Measurement of Small Intestinal Damage

Koji Takeuchi1, Hiroshi Satoh1

1 Kyoto Pharmaceutical University, Kyoto, Japan
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 21.7
DOI:  10.1002/0471140856.tx2107s45
Online Posting Date:  August, 2010
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Abstract

Many animal models have been devised for investigating the pathogenesis of intestinal lesions and for screening drugs for the treatment of intestinal ulcers in humans. Recently, particular attention has been focused on NSAID‐induced intestinal lesions as a result of the development of the capsule endoscope and double‐balloon endoscope. Ischemic enteritis, one of the most dramatic abdominal emergencies, is known to cause severe damage to the small intestine by a significant decrease of arterial blood flow in the small intestine. In this unit, two animal models for small intestinal damage induced by NSAIDs or intestinal ischemia are described. Also included are methods for lesion induction and evaluation of the damage as well as the measurement of pathogenic functional and biochemical changes. Curr. Protoc. Toxicol. 45:21.7.1‐21.7.31. © 2010 by John Wiley & Sons, Inc.

Keywords: small intestinal damage; NSAID; ischemia; functional change; biochemical alteration

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Small Intestinal Damage in Rats by NSAIDs
  • Alternate Protocol 1: NSAID‐Induced Small Intestinal Damage in Dogs and Cats
  • Basic Protocol 2: Induction of Damage to the Rat Small Intestine by Ischemia
  • Measurements of Various Functions and Biochemical Events
  • Support Protocol 1: Measuring Intestinal Motility in Rats
  • Support Protocol 2: Measuring Gastrointestinal Motility in Cats
  • Support Protocol 3: Measuring Mucus Secretion in the Small Intestine
  • Support Protocol 4: Measuring Enterpooling (Fluid Secretion) in the Small Intestine
  • Support Protocol 5: Measuring Microvasular Permeability in the Small Intestine
  • Support Protocol 6: Measuring Lipid Peroxidation in the Small Intestine
  • Support Protocol 7: Measuring Myeloperoxidase (MPO) Activity in the Small Intestine
  • Support Protocol 8: Measuring Mucosal PGE2 Content in the Small Intestine
  • Support Protocol 9: Measuring COX and NOS mRNA Expression by RT‐PCR
  • Support Protocol 10: Measuring iNOS Protein Expression by Immunoblotting
  • Support Protocol 11: Measuring NOS Activity in the Small Intestine
  • Support Protocol 12: Measuring NO Content in Small Intestine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of Small Intestinal Damage in Rats by NSAIDs

  Materials
  • Male Sprague‐Dawley rats (6 to 8 weeks old; ∼200 to 260 g; Charles River)
  • Drugs to be tested:
    • 40 mg/kg diclofenac (Sigma chemicals)
    • 20 mg/kg flurbiprofen (Sigma chemicals)
    • 40 mg/kg naproxen (Sigma chemicals)
  • Hydroxylpropyl cellulose or physiological saline
  • 10 mg/kg indomethacin (Sigma chemicals)
  • 1% (w/v) Evans blue (Sigma Chemicals) in physiological saline
  • Ethyl ether (Wako)
  • 2% formalin
  • Scalpel or blunt‐end scissors
  • Forceps
  • Dissecting microscope (Olympus)

Alternate Protocol 1: NSAID‐Induced Small Intestinal Damage in Dogs and Cats

  Materials
  • Adult male and female dogs and cats bred for the experiment (cats: >2.5 kg; dogs: >8 kg; Narc Co.)
  • Indomethacin (Sigma Chemicals)
  • Regular dry food containing ≥3% dietary fiber
  • Drug or test compound
  • Flunixin meglumine (Dainippon‐Sumitomo)
  • Ketoprofen (Nihonzennyaku)
  • Carboxymethylcellulose (Wako)
  • Physiological saline
  • Xylazine (Bayer)
  • Pentobarbital sodium (Dainippon‐Sumitomo)
  • 10% formalin solution
  • Scalpel or blunt‐end scissors
  • Forceps
  • Dissecting microscope

Basic Protocol 2: Induction of Damage to the Rat Small Intestine by Ischemia

  Materials
  • Male rats (6 to 8 weeks old)
  • Ethyl ether (Wako)
  • Ampicillin or aztreconam, optional
  • Evans blue (Sigma Chemicals)
  • 2% formalin
  • Rat cages with raised mesh bottoms
  • Scalpel or blunt‐end scissors
  • Forceps
  • Needle holder
  • 23‐G needles
  • No. 5 sutures
  • Dissecting microscope (Olympus)

Support Protocol 1: Measuring Intestinal Motility in Rats

  Materials
  • Male rats (6 to 8 weeks old)
  • Urethane (Tokyo Kasei)
  • Miniature saline‐filled balloon (thin balloon made from silicone rubber)
  • NSAIDs
  • Hydroxylpropyl cellulose (Wako) or physiological saline
  • Indomethacin (10 mg/kg)
  • Diclofenac (40 mg/kg; Sigma Chemicals)
  • Scalpel or blunt‐end scissors, sterile
  • Cannula
  • Polyethylene catheter
  • Fine, sterile forceps
  • Needle holder, sterile
  • No. 5 sutures
  • Recorder (Tokai‐Irika, model no. U‐228)
  • Pressure transducer
  • Polygraph device (Nihon Koden)
  • NIH image 1.61 software (NIH)

