Rodent Models of Focal 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 9.6
DOI:  10.1002/0471142301.ns0906s12
Online Posting Date:  May, 2001
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Abstract

This unit presents models that are both used to study ischemic mechanisms and to test for neuroprotective agents or agents that enhance recovery from stroke. The Tamura model is one of the best characterized focal ischemia models in which the middle cerebral artery is occluded by electrocoagulation. Also described is the intraluminal monofilament model, the spontaneously hypertensive rat (SHR), and the newer endothelinā€1 model. 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

  • Basic Protocol 1: The Intraluminal Suture Model of Middle Cerebral Artery Occlusion (MCAO) to Test Neuroprotective Agents
  • Support Protocol 1: Fabrication of Intraluminal Monofilament Occluders
  • Basic Protocol 2: Middle Cerebral Artery Occlusion (MCA) Using Stereotaxic Infusion of Endothelin 1
  • Support Protocol 2: Use of Horizontal and Inclined Balance Beam to Assess Sensorimotor Performance After Et‐1 MCAO
  • Support Protocol 3: Use of the Staircase Test to Measure Skilled Paw Use After Et‐1 MCAO
  • Basic Protocol 3: The Tamura Model of Middle Cerebral Artery Occlusion (MCAO) to Test Neuroprotective Agents
  • Basic Protocol 4: The Spontaneously Hypertensive Rat Model of Middle Cerebral Artery Occlusion (MCAO) to Test Neuroprotective Agents
  • Support Protocol 4: Cresyl Violet Staining of Brain Tissue
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Intraluminal Suture Model of Middle Cerebral Artery Occlusion (MCAO) to Test Neuroprotective Agents

  Materials
  • Male Sprague‐Dawley rat, 270 to 300 g
  • Isoflurane (Abbott Laboratories)
  • 30% oxygen/70% nitrogen
  • Cyanoacrylate glue
  • Test compounds of interest (optional)
  • Anesthetic (e.g., pentobarbital or chloral hydrate)
  • 10% buffered formalin (see recipe)
  • Small PE20 jugular cannula (polyethylene tubing; Becton Dickinson)
  • Wound clips (e.g., autoclip; Stoelting) with applier and remover (Fisher Scientific)
  • Anesthetic vaporizer and flowmeter (e.g., Vetmac)
  • Dissecting microscope (e.g., stereomaster zoom microscope, Fisher Scientific) with 10× wide‐field eye piece and fiber optic light source
  • Homeothermic temperature system (Harvard Apparatus)
  • Assorted surgical instruments:
  •  Animal clippers (Fisher Scientific)
  •  Microdissecting tweezers (RS‐5005, Roboz Surgical Instruments)
  •  McPherson‐Vannas microdissecting scissors (RS‐5630; Roboz Surgical Instruments)
  •  Straight 6‐mm microaneurysm clip and 5.75‐in. (14.6‐cm) applying forceps with lock (Roboz Surgical Instruments)
  •  Bone‐cutting forceps (Roboz Surgical Instruments)
  •  Microdissecting forceps (Roboz Surgical Instruments)
  •  Fine straight 11‐cm iris scissors (Fine Science Tools)
  •  Dumont forceps (no. 11297‐00 and 11251‐30; Fine Science Tools)
  •  10‐cm curved and 14‐cm straight hemostatic forceps (Fine Science Tools)
  •  Fine forceps (no. 111500‐10; Fine Science Tools)
  •  Needle holders (Fine Science Tools)
  •  Retractors (Fine Science Tools)
  • Portable hot bead sterilizer (Fine Science Tools)
  • Laser Doppler monitor (optional; e.g., Perimed Periflux 4001) with probe (Probe 407) and probe miniholder (PH 07‐5)
  • Double‐sided tape (Perimed PH 105‐3)
  • Electrocautery unit (e.g., Geiger NY model 100 with light‐duty cautery tips, style B; George Tiemann)
  • 5‐0 silk black braided suture (Roboz Surgical Instruments)
  • Intraluminal monofilament occluder (see protocol 2)
  • Cryostat or microtome for sectioning
  • Glass microscope slides
  • Computerized image analysis system (e.g., Optimus 5.2 or ImageProplus; Datacell Software)
  • 16‐G needle
  • Peristaltic pump (e.g., Master‐flex, Cole Palmer)
  • Additional reagents and equipment for cresyl violet staining (see protocol 8) and histological preparation of fixed brain tissue (unit 9.5)
NOTE: Sterilize surgical instruments (i.e., forceps and scissors) using the hot bead sterilizer.

