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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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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Basic Protocol 1:

  • 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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Literature Cited

Literature Cited
   Baldwin, H.A., Jones, J.A., Snares, M., Williams, J.L., Cross, A.J., and Green, A.R. 1993a. Investigation of the role of 5‐hydroxytryptamine in a model of focal cerebral ischaemia in the rat. Neurodegeneration 2:33‐40.
   Baldwin, H.A., Snape, M.F., Williams, J.L., Misra, A., Jones, J.A., Snares, M., Green, A.R., and Cross, A.J. 1993b. Role of glutamate and GABA in a rat model of focal cerebral ischemia: Biochemical and pharmacological investigations. Neurodegeneration 2:129‐138.
   Benham, C.D., Brown, T.H., Cooper, G.D., Evans, M.L., Harries, M.H., Herdon, H.J., Meakin, J.E., Murkitt, K.L., Patel, S.R., Roberts, J.C., Rothaul, A.L., Smith, S.J., Wood, N., and Hunter, A.J. 1993. SB 201823‐A, a neuronal Ca2+ antagonist is neuroprotective in two models of cerebral ischemia. Neuropharmacology 32:1249‐1257.
   Bergeron, M., Ni, B., and Clemens, J.A. 2001. GSK3β expression in the penumbra following photothrombotic lesion in rat brain. Soc. Neurosci. Abstr. 27:203.4.
   Cameron, T., Prado, R., Watson, B.D., Gonzalez‐Carvajal, M., and Holets, V.R. 1990. Photochemically induced cystic lesion in the rat spinal cord. I. Behavioral and morphological analysis. Exp. Neurol. 109:214‐223.
   Chang, Y.‐Y., Fujimura, M., Morita‐Fujimura, Y., Kim, G.W., Huang, C.‐Y., Wu, H.‐S., Kawase, M., Copin, J.‐C., and Chan, P.H. 1999. Neuroprotective effects of an antioxidant in cortical cerebral ischemia: Prevention of early reduction of the apurinic/apyrimidinic endonuclease DNA repair enzyme. Neurosci. Lett. 277:61‐64.
   De Ryck, M., van Reempts, J., Borgers, M., Wauquier, A., and Janssen, P.A.J. 1989. Photochemical stroke model: Flunarizine prevents sensorimotor deficits after neocortical infarct in rats. Stroke 20:1383‐1390.
   De Ryck, M., Keersmaekers, R., Duytschaever, H., Claes, C., Clincke, G., Janssen, M., and Van Reet, G. 1996. Lubeluzole protects sensorimotor function and reduces infarct size in a photothrombotic stroke model in rats. J. Pharmacol. Exp. Ther. 279:748‐758.
   Dietrich, W.D., Ginsberg, M.D., Busto, R., and Watson, B.D. 1986. Photochemically induced cortical infarction in the rat. 1. Time course of hemodynamic consequences. J. Cerebr. Blood Flow Metab. 6:184‐194.
   Dietrich, W.D., Busto, R., Watson, B.D., Scheinberg, P., and Ginsberg, M.D. 1987a. Photochemically induced cerebral infarction. II. Edema and blood‐brain barrier disruption. Acta Neuropathol. (Berl.) 72:326‐334.
   Dietrich, W.D., Watson, B.D., Busto, R., Ginsberg, M.D., and Bethea, J.R. 1987b. Photochemically induced cerebral infarction. I. Early microvasculature alterations. Acta Neuropathol. (Berl.) 72:315‐325.
   Gannon, K.S., Robichaud, P.J., Gregersen, B., Clemens, J.A., and Bergeron, M. 2000. Assessment of beam‐walking performance as a function of lesion size and location in rats with focal ischemia. Soc. Neurosci. Abstr. 26:860.16
   Ginsberg, M.D., Sternau, L.L., Globus, M.Y., Dietrich, W.D., and Busto, R. 1992. Thereapeutic modulation of brain temperature: Relevance to ischemic brain injury. Cerebrovasc. Brain Metab. Rev. 4:189‐225.
   Gladilin, S., Bidmon, H.‐J., Divanach, A., Arteel, G.E., Witte, O.W., Zilles, K., and Sies, H. 2000. Ebselen lowers plasma interleukin‐6 levels and glial heme oxygenase‐1 expression after focal photothrombotic brain ischemia. Arch. Biochem. Biophys. 380:237‐242.
   Gu, W., Jiang, W., and Wester, P. 2000. Cortical neurogenesis in adult rats after reversible photothrombotic stroke. J. Cerebr. Blood Flow Metab. 20:1166‐1173.
   Hu, X., Wester, P., Brännström, T., Watson, B.D., and Gu, W. 2001. Progressive and reproducible focal cortical ischemia with or without late spontaneous reperfusion generated by a ring‐shaped, laser‐driven photothrombotic lesion in rats. Brain Res. Brain Res. Protoc. 7:76‐85.
   Huang, A.J., Watson, B.D., Hernandez, E., and Tseng, S.C. 1988. Photothrombosis of corneal neovascularization by intravenous rose bengal and argon laser irradiation. Arch. Ophthalmol. 106:680‐685.
   Hurwitz, B.E., Dietrich, W.D., McCabe, P.M., Watson, B.D., Ginsberg, M.D., and Schneiderman, N. 1990. Sensory‐motor deficit and recovery from thrombotic infarction of the vibrissal barrel‐field cortex. Brain Res. 512:210‐220.
