Name: two-vessel occlusion mouse model of cerebral ischemia-reperfusion
Uploaded: 2019-03-01T13:30:00
Description: Watch this Scientific Journal Video about Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion at JoVE.com

This work was supported by the Ministry of Science and Technology, Taiwan (MOST 106-2320-B-038-024, MOST 105-2221-E-038-007-MY3, and MOST 104-2320-B-424-001) and Taipei Medical University Hospital (107TMUH-SP-01). This manuscript was edited by Wallace Academic Editing.

The institutional animal care and use committees of Academia Sinica and Taipei Medical University approved this protocol for the use of experimental animals.
1. MCAO/reperfusion model
<ol>
	<li>Provide the mice with free access to water and chow until the surgery.</li>
	<li>Autoclave the surgical tools and sanitize the surgery table and equipment using 70% ethanol. Wear a surgical mask and sterile gloves. Use a dry bead sterilizer to resterilize the surgical tools if multiple mouse surgeries...

<ol>
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X., Shuaib, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hyperthermia+on+infarct+volume+in+focal+embolic+model+of+cerebral+ischemia+in+rats.">Effects of hyperthermia on infarct volume in focal embolic model of cerebral ischemia in rats.</a> Neuroscience Letters. 349 (2), 130-132 (2003).</li><li>Florian, B., 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=Long-term+hypothermia+reduces+infarct+volume+in+aged+rats+after+focal+ischemia.">Long-term hypothermia reduces infarct volume in aged rats after focal ischemia.</a> Neuroscience Letters. 438 (2), 180-185 (2008).</li><li>Carmichael, S. 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The MCAO/reperfusion mouse model is an animal model commonly employed to mimic transient ischemia in humans. This animal model can be applied to transgenic and knockout mice strains to investigate the pathophysiology of stroke. Several steps in the protocol are especially critical. (1) The microdrill must be carefully used when creating a hole in the skull, with inappropriate action easily causing bleeding from the MCA. (2) The MCA should not be damaged, and bleeding must be avoided before and after the ligation procedur...

The cerebral ischemia-reperfusion mouse model is one of the most widely used experimental approaches for investigating the pathophysiology of ischemia-induced brain injury1. Because cerebral ischemia-reperfusion activates the peripheral immune system, peripheral immune cells infiltrate into the ischemic brain and cause neuronal damage2. Thus, a reliable and reproducible mouse model that mimics cerebral ischemia-reperfusion is required to understand the pathophysiology of stroke.
C57BL/6J (B6) mice are the most commonly used strain in stroke experiments because they can easily be genetically manipulated. Two common models of MCAO/reperfusion that mimic the condition of cerebral ischemia-reperfusion are available. The first is the intraluminal filament model of proximal MCAO, where a silicon-coated filament is employed to intravascularly occlude the blood flow in the MCA; the occluding filament is subsequently removed to restore blood flow3. A short occlusion duration results in a lesion of the subcortical region, whereas a longer occlusion duration causes infarcts in the cortical and subcortical areas. The second model is the ligation model of distal MCAO, which involves extravascular ligation of the MCA and CCA to reduce the blood flow through the MCA, after which blood flow is restored through the removal of the suture and aneurysm clip4. In this model, an infarct is caused in the cortical areas, and the mortality rate is low. Because the ligation of MCAO/reperfusion model requires craniectomy to expose the site of the distal MCA, the site can be easily confirmed, and examining whether the blood flow in the distal MCA is disrupted during the procedure is straightforward.
B6 mice exhibit considerable variations in the anatomy of their circle of Willis; this might affect the infarct volume following cerebral ischemia-reperfusion5,6,7. Currently, this problem can be overcome through ligation of the distal MCA8. In this study, we establish a method for occluding the MCA blood flow and enabling reperfusion after a predetermined period of ischemia. Two-vessel occlusion of the cerebral ischemia-reperfusion model induces transient ischemia of the MCA territory through ligation of the right distal MCA and right CCA, with blood flow restored after a certain period of ischemia.This MCAO/reperfusion model induces an infarct of stable size, a bulk of brain-infiltrating immune cells in the ischemic brain, and a behavioral deficit after cerebral ischemia&#8211;reperfusion4.

<table>
	<tbody>
		<tr>
			<td>Bone rongeur</td>
			<td>Diener</td>
			<td></td>
			<td>Friedman</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Sigma</td>
			<td>B-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>Sigma</td>
			<td>1097603</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate</td>
			<td>Sigma</td>
			<td>C8383</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-745</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric clippers</td>
			<td>Petpro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% formalin</td>
			<td>Sigma</td>
			<td>F5304</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator dry bead sterilizer</td>
			<td>Braintree Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Forceps</td>
			<td>Karl Klappenecker</td>
			<td></td>
			<td>10 cm</td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>Diener</td>
			<td></td>
			<td>9 cm</td>
		</tr>
		<tr>
			<td>Iris Scissors STR</td>
			<td>Karl Klappenecker</td>
			<td></td>
			<td>11 cm</td>
		</tr>
		<tr>
			<td>Microdrill</td>
			<td>Stoelting</td>
			<td>FOREEDOM K.1070</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-scissors-Vannas</td>
			<td>HEISS</td>
			<td>H-4240</td>
			<td>blade 7mm, 8 cm</td>
		</tr>
		<tr>
			<td>Mouse brain matrix</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Non-invasive blood pressure system</td>
			<td>Muromachi</td>
			<td>MK-2000ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors STR</td>
			<td>Karl Klappenecker</td>
			<td></td>
			<td>14 cm</td>
		</tr>
		<tr>
			<td>Physiological Monitoring System</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Ever-Ready</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stoelting Rodent Warmers</td>
			<td>Stoelting</td>
			<td>53810</td>
			<td>Heating pad</td>
		</tr>
		<tr>
			<td>Suture clip</td>
			<td>Stoelting</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>IDEALTEK</td>
			<td></td>
			<td>No.3</td>
		</tr>
		<tr>
			<td>Vetbond</td>
			<td>3M</td>
			<td>15672</td>
			<td>Surgical glue</td>
		</tr>
		<tr>
			<td>10-0 suture</td>
			<td>UNIK</td>
			<td>NT0410</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-Triphenyltetrazolium chloride</td>
			<td>Sigma</td>
			<td>T8877</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This MCAO/reperfusion procedure produced a cortical infarct in the vicinity of the right MCA and caused a behavioral deficit. Different degrees of ischemia-induced infarct volume (Figure 1A,B) and neuronal loss (Figure 1C,D) were created in the cerebral cortex of the right MCA area through an increase in ligation duration. This MCAO/reperfusion injury decreased the animal&#39;s locomotor activity...

Watch this Scientific Journal Video about Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion at JoVE.com

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

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 ...

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