This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida and is in compliance with the 
 "Principle of Laboratory Animal Care" (NIH publication No. 86-23, revised 1985).
 1. Materials
 <ol>
 <li>Animals: Male C57BL/6 mice (Harlan Laboratories, Indianapolis, IN, USA) weighing 20&#x2013;26 grams at the time of surgery.</li>
 <li> Anesthesia: 
 <ol>
 <li>Anesthesia machine: VetEquip inhalation anesthesia system (VetEquip Inc., Pleasanton, CA, USA).</li>
 <li>Inhalation anesthetic: Isoflurane (Baxter Pharmaceutics, Deerfield, IL).</li>
 </ol>
 </li>
 <li>Surgical tools: SuperCut Iris scissors, straight (World Precision Instruments, Inc., Sarasota, FL, USA); Vannas micro-scissors, straight (World Precision 
 Instruments, Inc., Sarasota, FL, USA); Dumont forceps (fine tip, 45&deg; bent tips, World Precision Instruments, Inc., Sarasota, FL, USA); Bipolar coagulation unit 
 (High Frequency Desiccator 900, World Precision Instruments, Inc., Sarasota, FL, USA); micro vessel clip (World Precision Instruments, Inc., Sarasota, FL, USA) </li>
 <li>Surgical supplies: Sterile cotton tips; Wipes; Gauze sponges; Suture (7-0 Silk suture, Ethicon, Inc., Somerville, NJ, USA); Tissue adhesive (Vetbond, 3M Company, St 
 Paul, MN, USA); Surgical scrub: Bethadine, 70% ethanol, and sterile 0.9% saline; Eye ointment (Puralube Vet Ointment, Pharmaderm, Melville, NY, USA).</li>
 <li>Occlusion suture preparation for MCAO:
 <ol>
 <li>Filament for MCA occlusion: 7-0 surgical Nylon monofilament suture (Ethicon, Inc., Somerville, NJ, USA).</li>
 <li>Heat source for blunting suture tip (Thermal Cautery Unit, World Precision Instruments, Inc., Sarasota, FL).</li>
 <li>Liquid silicone rubber (Silicone RTV adhesive, World Precision Instruments, Inc., Sarasota, FL, USA).</li>
 </ol>
 </li>
 <li>Surgical/Dissecting microscope (Leica Stereo Microscope MZ6).
 <ol>
 <li>Fiber optic illuminator (Fiber Optic illuminator FO1&#x2013;150, TechniQuip Corp., Livermore, CA, USA).</li>
 </ol>
 </li>
 <li>Laser doppler flowmetry (PeriFlux System 5000, Perimed, Stockholm, Sweden).
 <ol>
 <li>Flexible microtip (MT B500-0 Straight Microtip, Perimed, Stockholm, Sweden)</li>
 </ol>
 </li>
 <li>Temperature parameters:
 <ol>
 <li>Rectal temperatures monitor (BAT-12, World Precision Instruments, Inc., Sarasota, FL).</li>
 <li>Thermal blanket (T/PUMP, TP600, Gaymar, NY, USA).</li>
 </ol>
 </li>
 <li>Staining and quantitative measurement of volume infarct:
 <ol>
 <li>2% solution 2,3,5-triphenyltetrazolium chloride (2% TTC, Sigma-Aldrich Co., St Louis, MO, USA).</li>
 <li>Mice brain matrix slicer (Zivic-Miller Lab., Inc., Allison Park, PA, USA).</li>
 <li>Digital camera (V1253, Kodak Co., Rochester, NY, USA).</li>
 <li>Image J software (ImageJ 1.42q software, U.S. National Institutes of Health, Bethesda, MA, USA).</li>
 </ol>
 </li>
</ol>
2. Pre-surgical steps
<ol>
 <li>Prior to surgery, the mice are housed under a 12:12 light/dark cycle with free access to water and rodent chow.</li>
 <li>Sutures for MCAO are prepared by blunting 1-2mm of the suture tip with heat and coating 3-4mm of suture shaft from its tip with liquid silicone rubber, allowing it 
 to dry (7-0; tip diameter, 0.20-0.25mm; coating length, 3-4mm; suture length 11-13mm). Sterility should be preserved in all of the aforementioned steps. </li>
 <li>After weighing the mouse, anesthesia is induced with 4% isoflurane in an induction chamber and maintained during the procedure with 1.5-2% isoflurane in 30% O2 
 and 70% N2O gas mixture via a facemask.</li>
 <li>The mouse is placed in a prone position on a gauze pad lying on a temperature-controlled operating surface (thermal blanket); the head fur is shaved.</li>
 <li>Lubricant ophthalmic ointment is applied to both eyes, and eyelids are closed to prevent eye desiccation during the surgical process.</li>
 <li>A midline incision (1 cm) is made on skin overlying the calvarium from superior nuchal line to the nasion and the skin is pulled laterally to affix a flexible 
