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 ...

This two-vessel occlusion mouse model of cerebral ischemia-reperfusion can investigate the pathophysiology of stroke. This model will induce a stable infarct volume of bulk of brain infiltrating immune cells in the ischemic brain and the behavioral deficit after cerebral ischemia-reperfusion. In this model, an infarct is caused in the cortical areas and the mortality rate is low.Because the ligation MCAO reperfusion model requires craniotomy to expose the size of distal MCA, the size can be easily confirmed, and the exam whether the blood flow in the distal MCAO is disrupted during the procedure is straightforward. Provide the mice with free access to water and food until the surgery. Autoclave the surgical tools, and use 70%ethanol to sanitize the surgery table and equipment.After anesthetizing an eight to 12-week-old mouse, make sure that it does not have a pedal reflex when tested using a firm toe pinch. Use vet ointment to prevent eye dryness while the mouse is under anesthesia. To monitor the blood pressure, first remove the hair, and then use a non-invasive blood pressure system and a physiological monitoring system to monitor rectal temperature and arterial blood gasses.Inject the mouse subcutaneously with a prophylactic antibiotic. Next, place the mouse in the supine position on the heating pad. To expose the skin, use electric clippers to shave the fur on the ventral neck region and in the region between the right eye and right ear.Clear the fur from the animal's body with epilating cream, and disinfect the surgical site with 70%ethanol. Then cut a one-centimeter-long midline incision at the neck using iris scissors. With iris forceps, carefully dissect the common carotid artery free from the vagus nerves without causing physical injury, and use 5-0 silk sutures to isolate the artery.Next, use iris scissors to make a 0.3-centimeter incision in the scalp at the midpoint between the right eye and right ear. Using micro scissors, cut the temporalis muscle to expose the zygomatic and squamosal bone. Place the mouse under a stereo dissecting microscope, and use a micro drill to create a two milliliter hole directly over the right-side distal middle cerebral artery.Use a 10-0 suture to ligate the trunk of the right-side distal MCA, and use a nontraumatic aneurism clip to occlude the right-side CCA. After desired duration of ischemia, remove the suture and aneurism clip to restore blood flow to the MCA and CCA. Use a suture clip to seal the skin incision on the head and seal the cervical skin incisions with surgical glue.Inject buprenorphine subcutaneously for pain relief. Maintain the animal's body temperature at 36.5 plus or minus 0.5 degrees Celsius on the heating pad, and do not leave unattended or return it to the company of other animals until it is fully recovered from the anesthesia. Finally, place the mouse into the autoclave cage so that it can freely access water and food after it has fully recovered.Use iris scissors to make an incision in the skin of the head of the anesthetized and decapitated mouse to expose the skull. Then use operating scissors to cut the anterior of the frontal bone, and with iris scissors, cut the skull along the sagittal suture. With a bone rongeur, push aside the frontal and parietal bone, and expose the brain.Remove the brain, and place it in a mouse brain matrix. Use razor blades to obtain two-milliliter coronal slices, and place them in 50-milliliter tubes filled with five milliliters of 2%TTC. When using mouse brain matrix to obtain coronal slices, the cutting size should be same in different brains.Cutting size affect the size of brain slices, which also affect the calculation of infarct size. Stain the brain slices with 2%TTC and 1X PBS for 10 minutes at 37 degrees Celsius, and then rinse two times with 10%formalin. Fix the brain slices in 10%formalin at room temperature for 24 hours.After drying the brain slices on the tissue paper to absorb excess formalin, arrange them on a clean plastic slide, orienting them from rostral to caudal, and cover them with another clean plastic slide. Scan the slide using a scanner. Flip the slide over, and scan the reverse side.Use ImageJ software to calculate the infarction area of each section, by first opening the image file and setting up the scale for the image. Then use freehand selection to select the infarct area, and use the regions of interest ROI manager to measure the area of interest. Finally, sum the infarct areas for each section, and multiply the result by the section thickness to estimate the total infarction volume, and then complete statistical analysis as described in the manuscript.Increase in ischemia duration during this MCAO reperfusion procedure produced different degrees of ischemia-induced infarct volume and neuronal loss in the cerebral cortex of the right MCA area. Furthermore, the ischemia decreased the animal's locomotor activity at 48 hours after the MCAO reperfusion. After the cerebral ischemia-reperfusion, a bulk of peripheral immune cells infiltrated the ipsilateral hemisphere of the ischemic brain.In addition, when this two-vessel occlusion model was compared with the MCAO model, their infarct volumes were not significantly different. The micro drill must be carefully used when creating a hole in the skull. With inappropriate action, easy causing bleeding from the MCA.The MCA should not be damaged, and the bleeding must be avoided before and after the ligation procedure. The MCA occlusion and the reperfusion status can be checked by the laser Doppler. This method can confirm the condition of the blood flow of MCA.

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11.5K Views.  Taipei Medical University. This two-vessel occlusion mouse model of cerebral ischemia-reperfusion can investigate the pathophysiology of stroke. This model will induce a stable infarct volume of bulk of brain infiltrating immune cells in the ischemic brain and the behavioral deficit after cerebral ischemia-reperfusion. In this model, an infarct is caused in the cortical areas and the mortality rate is low.Because the ligation MCAO reperfusion model requires craniotomy to expose the size of distal MCA, the size c...