We would like to thank Dr. Lei Chen (Icahn School of Medicine at Mount Sinai, NY) who first established this model in our laboratory. Supported in part by HL126559, DA039576, MH098891, MH63022, MH072567, DA027569, and NSC 2015/17/B/NZ7/02985. Dr. Luc Bertrand is supported in part by a postdoctoral fellowship from the American Heart Association (16POST31170002).

All procedures were approved by the University of Miami Institutional Animal Care and Use Committee (IACUC) in accordance with the National Institutes of Health (NIH) guidelines.&#160;The use to sterile equipment and aseptic techniques is required.
1. Preparing the Occlusion Suture
<ol>
	<li>Use a suture of 0.21 mm diameter for mice between 20 - 25 g and 0.23 mm for mice between 25 - 35 g of body weight. The choice of the type of sutures for the MCAO procedure depends on animal weight.</li>
	<li>Using a silver pen, mark the suture at 9 mm starting from the silicone coated tip. This will serve as a guide for insertion length.</li>
</ol>
2. Preparation for Surgery
<ol>
	<li>Anesthetize mouse with isoflurane mixed with oxygen, using a laboratory anesthesia system. Use isoflurane at setting 5 and oxygen flow at 2 on the commercial machine (See Materials Table). Transfer the animal to the surgery surface and sustain anesthesia using a nose cone (use isoflurane setting 1.5 - 2.5 and oxygen flow at 2).
	<ol>
		<li>Ensure that the mouse respiratory rate is around 1 - 2 respiration per sec without gasping. In addition, ensure that the animal does not exhibit whiskers stimulation reaction and pedal reflex (toe pinch). Monitor respiration rate and effort during the surgery, at least every 5 min.</li>
	</ol>
	</li>
	<li>Put a drop of ophthalmic lubricant on each eye using a sterile swab to prevent them from drying during the procedure. Alternatively, apply ophthalmic ointment to the eye from a sterile pharmaceutical tube.</li>
	<li>Flip the animal on its back and shave the incision area. Afterward, thoroughly disinfect the surgery area using 70% ethanol, followed by Chlorhexidine, and the final swab with 70% ethanol.&#160;Place the animal on a warm surgical surface under a stereomicroscope.</li>
</ol>
3. Dissection of the Common Carotid Artery and Internal/External Branching
<ol>
	<li>Using surgical scissors and forceps, perform a shallow midline incision in the neck, from above the breast bone to below the jaw (approximately 3 - 4 cm).&#160;&#160;Surgical drape should be placed around the surgery area to prevent instruments from coming in contact with non-sterile surfaces. The surgical drape was omitted in the current video to aid filming.</li>
	<li>Using forceps, carefully separate fatty and connective tissue to expose the trachea. Tissue should easily and naturally separate to both sides.</li>
	<li>Place a pillow (round object, about 0.5 cm in diameter) on the back of the neck of the mice to extend the neck, further exposing the area for surgery.</li>
	<li>Open the incision using either a tissue retractor or hooks.</li>
	<li>On the animal&#39;s left side of the trachea, carefully tweeze apart the connective tissue to expose the left common carotid artery (CCA). Be careful not to damage nerves and major veins bundled with the CCA.</li>
	<li>Continue exposing the CCA, being careful to detach from underlying tissue, and expose the top &#34;Y&#34; branching of the internal cerebral artery (ICA) and external cerebral artery (ECA). To aid in detaching the CCA, insert the curved forceps under the CCA to pierce the connective tissue and then slowly allow them to open.</li>
</ol>
4. Preparation of the CCA for the MCAO Suture Insertion
<ol>
	<li>Insert 3 segments of nylon suture of about 4 cm in length under the CCA. Ensure that the CCA is not twisted as this would drastically complicate the insertion of the suture.</li>
	<li>At the lowest point possible, close the bottom suture using a permanent knot.</li>
	<li>Tie the top suture just below the ICA/ECA branching using a removable slip knot.</li>
	<li>Tie the middle suture using a removable slip knot, but keep it wide open. It is important to leave plenty of space as not to hinder suture insertion.</li>
	<li>Using microdissection spring scissors, perform an incision in the CCA between the bottom and middle sutures. Perform the 0.2 mm incision close to the bottom suture.</li>
</ol>
5. Middle Cerebral Artery Occlusion
<ol>
	<li>Using forceps, insert the MCAO suture into the CCA incision, guiding it up to the top. Perform this step quickly after step 4.5 to prevent clotting and closing of the CCA opening. In the event that suture insertion is blocked, use the tips of forceps to reopen the incision.</li>
	<li>Gently tie down the middle suture on the MCAO suture silicone part using a slip knot to restrict blood flow around it, but loose enough to allow it to move freely.</li>
	<li>Carefully undo the top suture, making sure that the MCAO suture does not slip out.</li>
	<li>Insert the MCAO suture in the ICA by a couple of millimeters and then reclose the top suture, in the same manner as described in step 5.2.</li>
