Study supported by grant PID2020-113202RB-I00 funded by Ministerio de Ciencia e Innovaci&#243;n (MCIN)/Agencia Estatal de Investigaci&#243;n (AEI), Gobierno de Espa&#241;a/10.13039/501100011033 and &#34;European Regional Development Fund (ERDF). A way of making Europe&#34;. NCC and MAR had predoctoral fellowships (PRE2021-099481 and PRE2018-085737, respectively) funded by MCIN/AEI/ 10.13039/501100011033 and by &#34;European Social Fund (ESF) Investing in your future&#34;.&#160;We thank Francisca Ruiz-Ja&#233;n and Leonardo M&#225;rquez-Kisinousky for their technical support. We acknowledge the support of the MRI imaging facility of Institut d&#39;Investigacions Biom&#232;diques August Pi i Sunyer (IDIBAPS). The Centres de Recerca de Catalunya (CERCA) Program of Generalitat de Catalunya supports IDIBAPS.

Animal work was conducted following the Catalan and Spanish laws (Real Decreto 53/2013) and the European Directives, with approval of the ethical committee (Comit&#233; &#200;tic d&#39;Experimentaci&#243; Animal, CEEA) of the University of Barcelona, and the local regulatory bodies of the Generalitat de Catalunya. Studies are reported in compliance with the ARRIVE guidelines. This procedure is designed to be performed in adult mice, starting at 8 weeks of age, with no age limit. Examples of the surgical procedure develop...

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

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C., 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=Therapeutic+relevance+of+elevated+blood+pressure+after+ischemic+stroke+in+the+hypertensive+rats.">Therapeutic relevance of elevated blood pressure after ischemic stroke in the hypertensive rats.</a> Hypertension. 75 (3), 740-747 (2020).</li><li>Yu, X., Feng, Y., Liu, R., Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia+protects+mice+against+ischemic+stroke+by+modulating+macrophage+polarization+through+upregulation+of+interferon+regulatory+factor-4.">Hypothermia protects mice against ischemic stroke by modulating macrophage polarization through upregulation of interferon regulatory factor-4.</a> Journal of Inflammation Research. 14, 1271-1281 (2021).</li><li>Denorme, F., Portier, I., Kosaka, Y., Campbell, R. 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The intraluminal tMCAo procedure is the most commonly used model of focal brain ischemia with reperfusion in basic research. Currently, mice are the preferred animal model due to the availability of genetically modified strains. However, it&#39;s essential to acknowledge that genetically modified mice and their genetic backgrounds can impact brain vascularization. The presence of collateral circulation and anastomoses between different arterial territories can significantly influence the outcomes of experimental procedur...

