Mouse Models of Focal Cerebral Ischemia

Nariman Panahian1

1 InforMax Inc., North Bethesda, Maryland
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.9
DOI:  10.1002/0471140856.tx1109s06
Online Posting Date:  May, 2001
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Abstract

As with models of global ischemia (UNIT), models of focal ischemia in the mouse are useful for understanding the pathophysiology of this condition. The models are also useful for assessing the neuroprotective effects of particular genes and pharmacologic agents. This unit describes two methods for inducing focal ischemia: one by proximal middle cerebral artery occlusion and one by permanent endovascular occlusion. Alternatives of transient endovascular occlusion and subarachnoid hemorrhage are also given. A number of protocols are described for determining the effects of ischemia induction: assessing physiological features, mapping of the circulation by perfusing the blood vessels with carbon black, determining stroke volume by staining with 2,3,5‐triphenyltetrazolium chloride, evaluating post‐treatment behavior, assessing stroke volume and cerebral edema, assessing the size of ischemic penumbra, and analyzing the statistics and presenting the data.

     
 
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Table of Contents

  • Basic Protocol 1: Subtemporal Craniectomy: Proximal MCA Occlusion
  • Basic Protocol 2: Permanent Endovascular Occlusion Model of Focal Ischemia
  • Alternate Protocol 1: Transient Endovascular Occlusion Focal Ischemia
  • Alternate Protocol 2: Inducing the Filament Model of Subarachnoid Hemorrhage
  • Alternate Protocol 3: Inducing Hemorrhagic Stroke Model by Collagenase Injection
  • Support Protocol 1: Assessment of Systemic Physiological Parameters
  • Support Protocol 2: Carbon Black Perfusions
  • Support Protocol 3: TTC Staining to Determine Stroke Volume
  • Support Protocol 4: Behavioral Assessment of Focal Ischemia
  • Support Protocol 5: Assessment of Hemispheric Volume of Stroke and Cerebral Edema
  • Support Protocol 6: Assessment of the Size of the Ischemic Penumbra
  • Support Protocol 7: Statistical Analysis and Data Presentation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Subtemporal Craniectomy: Proximal MCA Occlusion

  Materials
  • Mice
  • 2% and 3% (v/v) halothane vaporized with 30% O 2/70% N 2 (Halocarbon Laboratories)
  • Halothane vaporizer (e.g., Fluotec model 3; Colonial Medical Supply)
  • Plexiglas anesthesia induction chamber for rodents (Stoelting)
  • Homeothermic blanket with feedback‐regulated maintenance of body temperature (Stoelting)
  • YSI thermocouple equipped with a rectal temperature probe (Harvard Apparatus)
  • Nose cone: plastic housing of 20‐G angiocatheter (Angiocath, Deseret Medical)
  • Surgical gauze
  • Surgical and microsurgical instruments (unit 11.8; Fine Science Tools):
  •  Student Vannas scissors
  •  Electrocautery device
  •  Friedman‐Pearson micro‐rongeurs
  •  Mini‐Goldstein retractor
  •  No. 5 forceps
  •  No. 55 microforceps
  • Stereomicroscope with 10× objective (e.g., Leica MZ‐8; Leica USA)
  • Microdrill and 0.35‐ to 0.42‐mm diameter ultrafine burrs (Fine Science Tools)
  • 27.5‐G blunted needle with tip bent 90° (Becton Dickinson)
  • 30‐G needle, bent (Becton Dickinson)
  • Gelfoam (Upjohn, Becton Dickinson)
  • 6‐0 monofilament nylon atraumatic sutures (Ethicon)
  • Rodent intensive care and temperature‐control unit (Thermocare)