	<li>Guide the MCAO suture to the occlusion area. Indication of successful insertion is evident by the low amount of blood backflow from the CCA incision and that the silver 9 mm mark is situated between the CCA incision and the ICA/ECA bifurcation. Further confirmation of successful occlusion can be obtained using monitoring methods such as a laser Doppler blood flow monitoring system15,16. 
	NOTE: A drop in 90% of blood flow to the middle brain area on the occluded area side indicates successful occlusion.</li>
	<li>After successful occlusion, tie down the middle and top suture tightly. If performing sham insertion, do not reclose sutures but instead immediately skip to step 7.3.</li>
</ol>
6. Incision Closing and Post-operative Care
<ol>
	<li>Tuck in the sutures into the incision area.</li>
	<li>Remove retractor or tissue hooks and pillow.</li>
	<li>Clean suture area using sterile saline and a cotton swab.</li>
	<li>Close the incision using nylon suture/needle and forceps.</li>
	<li>Administer anti-inflammatory (e.g., Carprofen 10 mg/kg, s.c.) and analgesic (e.g., Buprenorphine 0.1 mg/kg s.c., twice a day) drugs to alleviate post-operative discomfort. Place the mice in a cage placed on a heated pad to prevent hypothermia and give ad labium access to water and softened food.</li>
	<li>Monitor the recovery of animals (5 - 10 min) and for signs of stroke. They can vary from mild lateral paralysis and circling to severe contraction of the contralateral flank and rolling. Several evaluation points are listed in the discussion.&#160;Animals displaying signs of respiratory distress or severe seizures should be euthanized.</li>
	<li>Depending on ischemia reperfusion protocol, leave the MCAO suture in place from 30 min to 120 min or more.&#160;Reducing occlusion time prevents mortality.</li>
	<li>For permanent occlusion, leave the MCAO suture in place for 24 hr, however significant mortality rate is to be expected. After this time lapse, proceed to step 7.
	<ol>
		<li>If using permanent occlusion model, keep mice in cage until end point. At that time, remove suture (steps 7.1 to 7.8). This procedure can also be performed after euthanasia.</li>
	</ol>
	</li>
</ol>
7. Reperfusion
<ol>
	<li>Anesthetize the animal again (follow instructions in 2.1) and remove wound closing sutures.</li>
	<li>Using forceps and tissue separators, re-open the incision and expose the CCA.</li>
	<li>Carefully remove the top suture and gently pull on the MCAO suture until the silicone coated part is situated at the middle knot.</li>
	<li>Redo the top knot on the suture to prevent blood from flowing pass the suture (it does not have to be a slip knot).</li>
	<li>Carefully undo the middle knot and pull the suture past the top knot, but keep it inside the CCA.</li>
	<li>Tightly close the top knot to block artery blood flow.</li>
	<li>Pull out completely the MCAO suture and close tightly the middle knot.</li>
	<li>The MCAO suture can be re-used several times. After each use, clean the suture carefully in 70% ethanol to remove contaminants (e.g., blood or tissue) using a sterile gauze. Afterwards, place the suture in a sterilizer pouch and sterilize it.</li>
	<li>Repeat steps 6.1 - 6.4 and monitor animal recovery.</li>
</ol>
8. Tissue Analysis
<ol>
	<li>To monitor stroke volume, dissect out the brain.
	<ol>
		<li>Euthanize the mouse using a two-step process. First, expose the animal to an overdose of isoflurane until breathing stops.&#160;For TTC staining, decapitate the mouse immediately and proceed to brain extraction. For brain sectioning (e.g., for immunostaining analyses), perform perfusion via a cardiac puncture prior to decapitation. &#160;</li>
		<li>Cut-off the head at the base of the skull and cut the scalp from the neck opening to the top of the skull up to between the eyes. With the skull exposed, cut the bone along both sides starting from the skull base opening up to the eye socket. Make this cut along the wide part of the skull. Be careful not to reach too deep with scissors to prevent damaging the brain.</li>
		<li>Afterwards, cut the bone between the two eye sockets.</li>
	</ol>
	</li>
	<li>Using forceps, lift up the top of the skull to expose the brain. There should be only a small resistance. If it does not lift easily, skull incisions may be incomplete. Pry out the brain gently using blunt curved forceps. The brain might still be attached by several nerves. Proceed slowly, removing attachments along the way.</li>
	<li>Place the fresh brain in a 1 mm brain matrix, add a couple of drops of PBS, and slice along the matrix using a razor blade or cryostat blade.</li>
	<li>Transfer the brain to a 100 mm dish and add staining solution (2% 2,3,5-triphenyltetrazolium chloride [TTC] dissolved in PBS) to cover the bottom of the dish. Using two forceps, separate all brain slices (front facing down) and place them in order at the bottom of the dish. 
	NOTE: Staining will turn viable tissue red, with non-viable tissue remaining white. Warming the solution to 37 &#176;C will accelerate staining. Signs of stroke area (white) can be seen as quickly as 90 min post stroke and remain visible for more than 7 days. Imaging should be done along with a ruler to allow calculation of stroke volume.</li>
	<li>Process dissected brains for sectioning, immuno-staining, protein isolation, or a variety of other procedures17,18,19.</li>
</ol>

