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>
...

<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. 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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 e...

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 border="0" cellpadding="0" cellspacing="0" width="544">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">MCAO suture 0.23mm</td>
			<td style="width:149px;">Doccol</td>
			<td style="width:119px;">702345PK5Re</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">MCAO suture 0.21mm</td>
			<td style="width:149px;">Doccol</td>
			<td style="width:119px;">702145PK5Re</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Silver pen</td>
			<td style="width:149px;">staples</td>
			<td style="width:119px;">503205</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Anesthesia machine</td>
			<td style="width:149px;">Vetequip</td>
			<td style="width:119px;">901806</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Surgical scissors</td>
			<td style="width:149px;">Fine science tool</td>
			<td style="width:119px;">14558-09</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Surgical forceps straight tip</td>
			<td style="width:149px;">Fine science tool</td>
			<td style="width:119px;">00108-11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Surgical forceps angled tip</td>
			<td style="width:149px;">Fine science tool</td>
			<td style="width:119px;">00109-11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Spring scissors</td>
			<td style="width:149px;">Fine science tool</td>
			<td style="width:119px;">15000-08</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Nylon suture</td>
			<td style="width:149px;">Braintree scintific</td>
			<td style="width:119px;">SUT-S 104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Closing suture</td>
			<td style="width:149px;">VWR</td>
			<td style="width:119px;">95057-036</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Isoflurane</td>
			<td style="width:149px;">Piramal</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">2,3,5-Triphenyltetrazolium chloride</td>
			<td style="width:149px;">FisherSci</td>
			<td style="width:119px;">50-121-8005</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Brain block</td>
			<td style="width:149px;">Braintree scintific</td>
			<td style="width:119px;">BS-A 5000C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Cryostat blade</td>
			<td style="width:149px;">VWR</td>
			<td style="width:119px;">89202-606</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Optional:</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Periflux Laser doppler system</td>
			<td style="width:149px;">Perimed</td>
			<td style="width:119px;">Periflux 5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Monitoring unit</td>
			<td style="width:149px;">Perimed</td>
			<td style="width:119px;">PF 5010 - LDPM</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 de...

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 ...

The overall goal of this surgical procedure is to officially and reliably induce Ischemic stroke while leaving the external cerebral artery intact. This method can help answer key questions in the field of stroke research, such as the impact of treatment on stroke severity. The main advantage of this technique is that following stroke induction, the external cerebral artery remains available to infuse treatment directly into the affected hemisphere.Place a mouse anesthetized with isoflurane onto the prepared surgery surface and fit a nose cone to administer isoflurane mixed with oxygen. Ensure that the respiratory rate is around one to two respirations per second without gasping. In addition, ensure that the animal does not exhibit a reaction to whisker stimulation or the pedal reflex.Put a drop of ophthalmic lubricant on each eye, using a steril swab to prevent them from drying during the procedure. After shaving the incision area, thoroughly disinfect the surgery area using 70%ethanol and a chlorhexidine-based solution. Keep the animal on a warm surgical surface under a stereo microscope.Next, using surgical scissors and forceps, perform a shallow midline incision in the neck from above the breast bone to below the jaw. Using forceps, carefully separate fatty and connective tissues to expose the trachea. The tissues should easily and naturally separate to both sides.Then place a pillow or other round object of about 0.5 centimeters in diameter under the back of the neck to extend the neck and further expose the area for surgery. Open the incision using either a tissue retractor or hooks. Then on the left side of the trachea, carefully tweeze apart the connective tissue to expose the left common carotid artery without damaging nearby nerves and major veins.Next, insert the curved forceps under the CCA to pierce the connective tissue and then slowly allow them to open to release the CCA. Detach the CCA from the underlying tissue and expose the top y branching of internal cerebral artery and external cerebral artery. Once the CCA is exposed, insert three segments of nylon suture under the CCA.Ensure that the CCA is not twisted, as this would drastically complicate the insertion of the suture. Then at the lowest point possible, close the bottom suture using a permanent knot. Tie the top suture just below the branching of the ICA and ECA using a removable slipknot.Next, tie the middle suture using a removable slipknot, but keep it wide open. Then, using micro dissection spring scissors, create a 0.2 millimeter in the CCA between the bottom and middle sutures closer to the bottom suture. Using forceps, insert the MCAO suture into the CCA incision, guiding it towards the top suture.Then gently tighten the slipknot of the middle suture on the CCA so that it restricts blood flow around the MCAO suture, but is loose enough to allow the MCAO suture to move freely. Now carefully undo the top suture, making sure that the MCAO suture does not slip out. Insert the MCAO suture a couple of millimeters into the ICA and then reclose the top suture with a slipknot as before.Next, guide the MCAO suture to the occlusion area, first by routing it into the ICA past the ECA, then by ensuring by ensuring that the suture stays in the ICA, bypassing the pterygopalatine artery. The insertion is successful if there is little blood backflow from the CCA incision and if the silver nine millimeter mark is situated between the CCA incision and the bifurcation of the ICA and ECA. After successful occlusion, tie down the middle and top suture tightly then tuck the sutures into the incision area.After removing the retractor or tissue hooks and the pillow, clean the suture area using sterile saline on a cotton swab. Then close the skin with nylon suture. Place the mouse in a clean cage situated on a heated pad to prevent hypothermia and allow ad libitum access to water and softened food.Monitor the animal for signs of stroke for at least five to 10 minutes. Depending on the ischemia reperfusion protocol, leave the MCAO suture in place from 30 minutes to 120 minutes or more. After anesthetizing the animal as before and removing the wound closing sutures, use forceps and tissue separators to reopen the incision and expose the CCA.Then carefully remove the top suture and gently pull on the MCAO suture until the silicon-coated part is situated at the middle knot. Redo the top knot suture to prevent blood from flowing pass the MCAO suture. Then carefully undo the middle knot and pull the MCAO suture past the top knot, keeping it inside the CCA.Tightly close the top knot to block artery blood flow. Next, completely pull out the MCAO suture and tightly close the middle knot. Close the wound and administer post operative care as before.This schematic shows the occlusion area that results from the procedure demonstrated in this video in blue. The surgery area is located at the bottom of the figure, indicated by the oval shape, and suture placement is indicated by black lines. The following images show TTC standing of brain sections.This image represents typical staining following sham insertion. The image is representative of the tissue damage caused by a 60 minute MCAO, follow by 90 minutes of reperfusion. Greater tissue damage is seen after 24 hours of reperfusion, following a 60 minute MCAO.Immunofluorescent staining for the neuronal marker MAP2 allows neuronal loss from 60 minutes of occlusion, followed by 23 hours of reperfusion to be clearly visualized in the image on the right. Astrogliosis is visible after immunofluorescent staining with GFAP. In the picture on the right, the astroglia are concentrated in the area adjacent to tissue damage in the ischemia reperfusion brain, whereas a diffused patter of staining is seen in the shame operated control.While attempting this procedure, it's important to monitor animal well-being and occlusion time. Following this procedure, other techniques such as odd behavior analysis and immunofluorescence can be performed to answer additional questions on tissue injury and recovery.

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JoVE Journal

Medicine

16.0K visualizações.  University of Miami. The overall goal of this surgical procedure is to officially and reliably induce Ischemic stroke while leaving the external cerebral artery intact. This method can help answer key questions in the field of stroke research, such as the impact of treatment on stroke severity. The main advantage of this technique is that following stroke induction, the external cerebral artery remains available to infuse treatment directly into the affected hemisphere.Place a mouse anesthetized with isofl...