Basic Protocol 2: Permanent Endovascular Occlusion Model of Focal Ischemia

  Materials
  • Coating material (Table 11.9.1)
  • Mice to be tested
  • 2% and 3% (v/v) halothane in 30% O 2/70% N 2 (Halocarbon Laboratories)
  • 6‐0, 7‐0, 8‐0, and 10‐0 monofilament nylon atraumatic sutures (Ethicon)
  • 27.5‐G needle (Becton Dickinson)
  • Halothane vaporizer (e.g., Fluotec Model 3; Colonial Medical Supply)
  • Plexiglas induction anesthesia chamber (Stoelting)
  • Homeothermic blanket for rodents with feedback‐regulated maintenance of body temperature (Stoelting)
  • YSI thermocouple equipped with a rectal temperature probe (Harvard Apparatus)
  • SAR‐830/P small animal ventilator, volume and pressure cycled, with built in pump, flowmeter, and internal valves (CWE)
  • Endotracheal tube: 20‐G, 17‐mm‐long angiocatheter on a curved stilette (Angiocath, Deseret Medical)
  • Catheter (i.e., PE‐10 polyethylene tubing; Intramedic)
  • Microcapnometer (Columbus Instruments)
  • PF‐3 laser Doppler flowmeter with masterprobe (Periflux, Perimed)
  • Cotton swabs
  • Microdrill and 0.35‐ to 0.42‐mm diameter, ultrafine burrs (Fine Science Tools)
  • Surgical instruments (Fine Science Tools):
  •  Student Vannas microscissors
  •  No. 5 and no. 55 forceps
  •  Curved Moria forceps
  • Stereotaxic frame, non‐ferromagnetic (Massachusetts General Hospital machine shop; optional)
  • Surgical glue (optional)
  • 16‐G tubing (optional)
  • Silicone lubricant (optional)
  • Gelfoam (Upjohn, Becton Dickinson; optional)
  • Macintosh Power PC laptop computer
  • MacLab/8e data acquisition system equipped with ETH‐400 and bridge preamplifiers running Scope and Chart software, with blood pressure transducers, EEG and EKG electrodes, BNC cables, and thermocouple (AD Instruments)
  • Blood gas analyzer (800 series, Ciba‐Corning)
  • Zen temporary clips (13 × 0.4–mm, 15 g closing force; Ohwa Tsusho)
  • Vessel cannulation forceps S&T (Fine Science Tools)
  • Additional reagents and equipment for inserting arterial catheters, positioning laser Doppler flowmeter probes for rCBF monitoring, and artificial ventilation (unit 11.8).

Alternate Protocol 1: Transient Endovascular Occlusion Focal Ischemia

  Materials
  • Mice
  • 2% (v/v) halothane in 30% O 2/70% N 2 (Halocarbon Laboratories)
  • 0.150 U/µl Clostridium histolyticum collagenase (Sigma)
  • Normal saline: 0.9% (w/v) NaCl
  • TTC staining solution (see recipe) or H&E stain (optional)
  • Homeothermic blanket for rodents with feedback‐regulated maintenance of body temperature (Stoelting)
  • YSI thermocouple equipped with a rectal temperature probe (Harvard Apparatus)
  • Ultraprecise stereotaxic apparatus with gas anesthesia nose cone (Kopf Instruments)
  • Stereotaxic drill
  • Microdrill with ultrafine drill bits slightly larger than 32‐G needle (Fine Science Tools)
  • Gas‐tight microsyringes (Hamilton) with 32‐G stainless steel needles without bevels
  • Rodent intensive care and temperature‐control unit (Thermocare)
  • Surgical wax (Ethicon)
  • 6‐0 monofilament nylon sutures (Ethicon)

Alternate Protocol 2: Inducing the Filament Model of Subarachnoid Hemorrhage

  • Mice treated to induce focal ischemia (see protocol 1 and protocol 22) and control mice
  • 2.5% and 1.0% to 1.5% (v/v) halothane vaporized with 30% O 2/70% N 2O (Halocarbon Laboratories)
  • α‐Chloralose (Rochester Hospital Supply)
  • Homeothermic blanket (Stoelting)
  • Overhead infrared lamp, YSI thermocouple with rectal temperature probe (Harvard Apparatus)
  • Macintosh Powerbook, MacLab/8e data acquisition system with four‐channel ETH‐400 transducer‐amplifier (AD Instruments), and appropriate software (e.g., Scope and Chart; AD Instruments)

