Models of Cardiac Ischemia‐Reperfusion Injury in Dogs and Rats

Garrett J. Gross1

1 Medical College of Wisconsin, Milwaukee, Wisconsin
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 5.27
DOI:  10.1002/0471141755.ph0527s16
Online Posting Date:  May, 2002
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A marked reduction in coronary blood flow produces a reduction in myocardial function, electrocardiographic abnormalities and eventually a myocardial infarction if the ischemic episode were to persist for more than 20 to 30 min. There have been numerous studies in animal models in search of a magic bullet or drug that can ameliorate these symptoms and result in a reduction in infarct size, improvement in the recovery of contractile function, and abrogation of malignant ventricular arrhythmias in humans. This unit describes two animal models of myocardial ischemia/reperfusion injury which are used to evaluate pharmacological agents that may eventually demonstrate cardioprotective activity in a clinical setting.

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Table of Contents

  • Basic Protocol 1: Dog Model of Ischemia‐Reperfusion Injury
  • Basic Protocol 2: Rat Model of Ischemia‐Reperfusion Injury
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Dog Model of Ischemia‐Reperfusion Injury

  • 15‐ to 25‐kg adult mongrel dogs of either sex
  • Sodium pentobarbital (Sigma)
  • Sodium barbital (Sigma)
  • 0.9% (w/v) NaCl solution (i.e., saline)
  • 10% (w/v) Patent blue dye (Sigma)
  • Triphenyltetrazolium chloride (TTC) solution (see recipe)
  • 10% formaldehyde solution (Fisher Scientific)
  • Surgical lamp
  • Thermoregulated heating pad with probe (Harvard Apparatus)
  • Surgical operating table
  • 9‐French endotracheal tube
  • Respirator with water trap (Harvard 607)
  • Tank of compressed 100% O 2 gas and connector for respirator in‐flow tubing
  • Blood gas analyzer (e.g., AVL Scientific 995)
  • Double‐tipped SPR‐277 pressure transducer catheter with cables and interface boxes (Millar Instruments)
  • PE 330 polyethylene tubing (Becton‐Dickinson)
  • PE‐240 Catheter
  • Electrocautery gun and tips (George Tieman)
  • Rib spreaders or retractors (e.g., Rubio Farrell adult rib spreader; Pilling Surgical Instruments)
  • Gould‐Statham 2202 flowmeter and calibrated 2‐ to 3‐mm‐i.d. flow probes (Statham SP 7515)
  • Vascular bull‐dog clamp (George Tieman)
  • Grass 7P4F tachograph and SD9 stimulator
  • 12‐channel polygraph (e.g., Grass 7)
  • 20‐G needles (e.g., Becton‐Dickinson)
  • Sonomicrometer (Valpey‐Fisher) and 2 sets of segment‐length piezoelectric crystals and connectors: before surgery, calibrate crystals in H 2O according to the manufacturer's specifications and monitor the tracings on a Soltec 520 oscilloscope
  • 15‐µm‐diameter carbonized, plastic, radiolabeled (e.g., 141Ce or 95Nb; 1 to 2 mCi each) microspheres (New England Nuclear) in isotonic saline containing 0.01% Tween 80
  • Electrical defibrillator (GE Medical Systems)
  • Scintillation vials
  • Searle Analytic 1195 autogamma spectrometer
NOTE: Dogs which fail to meet certain criteria are excluded from data analysis if (1) heartworms are found when removing the heart, (2) transmural collateral blood flow is greater than 0.20 ml/min/g, (3) the resting heart rate is >180 beats/min at the initiation of the experimental protocol, or (4) more than three consecutive attempts are needed to convert ventricular fibrillation during the ischemic period or following reperfusion. The dogs are most likely to fibrillate immediately upon reperfusion and throughout the first 30 min of reperfusion.

Basic Protocol 2: Rat Model of Ischemia‐Reperfusion Injury

  • 250‐ to 350‐g adult male Wistar or Sprague‐Dawley rat
  • 1 g inactin (Sigma RBI)/10 ml distilled water
  • 10% (w/v) Patent blue dye (Sigma) solution
  • 15% (w/v) KCl
  • Triphenyltetrazolium chloride (TTC) solution, 37°C (see recipe)
  • 10% (v/v) formaldehyde solution (Fisher Scientific)
  • Heating pad, rectal temperature probes, and recorder (Physitemp Instruments, Inc., TH‐8)
  • Heat lamps
  • Tracheal cannula
  • Rodent ventilator with water trap (Harvard Apparatus 683 or Columbus Instruments)
  • Tank of compressed 100% O 2 and connectors for respirator in‐flow tubing
  • Blood gas system (AVL Scientific 995)
  • Disposable pressure transducer and adapter for Grass polygraph (Maxim Medical)
  • PE 50 polyethylene tubing (Becton Dickinson)
  • Rat rib spreaders (Pilling, Cat.#165086)
  • 6‐0 prolene suture (Ethicon)
  • PE 160 polyethylene tubing with a fire‐polished blunt end
  • Hemostat
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Literature Cited

Literature Cited
   Bolli, R. 1995. Common methodological problems and artifacts associated with studies of myocardial stunning in vivo. Basic Res. Cardiol. 90:257‐262.
   Fishbein, M.C., Meerbaum, S., Rit, J., Lando, U., Kanmatsuse, K., Mercier, J. C., Corday, E., and Ganz, W. 1981. Early phase acute myocardial infarct size quantification: Validation of the triphenyltetrazolium chloride tissue enzyme staining technique. Am. Heart J. 100:593‐600.
   Fryer, R.M., Hsu, A.K., Nagase, H., and Gross, G.J. 2000. Opioid‐induced cardioprotection against myocardial infarction and arrhythmias: Mitochondrial versus sarcolemmal ATP‐sensitive potassium channels. J. Pharmacol. Exp. Ther. 294:451‐457.
   Gumina, R. J., Buerger, E., Eickmeier, C., Moore, J., Daemmgen, J., and Gross, G. J. 1999. Inhibition of the Na+/H+ exchanger confers greater cardioprotection against 90 minutes of myocardial ischemia than ischemic preconditioning in dogs. Circulation. 100:2519‐2526.
   Hoemeister, J.W., Hoff, P.T., Fletcher, D.D., and Lucchesi, B.R. 1990. Combined adenosine and lidocaine administration limits myocardial reperfusion injury. Circulation 82:595‐608.
   Schwartz, L.M., Verbinski, S.G., Vander Heide, R.S., and Reimer, K.A. 1997. Epicardial temperature is a major predictor of myocrdial infarct size in dogs. J. Mol. Cell. Cardiol. 29:1577‐1583.
   Verdouw, P.D., van den Doel, M.A., de Zeeuw, S., and Duncker, D.J. 1998. Animal models in the study of myocardial ischemia and ischemic syndromes. Cardiovasc. Res. 39:121‐135.
Key References
   Hearse, D.J. and Sutherland, F.J. 2000. Experimental models for the study of cardiovascular function and disease. Pharmacol. Res. 41:597‐603.
  This reference is particularly relevant since it describes other cardiovascular animal models not discussed in this unit.
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