Support Protocol 2: Measuring Gastrointestinal Motility in Cats

  Materials
  • Adult male and female cats bred for the experiment
  • Regular dry food containing ≥3% dietary fiber
  • Xylazine (Bayer)
  • Pentobarbital sodium (Dainippon‐Sumitomo)
  • Povidone‐iodine (Iwaki)
  • Drug or test compound:
    • Ampicillin sodium (Kyoritsu)
    • Atropine sulfate (Mitsubishi Tanabe)
    • Benzarkonium chloride (Takeda)
    • Meloxicam (Boehringer Ingelheim)
  • Positive controls:
    • Indomethacin (Sigma Chemicals)
    • Ketoprofen (Nihonzennyaku)
    • Flunixin meglumine (Dainippon‐Sumitomo)
  • Carboxymethylcellulose (Wako) or physiological saline
  • Sterile surgical instruments: scalpel, scissors, forceps
  • Surgical sutures, sterile
  • Surgical needles, sterile
  • Needle holders, sterile
  • Strain‐gauge force transducer (8 × 5–mm; Star Medical, cat. no. F‐08IS‐P)
  • Jacket‐type protector (for cat; Star Medical)
  • Transmitter (Star Medical, cat. no. IMT‐40TA)
  • Telemetry system (Star Medical, model GTS‐400)
  • Receiver (Star Medical, cat. no. IMT‐40RA)
  • Personal computer (Dell Inspiron 2200)
  • Computer program (Analize II.dll; Star Medical)

Support Protocol 3: Measuring Mucus Secretion in the Small Intestine

  Materials
  • Male rats (6 to 8 weeks old)
  • Drugs affecting the mucus secretion in hydroxylpropyl cellulose (Wako) or physiological saline
  • Ethyl ether
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 2% (w/v) N‐acetylcysteine (Wako)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 50 mM Tris⋅Cl, pH 7.2 ( appendix 2A) containing 2% (v/v) Triton X‐100
  • Sepharose 2B 50‐cm column (Pharmacia Biotech)
  • Indomethacin (Sigma Chemicals)
  • Carnoy's fluid: 6:1:3 (v/v) ethanol/acetic acid/chloroform, optional
  • Paraffin, optional
  • Scalpel or blunt‐end scissors, sterile
  • Forceps, sterile
  • Magnetic stirrer (Iwaki) and plate
  • Lyophilizer
  • Polytron tissue homogenizer (Ika)
  • Microtome, optional

Support Protocol 4: Measuring Enterpooling (Fluid Secretion) in the Small Intestine

  Materials
  • Male rats (6 to 8 weeks old)
  • Indomethacin (Sigma Chemicals) or drugs affecting fluid secretion
  • Hydroxylpropyl cellulose (Wako) or physiological saline
  • Ethyl ether
  • Sterile surgical instruments: scalpel or blunt‐end scissors, forceps
  • 15‐ml graduated tube
  • Centrifuge (Kubota5010)

Support Protocol 5: Measuring Microvasular Permeability in the Small Intestine

  Materials
  • Male rats (6 to 8 weeks old)
  • Indomethacin (Sigma Chemicals)
  • Hydroxypropyl cellulose (Wako)
  • Physiological saline
  • Evans blue (Sigma Chemicals)
  • Ethyl ether
  • Sterile surgical instruments: scalpel or blunt‐end scissors, forceps
  • Polytron tissue homogenizer (Ika)
  • Spectrophotometer (Hitachi, model no. U‐2000)

Support Protocol 6: Measuring Lipid Peroxidation in the Small Intestine

  Materials
  • Male rats (6 to 8 weeks old)
  • Drugs to be tested:
    • Indomethacin (Sigma Chemicals)
  • Hydroxypropyl cellulose (Wako)
  • Physiological saline
  • Ethyl ether
  • 1.15% (w/v) KCl ( appendix 2A)
  • Thiobarbituric acid (see recipe)
  • 15:1 (v/v) n‐butanol/pyridine
  • Sterile surgical instruments: scalpel or blunt‐end scissors, forceps
  • Polytron tissue homogenizer (Ika)
  • Centrifuge
  • Spectrophotometer (Hitachi, model no. U‐2000)

Support Protocol 7: Measuring Myeloperoxidase (MPO) Activity in the Small Intestine

