Assessment of Gastrointestinal Motility Using Three Different Assays In Vitro

Cristina Pozzoli1, Enzo Poli1

1 University of Parma Medical School, Parma, Italy
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 21.8
DOI:  10.1002/0471140856.tx2108s46
Online Posting Date:  November, 2010
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Abstract

The protocols detailed in this unit are designed to assess the motor activity of different gastric and intestinal muscle preparations in vitro and the effects of drugs that modulate gastrointestinal motility. The preparations described are characterized by different contractile behaviors, consisting of spontaneous (duodenum), neurogenic (ileum), and drug‐stimulated (fundus, ileum) motility; these reproduce motility patterns occurring in the gut wall in vivo. These protocols document the variety of factors that can influence the responses of isolated tissues and describe how such tissues can be used for testing substances that affect gut movements. These preparations allow evaluation of direct interactions with the processes that control contractile machinery, as well as indirect effects resulting from the modification of neurotransmitter release from myenteric neurons. These models can be exploited to assay novel compounds undergoing preclinical development or to evaluate the functional toxicity exerted by environmental or alimentary pollutants, like xenobiotics and naturally occurring toxins, as well as the mechanisms underlying these effects.Curr. Protoc. Toxicol. 46:21.8.1‐21.8.33. © 2010 by John Wiley & Sons, Inc.

Keywords: gastrointestinal tract; smooth muscle contraction; duodenal motility; guinea pig ileum; electrical field stimulus; gastric fundus; spasmolytic activity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Rat Gastric Fundus Contractility
  • Basic Protocol 2: Evaluation of Spontaneous Motility in the Rabbit Duodenum
  • Basic Protocol 3: Evaluation of Neurogenic Responses in the Guinea Pig Ileum
  • Alternate Protocol 1: The Longitudinal Muscle‐Myenteric Plexus (LMMP) Preparation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Rat Gastric Fundus Contractility

  Materials
  • Adult male Wistar rats (180 to 220 g), fasted overnight with free access to water
  • Standard rodent food
  • Modified Krebs solution freshly prepared (see recipe) and continuously gassed with 5% CO 2 in O 2 through a fine glass sinter (Sigma‐Aldrich)
  • Diethylether (ether; Fluka)
  • 100 mM bethanechol stock solution (see recipe)
  • Stock solutions of test compounds under investigation, prepared as indicated by suppliers (see recipe)
  • 4 M KCl stock solution (see recipe)
  • 3 M BaCl 2 stock solution (see recipe) prewarmed and continuously stirred
  • 10 mM atropine (see recipe)
  • 10 mM verapamil hydrochloride (see recipe)
  • Polypropylene cages (approximate floor area 1000 cm2)
  • Jacketed organ baths (10‐ml), connected to a thermostatic water circulator (2Biological Instruments)
  • Cylinder containing 5% (v/v) CO 2 in oxygen (carboxygen)
  • Pressure regulators, connected to the distribution system of carboxygen, delivering gas at 5 to 15 psi (3.5 to 10 millibar)
  • Recording apparatus including:
    • Force (isotonic) transducers (e.g., Ugo Basile 7006)
    • Transducer interface to the recorder, if necessary (e.g., Ugo Basile, model 7082)
    • Polygraph/chart recorder (e.g., 2‐channel recorder, Ugo Basile, model 7070)
    • PC‐based acquisition system (optional, e.g., BIOPAC, Biological Instruments)
  • Animal balance (accuracy ±0.1 g; e.g., Mettler)
  • Glass bell, large enough to accommodate the animal
  • Glassware: beakers, graduated cylinders, conical flasks, and volumetric flasks of different capacity
  • Hydrophilic cotton (standard product for medication)
  • Surgical instruments including:
    • Hemostat (curved tip, size 20 cm; Rudolf Surgical Instruments)
    • Straight scissors, 16‐cm long (Rudolf Surgical Instruments)
    • Fine scissors (Rudolf Surgical Instruments)
    • Pins
    • Fine forceps (10‐cm eye‐dressing forceps, tip width 0.8 mm; Rudolf Surgical Instruments)
    • Surgical clamps
  • Petri dishes with attached silicon or cork disk, where the tissue may be pinned
  • White 50‐wt cotton thread
  • Thin hooked rod
  • Test tubes
  • 20‐, 200‐, 1000‐, and 5000‐µl automatic pipettors (e.g., Gilson)
  • Dedicated computer program (e.g., GraphPad Prism Software, Version 3.0 or higher)
  • Calculator