<ol>
	<li>Cuomo, O., 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=Antithrombin+reduces+ischemic+volume,+ameliorates+neurologic+deficits,+and+prolongs+animal+survival+in+both+transient+and+permanent+focal+ischemia.">Antithrombin reduces ischemic volume, ameliorates neurologic deficits, and prolongs animal survival in both transient and permanent focal ischemia.</a> Stroke. 38 (12), 3272-3279 (2007).</li><li>Wauquier, A., Melis, W., Janssen, 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=Long-term+neurological+assessment+of+the+post-resuscitative+effects+of+flunarizine,+verapamil+and+nimodipine+in+a+new+model+of+global+complete+ischaemia.">Long-term neurological assessment of the post-resuscitative effects of flunarizine, verapamil and nimodipine in a new model of global complete ischaemia.</a> Neuropharmacology. 28 (8), 837-846 (1989).</li><li>Liu, F., McCullough, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle+cerebral+artery+occlusion+model+in+rodents:+methods+and+potential+pitfalls.">Middle cerebral artery occlusion model in rodents: methods and potential pitfalls.</a> J Biomed Biotechnol. 2011, 464701 (2011).</li><li>Chiu, 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=Intravenous+tissue+plasminogen+activator+for+acute+ischemic+stroke:+feasibility,+safety,+and+efficacy+in+the+first+year+of+clinical+practice.">Intravenous tissue plasminogen activator for acute ischemic stroke: feasibility, safety, and efficacy in the first year of clinical practice.</a> Stroke. 29 (1), 18-22 (1998).</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. 20 (1), 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>Liu, F., Schafer, D. P., McCullough, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TTC,+fluoro-Jade+B+and+NeuN+staining+confirm+evolving+phases+of+infarction+induced+by+middle+cerebral+artery+occlusion.">TTC, fluoro-Jade B and NeuN staining confirm evolving phases of infarction induced by middle cerebral artery occlusion.</a> J Neurosci Methods. 179 (1), 1-8 (2009).</li><li>Liu, F., Yuan, R., Benashski, S. E., McCullough, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+experimental+stroke+outcome+across+the+life+span.">Changes in experimental stroke outcome across the life span.</a> J Cereb Blood Flow Metab. 29 (4), 792-802 (2009).</li><li>Wang, R. Y., Wang, P. S., Yang, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+age+in+rats+following+middle+cerebral+artery+occlusion.">Effect of age in rats following middle cerebral artery occlusion.</a> Gerontology. 49 (1), 27-32 (2003).</li><li>Baskerville, T. A., Macrae, I. M., Holmes, W. M., McCabe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+gender+on+'tissue+at+risk'+in+acute+stroke:+A+diffusion-weighted+magnetic+resonance+imaging+study+in+a+rat+model+of+focal+cerebral+ischaemia.">The influence of gender on 'tissue at risk' in acute stroke: A diffusion-weighted magnetic resonance imaging study in a rat model of focal cerebral ischaemia.</a> J Cereb Blood Flow Metab. 36 (2), 381-386 (2016).</li><li>Cai, Q., 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=Co-transplantation+of+hippocampal+neural+stem+cells+and+astrocytes+and+microvascular+endothelial+cells+improve+the+memory+in+ischemic+stroke+rat.">Co-transplantation of hippocampal neural stem cells and astrocytes and microvascular endothelial cells improve the memory in ischemic stroke rat.</a> Int J Clin Exp Med. 8 (8), 13109-13117 (2015).</li><li>Cheng, Y., 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=Intravenously+delivered+neural+stem+cells+migrate+into+ischemic+brain,+differentiate+and+improve+functional+recovery+after+transient+ischemic+stroke+in+adult+rats.">Intravenously delivered neural stem cells migrate into ischemic brain, differentiate and improve functional recovery after transient ischemic stroke in adult rats.</a> Int J Clin Exp Pathol. 8 (3), 2928-2936 (2015).</li><li>Nagai, N., 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=Intravenous+Administration+of+Cilostazol+Nanoparticles+Ameliorates+Acute+Ischemic+Stroke+in+a+Cerebral+Ischemia/Reperfusion-Induced+Injury+Model.">Intravenous Administration of Cilostazol Nanoparticles Ameliorates Acute Ischemic Stroke in a Cerebral Ischemia/Reperfusion-Induced Injury Model.