Inducing Photochemical Cortical Lesions in Rat Brain

Marcelle Bergeron1

1 Lilly Research Laboratories, Indianapolis, Indiana
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 9.16
DOI:  10.1002/0471142301.ns0916s23
Online Posting Date:  July, 2003
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Abstract

In the rat photochemical cortical lesion model described in this unit, an intravascular photochemical reaction induces endothelial damage resulting in platelet aggregation, thrombosis, thrombotic response (secretion of factors by the platelets) and permanent cerebral vascular occlusion. Because thrombosis is produced in pial vessels, the resulting cortical infarct is generally smaller and more reproducible than in the models involving occlusion of the middle cerebral artery. The surgical procedures involved are limited, making this model generally easier to perform and less invasive than most other models of permanent focal ischemia that involve mechanical occlusion of major cerebral arteries.

     
 
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Table of Contents

  • Reagents and Solution
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • 275‐ to 300‐g male or female rats (e.g., Charles River Laboratories, Harlan)
  • 4% (v/v) isoflurane (Henry Schein) in 30% (v/v) oxygen/70% (v/v) nitrogen gas mixture
  • Lubricant gel (e.g., Henry Schein, Owens and Minor)
  • Lubricant ophthalmic ointment or artificial tears (e.g., The Butler Company, Henry Schein)
  • Antiseptic scrubbing solution (e.g., Betadine; Henry Schein, Owens and Minor)
  • 70% (v/v) ethanol
  • Diluted styptic pencil solution, made by soaking tip of styptic pencil (generally available from pharmacies) in 10 ml sterile water for 2 to 4 min
  • Water, 37 to 40°C
  • 1‐ml syringe filled with 0.2 ml sterile saline (0.9% [w/v] NaCl)
  • Mask with desired aperture for lesion cut with heavy‐duty punch or knife from ≤0.25‐mm‐thick brass sheet
  • Superglue
  • Mineral oil, optional
  • recipePhotosensitizing dye solution (see recipe)
  • Saline (0.9% [w/v] NaCl), sterile
  • Topical antibiotic ointment (e.g., Panolog; Henry Schein)
  • Topical anesthetic cream, such as 2.5% lidocaine and 2.5% prilocaine (e.g., ELMA cream; Henry Schein)
  • Dry ice/isopentane bath, for infarct assessment without fixation
  • Laser safety alignment eyewear, with an optical density (O.D.) of 2 to 3 and wavelength range of 488 to 515 nm (e.g., Ritz Safety Equipment, Melles Griot, UVEX Safety, Kentek, Rockwell Laser Industries)
  • Argon ion laser (multiline or 514.5 nm), available new (e.g., Lexel, Coherent Laser, Melles Griot) or as refurbished equipment (e.g., Evergreen Laser)
  • Optical components for photochemical lesioning, including:
  •  Kinematic mount with mirror (e.g., Melles Griot)
  •  Plano‐convex BK7 glass lens with 250‐mm focal length and 30‐mm diameter (e.g, Melles Griot)
  •  Lens holder with 30‐mm nominal lens diameter (e.g., Melles Griot)
  •  Mounting post, 20 mm in diameter and 150 mm long (e.g., Melles Griot)
  •  Collet post holder (e.g., Melles Griot) for mounting post
  •  Optical rail carrier (e.g., Melles Griot) for 50‐mm optical rail
  •  50 × 500–mm optical rail (e.g., Melles Griot)
  • Indoor refrigerated, recirculating cooling unit, high capacity (Electro Impulse)
  • Ruler
  • Surgical table or other suitable nonreflective surface
  • Transparent anesthesia‐induction chamber (e.g., Stoelting, VetEquip, Harvard Apparatus)
  • Anesthetic vaporizer and flow meter for use with isoflurane (e.g., VetEquip, Vetamac, Stoelting, Harvard Apparatus)
  • Electric hair clippers (e.g., Jeffers, Harvard Apparatus, Stoelting, Roboz Surgical Instruments)
  • Thermocouple rectal temperature probe and feedback‐controlled homeothermic heating blanket (e.g., Harvard Apparatus)
  • Stereotaxic frame (e.g., David Kopf Instruments) equipped with rat anesthesia nose cone (e.g., David Kopf Instruments, Stoelting, Harvard Apparatus) fitted over the incisor bar
  • Gauze, sterile (e.g., Henry Schein, Owens and Minor)
  • Dual fiber‐optic illuminator (e.g., Stoelting, Harvard Apparatus)