Support Protocol 1: Fabrication of Intraluminal Monofilament Occluders

  Materials
  • 0.1% (w/v) poly‐L‐lysine (Sigma)
  • Nylon monofilament:
  •  Stren Monofilament fishing line, 6‐ to 8‐lb test, 0.28‐mm diameter (DuPont) with tip rounded by dipping in fingernail polish (e.g., Double Magic Cutex, base coat/topcoat), or
  •  3‐0 Dermalon blue nylon monofilament (The Butler Company)
  • 60°C incubator

Basic Protocol 2: Middle Cerebral Artery Occlusion (MCA) Using Stereotaxic Infusion of Endothelin 1

  Materials
  • Et‐1 solution (see recipe)
  • Male Sprague‐Dawley rat, 280 to 320 g
  • Isoflurane (Vet Drug)
  • Test compounds of interest (optional)
  • 28‐G steel infusion cannula (e.g., Plastics One, c/o Bilaney Consultants)
  • Stereotaxic frame (e.g., David Kopf Instruments or Bilaney Consultants)
  • 25‐µl Hamilton syringe (e.g., Alltech Associates Applied Science)
  • FEP tubing (CMA, Biotech Instruments Ltd.)
  • Infusion pump (e.g., World Precision Instruments)
  • Rat anesthesia mask (e.g., David Kopf Instruments or Bilaney Consultants)
  • Rectal temperature monitor and probe (e.g., RS Components)
  • Heating pad/blanket (e.g., International Market Supply) to which rectal probe can be attached
  • Electric hair clippers
  • Surgical instruments (e.g., see protocol 1)
  • Large artery clips
  • Small clips
  • Bone chisel
  • Cotton swabs
  • Surgical spears (e.g., Interfocus)
  • Dental drill (e.g., Biotech Instruments)
  • 0.9‐mm steel drill bit/burr (e.g., Interfocus)
  • Mersilk 410 W536 Ethicon braided silk sutures (International Market Supply)
  • Thermacage (Beta Medical & Scientific)
  • Additional reagents and equipment for evaluating effects of MCAO (see protocol 4 and protocol 5), and analysis by cresyl violet staining or other histological procedures (see protocol 1)

Support Protocol 2: Use of Horizontal and Inclined Balance Beam to Assess Sensorimotor Performance After Et‐1 MCAO

  Materials
  • Rat: untreated for training, and then Et‐1‐treated (see protocol 3) for testing
  • Two pieces of wood, 2 cm wide and 1 m long

Support Protocol 3: Use of the Staircase Test to Measure Skilled Paw Use After Et‐1 MCAO

  Materials
  • Rat: untreated for training, and then Et‐1‐treated (see protocol 3) for testing
  • Staircase apparatus (Fig. ; constructed as described in Montoya et al., )
  • Food pellets (e.g., 45‐mg Noyes sucrose reward pellets; Sandown Scientific)

Basic Protocol 3: The Tamura Model of Middle Cerebral Artery Occlusion (MCAO) to Test Neuroprotective Agents

  Materials
  • Male Sprague‐Dawley rat, 275 to 300 g
  • Cefazolin (The Butler Company)
  • Isofluorane (Abbott Laboratories)
  • 30% oxygen/70% nitrogen
  • Test compounds of interest (optional)
  • Artificial tears (The Butler Company)
  • Surgical scrub: Betadine and 70% (v/v) ethanol
  • EMLA cream (2.5% lidocaine and 2.5% prilocaine; Henry Schein)
  • Polysporin topical ointment (Henry Schein)
  • Anesthetic vaporizer and flow meter (e.g., Vetamac)
  • Rat anesthesia mask designed so that the rat can lay on its side with head held in place by an incisor bar
  • PE50 jugular cannula (polyethylene tubing; Becton Dickinson; optional)
  • Compact syringe pump (infusion pump; e.g., Harvard Apparatus model 975)
  • Flow‐through swivel (e.g., Harvard Apparatus)
  • Homeothermic blanket system with rectal probe (Harvard Apparatus)
  • Elastikon porous tape
  • Surgical instruments (e.g., see protocol 1)
  • Portable hot bead sterilizer (Fine Science Tools)
  • Dissecting microscope (e.g., aus Jena 212T OPM) or other suitable model
  • Hand‐held drill with a 1.4‐mm steel burr (Fine Science Tools)
  • Curved Fredman‐Peason microrongeurs (Fine Science Tools)
  • 440E bipolar coagulation unit (Radionics)
  • Extra‐delicate mini‐Vannas scissors (Fine Science Tools)
  • Surgical 3‐0 nylon suture (Harvard Apparatus)
  • Additional reagents and equipment for analysis by cresyl violet staining or other histological procedures (see protocol 1)
NOTE: Sterilize surgical instruments (i.e., forceps and scissors) between surgery for each rat using the hot bead sterilizer.