</a> Int J Mol Sci. 16 (12), 29329-29344 (2015).</li><li>Liu, Y., 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=Intravenous+PEP-1-GDNF+is+protective+after+focal+cerebral+ischemia+in+rats.">Intravenous PEP-1-GDNF is protective after focal cerebral ischemia in rats.</a> Neurosci Lett. 617, 150-155 (2016).</li><li>Hedna, V. S., 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=Validity+of+Laser+Doppler+Flowmetry+in+Predicting+Outcome+in+Murine+Intraluminal+Middle+Cerebral+Artery+Occlusion+Stroke.">Validity of Laser Doppler Flowmetry in Predicting Outcome in Murine Intraluminal Middle Cerebral Artery Occlusion Stroke.</a> J Vasc Interv Neurol. 8 (3), 74-82 (2015).</li><li>Tajima, Y., 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=Reproducibility+of+measuring+cerebral+blood+flow+by+laser-Doppler+flowmetry+in+mice.">Reproducibility of measuring cerebral blood flow by laser-Doppler flowmetry in mice.</a> Front Biosci (Elite Ed). 6, 62-68 (2014).</li><li>Fang, 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=Scutellarin+regulates+microglia-mediated+TNC1+astrocytic+reaction+and+astrogliosis+in+cerebral+ischemia+in+the+adult+rats.">Scutellarin regulates microglia-mediated TNC1 astrocytic reaction and astrogliosis in cerebral ischemia in the adult rats.</a> BMC Neurosci. 16, 84 (2015).</li><li>Evilsizor, M. N., Ray-Jones, H. F., Lifshitz, J., Ziebell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+for+immunohistochemistry+on+cryosectioned+rat+brain+tissue:+example+staining+for+microglia+and+neurons.">Primer for immunohistochemistry on cryosectioned rat brain tissue: example staining for microglia and neurons.</a> J Vis Exp. (99), e52293 (2015).</li><li>Kramer, 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=TTC+staining+of+damaged+brain+areas+after+MCA+occlusion+in+the+rat+does+not+constrict+quantitative+gene+and+protein+analyses.">TTC staining of damaged brain areas after MCA occlusion in the rat does not constrict quantitative gene and protein analyses.</a> J Neurosci Methods. 187 (1), 84-89 (2010).</li><li>Wu, L., 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=Keep+warm+and+get+success:+the+role+of+postischemic+temperature+in+the+mouse+middle+cerebral+artery+occlusion+model.">Keep warm and get success: the role of postischemic temperature in the mouse middle cerebral artery occlusion model.</a> Brain Res Bull. 101, 12-17 (2014).</li><li>Liu, S., Zhen, G., Meloni, B. P., Campbell, K., Winn, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+Stroke+Model+Guidelines+for+Preclinical+Stroke+Trials+(1st+Edition).">Rodent Stroke Model Guidelines for Preclinical Stroke Trials (1st Edition).</a> J Exp Stroke Transl Med. 2 (2), 2-27 (2009).</li><li>Tsuchiya, D., Hong, S., Kayama, T., Panter, S. S., Weinstein, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+suture+size+and+carotid+clip+application+upon+blood+flow+and+infarct+volume+after+permanent+and+temporary+middle+cerebral+artery+occlusion+in+mice.">Effect of suture size and carotid clip application upon blood flow and infarct volume after permanent and temporary middle cerebral artery occlusion in mice.</a> Brain Res. 970 (1-2), 131-139 (2003).</li><li>Wolff, G., Davidson, S. J., Wrobel, J. K., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+maintains+blood-brain+barrier+integrity+during+early+stages+of+brain+metastasis+formation.">Exercise maintains blood-brain barrier integrity during early stages of brain metastasis formation.</a> Biochem Biophys Res Commun. 463 (4), 811-817 (2015).</li><li>Wrobel, J. K., Wolff, G., Xiao, R., Power, R. F., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+Selenium+Supplementation+Modulates+Growth+of+Brain+Metastatic+Tumors+and+Changes+the+Expression+of+Adhesion+Molecules+in+Brain+Microvessels.">Dietary Selenium Supplementation Modulates Growth of Brain Metastatic Tumors and Changes the Expression of Adhesion Molecules in Brain Microvessels.</a> Biol Trace Elem Res. , (2015).</li><li>Chen, L., Swartz, K. R., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+microport+technique+for+applications+in+cerebrovascular+research.">Vessel microport technique for applications in cerebrovascular research.</a> J Neurosci Res. 87 (7), 1718-1727 (2009).</li></ol>

