Name: induction of ischemic stroke and ischemia-reperfusion in mice using the middle artery occlusion technique and visualization of infarct area
Uploaded: 2017-02-02T12:30:00
Description: 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

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. 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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 ...

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