Rodent Models of Global Cerebral Ischemia

Michael J. O'Neill1, James A. Clemens2

1 Eli Lilly and Co., Windlesham, Surrey, United Kingdom, 2 Lilly Corporate Center, Indianapolis, Indiana
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 9.5
DOI:  10.1002/0471142301.ns0905s12
Online Posting Date:  May, 2001
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Brain damage after stroke and head injury remains a huge clinical problem. In stroke, the initial cause of the damage is a blockage in a blood vessel (often the middle cerebral artery) and this sets off several pathways that ultimately lead to cell death. Recent studies have demonstrated that several new mechanisms are involved in neuronal death and this has led to an increase in research into novel molecules that might prevent brain damage or improve recuperation post‐stroke. There are several models of global cerebral ischemia. Two of the most widely‐used models are discussed in detail in UNIT, the gerbil bilateral carotid artery occlusion (BCAO) model and rat 4‐vessel occlusion (4‐VO) model. Additionally, several models of focal cerebral ischemia have been developed to mimic the effects of human stroke. The rationale behind the use of animal models, the various types of models and advantage and disadvantages of each model are presented.

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Table of Contents

  • Why Use Animal Models?
  • Basic Protocol 1: Gerbil Bilateral Carotid Artery Occlusion (BCAO) Model to Test Systemically Active Neuroprotective Agents
  • Alternate Protocol 1: Gerbil BCAO Model to Test Neuroprotective Agents that Do Not Penetrate the Brain
  • Alternate Protocol 2: Gerbil BCAO to Induce Ischemic Tolerance
  • Basic Protocol 2: Use of 4‐Vessel Occlusion (4‐VO) Model to Study Neuronal Degeneration and Test the Effects of Neuroprotective Agents Against Global Cerebral Ischemia
  • Support Protocol 1: Hematoxylin and Eosin Staining of Brain Tissue
  • Support Protocol 2: Measurement of Locomotor Activity After Ischemia in Gerbils
  • Support Protocol 3: Fabrication of Atraumatic Clasps for Rat 4‐VO
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Gerbil Bilateral Carotid Artery Occlusion (BCAO) Model to Test Systemically Active Neuroprotective Agents

  • Male Mongolian gerbils, 60 to 80 g (Bantin and Kingman)
  • Test compounds
  • Inhalational anesthetic (halothane or isoflurane)
  • Saline: 0.9% NaCl
  • 10% buffered formalin (see recipe)
  • Pentobarbital or chloral hydrate
  • 60%, 80%, and 90% industrial methylated spirits (IMS; e.g., Fisher) in distilled H 2O
  • Syringes and needles suitable for route of compound administration (e.g., intramuscular, intravenous, or oral)
  • Inhalation anesthetic apparatus comprising, e.g., oxygen, vaporizer, tubing, and vacuum trap (International Market Supply)
  • Heating pad/blanket
  • Rectal temperature monitor (RS Components)
  • Electric hair clippers
  • Methiolate tincture (Lilly)
  • Fiber optic light source
  • Surgical instruments: including scissors, scalpel, and forceps (e.g., John Weiss & Sons)
  • Vascular clamps (Holborn Surgical Instruments)
  • Silk thread (6/6 braided suture; International Market Supply)
  • Suture (Ethicon W529 6/0 Mersilk; A.C. Daniels)
  • Thermacages (Beta Medical & Scientific) or thermostatically controlled incubators
  • Dram vials, glass scintillation vials, or similar glass vials
  • Rodent brain matrix, coronal gerbil (ASI Instruments)
  • Automated tissue processor (e.g., Tissue‐Tek VIP 2000 vacuum infiltrator processor; Miles Scientific or Bayer Diagnostics)
  • Sledge microtome (Leitz 1400)
  • Additional reagents and equipment for hematoxylin/eosin staining of brain tissue (see protocol 5) and perfusion fixation (see unit 1.1)

Alternate Protocol 1: Gerbil BCAO Model to Test Neuroprotective Agents that Do Not Penetrate the Brain

  • Stereotaxic frame (e.g., model 900 from David Kopf Instruments or Bilaney Consultants)
  • 28‐G injection cannula (Plastics One)
  • Electrode holders/cannula holder (e.g., model 1770,1771 from David Kopf Intruments)
  • 25‐µl Hamilton syringe
  • Infusion pump (World Precision Instruments)
  • Anesthesia mask (David Kopf Instruments or Bilaney Consultants)
  • Fine‐tipped felt pen
  • Dental drill (Biotech Instruments) and 0.9‐mm steel drill bits/burrs (Interfocus Ltd.)