Authors have no competing or conflicts of interest.

The successful utilization of the described MCAO method is highly dependent on an understanding of cerebral blood flow anatomy. Since the correct placement of the suture is hard to discern due to the lack of direct visual clues, repeated practice is important to master the procedure before using it for investigative studies. Stroke volume should be analyzed to ensure consistent results. The addition of a laser Doppler system can help to determine the successful occlusion of blood flow and should be used periodically to ensure that the procedure is done correctly. Routing the MCAO suture to the occlusion area can be facilitated by manipulating the artery. To help in guiding the suture to the MCA, once it has just passed the ECA/ICA branching, press against the ICA slightly above the branching (2-3 mm) with forceps to guide the suture to the left/down and prevent it from going into the pterygopalatine artery. In addition, moving the pillow placed in step 3.3 or pulling the bottom knot up and to the right can help in orienting the artery to ease the insertion. The remaining steps of the protocol involve microsurgery under stereomicroscope and are fairly straightforward. The success rate of the method described, as obtained by our group and reported by others, is of around 80 - 90%. Nevertheless, several factors can influence the survival, including body temperature control and suture selection 20,21,22.&#160;The maintenance of animal body temperature during surgery is important to improve animal survival.
Depending on the experience of the user, this method can be employed in a high throughput manner to study a large cohort of animals. The statistical significance of animal studies depends on the usage of sufficient number of subjects to discern between treatment groups despite intrinsic variation between animal subjects. The protocol presented enables such studies while recreating closely parts of the disease process.
The advantage of the MCAO technique is that, while it involves some surgical procedures, it does not require highly invasive procedures as in the craniotomy model. In addition, it is highly reproducible and the reperfusion is highly controllable, which is not possible using the endothelin-1 or the embolic stroke models. The technique closely mimics the process of a human ischemic stroke and produces characteristic tissue injury seen in humans, which is not the case for the photothrombosis model. As compared to other published techniques, the MCAO procedure does not require the cauterization of the ECA. This is an advantage since it can be combined with our ICA infusion model to deliver therapeutic agents to the affected hemisphere following stroke induction 23,24,25.
In addition to tissue analysis, animal behavior can be monitored to assess longitudinally the stroke severity and recovery. This can help compare recovery between different treatment groups. Several approaches have been developed in order to evaluate behavior. A simple 5 point scale can be used to evaluate neurological deficit post stroke (0: No behavior deficit; 1: No contralateral forepaw extension; 2: Circling on the contralateral side of infarct; 3: Falling on the contralateral side of infarct; 4: Low consciousness level and spontaneous movement) 5. In addition, in depth analysis of animal condition and behavior can be conducted to assess recovery as described in Table 11,2.
The surgical procedure presented uses a mechanical method for the induction of stroke, which limits utilization of this technique in studies on stroke susceptibility and/or causative agents. However, this protocol can be remarkably useful in analyzing stroke severity, preventive or mitigating strategies, aggravating factors and possible therapeutic approaches post-stroke. Comparison between treatment groups can help in identifying potential treatment that may minimize stroke damage or accelerate recovery. Indeed, enhancing recovery would be of great importance in helping stroke patients.