   Hurwitz, B.E., Dietrich, W.D., McCabe, P.M., Alonso, O., Watson, B.D., Ginsberg, M.D., and Schneiderman, N. 1991. Amphetamine promotes recovery from sensory‐motor integration deficit after thrombotic infarction of the primary somatosensory rat cortex. Stroke 22:648‐654.
   Kim, G.W., Lewén, A., Copin, J.‐C., Watson, B.D., and Chan, P.H. 2001. The cytosolic antioxidant, copper/zinc superoxide dismutase, attenuates blood‐brain barrier disruption and oxidative cellular injury after photothrombotic cortical ischemia in mice. Neuroscience 105:1007‐1018.
   Klaassen, C.D. 1976. Pharmacokinetics of rose bengal in the rat, rabbit, dog, and guinea pig. Toxicol. Appl. Pharmacol. 38:85‐100.
   Liang, J.D., Robichaud, P.J., and Bergeron, M. 2000. Upregulation of plasticity markers in rat brain after focal ischemia. Soc. Neurosci. Abstr. 26:86.4
   Liang, J.D., Liu, J., McClelland, P., and Bergeron, M. 2001. Cellular localization of BM88 mRNA in paraffin‐embedded rat brain sections by combined immunohistochemistry and non‐radioactive in situ hybridization. Brain Res. Brain Res. Protoc. 7:121‐130.
   Maier, C.M., Ahern, K., Cheng, M.L., Lee, J.E., Yenari, M.A., and Steinberg, G.K. 1998. Optimal depth and duration of mild hypothermia in a focal model of transient cerebral ischemia: Effects on neurologic outcome, infarct size, apoptosis and inflammation. Stroke 29:2171‐2180.
   Markgraf, C.G., Kraydieh, S., Prado, R., Watson, B.D., Dietrich, W.D., and Ginsberg, M.D. 1993. Comparative histopathological consequences of photothrombotic occlusion of the distal middle cerebral artery in Sprague‐Dawley and Wister rats. Stroke 24:286‐293.
   Onesti, S.T., Baker, C.J., Sun, P.P., and Solomon, R.A. 1991. Transient hypothermia reduces focal ischemic brain injury in the rat. Neurosurgery 29:369‐373.
   Ostrovskaya, R.U., Romanova, G.A., Barskov, I.V., Shanina, E.V., Gudasheva, T.A., Victorov, I.V., Voronina, T.A., and Seredenin, S.B. 1999. Memory restoring and neuroprotective effects of the proline‐containing dipeptide, GVS‐111, in a photochemical stroke model. Behav. Pharmacol. 10:549‐553.
   Paxinos, G. and Watson, C. 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney, Australia.
   Plumier, J.‐C.L., Armstrong, J.N., Wood, N.I., Babity, J.M., Hamilton, T.C., Hunter, A.J., Robertson, H.A., and Currie, R.W. 1997. Differential expression of c‐fos, Hsp70 and Hsp27 after photothrombotic injury in the rat brain. Mol. Brain Res. 45:239‐246.
   Schroeter, M., Jander, S., Witte, O.W., and Stoll, G. 1999. Heterogeneity of the microglial response in photochemically induced focal ischemia of the rat cerebral cortex. Neuroscience 89:1367‐1377.
   Shivers, R.R. and Wijsman, J.A. 1998. Blood‐brain barrier permeability during hyperthermia. Prog. Brain. Res. 115:413‐424.
   Snape, M.F., Baldwin, H.A., Cross, A.J., and Green, A.R. 1993. The effects of chlormethiazole and nimodipine on cortical infarct area after focal cerebral ischaemia in the rat. Neuroscience 53:837‐844.
   Urakawa, M., Yamaguchi, K., Tsuchida, E., Kashiwagi, S., Ito, H., and Matsuda, T. 1995. Blood‐brain barrier disturbance following localized hyperthermia in rats. Int. J. Hyperthermia 11:709‐718.
   Watson, B.D. 1998. Animal models of photochemically induced brain ischemia and stroke. In Cerebrovascular Disease, Part I, Introduction, Models, and Neuropathology. (M.D. Ginsberg and J. Bogousslavsky eds.) pp. 52‐73. Blackwell Science, Malden, Mass.
   Watson, B.D., Dietrich, W.D., Busto, R., Wachtel, M.S., and Ginsberg, M.D. 1985. Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann. Neurol. 17:497‐504.
   Watson, B.D., Prado, R., Dietrich, W.D., Ginsberg, M.D., and Green, B.A. 1986. Photochemically induced spinal cord injury in the rat. Brain Res. 367:296‐300.
   Watson, B.D., Dietrich, W.D., Prado, R., Nakayama, H., Kanemitsu, H., Futrell, N.N., Yao, H., Markgraf, C.G., and Wester, P. 1995. Concepts and techniques of experimental stroke induced by cerebrovasculature photothrombosis. In Central Nervous System Trauma: Research Techniques (S.T. Ohnishi and T. Ohnishi, eds.) pp. 169‐194. CRC Press, Boca Raton, Fla.
   Wood, N.I., Sopesen, B.V., Roberts, J.C., Pambakian, P., Rothaul, A.L., Hunter, A.J., and Hamilton, T.C. 1996. Motor dysfunction in a photothrombotic focal ischaemia model. Behav. Brain Res. 78:113‐120.
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
  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.
  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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