  • Sterile surgical instruments (e.g., Roboz Surgical Instruments, Fine Science Tools, Miltex), including:
  •   Scalpel
  •   4.4‐cm (1.75‐in.) barraquer retractor (e.g., Roboz Surgical Instruments)
  •   Hemostats
  • Cotton swabs, sterile (e.g., Fisher, Henry Schein)
  • 24‐G, 1.9‐cm (0.75‐in.) intravenous catheter needle with needle guide (e.g., Caligor), sterile
  • 1‐ml syringes
  • High–optical density argon laser safety eyewear, with O.D. of 7 to 8 and wavelength range of 190 to 532 nm (e.g., Ritz Safety Equipment, Melles Griot, Uvex Safety, Kentek, Rockwell Laser Industries)
  • Syringe pump (e.g., Harvard Apparatus, Stoelting)
  • Braided–silk or nylon suture (gauge 4–0 or 3–0; length, 46 cm) with 1/4 or 3/8 curved, reverse cutting needle (e.g., Roboz Surgical Instruments, Henry Schein, Harvard Apparatus), sterile
  • Cage fitted with heating pad (e.g., Baxter, Harvard Apparatus), 37°C
  • Additional reagents and equipment for removing and cryosectioning rat brains (unit 1.1) and cresyl violet staining and determining tissue area (unit 9.6) or for perfusion fixation (unit 1.1) and fixing, staining, and sectioning fixed brain tissue (unit 9.5)
CAUTION: There are two main potential hazards associated with argon lasers: the laser beam and the high‐voltage power supply. Before operating a laser, appropriate safety measures should be implemented to prevent serious and irreversible injury to the eyes, skin, and other parts of the body. In addition, the surgery room should be approved for the use of a laser by the local non‐ionizing radiation safety officer and should conform to Occupational Safety and Health Administration (OSHA) regulations. Safety measures include but are not limited to understanding the concepts of non‐ionizing radiations, appropriate training on the particular laser to be used, using approved laser safety eyewear, appropriate shielding of refractive surfaces and instruments during laser operation, and using an interlock safety system that shuts off the laser power if, for example, the cover is removed, the electrical components malfunction, the internal laser temperature is too high, or cooling water flow too low.NOTE: All surgical instruments and sutures should be sterilized before each surgery by autoclaving. Alternatively, soaking the instruments in glutaraldehyde or other noncorrosive antiseptic sterilizing solutions for ≥1 hr (before surgery) and rinsing them thoroughly in sterile water before use will achieve a similar degree of aseptic protection. The use of a portable hot‐bead sterilizer (e.g., Fine Science Tools, Harvard Apparatus, Stoelting) to sterilize instruments between animals is also useful.
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Figures

Videos

Literature Cited

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Key References
   Hecht, J. 1993. Understanding Lasers. An Entry‐Level Guide. 2nd ed. The Institute of Electrical and Electronics Engineers Press, New York.
  These books are a perfect introduction to laser principals and safety issues.
  Laser Institute of America (LIA) Laser Safety Committee. 1993. Laser Safety Guide. 9th ed. (D.H. Sliney ed.) LIA, Orlando, Fla.
  This paper was the first to describe the technique to produce focal cortical ischemia following photochemically induced thrombosis.
   Watson et al., 1985. See above.
  These two papers present essential information on the principles of photochemically induced thrombosis for the study of stroke.
   Watson, 1998. See above.
   Watson et al., 1995. See above
Internet Resources
   http://www.cbfm.org/
  Web site of International Society for Cerebral Blood Flow and Metabolism. The official journal of this society is the Journal of Cerebral Blood Flow and Metabolism, which publishes detailed manuscripts on various animal models of cerebral ischemia.
   http://stroke.ahajournals.org/
  Web site for the journal Stroke. This is the official journal of the American Heart Association, which publishes research reports on animal models of ischemia as well as clinical reports and updates on current clinical trials in stroke.
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