The study of cardiovascular disease, such as stroke, relies on the use of in vivo models. To understand the possible implication of ischemia, drug toxicity, and/or treatment, there is a need to use a suitable, standardized, reliable, and reproducible model of the disease, which enables comparative studies between treatment groups. In this manuscript, we are using mice, given the availability of a large number of transgenic mice and standardized assessment models. Raking scores to assess motor and behavior deficits following experimental ischemic stroke and the following recovery have been developed1,2.
Several ischemic stroke models are available, such as complete global cerebral ischemia, incomplete global ischemia, multifocal cerebral ischemia and focal cerebral ischemia. The latter group is also the category of stroke most prevalent in patients. The majority of events are initiated by the formation of an embolic or thrombotic occlusion at or near the middle cerebral artery (MCA). Given these parameters, the model presented closely mimics disease etiology of human stroke and makes results obtained highly relevant3. Nevertheless, the translation of discoveries from animal models to disease treatment in humans has proven to be challenging. Up to now, only the use of thrombolytic tissue plasminogen activator has been approved for treatment of acute ischemic stroke4.
Among models of focal cerebral ischemia in mouse, posterior cerebral circulation stroke model and cerebral venous thrombosis model are highly invasive, diminishing their applicability and restricting the range of analyses that can be performed. However, other techniques, such as the embolic model, photothrombosis model, endothelin-1 induced stroke model, and intraluminal suture middle cerebral artery occlusion (MCAO) model, are available for use without such limitations. The MCAO model is a technique described in this protocol. It offers a reliable method of inducing focal cerebral ischemia that can be readily reperfused and performed in a high-throughput manner. There are two approaches to this model, namely, the Zea-Longa and Koizumi methods. They differ slightly in the way the occlusion suture is inserted in the vasculature. In the Zea-Longa technique, the suture is inserted via the external carotid artery5. The technique presented here is modified from the Koizumi method in which the occluding suture is inserted via the common carotid artery6.
The MCAO model has been successfully applied to evaluate different events occurring during ischemic stroke. Following reperfusion, brain edema can be observed along with the breakdown of the blood-brain barrier. Peak neuronal death is usually observed at 24 hr; however, it returns to baseline levels after 7 days7. In humans, sex and age are important variables when determining stroke outcome, this is also observed in mice and rats8,9,10. Several publications have used the MCAO model to demonstrate treatment efficiency11,12,13,14.