 microtip (MT B500-0 Straight Microtip; 0.5mm diameter). The microtip is placed perpendicular to the surface of the right parietal skull (1 mm posterior and 5 mm 
 lateral to the bregma) to monitor blood flow in the MCA territory by laser doppler flowmetry.</li>
 <li>Make sure that the flexible microtip is attached to skull firmly before the animal is turned to a supine position. The animal&#x2019;s neck is placed on a soft pad 
 and its nose is held in nose cone of the anesthetic apparatus. This will result in having enough space for the flexible microtip. </li>
 <li>A rectal temperature probe is inserted to maintain a constant animal core temperature of 37&plusmn;0.5&deg;C.</li>
 <li>Both forepaws and hindpaws are taped to the operating platform surface, and the tail is taped to the rectal tube.</li>
</ol>
3. MCA occlusion surgery:
<ol>
 <li>Under the operating microscope, a midline neck incision is made with scissors between the manubrium and the jaw by gently lifting the loose skin.</li>
 <li>The paired underlying submandibular glands are bluntly divided, with the left gland left in situ. The right submandibular gland is retracted cranially and secured along with the sternocleidomastoid muscle.</li>
 <li>The exposed division of the omohyoid muscle covering the carotid sheath is carefully divided with pickups.</li>
 <li>The right common carotid artery (CCA) is carefully separated and isolated from the vagus nerve and its sheath (located lateral to CCA).</li>
 <li>Hereafter, the 2 surgical methods that have been used frequently to perform MCA occlusion diverge:</li>
</ol>
Koizumi's4 method 
<ol class="lroman" type="i">
 <li>Three loose collar sutures (7-0 silk) are prepared around the right CCA.</li>
 <li>The external carotid artery (ECA) is identified, isolated, and cauterized (2-3mm distal to bifurcation). It can subsequently be divided with micro-scissors. 
 To avoid inadvertent peripheral damages at the cauterization site, the power should be set at 1 using the power knob. This is consistent for all cauterizations.</li>
 <li>Sufficient tension is applied to the most proximal collar suture on the CCA to occlude the blood flow (permanent ligation). The flow occlusion is verified by a 
 ~50% drop of baseline cortical perfusion values.</li>
 <li>Then a vessel clip is placed just before the CCA bifurcation to avoid retrograde flow at the time of arteriotomy.</li>
 <li>An arteriotomy is performed between the proximal ligated suture and the vessel clip on the CCA with micro-scissors (closer to ligation).</li>
 <li>The heat-blunted silicone-coated 7-0 nylon suture is introduced via the arteriotomy and advanced till it reaches the vessel clip. The 2 remaining loose 
 collar sutures are gently tightened around the CCA between the arteriotomy and the vessel clip to avoid back flow leakage through CCA, with caution not to 
 traumatize the arterial wall; then the vessel clip is withdrawn.</li>
</ol>
Longa's3 method 
<ol class="lalpha" type="a">
 <li>The ECA is identified, dissected as much as possible (near its bifurcation into lingual and maxillary arteries), cauterized (with power set to 1), 
 and divided with micro-scissors. </li>
 <li>Two loose collar sutures (7-0 silk) are prepared around the proximal ICA just above the CCA bifurcation.</li>
 <li>The CCA is temporarily closed with a vessel clip. The flow occlusion is verified by a ~50% drop of baseline cortical perfusion values.</li>
 <li>Then the ICA is temporarily closed with a vessel clip above the two loose collar sutures to avoid retrograde flow at the time of arteriotomy.</li>
 <li>An arteriotomy is performed using micro-scissors in the reflected ECA close to the stump.</li>
 <li>The heat-blunted silicone-coated 7-0 nylon suture is introduced via the arteriotomy in ECA and advanced till it reaches the applied vessel clip in ICA. The 2 loose 
 collar sutures in proximal ICA are gently tightened around the proximal ICA to avoid reflux blood flow through ICA, with enough attention not to traumatize the arterial 
 wall; then the vessel clip is withdrawn.</li>
</ol>
