Assessment of Intestinal Peristalsis in Vitro

Cristina Pozzoli1, Enzo Poli1

1 University of Parma Medical School, Parma, Italy
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 21.11
DOI:  10.1002/0471140856.tx2111s54
Online Posting Date:  November, 2012
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Abstract

The protocol detailed in this unit is designed to assess intestinal peristaltic motility in the isolated small intestine in vitro and to measure the effects of drugs able to interfere with gut propulsive activity. The procedure is based on Trendelenburg's classic technique, described at the beginning of the 20th century in the isolated guinea pig ileum and, later on, extended to other intestinal preparations from the same animal and other animal species. This unit illustrates the basic procedures for setting up the intestinal preparation, recording peristalsis under near‐physiologic conditions, and testing the pharmaco‐toxicological effects of drugs and pollutants on the contractile behavior of the gut wall. The protocol allows evaluating the action of drugs affecting sensory and/or motor neurons of the enteric nervous system and how these neurons control the development of the motor program of the gut wall. This model can be exploited to investigate novel compounds undergoing preclinical development and both inhibitors and stimulants of gastrointestinal peristaltic activity, as well as environmental or alimentary pollutants, like xenobiotics and naturally‐occurring toxins, endowed with noxious activity with regard to digestive functions. Curr. Protoc. Toxicol. 54:21.11.1‐21.11.14. © 2012 by John Wiley & Sons, Inc.

Keywords: guinea pig ileum; isolated intestine; perfused ileum; ileal peristalsis; gut propulsion

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Male Hartley guinea pigs (300 to 400 g, Harlan Labs)
  • Standard pellet guinea pig diet
  • Modified Krebs' solution (see recipe), freshly prepared and continuously gassed with 95% CO 2 in oxygen (O 2) (carbogen); prepare from modified Krebs' buffer stock solution (see recipe)
  • Cylinder containing 5% (v/v) CO 2 in oxygen (carbogen)
  • Diethyl ether (Fluka)
  • Test compounds (see recipe)
  • Tetrodotoxin (see recipe)
  • RBS 35 Solution Concentrate (Sigma‐Aldrich, cat. no. 83461); use this product diluted 1:10 (v/v) as a detergent for general cleaning purposes (equivalent products are available)
  • Polypropylene animal cages (approximate floor area 2000 cm2) with wire mesh grids at bottom
  • Oxygenation set, to deliver CO 2/O 2 mixture: when a centralized distribution system is not available, the mixture can be delivered from cylinders kept in a safe place near the lab bench and connected to the dispensing line (use a pressure regulator on the cylinder, e.g. AirCom model RH200, http://www.aircom.net/, and adjust the pressure of gas mixture in the line)
  • Perfusion setup (e.g., Horizontal Tissue Bath IPR, Type 813/2, Harvard Apparatus) including:
    • Jacketed organ bath, volume 30 ml (inner dimensions 10 × 2 × 1.5 cm for horizontal accommodation of preparation; the organ bath used in the authors' lab is made by a local glassworker; complete organ bath settings, which include peristaltic pump, thermostat, tubing and cannulas, are commercially available, e.g., from Harvard Apparatus)
    • Thermostatically regulated water circulator (e.g., Julabo heating immersion circulator mod. 9116000; http://www.julabo.com/)
    • Peristaltic pump, capable of delivering 0.1 to 3 ml/min (e.g., Gilson Minipuls series)
    • Perfusion rubber tubes, specific for peristaltic pump; use tubes suitable to obtain a perfusion rate ranging from 0.1 to 3 ml/min
    • 500‐ml flasks
    • T‐shaped inflow cannula (3 mm internal diameter; e.g., T‐shape connector, cat. no. 1528 00, BrandTech Scientific, http://www.brandtech.com/)
    • Outflow cannula (1‐mm internal diameter)
  • Fine glass sinter
  • Thermometer
  • Magnetic stirrers
  • Pressure transducer: a standard thin‐layer transducer can be used; the transducer should be sensitive enough to measure change in pressure between 10 and 2 kPa (Statham model P23XL could be suitable; Grass Instruments)
  • 5‐ml plastic syringes
  • Polygraph/chart recorder: use a pen‐writing polygraph (the two‐channel recorder model Gemini, from Ugo Basile, equipped with interface with PC connection, is suitable to record a single experiment)
  • Mercury sphygmomanometer
  • Glass bell large enough to accommodate guinea pig
  • 50‐ml plastic beaker
  • Hydrophilic cotton
  • Hemostat (curved, 15 cm long)
  • Dissecting instruments:
    • Straight tip scissors (5 cm)
    • Fine forceps (3 cm)
    • Pins
  • White 50‐wt cotton wool thread
  • Petri dishes with attached a silicon or cork disk
  • PC‐based acquisition system (optional, e.g., BIOPAC, Biological Instruments)
  • Dedicated computer program (e.g., GraphPad Prism Software, Version 3.0 or higher)
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Figures

Videos

Literature Cited

Literature Cited
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   Bartho, L. and Holzer, P. 1995. The inhibitory modulation of guinea‐pig intestinal peristalsis caused by capsaicin involves calcitonin gene‐related peptide and nitric oxide. Naunyn Schmiedebergs Arch. Pharmacol. 353:102‐109.
   Beckett, E.A.H., McCloskey, C., O'Kane, N., Sanders, K.M., and Koh, S.D. 2006. Effects of female steroid hormones on A‐type K+ currents in murine colon. J. Physiol. 573:453‐468.
   Bennett, A. 1968. Relationship between the in vitro studies of gastrointestinal muscle and motility of the alimentary tract in vivo. Am. J. Dig. Dis. 13:410‐414.
   Bennett, A. 1973. The pharmacology of isolated gastrointestinal muscle. In Pharmacology of Gastrointestinal Motility and Secretion, I.E.P.T. Section 39A Volume 2. (P. Holton ed.) Pergamon Press, New York.
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   Buchheit, K.H. and Buhl, T. 1991. Prokinetic benzamides stimulate peristaltic activity in the isolated guinea pig ileum by activation of 5‐HT4 receptors. Eur. J. Pharmacol. 205:203‐208.
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   Herbert, M.K., Roth‐Goldbrunner, S., Holzer, P., and Roewer, N. 2002. Clonidine and dexmedetomidine potently inhibit peristalsis in the guinea pig ileum in vitro. Anesthesiology 97:1491‐1499.
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   Percy, W.H. 1996. In vitro techniques for the study of gastrointestinal motility. In Handbook of Methods in Gastrointestinal Pharmacology. (T.S. Gaginella, ed.) pp. 189‐224. CRC Press, Inc., Boca Raton, Florida.
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