Experimental Models of Hypoxic‐Ischemic Encephalopathy: Hypoxia‐Ischemia in the Immature Rat

Silvia Carloni1, Walter Balduini1

1 Istituto di Farmacologia e Farmacognosia, Università di Urbino “Carlo Bo”, Urbino, Italy
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.15
DOI:  10.1002/0471140856.tx1115s35
Online Posting Date:  February, 2008
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The development of experimental models to study the mechanisms of perinatal hypoxic‐ischemic encephalopathy and stroke and effective therapies represents an important goal in perinatal medicine. However, due to the complexity of this pathological condition in humans, to date there is no ideal animal model that completely reproduces this condition. This unit describes the most widely used rodent animal model for the study of hypoxic‐ischemic encephalopathy during development. The model consists of 7‐day‐old pup rats subjected to unilateral carotid artery ligation followed by timed hypoxia exposure, and incorporates both focal cerebral ischemia and reperfusion. Its strength lies in the relative ease of the surgical procedure, the low mortality rate, and the possibility of performing long‐term experiments, a necessity for preclinical therapeutic trials. Over the years, this model has been extensively characterized, and most information on the mechanisms responsible for neurodegeneration and possible therapeutic approaches following hypoxia‐ischemia during brain development derives from studies performed using this model. Curr. Protoc. Toxicol. 35:11.15.1‐11.15.11. © 2008 by John Wiley & Sons, Inc.

Keywords: neonatal hypoxia‐ischemia; stroke; rat; development

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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Cerebral Hypoxia‐Ischemia in 7‐Day‐Old Rats
  • Support Protocol 1: Measurement of Infarct Area and Volume
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Induction of Cerebral Hypoxia‐Ischemia in 7‐Day‐Old Rats

  • Pregnant female rats (e.g., Sprague Dawley, Wistar)
  • 2% to 4% isoflurane in O 2
  • Hypoxic gas mixture (8% O 2/92% N 2)
  • Retraction system (to stretch the animal out in the supine position; made in laboratory)
  • Thermometer with rectal probe
  • Warming pad/table capable of maintaining rats’ core temperature at 36.5° ± 1°C
  • Desk magnifier lamp
  • Surgery tools:
    • Delicate scissors (preferably angled to side)
    • Delicate curved forceps (2 forceps, 0.3 mm)
    • Suture needle (3/8 circle, curved, round and fitted with standard eye; needle diameter ∼0.3 to 0.4 mm)
    • Nonsterile surgical silk (size 4–0), cut in 10‐ to 15‐cm lengths
    • Surgical micro needle holder
  • Overhead heating (infrared) lamp
  • Induction chamber for hypoxia (volume of 1700 ml; appropriate for inducing hypoxia in up to ten ischemic rat pups; see Fig. )
  • Temperature‐controlled water bath
  • Additional reagents and equipment for inhalant anesthesia of rodents (Donovan and Brown, )

Support Protocol 1: Measurement of Infarct Area and Volume

  • 14‐day‐old (or older) ischemic rats (7 days after hypoxia‐ischemia; see protocol 1)
  • Fixative: 4% (w/v) paraformaldehyde in phosphate‐buffered saline (PBS)
  • 0.6% (w/v) toluidine blue in H 2O
  • 50%, 95%, 100% ethanol
  • 100% acetic acid
  • Xylenes
  • DPX mountant for histology (Canemco; http://www.canemco.com)
  • Large scissors or guillotine
  • Cryostat
  • Acid‐washed, gelatin‐subbed slides (gelatin and chrome alum; see recipe)
  • Staining racks (Kartell Labware Division, model no. 354; http://www.kartell.it)
  • Staining jars with lids (Kartell Labware Division, model no. 353; http://www.kartell.it)
  • Computerized video camera–based image analysis system
  • Image‐analysis software (e.g., NIH Image)
  • Additional reagents and equipment for pentobarbital anesthesia of rodents (Donovan and Brown, )
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Literature Cited

Literature Cited
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