The rest of the procedure is largely convergent:
<ol>
 <li>The occluding suture is slowly advanced through internal carotid artery (ICA) toward the cranial base until a mild resistance is felt. A concurrent drop in cerebral 
 blood flow value as a percentage (= 80%) relative to baseline is confirmed by laser doppler flowmetry. The 2 collar sutures are tightened around the inserted filament.</li>
 <li>For transient cerebral ischemia experiments, the occluding suture is withdrawn gently after 45 minutes. This time point can be altered for various experimental aims.</li>
 <li>After withdrawing the suture filament, the arteriotomy site is closed to avoid post-surgical bleeding. In Koizumi&#x2019;s method, this involves securely tightening the 
 collar sutures around the CCA. In Longa&#x2019;s technique, the arteriotomy site at the base of ECA is cauterized, then the collar sutures at the proximal ICA are loosened and removed to restore the carotid flow.</li>
 <li>To close the incision, the submandibular gland and SCM are laid over the operative field. The wound edges are then approximated with tissue adhesive in cervical and 
 scalp areas. Alternatively, an absorbable suture or metal staple could be used to close the wound especially when long-term survival of the animal is intended.</li>
 <li>After incision closure, the animal receives 1 ml warm saline subcutaneously as volume replenishment. A dose of appropriate analgesics (i.e. Buprenorphine at 
 0.05-0.1 mg/kg) should be used to minimize animal&#x2019;s pain and discomfort during recovery period. The animal is removed from the surgical suite and placed in a warm 
 recovery area/cage to prevent hypothermia, with free, easy access to soft food and water. </li>
</ol>
4. Post-Op neurological evaluation
After the animal regains total consciousness, neurological deficits can be evaluated by a simple scale (five-point scale) as follows3:
<ol start="0">
 <li>no observable deficits</li>
 <li>failure to extend contralateral forepaw (mild focal neurologic deficit)</li>
 <li>circling in a direction contralateral to infarct (moderate focal neurologic deficit)</li>
 <li>falling in a direction contralateral to infarct (severe focal neurologic deficit)</li>
 <li>depressed level of consciousness without spontaneous movement</li>
</ol>
5. Staining and quantitative measurement of brain infarct volume
<ol>
 <li>After 24 hours of MCA reperfusion, the surviving mouse is weighed, deeply anesthetized with 4% isoflorane, and sacrificed according to approved protocol.</li>
 <li>The brain is carefully removed and placed in a mouse brain matrix slicer, and the entire cerebrum is coronally sectioned at 2 mm intervals.</li>
 <li>The sections are immersed in 2% TTC in phosphate buffered saline (PBS) at 37 &deg;C for 20 minutes. After TTC staining, infracted area is shown as a white (unstained) tissue adjacent to red brick (viable) tissue (Figure 1).</li>
 <li>Serial sections are photographed using a digital camera and the area of infarct is measured with the image J program.</li>
 <li>The area of infarct (white, unstained), the area of ipsilateral hemisphere (white and red brick, stained), and the area of the contralateral hemisphere (red brick, stained) are measured for each section. The volume is calculated by summing the representative areas in all sections and multiplying by the slice thickness. After correcting for edema, the volume of infarction is calculated as follows9: 
 Corrected Infarct Volume (CIV), % = [contralateral hemisphere volume - (ipsilateral hemisphere volume - infarct volume)]/contralateral hemisphere volume x 100</li>
</ol>
6. Representative Results
After occlusion and reperfusion of the MCA via left CCA to establish transient cerebral ischemia, animals are sacrificed and their coronally sectioned brains are stained with TTC to evaluate the volume of infarct (Figure 1). 
<img src="/files/ftp_upload/2879/2879fig1.jpg" alt="Figure 1" /> 
Figure 1. TTC-stained serial coronal brain sections (2 mm) from mice subjected to MCA occlusion.

