences, University of Vermont, Burlington, VT 05405, USA
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Kornyushin O, Galagudza M, Kotslova A, Nutfullina G, Shved N, Nevorotin A, Sedov V, Vlasov T. Intestinal injury can be reduced by intra-arterial postischemic perfusion with hypertonic saline. World J Gastroenterol 2013; 19:209-218. [PMID: 23345943 PMCID: PMC3547561 DOI: 10.3748/wjg.v19.i2.209] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/30/2012] [Accepted: 11/15/2012] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the effect of local intestinal perfusion with hypertonic saline (HTS) on intestinal ischemia-reperfusion injury (IRI) in both ex vivo and in vivo rat models.
METHODS: All experiments were performed on male Wistar rats anesthetized with pentobarbital sodium given intraperitoneally at a dose of 60 mg/kg. Ex vivo vascularly perfused rat intestine was subjected to 60-min ischemia and either 30-min reperfusion with isotonic buffer (controls), or 5 min with HTS of 365 or 415 mOsm/L osmolarity (HTS365mOsm or HTS415mOsm, respectively) followed by 25-min reperfusion with isotonic buffer. The vascular intestinal perfusate flow (IPF) rate was determined by collection of the effluent from the portal vein in a calibrated tube. Spontaneous intestinal contraction rate was monitored throughout. Irreversible intestinal injury or area of necrosis (AN) was evaluated histochemically using 2.3.5-triphenyltetrazolium chloride staining. In vivo, 30-min ischemia was followed by either 30-min blood perfusion or 5-min reperfusion with HTS365mOsm through the superior mesenteric artery (SMA) followed by 25-min blood perfusion. Arterial blood pressure (BP) was measured in the common carotid artery using a miniature pressure transducer. Histological injury was evaluated in both preparations using the Chui score.
RESULTS: Ex vivo, intestinal IRI resulted in a reduction in the IPF rate during reperfusion (P < 0.05 vs sham). The postischemic recovery of the IPF rate did not differ between the controls and the HTS365mOsm group. In the HTS415mOsm group, postischemic IPF rates were lower than in the controls and the HTS365mOsm group (P < 0.05). The intestinal contraction rate was similar at baseline in all groups. An increase in this parameter was observed during the first 10 min of reperfusion in the control group as compared to the sham-treated group, but no such increase was seen in the HTS365mOsm group. In controls, AN averaged 14.8% ± 5.07% of the total tissue volume. Administration of HTS365mOsm for 5 min after 60-min ischemia resulted in decrease in AN (5.1% ± 1.20% vs controls, P < 0.01). However, perfusion of the intestine with the HTS of greater osmolarity (HTS415mOsm) failed to protect the intestine from irreversible injury. The Chiu score was lower in the HTS365mOsm group in comparison with controls (2.4 ± 0.54 vs 3.2 ± 0.44, P = 0.042), while intestinal perfusion with HTS415mOsm failed to improve the Chiu score. Intestinal reperfusion with HTS365mOsm in the in vivo series secured rapid recovery of BP after its transient fall, whereas in the controls no recovery was seen. The Chiu score was lower in the HTS365mOsm group vs controls (3.1 ± 0.26 and 3.8 ± 0.22, P = 0.0079 respectively,), although the magnitude of the effect was lower than in the ex vivo series.
CONCLUSION: Brief intestinal postischemic perfusion with HTS365mOsm through the SMA followed by blood flow restoration is a protective procedure that could be used for the prevention of intestinal IRI.
