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. J., Afzal, A., Mocco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraluminal+middle+cerebral+artery+occlusion+(MCAO)+model+for+ischemic+stroke+with+laser+doppler+flowmetry+guidance+in+mice.">Intraluminal middle cerebral artery occlusion (MCAO) model for ischemic stroke with laser doppler flowmetry guidance in mice.</a> J. Vis. Exp. (51), e2879 (2011).</li><li>Sharkey, J., Butcher, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+an+experimental+model+of+stroke+produced+by+intracerebral+microinjection+of+endothelin-1+adjacent+to+the+rat+middle+cerebral+artery.">Characterisation of an experimental model of stroke produced by intracerebral microinjection of endothelin-1 adjacent to the rat middle cerebral artery.</a> J. Neurosci. Methods. 60, 125-131 (1995).</li><li>Macrae, I. M., Robinson, M. J., Graham, D. I., 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=Endothelin-1-induced+reductions+in+cerebral+blood+flow:+dose+dependency,+time+course,+and+neuropathological+consequences.">Endothelin-1-induced reductions in cerebral blood flow: dose dependency, time course, and neuropathological consequences.</a> J. Cereb. Blood Flow Metab. 13, 276-284 (1993).</li><li>Mecca, A. P., 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=Cerebroprotection+by+angiotensin-(1-7)+in+endothelin-1-induced+ischaemic+stroke.">Cerebroprotection by angiotensin-(1-7) in endothelin-1-induced ischaemic stroke.</a> Exp. 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...

Watch this Scientific Journal Video about Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat at JoVE.com

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.

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

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...