<ol>
	<li>. <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; a journal of cerebral circulation. 20, 1407-1431 (1989).</li><li>Lloyd-Jones, D. <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, e21-e181 (2009).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke; a journal of cerebral circulation. 20, 84-91 (1989).</li><li>Koizumi, J., Yoshida, Y., Nakazawa, T., Ooneda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+ischemic+brain+edema.+I.+A+new+experimental+model+of+cerebral+embolism+in+rats+in+which+recirculation+can+be+introduced+in+the+ischemic+area.">Experimental studies of ischemic brain edema. I. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area.</a> Jpn J Stroke. 8, 1-8 (1986).</li><li>Clark, W. M., Lessov, N. S., Dixon, M. P., Eckenstein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monofilament+intraluminal+middle+cerebral+artery+occlusion+in+the+mouse.">Monofilament intraluminal middle cerebral artery occlusion in the mouse.</a> Neurol Res. 19, 641-648 (1997).</li><li>Fujii, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-related+differences+in+susceptibility+to+transient+forebrain+ischemia+in+SV-129+and+C57black/6+mice.+.">Strain-related differences in susceptibility to transient forebrain ischemia in SV-129 and C57black/6 mice. .</a> Stroke; a journal of cerebral circulation. 28, 1805-1810 (1997).</li><li>Bederson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+2,3,5-triphenyltetrazolium+chloride+as+a+stain+for+detection+and+quantification+of+experimental+cerebral+infarction+in+rats.">Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats.</a> Stroke; a journal of cerebral circulation. 17, 1304-1308 (1986).</li><li>Qi, X., Hosoi, T., Okuma, Y., Kaneko, M., Nomura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+4-phenylbutyrate+protects+against+cerebral+ischemic+injury.">Sodium 4-phenylbutyrate protects against cerebral ischemic injury.</a> Mol Pharmacol. 66, 899-908 (2004).</li><li>Barone, F. C., Knudsen, D. J., Nelson, A. H., Feuerstein, G. Z., Willette, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+strain+differences+in+susceptibility+to+cerebral+ischemia+are+related+to+cerebral+vascular+anatomy.">Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy.</a> J Cereb Blood Flow Metab. 13, 683-692 (1993).</li><li>Tureyen, K., Vemuganti, R., Sailor, K. A., Dempsey, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ideal+suture+diameter+is+critical+for+consistent+middle+cerebral+artery+occlusion+in+mice.">Ideal suture diameter is critical for consistent middle cerebral artery occlusion in mice.</a> Neurosurgery. 56, 196-200 (2005).</li><li>Barber, P. A., Hoyte, L., Colbourne, F., Buchan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-regulated+model+of+focal+ischemia+in+the+mouse:+a+study+with+histopathological+and+behavioral+outcomes.">Temperature-regulated model of focal ischemia in the mouse: a study with histopathological and behavioral outcomes.</a> Stroke; a journal of cerebral circulation. 35, 1720-1725 (2004).</li><li>Kapinya, K. J., Prass, K., Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induced+prolonged+protection+against+cerebral+ischemia+in+mice:+a+redox+sensitive+mechanism?.">Isoflurane induced prolonged protection against cerebral ischemia in mice: a redox sensitive mechanism?.</a> Neuroreport. 13, 1431-1435 (2002).</li></ol>