Basic Protocol 2: Evaluation of Spontaneous Motility in the Rabbit Duodenum

  Materials
  • New Zealand rabbits of either sex (2 to 2.5 kg; Harlan Laboratories)
  • Sodium pentobarbital (controlled substance; see recipe)
  • Tyrode's solution (see recipe), freshly prepared and continuously gassed with 5% CO 2 in oxygen through a fine glass sinter (Sigma‐Aldrich)
  • Stock solutions of test compounds under investigation, prepared as indicated by suppliers (see recipe)
  • 10 mM verapamil hydrochloride (see recipe)
  • Stainless steel cages with approximate floor area of 2500 cm2
  • Glassware: beakers, graduate cylinders, conical flasks, and volumetric flasks of different capacity
  • 20‐, 200‐, 1000‐, and 5000‐µl automatic pipettors (e.g., Gilson)
  • Recording apparatus including:
    • Force (isotonic) transducers (e.g., Ugo Basile, model 7006)
    • Transducer interface to the recorder, if necessary (e.g., Ugo Basile, model 7082)
    • Polygraph/chart recorder (e.g., 2‐channel recorder Ugo Basile, model 7070)
    • PC‐based acquisition system (optional, e.g., BIOPAC, 2Biological Instruments)
  • Animal balance (accuracy ±0.1 g, e.g., Mettler)
  • Surgical instruments including:
    • Hemostat (20‐cm long)
    • Fine scissors
    • Fine forceps (10‐cm eye‐dressing forceps, tip width 0.8 mm; Rudolf Surgical Instruments)
    • Surgical needles (3/8 circle curved)
    • Pins
    • Straight scissors, 16‐cm long
    • Surgical clamps
  • Petri dishes with attached silicon or cork disk for fixing the tissue using pins
  • Cylinder containing 5% (v/v) CO 2 in oxygen (carboxygen)
  • Pressure regulators, connected to the distribution system of carboxygen, delivering gas at 5 to 15 psi (3.5 to 10 millibar)
  • 10‐ml medical syringes equipped with 20‐G needles
  • White 50 wt cotton thread
  • Thin hooked rod
  • 10‐ml jacketed organ baths, connected to the thermostatic water circulator
  • 10‐ml test tubes
  • Dedicated computer program (e.g., GraphPad Prism Software, Version 3.0 or higher)

Basic Protocol 3: Evaluation of Neurogenic Responses in the Guinea Pig Ileum

  Materials
  • Male Hartley guinea pig (300 to 330 g; e.g., Harlan Laboratories)
  • Diethyl‐ether (Fluka) or dry ice pellets
  • Modified Krebs solution (see recipe) freshly prepared and continuously gassed with 5% CO 2 in oxygen
  • Stock solutions of test compounds under investigation prepared as indicated by suppliers (see recipe)
  • 100 mM methacholine stock solution (see recipe)
  • 4 M KCl stock solution (see recipe), optional
  • 10 mM atropine sulfate (see recipe)
  • Polypropylene cages (approximate floor area 2000 cm2)
  • Recording apparatus including:
    • Force (isotonic) transducers (e.g., Ugo Basile, model 7006)
    • Transducer interface to the recorder, if necessary (e.g., Ugo Basile, model 7082)
    • Polygraph/chart recorder (e.g., 2‐channel recorder Ugo Basile, model 7070)
    • PC‐based acquisition system (optional, e.g., BIOPAC, 2Biological Instruments)
  • Glass bell large enough to accommodate guinea pig or airtight chamber
  • Hydrophilic cotton
  • 50‐ml glass beakers
  • Glassware: beakers, graduate cylinders, conical flasks and volumetric flasks
  • 20‐, 200‐, 1000‐, and 5000‐µl automatic pipettors (e.g., Gilson)
  • Surgical instruments including:
    • Surgical needles (3.8 circle curved)
    • Straight tip scissors (16 cm long)
    • Fine forceps
    • Pins
    • Haemostat 20 cm (straight tip)
    • Fine scissors
    • Surgical clamps
  • Petri dishes with attached silicon or cork disk for fixing the tissue using pins
  • Cylinder containing 5% (v/v) CO 2 in oxygen (carboxygen)
  • Pressure regulators, connected to the distribution system of carboxygen, delivering gas at 5 to 15 psi (3.5 to 10 millibar)
  • 2.5‐ml medical syringes
  • White 50‐wt cotton thread
  • Thin hooked rod
  • Jacketed organ bath (10‐ml), connected to the thermostatically regulated water circulator
  • Thermostatically regulated water circulator
  • One pair of platinum electrodes (3.5‐ to 4‐cm long) for each preparation
  • Electrical stimulator and constant current isolator (e.g., four‐channel isolator “Multiplexing Pulse Booster;” Ugo Basile)
  • Dedicated computer program (e.g., GraphPad Prism Software, Version 3.0 or higher)

Alternate Protocol 1: The Longitudinal Muscle‐Myenteric Plexus (LMMP) Preparation

  • Glass Pasteur pipet (1‐ml, 13.8‐cm length, 0.65‐cm external diameter)
  • Cotton wool
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Figures

Videos

Literature Cited

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