Alternate Protocol 3: Inducing Hemorrhagic Stroke Model by Collagenase Injection

  Materials
  • Mice treated to induce focal ischemia (see protocol 1 or protocol 22) and control mice
  • Heparinized saline: 100 µl heparin in 100 ml saline (0.9% w/v NaCl; use immediately)
  • 1.6% to 1.8% (v/v) Higgins Black Magic waterproof drawing ink (Eberhard Faber)
  • 4% (w/v) paraformaldehyde (unit 11.8)
  • MZ‐8 Leica stereomicroscope
  • Styrofoam board and pins or tacks
  • 26‐G plastic catheter mounted on a 10‐ml syringe
  • Camera (e.g., MicroCam; Polaroid)

Support Protocol 1: Assessment of Systemic Physiological Parameters

  Materials
  • Brains of control mice and mice treated to induce focal ischemia (see Basic Protocols protocol 11 and protocol 22)
  • PBS ( appendix 2A), 4°C
  • 2% (w/v) 2,3,5‐triphenyltetrazolium chloride staining solution (TTC; see recipe), prewarmed to 35° to 36°C
  • Brainslicer (EM Corp.) and razor blades, 4°C (stainless steel)
  • Spatula
  • Weigh boats or Petri dishes
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Figures

  •   FigureFigure 11.9.1 Hemorrhagic stroke 24 hr after stereotaxic administration of collagenase (0.075 U in 0.5 µl) into the caudate nucleus according to Clark et al. (). Hemorrhagic transformation and edema within this 2‐mm‐thick, TTC‐stained coronal specimen of the mouse brain. Scale: 1 mm; magnification: 10×.
  •   FigureFigure 11.9.2 Carbon black vascular mapping of the brains of intact mice for demonstration of vascular anomalies (A, C, D, E, F) and mapping of arterial cortical collaterals (B, marked as small single arrows) between branches of pericallosal artery (marked with single arrow‐heads) and middle cerebral artery (mca; marked with small double arrows). The level of the bregma is marked with big double arrows. Note duplications of MCA marked by small double arrows (C and F); absence of one (or both) of the posterior communicating arteries (pcom; C versus A); presence of increased posterior vascular arborizations of the MCA (D and E); absence of right anterior inferior cerebellar artery (aica, C). (G, H, I, J) TTC staining of mouse brains after 24 hr MCAo using either filament (G and J), Brint (H) or Tamura‐type models (I). Note decreased cerebral edema in the Tamura (I) versus the filament model (J). Upper cortical regions overlying the caudate nucleus and corresponding to watershed areas (marked with arrowheads) are preserved in the Brint and Tamura models (H and I), but are compromised in the filament model (J). SV‐129 mice were used in these experiments. As a result of partial filament insertion due to excessive coating material, occlusion of the posterior hippocampal artery (pha) resulted in a lesion in the upper posterior portions of the hippocampus in a C57Bl/6 mouse (G). Abbreviations: ac, anterior communicating artery; aca, anterior cerebral artery; aica, anterior inferior cerebellar artery; asa, anterior spinal artery; ba, basilar artery; ica, internal carotid artery; mca, middle cerebral artery; pca, posterior cerebral artery; pcla, posterior cerebellar artery; pcom, posterior communicating artery; pha, posterior hippocampal artery; pica, posterior inferior cerebellar artery; sca, superior cerebellar artery; va, vertebral artery.
  •   FigureFigure 11.9.3 Analysis of stroke volume. (A) Cumulative stroke volume in DNX mice derived from seven representative coronal brain sections, multiplied by the distance between them. Animals were subjected to permanent MCA occlusion (pMCAo) using the subtemporal approach. (B) Anterior‐posterior distribution of injury at 6 hr (crosses) and at 24 hr (diamonds) in DNX mice. The volume of stroke and anterior‐posterior distribution was reconstructed based on hematoxylin and eosin–stained specimens. (C) H&E–stained coronal sections 1 to 7 were used to plot anterior‐posterior distribution of the ischemic lesion at 6 hr after MCAo in DNX mice. The lesion appears to be much smaller in heme oxygenase‐1 overexpressing mice (D).

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Literature Cited

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