  Materials
  • Male rats (6 to 8 weeks old)
  • Indomethacin (Sigma Chemicals)
  • Hydroxypropyl cellulose (Wako)
  • Physiological saline
  • Ethyl ether
  • 50 mM phosphate buffer, pH 6.0, containing 0.5% (w/v) hexadecyltrimethylammoniumbromide (Sigma Chemicals)
  • Phosphate buffer containing 0.17 mg/ml o‐danisidine dihydrochloride (Sigma Chemicals) and 0.0005% (v/v) H 2O 2 (pH 6.0)
  • Sterile surgical instruments: scalpel or blunt‐end scissors, forceps
  • Polytron tissue homogenizer (Ika)
  • Microtiter plates
  • Microtiter plate reader (Thermo Max; Molecular Devices)

Support Protocol 8: Measuring Mucosal PGE2 Content in the Small Intestine

  Materials
  • Male rats (6 to 8 weeks old)
  • Indomethacin (Sigma Chemicals) or other NSAIDs
  • Hydroxypropyl cellulose (Wako) or physiological saline
  • Ethyl ether
  • 0.1 M indomethacin in 99.8% methanol
  • PGE 2 enzyme immunoassay (EIA) kit (Cayman, cat. no. 514010)
  • Sterile surgical instruments: scalpel or blunt‐end scissors, forceps
  • Polytron tissue homogenizer (Ika)

Support Protocol 9: Measuring COX and NOS mRNA Expression by RT‐PCR

  Materials
  • Male rats (6 to 8 weeks old)
  • NSAIDs or COX inhibitors
    • Indomethacin (10 mg/kg)
    • Dicrofenac (40 mg/kg)
    • Flurbiprofen (20 mg/kg)
    • Naproxen (40 mg/kg; Sigma Chemicals)
    • SC‐560 (10 mg/kg; Cayman Chemical)
    • Rofecoxib (10 mg/kg; Merck)
  • Hydroxypropyl cellulose (Wako) or physiological saline
  • Ethyl ether
  • Liquid nitrogen
  • Phenol/chloroform
  • Trizol (Invitrogen)
  • Sepasol RNA‐I (Nacalai Tesque)
  • Superscript preamplification system (Invitrogen)
  • Agarose
  • TAE buffer (see recipe)
  • Ethidium bromide
  • Sterile surgical instruments: scalpel or blunt‐end scissors, forceps
  • Polytron tissue homogenizer (Ika)
  • Apparatus for electrophoresis (Atto)
  • Gel photographic equipment

Support Protocol 10: Measuring iNOS Protein Expression by Immunoblotting

  Materials
  • Male rat (6 to 8 weeks old)
  • Drugs to be tested and controls
  • 0.5% hydroxylpropyl cellulose or physiological saline
  • Ethyl ether (Wako)
  • Homogenization buffer I (see recipe), ice cold
  • Bicinchoninic acid protein assay kit (Pierce)
  • 7.5% SDS‐polyacrylamide gel
  • 5% (w/v) skim milk in PBST (0.1% Triton X 100/phosphate buffered saline)
  • Anti‐iNOS antibody (Santa Cruz Biotechnology)
  • Horseradish peroxidase‐conjugated anti‐rabbit IgG (Santa Cruz Biotechnology)
  • Enhanced chemiluminescence detection system (Western Blot Chemiluminescence Reagent Plus; NEN)
  • Sterile surgical instruments: scalpel or blunt‐end scissors, forceps, needle holder, no. 5 sutures, 23‐G sterile needles
  • Polytron tissue homogenizer (Ika)
  • Sonicator (Tomy‐Seiko)
  • Refrigerated centrifuge (Sigma Laboratories)
  • Apparatus for electrophoresis (Atto)
  • Nitrocellulose membranes (Protran; Schleicher & Schuell)

Support Protocol 11: Measuring NOS Activity in the Small Intestine

  Materials
  • Male rats (6 to 8 weeks old)
  • Indomethacin (Sigma Chemicals)
  • Hydroxypropyl cellulose (Wako)
  • Physiological saline
  • Ethyl ether (Wako)
  • Homogenization buffer II (see recipe), ice cold
  • [3H]L‐arginine
  • EGTA ( appendix 2A)
  • Sterile surgical instruments: scalpel or blunt‐end scissors, forceps
  • Glass histological slides
  • Polytron tissue homogenizer (Ika)
  • Refrigerated centrifuge (Sigma Laboratories)
  • Spectrophotometer (Hitachi, model no. U‐2000)
  • 37°C water bath

Support Protocol 12: Measuring NO Content in Small Intestine

  Materials
  • Male rats (6 to 8 weeks old)
  • Indomethacin (Sigma Chemicals)
  • Hydroxypropyl cellulose (Wako)
  • Physiological saline, ice cold
  • Aminoguanidine (Sigma Chemicals)
  • Ethyl ether (Wako)
  • 50 mM KHPO 4
  • Nitrate reductase (Sigma Chemicals)
  • Griess reagent (see recipe)
  • Sodium nitrate
  • Sterile surgical instruments: scalpel or blunt‐end scissors, forceps
  • Glass histological slides
  • Polytron tissue homogenizer (Ika)
  • Refrigerated centrifuge (Sigma Laboratories)
  • Spectrophotometer (Hitachi, model no. U‐2000)
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Figures

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Literature Cited

Literature Cited
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