Stroke is a devastating disease that affects approximately 15 million people worldwide annually, according to the WHO. Around one-third of patients succumb to the condition, while another third experience permanent disability. Stroke is a complex pathology involving various cell types, such as neural and peripheral immune cells, vasculature, and systemic responses1. The intricate network of reactions triggered by stroke at the systems level cannot be currently replicated using in vitro models. Thus, experimental animal models are essential to delve into the disease&#39;s mechanisms and to develop and test new therapies. Currently, early tissue reperfusion is the only approved intervention, either through thrombolysis with tissue-type plasminogen activator (tPA) or endovascular thrombectomy1.
Occlusions of the middle cerebral artery (MCA) are frequent in stroke patients. Consequently, rodent models of transient MCA occlusion (tMCAo) were initially developed in rats2,3,4. Nowadays, genetically modified mice are the most commonly used animals in experimental stroke models. In this study, we describe a minimally invasive model of intraluminal tMCAo in mice. The approach is performed via the carotid artery at the neck level, without craniectomy.
The duration of the occlusion period is a critical factor that determines the extent of the ischemic lesion. Even short occlusions of 10 min can cause selective neuronal death without an apparent infarction, while longer occlusions, typically lasting 30 to 60 min, result in some degree of cerebral infarction. Unlike the proximal and distal branches of the MCA that supply the cortex and have collaterals, the lenticulo-striatal arteries providing blood to the striatum lack collaterals5. As a consequence, there is a greater reduction of blood flow in the striatum than in the cortex after tMCAo. Thus, occlusions of 30 min or less generally affect the striatum but not the cortex, whereas longer occlusions, from 45 min onwards, often generate an ischemic lesion in the entire MCA territory, including the striatum and dorsolateral cortex.
To ensure the well-being of the mice, we administer analgesics prior to the procedure and use anesthesia during surgery. Nevertheless, anesthesia can potentially introduce artificial alterations in the physiology of the mouse and affect some outcome measures6. The surgical intervention, when performed by experienced personnel, usually lasts about 15 min for inducing MCAo. Subsequently, the total time under anesthesia depends on the occlusion period. For experiments where minimizing anesthesia is crucial, an alternative step in the procedure involves discontinuing anesthesia during the occlusion period and limiting it only to the surgical steps for inserting and withdrawing the filament occluding the MCA. This approach reduces the duration of anesthesia and minimizes its potential artifactual effects on the experimental model7,8. Therefore, the method of inducing transient focal ischemia is presented by intraluminal occlusion of the MCA with two variants: with the mouse anesthetized during the entire occlusion period or with the mouse awake during this period. In either case, a sham surgery should be performed in parallel with the intervention carried out on the ischemic mice. Additionally, data on outcome assessment is provided as measured by behavioral tests and MRI at various time points after reperfusion. Finally, the main factors to consider when implementing the experimental procedure are discussed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6/0 suture&#160;</td>
			<td>Arago</td>
			<td></td>
			<td>Vascular ligatures</td>
		</tr>
		<tr>
			<td>6/0 suture with curved needle</td>
			<td>Arago</td>
			<td></td>
			<td>Skin sutures</td>
		</tr>
		<tr>
			<td>9 mg/mL Saline</td>
			<td>Fresenius Kabi</td>
			<td>CN616003 EC</td>
			<td>For hydration</td>
		</tr>
		<tr>
			<td>Anaesthesia system</td>
			<td>SurgiVet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt retractors, 1 mm wide</td>
			<td>Fine Science Tools</td>
			<td>18200-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorfine</td>
			<td>Buprex</td>
			<td></td>
			<td>For pain relief</td>
		</tr>
		<tr>
			<td>Clamp applying forceps</td>
			<td>Fine Science Tools</td>
			<td>S&#38;T CAF4</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont mini forceps</td>
			<td>Fine Science Tools</td>
			<td>M3S 11200-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>91106-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>Loctite</td>
			<td></td>
			<td>To stick LDF probe to the skull</td>
		</tr>
		<tr>
			<td>Grip Strength Meter</td>
			<td>IITC Life Science Inc.</td>
			<td>#2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>B-Braun</td>
			<td>CN571105.8</td>
			<td></td>
		</tr>
		<tr>
			<td>LDF Perimed</td>
			<td>Perimed</td>
			<td>Periflux System 5000</td>
			<td></td>
		</tr>
		<tr>
			<td>LDF Probe Holders</td>
			<td>Perimed</td>
			<td>PH 07-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRI magnet</td>
			<td>Bruker BioSpin, Ettlingen, Germany</td>
			<td></td>
			<td>BioSpec 70/30 horizontal animal scanner&#160;</td>
		</tr>
		<tr>
			<td>Needle Holder with Suture Cutter</td>
			<td>Fine Science Tools</td>
			<td>12002-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon filament</td>
			<td>Doccol</td>
			<td>701912PK5Re</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery cage with heating pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sirgical scissors</td>
			<td>Fine Science Tools</td>
			<td>91401-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Small vessel cauterizer kit</td>
			<td>Fine Science Tools</td>
			<td>18000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope and cold light</td>
			<td>Leica</td>
			<td>M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture tying forceps</td>
			<td>Fine Science Tools</td>
			<td>18025-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostat, rectal probe and mouse pad</td>
			<td>Letica Science Instruments</td>
			<td>LE 13206</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas spring scissors (4mm cutting edge)</td>
			<td>Fine Science Tools</td>
			<td>15019-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular clamps</td>
			<td>Fine Science Tools</td>
			<td>00396-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 

Transient Middle Cerebral Artery Occlusion Model of Stroke

The middle cerebral artery occlusion model is widely used to simulate ischemic stroke in mice. This model allows to investigate a temporal progression of ischemic damage, evaluate effectiveness of neuroprotective agents, and explore the role of specific genes and pathways in stroke pathology with a final aim of finding new potential treatments. The implementation of neuroimaging techniques such as MRI allows to make better diagnosis, improve the assessment of outcomes, and reduce the number of animals needed in each study.This protocol offers a clear and detailed explanation of the most commonly used technique for inducing transient middle cerebral artery occlusion. Moreover, it implements the technique by introducing the precision modification of anesthesia withdrawal during occlusion time, reducing the detrimental effect of prolonged anesthesia time.

