This work was supported by grants from the American Heart Association Greater Southeast Affiliate (09GRNT2060421), the American Medical Association, and from the University of Florida Clinical and Translational Science Institute. Adam Mecca is a NIH/NINDS, NRSA predoctoral fellow (F30 NS-060335). Robert Regenhardt received predoctoral fellowship support from the University of Florida Multidisciplinary Training Program in Hypertension (T32 HL-083810).

This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida and is in compliance with the "Guide for the Care and Use of Laboratory Animals" (eighth edition, National Academy of Sciences, 2011).
Materials
<ol>
 <li>Animals: Eight-week-old, male, Sprague Dawley rats (Charles River Farms, Wilmington, MA, USA) weighing 250-300 g at the time of surgery.
 </li>
 <li>Anesthesia
 <ol class="lalpha">
 <li>Inhalation anesthesia system (...

<ol>
	<li>Stroke--1989. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+on+stroke+prevention,+diagnosis,+and+therapy.+Report+of+the+WHO+Task+Force+on+Stroke+and+other+Cerebrovascular+Disorders.">Recommendations on stroke prevention, diagnosis, and therapy. Report of the WHO Task Force on Stroke and other Cerebrovascular Disorders.</a> Stroke. 20, 1407-1431 (1989).</li><li>Lloyd-Jones, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics--2009+update:+a+report+from+the+American+Heart+Association+Statistics+Committee+and+Stroke+Statistics+Subcommittee.">Heart disease and stroke statistics--2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.</a> Circulation. 119, 480-486 (2009).</li><li>Mohr, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+microapplication+of+endothelin-1:+a+new+model+of+focal+cerebral+ischaemia+in+the+rat.">Perivascular microapplication of endothelin-1: a new model of focal cerebral ischaemia in the rat.</a> J. Cereb. Blood Flow Metab. 13, 865-871 (1993).</li><li>O'Neill, M. J., Clemens, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+cerebral+ischemia.">Rodent models of focal cerebral ischemia.</a> Curr. Protoc. Neurosci. Chapter 9 (Unit 9), (2001).</li><li>Mecca, A. P., O'Connor, T. E., Katovich, M. J., Sumners, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candesartan+pretreatment+is+cerebroprotective+in+a+rat+model+of+endothelin-1-induced+middle+cerebral+artery+occlusion.">Candesartan pretreatment is cerebroprotective in a rat model of endothelin-1-induced middle cerebral artery occlusion.</a> Exp. Physiol. 94, 937-946 (2009).</li><li>Braeuninger, S., Kleinschnitz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+cerebral+ischemia:+procedural+pitfalls+and+translational+problems.">Rodent models of focal cerebral ischemia: procedural pitfalls and translational problems.</a> Exp. Transl. Stroke Med. 1, 8 (2009).</li><li>Tomsick, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+thrombolysis+for+acute+ischemic+stroke.">Intravenous thrombolysis for acute ischemic stroke.</a> J. Vasc. Interv. Radiol. 15, 67-76 (2004).</li><li>Olsen, T. S., Lassen, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+concept+of+middle+cerebral+artery+occlusion+and+cerebral+infarction+in+the+acute+state+based+on+interpreting+severe+hyperemia+as+a+sign+of+embolic+migration.">A dynamic concept of middle cerebral artery occlusion and cerebral infarction in the acute state based on interpreting severe hyperemia as a sign of embolic migration.</a> Stroke. 15, 458-468 (1984).</li><li>Pritchett-Corning, K. R., Luo, Y., Mulder, G. B., White, W. 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The ET-1 induced MCAO is an established model of experimental ischemic stroke that is regularly used in multiple rat strains. Many variables, such as rat strain, animal age, body temperature, anesthesia method, and operator expertise can lead to increased variability in infarct volumes when using this model5, 14. However, several investigators have shown that advantages of this model include the relatively non-invasive approach, dose response of cerebral blood flow to ET-1, and ability to avoid anesthesia a...

