Mouse Models of Global Cerebral Ischemia

Nariman Panahian1

1 University of Rochester, Rochester, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.8
DOI:  10.1002/0471140856.tx1108s05
Online Posting Date:  May, 2001
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Abstract

Use of rodent models of hippocampal neurodegeneration are recommended for use in studies to understand the pathophysiology and molecular mechanisms involved. This unit includes protocols for two‐vessel occlusion, three‐vessel occlusion, and permanent bilateral vertebral artery occlusion plus methods for histological techniques for preparation of brain tissue and analysis of hippocampal injury.

     
 
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Table of Contents

  • Basic Protocol 1: Three‐Vessel Occlusion Model of Global Cerebral Ischemia
  • Basic Protocol 2: Four‐Vessel Occlusion Model of Global Cerebral Ischemia
  • Basic Protocol 3: Two‐Vessel Occlusion Model of Global Cerebral Ischemia
  • Support Protocol 1: Preparation and Staining of Frozen Sections
  • Support Protocol 2: Perfusion Fixation of Brains for Immunocytochemical Experiments
  • Support Protocol 3: Quantitative and Qualitative Methods of Evaluatiang Hippocampal Injury
  • Support Protocol 4: Morphological Analysis of Hippocampal Injury
  • Support Protocol 5: Physiological Parameters Confirming Hippocampal Injury
  • Support Protocol 6: Clinical Criteria of Hippocampal Injury
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Three‐Vessel Occlusion Model of Global Cerebral Ischemia

  Materials
  • Adult mice, 20 to 28 g (e.g., Harlan Bioproducts for Science, Jackson Laboratory, Taconic Farms)
  • 3% and 1.5% (v/v) halothane in 70% N 2O/30% O 2
  • Acrylic dental cement (Stoelting)
  • Surgical glue
  • Xylocaine solution: 2.5% (v/v) lidocaine solution
  • 1000 U/ml heparin
  • Heparinized saline: 100 µl heparin in 100 ml saline, use immediately
  • Fluotec 3 halothane vaporizer equipped with air, N 2O, and O 2 flowmeter assembly (Colonial Medical Supply)
  • Plexiglas halothane induction chamber for rodents (Stoelting)
  • Homeothermic blanket for rodents with feedback‐regulated maintenance of body temperature (Stoelting)
  • YSI thermocouple equipped with a microprobe (model 511, S.N. K076, Harvard Apparatus)
  • SEP/EEG gold screws: gold‐wire electrophysiology contacts for EEG and/or SEP monitoring (Fine Science Tools #19003‐02)
  • Stereotaxic frame: dual‐manipulator model with swivel mouse nose adaptor (David Kopf Instruments; optional)
  • Periflux PF‐3 laser Doppler flowmeter equipped with PF318 master probe and MTB 500‐0 straight fiberoptic microtips (Perimed)
  • Cotton swabs
  • 8‐0, 10‐0, and 11‐0 black monofilament nylon atraumatic sutures (Ethicon)
  • Absorbtion spears, 1 box
  • PE‐10 intramedic polyethylene tubing (Clay Adams)
  • Lucr‐Lok stopcock (Becton Dickinson)
  • 27.5‐G needle (Becton Dickinson)
  • 1‐ml tuberculin syringes (Becton Dickinson)
  • 20‐G, 17‐mm‐long angiocatheter (Angiocath, Deseret Medical) and curved stilette
  • SAR‐830/P small animal ventilator, volume and pressure cycled, with built‐in pump, flowmeter, and internal valves (CWE)
  • Microcapnometer (Columbus Instruments)
  • Blood gas analyzer (800 series; Ciba‐Corning)
  • Heparinized microhematocrit capillary tubes (Fisher Scientific)
  • Overhead infrared reflector lamp (120 V, 250 W, GE; Harvard Apparatus)
  • 6‐0 silk black braided monofilament suture on a cutting G‐7 needle (Ethicon)
  • Transpore surgical adhesive tape (Baxter Medical Supply)
  • Rodent intensive care and temperature control unit (DW‐1, ThermoCare)
  • Stereomicroscope consisting of:
  • MZ‐8 Leica stereomicroscope on a three‐dimensional SMS‐20 ball‐bearing boom stand, WF 10×/21M HP eyepieces, and 0.8× achromatic main objective lens, FWD = 112 mm (Kramer Scientific)
  • Optical carrier equipped with a double‐barrel coaxial surgical illuminator integrated with EKE rheostat light source
  • MacLab 8/e data acquisition system equipped with ETH‐400 and Bridge preamplifiers running Chart and Scope software, with blood pressure transducers, EEG, EKG electrodes, BNC cables, thermocouple (AD Instruments)
  • Macintosh Power PC Laptop computer
  • Statview v. 5 statistical package for the Macintosh (SAS Institute, Inc.)
  • Statistical software for power analysis: Primer of Biostatistics (McGraw‐Hill)
  • Viking, Nicolet Pathfinder four‐channel evoked potential unit (Nicolet Biomedical)
  • 440‐E animal laboratory bipolar coagulator with McPherson bipolar miniature forceps and cords (Medifor)
  • Software packages for image analysis: Image ProPlus v. 3.0 (Media Cybernetics)
  • XLC‐140 caposil or calcium‐filled liquid nitrogen (vapor releasing) freezing chamber (LabRepco)
  • Timer (VWR)
  • Microsurgical instruments (Fine Science Tools):
    • Vibration‐free microdrill (11,000 rpm) with 400‐µm drill bits (#18000‐17)
    • Moria miniature scalpel (#10315‐12)
    • Straight titanium forceps (#11602‐16) or suture‐tying forceps (#18025‐10)
    • Angled fine forceps (#11063‐07)
    • Biologie no. 5 titanium forceps, 0.05 × 0.02–mm tip (#11252‐40)
    • Dumont no. 4 forceps (#11241‐30)
    • Dumont microsurgery forceps with atraumatic 0.1 × 0.06–mm tips (no. 55; #11253‐20)
    • Dumont straight‐tip forceps, 0.2 × 0.12–mm tip (#11203‐23)
    • Dumont straight ultrafine forceps, 0.05 × 0.01–mm tip (#11254‐20)
    • Epoxy‐covered forceps (#11220‐21)
    • Moria MC40 ultrafine forceps (#11370‐40)
    • Vessel cannulation forceps S&T (#00571‐13)
    • Vannas‐Tubingen straight titanium scissors (#150007‐08)
    • Small‐blade scissors (#15000‐08) or extra‐delicate mini‐Vannas scissors (#15000‐00)
    • Student Vannas spring scissors, straight (#15100‐09)
    • Mini‐Goldstein retractor (#17002‐02)
    • Yasargil vascular clip (Aesculap FD722), closing force = 0.98 N
    • Battery‐operated small vessel cauterizer with angled and straight 0.2‐mm‐diameter tips made of platinum‐iridium alloy (#18000‐00)
    • Zen temporary clips (13 × 0.4–mm, 15‐g closing force; Ohwa Tsusho)
  • Additional reagents and equipment for assessment of hippocampal injury (Support Protocols protocol 41 to protocol 96)