Alternate Protocol 2: Gerbil BCAO to Induce Ischemic Tolerance

  • Male Wistar rats, 280 to 300 g
  • Isoflurane
  • Antiseptic solutions (e.g., Betadine and 70% ethanol)
  • Pentobarbital or chloral hydrate
  • 10% buffered formalin (see recipe)
  • Inhalation anesthetic apparatus suitable for use with isoflurane (Vetamac)
  • Stereotaxic frame (David Kopf Instruments)
  • Rat anesthesia mask (David Kopf Instruments)
  • Electric hair clippers
  • Surgical instruments including scissors, scalpel, forceps (Miltex Surgical Instruments)
  • Operating microscope
  • Electrocautery unit no. 160‐1370 (hand piece with electrocautery needle, 0.5 mm diameter; Tiemann & Co.)
  • Wound clips
  • Atraumatic carotid clasps (constructed according to protocol 7)
  • PE20 polyethylene tubing (Fisher)
  • Bulldog artery clamp, Johns Hopkins, straight, 1.5‐in. length (Roboz Surgical)
  • Rectal thermistor (Yellow Springs Instrument)
  • YSI Temperature Controller Model 73A (Yellow Springs Instrument)
  • Harvard Compact Infusion Pump Model 975 (Harvard Instruments)
  • Heat lamp (from local hardware store)
  • Physitemp temperature controller (Physitemp)
  • Physitemp type IT‐21 tissue implantable thermocouple (Physitemp)
  • Flow‐through swivel (Harvard Instrument)
  • Masterflex peristaltic pump (Cole Parmer)
  • Additional reagents and equipment for fixing and staining brain tissue (see protocol 1).

Basic Protocol 2: Use of 4‐Vessel Occlusion (4‐VO) Model to Study Neuronal Degeneration and Test the Effects of Neuroprotective Agents Against Global Cerebral Ischemia

  • Slides of paraffin‐embedded brain tissue (see protocol 1)
  • Xylene (Fisher)
  • 70%, 75%, and 100% industrial methylated spirits (IMS; e.g., Fisher) in Coplin jars
  • Gill's hematoxylin (available as ready‐to‐use preparation from Surgipath)
  • 2% eosin (see recipe)
  • DPX mountant (BDH)
  • Coverslips
  • Slide trays

Support Protocol 1: Hematoxylin and Eosin Staining of Brain Tissue

  • Automated locomotor activity apparatus (e.g., Greenacre; also see unit 8.1)
  • SAS Software for gathering and analyzing data (e.g., Misac Instruments)

Support Protocol 2: Measurement of Locomotor Activity After Ischemia in Gerbils

  • White baby buttons (Blumenthal Industries)
  • Silastic tubing, 0.012 in. i.d. × 0.25 in. o.d. (Dow‐Corning)
  • PE90 polyethylene tubing (Fisher)
  • 5‐min epoxy glue (from local hardware store)
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Key References
Internet Resources
  This is the Web site for the journal Stroke (Journal of American Heart Association), which publishes original research papers using animal models, but also publishes clinical papers and updates on current clinical trials in stroke.
  This is the Web site of International Society of Cerebral Blood Flow and Metabolism. The official journal of this society is the Journal of Cerebral Blood Flow and Metabolism, which publishes detailed papers using various animal models of cerebral ischemia.
  This is the Web site for the U.S. NIH, National Institute of Neurological Disorders and Stroke. This government agency also supports basic research through grant funding.
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