<table border="1">
<tbody>
<tr>
 <td></td>
 <td></td>
<td></td>
<td>
 <ol>
 <li>Animals: Eight-week-old, male, Sprague Dawley rats (Charles River Farms, Wilmington, MA, USA) weighing 250-300 g at the time of surgery.
 </li>
 <li>Anesthesia
 <ol class="lalpha">
 <li>Inhalation anesthesia system (VetEquip Inc., Pleasanton, CA, USA) </li>
 <li>Isoflurane anesthetic (Baxter Pharmaceutics, Deerfield, IL, USA)</li>
 </ol>
 </li>
 <li>Stereotaxic system (David Kopf Instruments, Tujunga, CA, USA)
 <ol class="lalpha">
 <li>Small animal stereotaxic system </li>
 <li>Non-rupture ear bars for rats </li>
 <li>Gas anesthesia head holder for rats </li>
 </ol>
 </li>
 <li>Temperature regulation
 <ol class="lalpha">
 <li>BAT-12 microprobe thermometer (World Precision Instruments, Inc., Sarasota, FL, USA) </li>
 <li>T/PUMP, TP600 Thermal blanket (Gaymar Industries, Inc., Orchard Park, NY, USA) </li>
 </ol>
 </li>
 <li>Surgical instruments 
 <ol class="lalpha">
 <li>Scalpel handle and #11 blade, iris forceps, Graefe forceps, bulldog clamp retractors, screwdriver, 10 &mu;l syringe with 26 gauge beveled needle (World Precision Instruments, Inc., Sarasota, FL, USA) 
 </li>
 <li>Micromotor drill and stereotaxic holder, Quintessential Stereotaxic Injector (Stoelting, Wood Dale, IL, USA) </li>
 <li>1.0 mm round drill bur, 1.0 mm inverted cone drill bur (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, USA) </li>
 </ol>
 </li>
 <li>Surgical Supplies 
 <ol class="lalpha">
 <li>Mounting screws 0-80 X 3/32 with 2.4 mm shaft length, 21-gauge guide cannula [4mm long below the pedestal] and cannula dummy (Plastics one, Roanoke, VA, USA)</li>
 <li>Jet denture acrylic and liquid (Lang Dental Manufacturing Co., Inc., Wheeling, IL, USA) </li>
 <li>3.0 nylon suture (Oasis, Mettawa, IL, USA) </li>
 <li>Cotton swabs, Puralube eye ointment (Fisher Scientific, Pittsburg, PA, USA)</li>
 <li>Electric hair clippers (Oster, Providence, RI, USA) </li>
 </ol>
 </li>
 <li>Chemicals
 <ol class="lalpha">
 <li>Endothelin-1 (American Peptide, Sunnyvale, CA, USA) </li>
 <li>Chlorhexidine 2% (Agrilabs, St. Joseph, MO, USA) </li>
 <li>Buprenorphine HCl (Hospira Inc., Lake Forest, IL, USA) </li>
 </ol>
 </li>
 <li>Visualization Equipment 
 <ol class="lalpha">
 <li>Surgical microscope (Seiler Instrument and Manufacturing; St. Louis, MO, USA) </li>
 <li>Fiber Optic illuminator (TechniQuip Corp., Livermore, CA, USA)</li>
 </ol>
 </li>
 <li>Laser Doppler flowmetry system (ADInstruments, Inc., Colorado Springs, CO, USA) 
 <ol class="lalpha">
 <li>Standard Pencil Probe </li>
 <li>Probe holder</li>
 <li>Blood FlowMeter </li>
 <li>Powerlab 4/30 with LabChart 7</li>
 </ol>
 </li>
 <li>Measurement of infarct volume
 <ol class="lalpha">
 <li>Rat brain matrix (Zivic-Miller Lab., Inc., Allison Park, PA, USA)</li>
 <li>2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich Co., St Louis, MO, USA) diluted to 0.05% in PBS</li>
 <li>Flatbed scanner (Epson Perfection V30, Epson America, Inc., Long Beach, CA, USA) </li>
 <li>Image J software (ImageJ 1.42q software, U.S. National Institutes of Health, Bethesda, MA, USA) </li>
 </ol>
 </li>
</ol>
</td>
</tr>
</tbody>
</table>

endothelin-1 induced middle cerebral artery occlusion model for ischemic stroke with laser doppler flowmetry guidance in rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

1. Post-Op neurological evaluation
After the animal regains consciousness, a wide variety of tests can be used to evaluate neurological deficits including balance, grip strength, paw placing, postural asymmetry and staircase climbing. The sunflower seed task is a gross assessment of motor and sensory function that has significant correlation with infarct volume7, 12. During this task, rats are timed while opening and consuming 5 sunflower seeds. The five seeds are placed in one corne...

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Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

endothelin-1-induced-middle-cerebral-artery-occlusion-model-for

Research

JoVE Journal

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19.7K visualizações.  University of Florida. Vários modelos animais de isquemia cerebral foi desenvolvido para simular a condição humana de acidente vascular cerebral. Este protocolo descreve a endotelina-1 (ET-1) induzida por oclusão da artéria cerebral média do modelo (MCAO) para acidente vascular cerebral isquêmico em ratos. Além disso, considerações importantes, vantagens e inconvenientes deste modelo são discutidos.