Basic Protocol 2: Four‐Vessel Occlusion Model of Global Cerebral Ischemia

  Materials
  • Adult mice, 20 to 28 g (e.g., Harlan Bioproducts for Science, Jackson Laboratory, Taconic Farms)
  • 2.0% (v/v) halothane in 70% N 2O/30% O 2
  • Surgical glue
  • Standard stereotaxic frame (Stoelting or David Kopf Instruments) equipped with mouse ear bars, micromanipulators, and a specially designed Narashigi head holder, and inhalation anesthesia nose cone
  • Transpore surgical adhesive tape (Baxter Medical Supply)
  • 6‐0 silk black monofilament nylon atraumatic sutures (Ethicon)
  • Cotton swabs
  • 1‐ml syringes (Becton Dickinson)
  • 6‐0 silk black braided suture on a cutting G‐7 needle (Ethicon)
  • Surgical gauze
  • Stereomicroscope consisting of:
  • MZ‐8 Leica stereomicroscope on a three‐dimensional SMS‐20 ball‐bearing boom stand, WF 10×/21M HP eyepieces, and 0.8× achromatic main objective lens, FWD = 112 mm (Kramer Scientific)
  • Optical carrier equipped with a double‐barrel coaxial surgical illuminator integrated with EKE rheostat light source
  • Fluotec 3 halothane vaporizer equipped with air, N 2O, and O 2 flowmeter assembly (Colonial Medical Supply)
  • Plexiglass halothane induction chamber for rodents (Stoelting)
  • 440‐E animal laboratory bipolar coagulator with McPherson bipolar miniature forceps and cords (Medifor)
  • YSI thermocouple equipped with a microprobe (model 511, S.N. K076; Harvard Apparatus)
  • Overhead infrared reflector lamp (120 V, 250 W, GE; Harvard Apparatus)
  • Rodent intensive care and temperature control unit (DW‐1, ThermoCare)
  • Hot glass bead dry surgical instrument sterilizer (Stoelting)
  • Timer (VWR)
  • Homeothermic blanket for rodents with feedback regulated maintenance of body temperature (Stoelting)
  • Microsurgical instruments (Fine Science Tools):
    • Vibration‐free microdrill (11,000 rpm) with 400‐µm drill bits (#18000‐17)
    • Scalpel blade
    • Biologie no. 5 titanium forceps, 0.05 × 0.02–mm tip (#11252‐40)
    • Dumont no. 4 forceps (#11241‐30)
    • Student Vannas spring scissors, straight (#15100‐09)
    • Mini‐Goldstein retractor (#17002‐02)
    • Battery‐operated small‐vessel cauterizer with angled and straight 0.2‐mm‐diameter platinum/iridium alloy tips (#18000‐00)
    • Suture‐tying forceps (#18025‐10)
    • Vessel cannulation forceps S&T (#00571‐13)
    • Zen temporary clips (13 × 0.4–mm, 15‐g closing force; Ohwa Tsusho) and clip applier
    • Absorbtion spears, 1 box
  • Additional reagents and equipment for assessment of hippocampal injury (Support Protocols protocol 41 to protocol 96)