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Hoffman JM, Tyler K, MacEachern SJ, Balemba OB, Johnson AC, Brooks EM, Zhao H, Swain GM, Moses PL, Galligan JJ, Sharkey KA, Greenwood-Van Meerveld B, Mawe GM. Activation of colonic mucosal 5-HT(4) receptors accelerates propulsive motility and inhibits visceral hypersensitivity. Gastroenterology 2012; 142:844-854.e4. [PMID: 22226658 PMCID: PMC3477545 DOI: 10.1053/j.gastro.2011.12.041] [Citation(s) in RCA: 216] [Impact Index Per Article: 16.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2011] [Revised: 12/07/2011] [Accepted: 12/13/2011] [Indexed: 12/19/2022]
Abstract
BACKGROUND & AIMS 5-hydroxytryptamine receptor (5-HT(4)R) agonists promote gastrointestinal motility and attenuate visceral pain, but concerns about adverse reactions have restricted their availability. We tested the hypotheses that 5-HT(4) receptors are expressed in the colonic epithelium and that 5-HT(4)R agonists can act intraluminally to increase motility and reduce visceral hypersensitivity. METHODS Mucosal expression of the 5-HT(4)R was evaluated by reverse-transcriptase polymerase chain reaction and immunohistochemical analysis of tissues from 5-HT(4)R(BAC)-enhanced green fluorescent protein mice. Amperometry, histology, and short-circuit current measurements were used to study 5-HT, mucus, and Cl(-) secretion, respectively. Propulsive motility was measured in guinea pig distal colon, and visceromotor responses were recorded in a rat model of colonic hypersensitivity. 5-HT(4)R compounds included cisapride, tegaserod, naronapride, SB204070, and GR113808. RESULTS Mucosal 5-HT(4) receptors were present in the small and large intestines. In the distal colon, 5-HT(4) receptors were expressed by most epithelial cells, including enterochromaffin and goblet cells. Stimulation of 5-HT(4)Rs evoked mucosal 5-HT release, goblet cell degranulation, and Cl(-) secretion. Luminal administration of 5-HT(4)R agonists accelerated propulsive motility; a 5-HT(4)R antagonist blocked this effect. Bath application of 5-HT(4)R agonists did not affect motility. Oral or intracolonic administration of 5-HT(4)R agonists attenuated visceral hypersensitivity. Intracolonic administration was more potent than oral administration, and was inhibited by a 5-HT(4)R antagonist. CONCLUSIONS Mucosal 5-HT(4) receptor activation can mediate the prokinetic and antinociceptive actions of 5-HT(4)R agonists. Colon-targeted, intraluminal delivery of 5-HT(4)R agonists might be used to promote motility and alleviate visceral pain, while restricting systemic bioavailability and resulting adverse side effects.
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MESH Headings
- Administration, Oral
- Analgesics/administration & dosage
- Analgesics/pharmacology
- Animals
- Chlorides/metabolism
- Chromosomes, Artificial, Bacterial
- Colon/drug effects
- Colon/innervation
- Colon/metabolism
- Disease Models, Animal
- Enterochromaffin Cells/drug effects
- Enterochromaffin Cells/metabolism
- Gastrointestinal Agents/administration & dosage
- Gastrointestinal Agents/pharmacology
- Gastrointestinal Motility/drug effects
- Goblet Cells/drug effects
- Goblet Cells/metabolism
- Green Fluorescent Proteins/genetics
- Green Fluorescent Proteins/metabolism
- Guinea Pigs
- Humans
- Hyperalgesia/metabolism
- Hyperalgesia/physiopathology
- Hyperalgesia/prevention & control
- Immunohistochemistry
- Intestinal Mucosa/drug effects
- Intestinal Mucosa/innervation
- Intestinal Mucosa/metabolism
- Male
- Membrane Potentials
- Mice
- Mice, Inbred BALB C
- Mice, Transgenic
- Mucus/metabolism
- Pain/metabolism
- Pain/physiopathology
- Pain/prevention & control
- Pain Threshold/drug effects
- Pressure
- Rats
- Rats, Sprague-Dawley
- Receptors, Serotonin, 5-HT4/drug effects
- Receptors, Serotonin, 5-HT4/genetics
- Receptors, Serotonin, 5-HT4/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Serotonin/metabolism
- Serotonin 5-HT4 Receptor Agonists/administration & dosage
- Serotonin 5-HT4 Receptor Agonists/pharmacology
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Affiliation(s)
- Jill M Hoffman
- Department of Anatomy & Neurobiology, University of Vermont, Burlington, Vermont 05405, USA
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