Authors declare that they have no conflict of interests.

The MCAO model is an established model that replicates cerebral ischemia in murine species. This model was first used in rats and thereafter 
adapted to mice. Avoiding craniectomy to eliminate the influence of surgical manipulation on blood brain barrier permeability and intracranial pressure is an advantage 
of this technique. However, many variables, such as strain-related differences10, size of suture tip11, duration of occlusion6, 
body temperature12, anesthesia13</su...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Type</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>Male C57BL/6 mice</td>
			<td>Animal</td>
			<td>Harlan Laboratories, Indianapolis, IN, USA</td>
			<td>&nbsp;</td>
			<td>20-26 grs</td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Microscope</td>
			<td>Leica stereo Microscope MZ6</td>
			<td>&nbsp;</td>
			<td>6.3:1 zoom</td>
		</tr>
		<tr>
			<td>Fiber optic illuminator</td>
			<td>Illuminator</td>
			<td>TechniQuip Corp., Livermore, CA, USA</td>
			<td>&nbsp;</td>
			<td>FO1&#x2013;150</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Anesthesia</td>
			<td>Baxter Pharmaceutics, Deerfield, IL</td>
			<td>1001936060</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SuperCut Iris scissor, straight</td>
			<td>Surgical tool</td>
			<td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
			<td>14218</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vannas micro-scissor, straight</td>
			<td>Surgical tool</td>
			<td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
			<td>14003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont fine tip forceps</td>
			<td>Surgical tool</td>
			<td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
			<td>503290</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vessel clip</td>
			<td>Surgical tool</td>
			<td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
			<td>14120</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>High Frequency Desiccator 900</td>
			<td>Surgical tool</td>
			<td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
			<td>500397</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>7-0 surgical Nylon monofilament suture</td>
			<td>Suture</td>
			<td>Ethicon, Inc., Somerville, NJ, USA</td>
			<td>1647G</td>
			<td>Suture for occlusion</td>
		</tr>
		<tr>
			<td>7-0 surgical Silk suture</td>
			<td>Suture</td>
			<td>Ethicon, Inc., Somerville, NJ, USA</td>
			<td>7733G</td>
			<td>Suture for ligation</td>
		</tr>
		<tr>
			<td>Silicone RTV adhesive</td>
			<td>Silicone rubber</td>
			<td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
			<td>1571</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vetbond</td>
			<td>Tissue adhesive</td>
			<td>3M Company, St Paul, MN, USA</td>
			<td>70200746587</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Puralube Vet Ointment</td>
			<td>Eye ointment</td>
			<td>Pharmaderm, Melville, NY, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochlorode</td>
			<td>Analgesics</td>
			<td>Hospira Inc., Lake Forest, IL, USA</td>
			<td>NDC 0409-2012-32</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermal Cautery Unit</td>
			<td>&nbsp;</td>
			<td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
			<td>501292</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PeriFlux System 5000</td>
			<td>TCD flowmetry</td>
			<td>Perimed, Stockholm, Sweeden</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BAT-12</td>
			<td>Rectal thermometer</td>
			<td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
			<td>BAT-12R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T/PUMP, TP600</td>
			<td>Thermal blanket</td>
			<td>Gaymar, NY, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride</td>
			<td>Reagent</td>
			<td>Sigma-Aldrich Co., St Louis, MO, USA</td>
			<td>T8877</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mice brain matrix slicer</td>
			<td>Brain slicer</td>
			<td>Zivic-Miller Lab., Inc., Allison Park, PA, USA</td>
			<td>BSMAS001-1</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice at JoVE.com

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.

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 ...

intraluminal-middle-cerebral-artery-occlusion-mcao-model-for-ischemic

Research

JoVE Journal

Medicine

49.6K Views.  University of Florida. 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.

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

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 ...

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 ...

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.

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 ...

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 ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...