Basic Protocol 3: Two‐Vessel Occlusion Model of Global Cerebral Ischemia

  Materials
  • Adult mice, 20 to 28 g (e.g., Harlan Bioproducts for Science, Jackson Laboratory, Taconic Farms)
  • 3% and 1.5% (v/v) halothane in 70% N 2O/30% O 2
  • Xylocaine solution
  • Heparinized saline: 100 µl heparin in 100 ml saline, use immediately
  • Surgical glue
  • Acrylic dental cement (Stoelting)
  • Fluotec 3 halothane vaporizer equipped with air, N 2O, and O 2 flowmeter assembly (Colonial Medical Supply)
  • Plexiglass halothane induction chamber for rodents (Stoelting)
  • Homeothermic blanket for rodents with feedback‐regulated maintenance body temperature (Stoelting)
  • YSI thermocouple equipped with a microphone (model 511, S.N. Ko76; Harvard Apparatus)
  • Omega thermocouple with 33‐G needle probe for direct brain temperature measurements (Harvard Apparatus)
  • Cotton swabs
  • 8‐0, 10‐0, and 11‐0 black monofilament nylon atraumatic sutures (Ethicon)
  • Absorption spears
  • PE‐10 intramedic polyethylene tubing (Clay Adams)
  • 27.5‐G needle (Becton Dickinson)
  • 1‐ and 10‐ml syringes (Becton Dickinson)
  • Periflux PF‐3 laser Doppler flowmeter equipped with PF318 master probe and MTB 500‐0 straight fiberoptic microtips (Perimed)
  • SEP/EEG gold screws: gold‐wire electrophysiology contacts for EEG and/or SEP monitoring (Fine Science Tools #19003‐02)
  • Stereotaxic frame: dual‐manipulator model with swivel mouse nose adaptor (David Kopf Instruments)
  • 20‐G and 26‐G, 17‐mm‐long angiocatheters (Angiocath, Deseret Medical) and curved stilette
  • SAR‐830/P small‐animal ventilator, volume and pressure cycled, with built‐in pump, flowmeter, and internal valves (CWE)
  • 6‐0 silk black braided suture on a cutting G‐7 needle (Ethicon)
  • 500‐µl gas‐tight Hamilton syringe, heparinized
  • Vascular occluders, 2‐mm diameter (OC2A; In Vivo Metric; optional)
  • Transpore surgical adhesive tape (Baxter Medical Supply)
  • Heparinized microhematocrit capillary tubes (Fisher Scientific)
  • Surgical gauze
  • Stereomicroscope consisting of:
  • MZ‐8 Leica stereomicroscope on a three‐dimensional SMS‐20 ball‐bearing boom stand, WF 10×/21M HP eyepieces, and 0.8× achromatic main objective lens, FWD = 112 mm (Kramer Scientific)
  • Optical carrier equipped with a double‐barrel coaxial surgical illuminator integrated with EKE rheostat light source
  • Microcapnometer (Columbus Instruments)
  • MacLab 8/e data acquisition system equipped with ETH‐400 and Bridge preamplifiers running Chart and Scope software, with blood pressure transducers, EEG, EKG electrodes, BNC cables, thermocouple (AD Instruments)
  • Macintosh Power PC Laptop computer
  • Viking, Nicolet Pathfinder four‐channel evoked potential unit (Nicolet Biomedical)
  • 800 series blood gas analyzer (Ciba‐Corning)
  • 440‐E animal laboratory bipolar coagulator with McPherson bipolar miniature forceps and cords (Medifor)
  • YSI thermocouple equipped with a microprobe (model 511, S.N. K076, Harvard Apparatus)
  • Omega thermocouple with 33‐G needle probe for direct brain temperature measurements (Harvard Apparatus)
  • Overhead infrared reflector lamp (120 V, 250 W, GE; Harvard Apparatus)
  • Rodent intensive care and temperature control unit (DW‐1, ThermoCare)
  • Hot glass bead dry surgical instrument sterilizer (Stoelting)
  • Timer (VWR)
  • Homeothermic blanket for rodents with feedback regulated maintenance of body temperature (Stoelting)
  • Laboratory scale for weighing mice
  • Micro‐surgical instruments (Fine Science Tools):
    • Vibration‐free microdrill (11,000 rpm) with 400‐µm drill bits (#18000‐17)
    • Biologie no. 5 titanium forceps, 0.05 × 0.02–mm tip (#11252‐40)
    • Dumont no. 4 forceps (#11241‐30)
    • Dumont microsurgery forceps with atraumatic 0.1 × 0.06–mm tips (no. 55; #11253‐20)
    • Dumont straight‐tip forceps, 0.2 × 0.12–mm tip (#11203‐23)
    • Dumont straight ultrafine forceps, 0.05 × 0.01–mm tip (#11254‐20)
    • Moria MC40 ultrafine forceps (#11370‐40)
    • Small‐blade scissors (#15000‐08) or extra‐delicate mini‐Vannas scissors (#15000‐00)
    • Student Vannas spring scissors, straight (#15100‐09)
    • Mini‐Goldstein retractor (#17002‐02)
    • Micro‐clip applicator with 18055 series micro‐serrefines (#18056‐14)
    • Battery‐operated small‐vessel cauterizer with angled and straight 0.2‐mm‐diameter platinum/iridium alloy tips (#18000‐00)
    • Vessel cannulation forceps S&T (#00571‐13)
    • Yasargil vascular clip (Aesculap FD722), closing force = 0.98 N
    • Zen temporary clips (13 × 0.4 mm, 15‐g closing force; Ohwa Tsusho)
  • Additional reagents and equipment for assessment of hippocampal injury ( protocol 4 to protocol 9)