<table><tbody><tr><td>MCAO suture 0.23 mm</td><td>Doccol</td><td>702345PK5Re</td><td></td></tr><tr><td>MCAO suture 0.21 mm</td><td>Doccol</td><td>702145PK5Re</td><td></td></tr><tr><td>Silver pen</td><td>staples</td><td>503205</td><td></td></tr><tr><td>Anesthesia machine</td><td>Vetequip</td><td>901806</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine science tool</td><td>14558-09</td><td></td></tr><tr><td>Surgical forceps straight tip</td><td>Fine science tool</td><td>00108-11</td><td></td></tr><tr><td>Surgical forceps angled tip</td><td>Fine science tool</td><td>00109-11</td><td></td></tr><tr><td>Spring scissors</td><td>Fine science tool</td><td>15000-08</td><td></td></tr><tr><td>Nylon suture</td><td>Braintree scintific</td><td>SUT-S 104</td><td></td></tr><tr><td>Closing suture</td><td>VWR</td><td>95057-036</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal</td><td></td><td></td></tr><tr><td>2,3,5-Triphenyltetrazolium chloride</td><td>FisherSci</td><td>50-121-8005</td><td></td></tr><tr><td>Brain block</td><td>Braintree scintific</td><td>BS-A 5000C</td><td></td></tr><tr><td>Cryostat blade</td><td>VWR</td><td>89202-606</td><td></td></tr><tr><td>Optional:</td><td></td><td></td><td></td></tr><tr><td>Periflux Laser doppler system</td><td>Perimed</td><td>Periflux 5000</td><td></td></tr><tr><td>Monitoring unit</td><td>Perimed</td><td>PF 5010 - LDPM</td><td></td></tr></tbody></table>

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.