Basic Protocol 4: The Spontaneously Hypertensive Rat Model of Middle Cerebral Artery Occlusion (MCAO) to Test Neuroprotective Agents

  Materials
  • Male spontaneously hypertensive rat, 300 to 325 g (Taconic or Harlan Bioproducts for Science)
  • Isofluorane (Abbott Laboratories)
  • 30% oxygen/70% nitrogen
  • Artificial tears (The Butler Company)
  • Surgical scrub: Betadine and 70% (v/v) ethanol
  • 0.9% (w/v) saline, sterile
  • EMLA cream (2.5% lidocaine and 2.5% prilocaine; Henry Schein)
  • Polysporin topical ointment (Henry Schein)
  • Test compounds of interest (optional)
  • Anesthetic vaporizer and flow meter (e.g., Vetmac)
  • Rat anesthesia mask designed so that the rat can lay on its side with head held in place by an incisor bar
  • Homeothermic blanket system with rectal probe (Harvard Apparatus)
  • Portable hot bead sterilizer (Fine Science Tools)
  • PE50 polyethylene tubing (Becton Dickinson)
  • Tygon tubing (0.010‐mm i.d. × 0.30‐mm o.d.)
  • Heart rate and blood pressure monitor (e.g., Gould model 11‐2927‐31 two‐channel ink recorder)
  • Surgical instruments (e.g., see protocol 1)
  • Small hand‐held retractor (Roboz Surgical Instruments)
  • Straight atraumatic aneurysm clamp, 0.75 × 4 mm (Roboz Surgical Instruments; optional)
  • 3‐0 silk suture (optional)
  • Dissecting microscope
  • Small hand‐held drill with 1.4‐mm diameter bit
  • Curved atraumatic aneurysm clamp, 1 × 6 mm (Roboz Surgical Instruments)
  • i‐STAT portable clinical analyzer and test cartridges for blood gas, pH, and glucose analysis (SDI‐sensor Device)
  • Light‐duty electrocautery unit (e.g., Geiger‐NY model 100 with style B cautery tips; George Tiemann)
  • Autoclip wound clips (Stoelting) with applier and remover (Fisher Scientific)
  • 3‐0 nylon suture
  • Compact syringe pump (infusion pump; e.g., Harvard Apparatus model 975)
  • Flow‐through swivel (e.g., Harvard Apparatus)
  • Additional reagents and equipment for analysis by cresyl violet staining or other histological procedures (see protocol 1)
NOTE: Sterilize surgical instruments (i.e., forceps and scissors) prior to surgery using the hot bead sterilizer.

Support Protocol 4: Cresyl Violet Staining of Brain Tissue

  Materials
  • Slides with 30‐µm cryostat sections from occluded brain (see protocol 1)
  • Industrial methylated spirits (IMS; Fisher Scientific)
  • Xylene (Fisher Scientific)
  • Cresyl violet solution (see recipe)
  • Acid formalin solution (see recipe)
  • DPX mountant (BDH)
  • Slide rack and staining dishes
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Figures

Videos

Literature Cited

Literature Cited
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Key References
Internet Resources
  http://stroke.ahajournals.org/
  Web site for the journal Stroke (journal of American Heart Association), which publishes original research papers using animal models, as well as clinical papers and updates on current clinical trials in stroke.
  http://cbfm.org/
  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.
  http://www.ninds.nih.gov/
  Web site for the National Institute of Neurological Disorders and Stroke (part of the U.S. National Institutes of Health). This government agency also supports basic research through grant funding.
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