Support Protocol 1: Preparation and Staining of Frozen Sections

  Materials
  • Mice from ischemia experiments (see Basic Protocols protocol 11 to protocol 33)
  • Isopentane (2‐methylbutane), −15° to −20°C
  • Fixative solution: 1:1:1:1 (v/v/v/v) glacial acetic acid/acetone/absolute ethanol/water
  • Harris hematoxylin solution, ready‐made (Polyscientific)
  • Thionin solution (see recipe)
  • 1% (v/v) acid alcohol (Polyscientific)
  • 0.05% (w/v) lithium carbonate (Polyscientific)
  • Eosin Y alcoholic working solution (Polyscientific)
  • 95% and 100% (v/v) ethanol
  • Cytoseal 60 (low‐viscosity mounting medium; VWR or Stephens Scientific) or Permount (Fisher Scientific)
  • Xylene
  • Dissection tools (e.g., see Basic Protocols protocol 11 to protocol 33 and protocol 5)
  • Dual‐compressor cryomicrotome (Jung CM 3000; Kramer Scientific)
  • 1600‐W hair dryer
  • Superfrost Plus glass slides (Fisher Scientific)
  • 25‐slide containers, sealed and dessicated (VWR)
  • Coplin jars
  • 24 × 50–mm coverslips (VWR)
  • Aluminum foil
  • 15‐ml plastic centrifuge tubes
  • Light microscope (e.g. Olympus AX‐70)

Support Protocol 2: Perfusion Fixation of Brains for Immunocytochemical Experiments

  Materials
  • Mice from ischemic experiment (see Basic Protocols protocol 11 to protocol 33)
  • Heparinized saline: 100 µl heparin in 100 ml saline, 4°C
  • 4% (w/v) paraformaldehyde, chilled to 4°C (see recipe)
  • Cryoprotectant solution (see recipe)
  • Powdered dry ice
  • Cytoseal
  • Mayo Toughcut tungsten‐carbide scissors (Fine Science Tools #14512‐15)
  • Adson Graefe tissue forceps (Fine Science Tools #11030‐12)
  • Fine straight serrated scissors (Fine Science Tools #14070‐12)
  • Moria fine forceps with microserrations (Fine Science Tools, Moria MC31#11370‐31)
  • Straight student Vannas scissors (Fine Science Tools #15000‐09)
  • 26‐G, 17‐mm‐long angiocatheter (Angiocath, Deseret Medical) and curved stilette
  • Friedman‐Pearson microrongeurs (Fine Science Tools #16020‐14)
  • 20‐ml scintillation vials
  • Sliding microtome (HM 400 R from MICROM Laborgeräte GmbH; distributed by Carl Zeiss)
  • Additional reagents and equipment for halothane anesthesia (see protocol 1)
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Figures

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Literature Cited

Literature Cited
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