The insertion route for the occlusion suture is demonstrated in Figure 1. The MCAO suture is to be routed to the occlusion area, bifurcating in the ICA. Successful occlusion of the MCA will lead to tissue injury, visible by TTC staining. Figure 2 presents images of staining from sham treated animal (Figure 2A) and from a 60 min MCAO ischemia reperfusion animal (staining at 90 min or 24 hr post-occlusion, Figure 2B). To determine stroke volume, first calculate the stroke area for each section, using the ruler included in the imaging procedure, by subtracting the non-infarcted area of the ipsilateral side from the total area of the contralateral side. This calculation can be performed using commercial software or open source software. Afterward, calculate stroke volume for each slice taking into consideration that slides are 1 mm thick and sum the stroke volumes for all slices. This procedure will provide the total stroke volume for each animal, which can then be compared between different animal groups and treatments. On average, a 60 min occlusion followed by 23 hr of reperfusion results in a stroke volume of about 21 &#177; 3 mm3 in wild type non-treated C57BL/6J mice of 15 weeks old.
In addition to investigating stroke volume, immunofluorescence can be performed for a multitude of targets, such as microtubule-associated protein-2 (MAP2) for neuronal injury (Figure 3A) or glial fibrillary acidic protein (GFAP) for astrogliosis (Figure 3B). Such analyses further enable the analysis of stroke progression and recovery as well as other processes involved in stroke tissue damage and repair.
<img alt="Figure 1" src="/files/ftp_upload/54805/54805fig1.jpg" /> 
Figure 1: Schematic Representation of Brain Arterial Physiology. Route of the MCAO insertion proceeds from the common carotid artery (CCA) to the occlusion area indicated in blue. The surgery area is located at the bottom of the figure (Oval shape) and suture placement is indicated by black lines (TS: Top suture; MS: Middle suture; BS: Bottom suture). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54805/54805fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54805/54805fig2.jpg" /> 
Figure 2: TTC Staining of Brain Sections. Top panel represent typical staining following sham insertion. Middle and bottom panels were obtained after a 60-minute MCAO, followed by 90 min or 24 hr of reperfusion. Scale bar is 1 mm.
<img alt="Figure 3" src="/files/ftp_upload/54805/54805fig3.jpg" /> 
Figure 3: Immunofluorescence Staining of Mouse Brain Following Stroke. Following a 60-minute occlusion, brains were reperfused for 23 hr, harvested, and processed for cryosectioning. Using antibodies to MAP2 (top) and GFAP (bottom), tissue was analyzed for neuronal injury and astroglyosis in either sham (left panels) or 90 min ischemia-reperfusion brains (right panels). Pictures presented are combined from multiple images taken by confocal microscopy using a 10X objective. Scale bar is 1 mm. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54805/54805fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Criteria of Evaluation</td>
			<td></td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>General Condition</td>
			<td>Hair condition</td>
			<td colspan="3">Self-cleaning behavior (0: None; 2: normal)</td>
		</tr>
		<tr>
			<td>(0: Worst; 2: Normal)</td>
			<td>Ears</td>
			<td colspan="3">Ears position (Droopy or raised)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="3">Hearing (Reactive or not)</td>
		</tr>
		<tr>
			<td></td>
			<td>Eye condition</td>
			<td colspan="3">Eyelid position</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="3">Response</td>
		</tr>
		<tr>
			<td></td>
			<td>Posture</td>
			<td colspan="3">Crawling, leaning, normal</td>
		</tr>
		<tr>
			<td></td>
			<td>Spontaneous activity</td>
			<td colspan="3">Unconscious/no movement, low activity, normal activity</td>
		</tr>
		<tr>
			<td>Neurodeficit</td>
			<td>Body symmetry</td>
			<td colspan="3">When mouse is still (No movement, crawling, circling, partial leaning, normal)</td>
		</tr>
		<tr>
			<td>(0: Worst; 4: Normal)</td>
			<td>Gait</td>
			<td colspan="3">No movement, rotating, circling, walking to one side, normal</td>
		</tr>
		<tr>
			<td></td>
			<td>Climbing</td>
			<td colspan="3">Walking behavior on angled path (Same criteria as Gait)</td>
		</tr>
		<tr>
			<td></td>
			<td>Circling behavior</td>
			<td colspan="3">Lifting animal by the tail (no movement, swirling, contracting to one side, both sides but with preference, normal)</td>
		</tr>
		<tr>
			<td></td>
			<td>Front limb symmetry</td>
			<td colspan="3">Look at grabbing behavior of front paws (No grabbing at all, only one side grabs, both grab but one paw rigid, both grab but one continually loose first, normal)</td>
		</tr>
		<tr>
			<td></td>
			<td>Compulsory circling</td>
			<td colspan="3">Put mouse on flat surface and push on shoulder from the side (No response/movement, rotating, falling to one side, leaning with resistance to push, normal)</td>
		</tr>
		<tr>
			<td></td>
			<td>Whisker response</td>
			<td colspan="3">Touch whiskers one side at the time (No response, whiskers movement only, turn head, turn trunk, normal)</td>
		</tr>
		<tr>
			<td></td>
			<td>Epileptic behavior</td>
			<td colspan="3">Behavior after sudden noise or light change (Unconscious, consistent general tonic spasm, transient general tonic spasm, transient focal tonic spasm, normal)</td>
		</tr>
	</tbody>
</table>
Table 1. Scale for the Evaluation of Mouse Condition and Neurodeficit Following Ischemia Stroke. Adapted from1,2.

Watch this Scientific Journal Video about Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area at JoVE.com

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

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 ...

induction-ischemic-stroke-ischemia-reperfusion-mice-using-middle

Universidad San Sebastian

Research

JoVE Journal

Medicine

16.2K Views.  University of Miami. 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.

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

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Neuroscience

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 ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

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 ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

A Model of Transient Cerebral Ischemia by Occlusion of Middle Cerebral Artery

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Author Spotlight: Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...