Assessment of Ischemic Vascular Damage

Viera Nosál'ová1, Ruzena Sotníková1

1 Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovak Republic
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 21.4
DOI:  10.1002/0471140856.tx2104s44
Online Posting Date:  May, 2010
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Abstract

This protocol describes a model convenient for acute experiments in anesthetized rats, performed by selective occlusion of the superior mesenteric artery. Such a model provides a means for assessing the role played by various pathophysiological mechanisms in the development of intestinal ischemic injury. It is especially suitable for studying different treatments, mainly pharmacological ones, to help cope with this problem in clinical practice. In the form of support protocols, this unit includes functional vascular and chemiluminescence studies, determination of vascular permeability and myeloperoxidase activity, transit time, and mortality. Curr. Protoc. Toxicol. 44:21.4.1‐21.4.21. © 2010 by John Wiley & Sons, Inc.

Keywords: ischemia; reperfusion; reactive oxygen species; rat; superior mesenteric artery; ileum

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Intestinal Ischemia/Reperfusion Study
  • Support Protocol 1: Macroscopic Evaluation of the Extent of I/R Injury
  • Support Protocol 2: Microscopic/Histologic Evaluation of the Extent of I/R Injury
  • Support Protocol 3: Measurement of Vascular Function in Mesenteric I/R Model
  • Support Protocol 4: Measurement of ROS Production Using a Chemiluminescence Method
  • Support Protocol 5: Measurement of Vascular Permeability in Mesenteric I/R Model
  • Support Protocol 6: Cannulation of the Carotid Artery
  • Support Protocol 7: Cannulation of the Femoral Vein
  • Support Protocol 8: Measurement of Gastrointestinal Transit in Mesenteric I/R Model
  • Support Protocol 9: Measurement of Myeloperoxidase Activity in Mesenteric I/R Model
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Intestinal Ischemia/Reperfusion Study

  Materials
  • Male Wistar rats weighing ∼250 to 280 g
  • Drugs to be studied (e.g., antioxidants: catalase, superoxide dismutase in concentrations of 10−4 to 10−5 mol/liter; antihistamines: loratadine, dithiaden, pheniramines 10 to 30 mg/kg; plant origin compounds: rosmarinic acid, arbutin, curcumin in concentration of 10−4 mol/liter)
  • Physiological saline: 0.9% (w/v) NaCl, ice cold
  • Thiopental (see recipe)
  • 70% (v/v) ethanol
  • Lamp
  • Pelleted rodent food
  • 2‐ml syringes
  • 23‐G needles
  • Animal scale
  • Electric shaver
  • Rat board
  • Surgical instruments including:
    • Scalpel
    • Hemostats
    • Jeweler's forceps, straight
    • Iridectomy scissors
    • Vessel clamp
    • Small straight anatomical forceps
    • Large straight surgical forceps
    • Scissors, sharp and blunt
  • Gauze
  • Sutures
  • Additional reagents and equipment sacrificing the rat (Donovan and Brown, )

Support Protocol 1: Macroscopic Evaluation of the Extent of I/R Injury

  • Male Wistar rats, control and treated
  • Gridded paper

Support Protocol 2: Microscopic/Histologic Evaluation of the Extent of I/R Injury

  Materials
  • Rat small intestine and other tissues, treated and control (see protocol 1)
  • 10% (w/v) formalin
  • Paraffin
  • Xylene
  • 50%, 70%, 80%, 90%, 95%, and 100% (v/v) ethanol
  • Acetone
  • Distilled water
  • Hematoxylin
  • Eosin
  • Xylene‐based mounting medium
  • Embedding cassettes
  • Automated tissue processor (Autotechnikon AT‐4, ZSSR)
  • Microtome with blades
  • Glass slides
  • 45° to 50°C oven
  • Coverslips
  • Microscope, e.g., Fluoval (Zeiss, DDR)

Support Protocol 3: Measurement of Vascular Function in Mesenteric I/R Model

  • Rats, male Wistar, treated and control ( protocol 1)
  • Physiological saline solution (PSS; see recipe)
  • Phenylephrine (see recipe)
  • Acetylcholine (see recipe)
  • NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 µM; see recipe)
  • Indomethacin (10 µM; see recipe)
  • 90‐mm petri dishes
  • 10‐ml jacketed organ bath (Experimetria)
  • Force transducer (Experimetria)
  • Graph paper or digital recorder
  • Precision balance

Support Protocol 4: Measurement of ROS Production Using a Chemiluminescence Method

  • Male Wistar rats, treated and control (see protocol 1)
  • Physiological saline solution (PSS; see recipe), pre‐oxygenated (95% O 2 and 5% CO 2)
  • Luminol (see recipe)
  • Beakers
  • Cuvettes
  • Lumi‐aggregometer (model 500; Chrono‐log)
  • Balance

Support Protocol 5: Measurement of Vascular Permeability in Mesenteric I/R Model

  Materials
  • Male Wistar rat
  • Thiopental (see recipe)
  • 1% Evans blue dye (see recipe)
  • Physiological saline: 0.9% (w/v) NaCl
  • Formamide (see recipe)
  • Animal scale
  • 2‐ml syringes with 23‐G needles
  • Filtration paper
  • 50°C shaking water bath
  • Spectrophotometer
  • Additional reagents and equipment for cannulating the carotid artery ( protocol 7) and femoral vein ( protocol 8) and sacrificing the rat (Donovan and Brown, )

Support Protocol 6: Cannulation of the Carotid Artery

  Materials
  • Male Wistar rat
  • Thiopental (see recipe)
  • 70% (v/v) ethanol
  • Heparinized saline (50 IU/ml), 0.9% NaCl
  • Electric shaver
  • Rat board
  • Scissors
  • Blunt forceps
  • Suture
  • Clamps
  • Cannula

Support Protocol 7: Cannulation of the Femoral Vein

  Materials
  • Male Wistar rat
  • Thiopental (see recipe)
  • Electric shaver
  • Scissors
  • Rat board
  • Cannula (with a fine metallic needle in its tip)
  • Gauze

Support Protocol 8: Measurement of Gastrointestinal Transit in Mesenteric I/R Model

  Materials
  • Male Wistar rats
  • Thiopental (see recipe)
  • Evans blue (see recipe)
  • Physiological saline: 0.9% (w/v) NaCl
  • Balance
  • Orogastric cannula (metal probe)
  • Clamps
  • Additional reagents and equipment for isolating the small intestine ( protocol 1)

Support Protocol 9: Measurement of Myeloperoxidase Activity in Mesenteric I/R Model

  • Intestinal mucosa (see step 17 of the protocol 1)
  • Homogenization medium (see recipe)
  • o‐dianisidine dihydrochloride reagent (see recipe)
  • Tissue homogenizer
  • Centrifuge
  • Spectrophotometer
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Figures

Videos

Literature Cited

Literature Cited
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