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 (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>
1. Pre-surgical Steps
<ol>
 <li>Prior to surgery, the rats are housed under a 12:12 light/dark cycle with free access to water and rodent chow.</li>
 <li>Anesthesia is induced with 4% isoflurane in 100% O2 gas mixture in an induction chamber until the rat no longer withdrawals to rear paw pinch. </li>
 <li>Aseptic technique should be maintained during this procedure including use of sterile gloves, sterile surgical instruments, and a sterile surgical drape11. </li>
 <li>The crown of the head is shaved with electric hair clippers.</li>
 <li>The rat is placed in the prone position on an absorbent pad lying on a temperature-controlled operating surface (thermal blanket).</li>
 <li>The head is placed in the stereotactic apparatus starting with placement of the gas anesthetic face mask.</li>
 <li>Next, the ear bars are inserted and tightened. </li>
 <li>During the procedure anesthesia is maintained with 2% isoflurane in 100% O2 gas mixture.</li>
 <li>Lubricant ophthalmic ointment is applied to both eyes, and the eyelids are closed to prevent eye desiccation during the surgical procedure. </li>
 <li>A rectal temperature probe is inserted to maintain a constant animal core temperature of 37&plusmn;0.5 &deg;C.
 </li>
 <li>With the head held firmly in the stereotaxic device, the surgical area is cleansed with alternating 2% chlorhexidine and saline three times. </li>
</ol>
2. Surgical Steps
<ol>
 <li>Using a scalpel, a midline incision is made on the skin overlying the calvarium from the most caudal aspects of the eyes (nasion) to between the ears (superior nuchal line). </li>
 <li>The skin is then retracted laterally with 3 bulldog clamps. </li>
 <li>Connective tissue is removed from the skull using dry cotton swabs so that multiple structures can be seen clearly. These include bregma, the coronal suture, and the right lateral skull ridge. Cotton swabs are used to remove blood from the surgical field.</li>
 <li>Using the surgical microscope, bregma is located, and the stereotaxic manipulators are adjusted until the 1.0 mm round drill bur is zeroed at bregma. </li>
 <li>The drill bur is then moved to 1.6 mm anterior and 5.2 mm lateral to bregma. </li>
 <li>A bur hole that penetrates the skull is drilled for cannula placement (Figure 1). Excess debris and blood are continually cleared using cotton swabs. </li>
</ol>
At this point, a guide cannula can be inserted (step 7) or a direct ET-1 injection through the bur hole can be performed (proceed directly to step 16). 
<ol start="7"> 
 <li>Next, bur holes for 3 mounting screws are drilled through partial thickness of the skull using a 1.0 mm inverted cone drill bur (Figure 1). One hole is drilled in each frontal bone about 1-2 mm anterior to the coronal suture and 1-2 mm lateral to the sagittal suture. One hole is drilled in the parietal bone about 2-3 mm posterior to the coronal suture and 2-3 mm lateral to the sagittal suture ipsilateral to the guide cannula bur hole. Three 0-80 x 3/32 mounting screws are placed in these bur holes and will provide support for the cement holding in the cannula. The screws should only be advanced 2 or 3 turns so as not to damage the dura matter.</li>
 <li>The guide cannula is placed in the stereotaxic cannula holder and bregma is located. </li>
 <li>The stereotaxic manipulators are adjusted until the guide cannula is zeroed at bregma. The cannula is moved to the bur hole located 1.6 mm anterior and 5.2 mm lateral to bregma. </li>
 <li>Finally, the guide cannula is lowered into the bur hole with the final tip position of 4.5 mm ventral to bregma. </li>
 <li>Laser Doppler flow probe placement (optional) 
 <ol class="lalpha">
 <li> In order to monitor cerebral blood flow using LDF, a probe holder can be placed into position prior to affixing the guide cannula with dental cement. </li>
 <li>The probe holder base is trimmed flush with the pedestal except for small wedge shaped tab. </li>
 <li> The probe holder is then placed posterior to the guide cannula and just medial to the lateral skull ridge with the tab oriented medially (Figure 1). </li>
 <li>The probe holder and guide cannula are affixed together using dental cement. </li>
 </ol>
 </li>
 <li>Dental cement is then used to secure the cannula in place. The cement is in contact with all three screws and surrounds the entire base of the cannula. </li>
 <li>The cement should be completely dry prior to removal of the cannula holder. This takes about 5 min. </li>
</ol>
After these steps, the surgical incision can be closed and the cannula dummy can be screwed into the guide cannula. Alternatively, ET-1 induced MCAO can be performed on the rat after a period of recovery from the cannula implantation surgery. For this method, step 19 should be performed next and steps 14-18 can be performed at a later time. To perform guide cannula implantation and ET-1 injection during the same surgery, step 14 should be performed next. 
<ol start="14">
<li>The infusion syringe is loaded with ET-1 (diluted to 80 &mu;M in PBS) and then mounted in the stereotaxic injector. </li>
<li>The stereotaxic manipulators are adjusted until the needle tip is zeroed at the rim of the guide cannula.</li>
<li>The needle tip is lowered through the guide cannula to a position of 17.2 mm ventral to the rim of the guide cannula. If a guide cannula is not used, the needle tip is zeroed at bregma and lowered through the bur hole to a position 8.7 mm ventral to bregma. </li>
<li>3 &mu;l of 80 &mu;M ET1 is infused at a rate of 1 &mu;l per min.</li>
<li>The syringe is left in place for 3 min after the infusion is complete and then slowly removed. </li>
<li>The incision is closed with 3.0 nylon suture and the cannula dummy is screwed into the cannula.</li>
<li>A dose of appropriate analgesic (i.e. buprenorphine at 0.05-0.1 mg/kg) should be used after the surgery to minimize pain and discomfort during the recovery period.</li>
<li>The rat is removed from the surgical suite and placed in a warm, dry recovery area to prevent hypothermia, with free, easy access to soft food and water.</li>
</ol>

<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. P., Gautier, J. C., Hier, D., Stein, R. W., Barnett, H. J. M., Stein, B. M., Mohr, J. P., Yatsu, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stroke: pathophysiology, diagnosis, and management. , 377-450 (1986).</li><li>Robinson, M. J., Macrae, I. M., Todd, M., Reid, J. L., McCulloch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+local+cerebral+blood+flow+to+pathological+levels+by+endothelin-1+applied+to+the+middle+cerebral+artery+in+the+rat.">Reduction of local cerebral blood flow to pathological levels by endothelin-1 applied to the middle cerebral artery in the rat.</a> Neurosci. Lett. 118, 269-272 (1990).</li><li>Sharkey, J., Ritchie, I. M., Kelly, P. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+rodent+surgery+for+the+new+surgeon.">Principles of rodent surgery for the new surgeon.</a> J. Vis. Exp. (47), e2586 (2011).</li><li>Gonzalez, C. L., Kolb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+different+models+of+stroke+on+behaviour+and+brain+morphology.">A comparison of different models of stroke on behaviour and brain morphology.</a> Eur. J. Neurosci. 18, 1950-1962 (2003).</li><li>Ansari, S., Azari, H., McConnell, D. 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Physiol. 96 (1-7), 1084-1096 (2011).</li><li>Fisher, M., 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=Update+of+the+stroke+therapy+academic+industry+roundtable+preclinical+recommendations.">Update of the stroke therapy academic industry roundtable preclinical recommendations.</a> Stroke. 40, 2244-2250 (2009).</li></ol>

No conflicts of interest declared.

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 ...

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19.8K Views.  University of Florida. 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.