There are different approaches to evaluate the outcome of the tMCAo procedure. In vivo neuroimaging methods (MRI) and behavioral testing are utilized here.
Mice develop ischemic lesions in the brain, mainly affecting the territory supplied by the MCA ipsilateral to the occlusion, such as the striatum and dorsolateral cortex. Several methods exist to determine the extent of the lesion, including 2,3,5-triphenyltetrazolium chloride (TTC) tissue staining, histological staining (hematoxyl...

Watch this Scientific Journal Video about Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model at JoVE.com

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 ...

transient-middle-cerebral-artery-occlusion-model-of-stroke

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JoVE Journal

Neuroscience

Surgical Procedure for Transient Middle Cerebral Artery Occlusion

To begin, position the anesthetized mouse on the operating table. Maintain body temperature using a rectal probe connected to a heating pad. Make an incision on the skin of the head in the direction of the sagittal suture, starting from the ears to the area between the eyes.Retract the skin and remove the periosteum on the right side of the skull. Then find the coordinates, 2.5 millimeters lateral from bregma. Apply cyanoacrylate to the doppler holder and attach it for laser-doppler flowmetry.After the glue has dried, connect the doppler probe. Then, check for the correct signal readout for assessing the cerebral blood flow. To perform artery occlusion, turn the mouse over to the supine position and fix it to the surgical table with medical tape.Then make a midline incision on the neck. Pull the skin laterally, followed by pulling away the salivary glands using retractors. This exposes the carotid territory.Identify the vascular anatomy of the common carotid artery, or CCA, the ICA, and the ECA, as well as the different arteries derived from them. For easier handling, detach CCA, followed by ECA from the connective tissue. Then cauterize the superior thyroid artery and detach ICA from the connective tissue.Check again to make sure the main arteries are fully detached from the connective tissue. Next, wrap 6-0 silk sutures around the ECA at the maxillary lingual bifurcation. Then suture around the CCA.Place a vascular clip interrupting blood circulation from the ICA. Make a small incision in the ECA close to the area where the tight knot is located. Insert the monofilament until the thick coating has completely entered the arterial lumen.Tighten the second knot to hold the monofilament inside the artery and to prevent the pressure exerted by the blood from pushing it out. Cut the ECA below the first knot and rotate the stump to orient it in the direction of the ICA. Advance the monofilament via the ICA until the point where the MCA branches out.The occlusion is reflected in an abrupt blood flow drop in the LDF readout. After 40 minutes, anesthetize the mouse again and place it back on the surgical table. Remove the stitches from the neck.After 45 minutes of occlusion, loosen the knot holding the monofilament in place. Pull slowly and gently on the filament and check that tissue recanalization occurs. Then pull out the filament and tighten the knot to prevent blood loss, clean the blood and untie the CCA knot.Ensure that there is no arterial wall damage. Remove the retractors and reposition the muscles, glands, and skin. Suture the skin using a 6-0 suture and apply disinfectant.Disconnect the Doppler probe and detach the holder. Finally, suture and disinfect the skin of the head. The outcome of the transient middle cerebral artery occlusion procedure was evaluated using in-vivo MRI neuroimaging.Lesion evolution assessed at different time points after reperfusion showed that progression of the lesion volume takes approximately two days to complete. Behavioral tests, such as grip strength test, showed a loss of strength in mice after 24 hours of transient middle cerebral artery occlusion. Also, upon stimulation in the corner test, the mice showed a preference to turn to the right side, which was ipsilateral to the lesion, indicating a successful middle cerebral artery occlusion on the right side.

1.1K Views.  Spanish National Research Council (CSIC).  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.

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

Summary