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 will be conducted in one experiment.</li>
	<li>Anesthetize an 8- to 12-week-old mouse (mass: 25&#8211;30 g) by using 0.8% chloral hydrate, via an intraperitoneal injection. Make sure the anesthetized mouse does not have a pedal reflex (as tested using a firm toe pinch) after the anesthetization.</li>
	<li>Use vet ointment to prevent eye dryness for the mouse while it is under anesthesia.</li>
	<li>Use a noninvasive blood pressure system to monitor the mouse&#8217;s blood pressure.</li>
	<li>Use a physiological monitoring system to monitor its rectal temperature and arterial blood gases. Maintain the body temperature at 36.5 &#177; 0.5 &#176;C.</li>
	<li>Subcutaneously inject the mouse with a prophylactic antibiotic (25 mg/kg cefazolin)8.</li>
	<li>Place the mouse in the supine position on the heating pad.</li>
	<li>Use electric clippers to expose the skin by shaving the mouse&#8217;s fur on the ventral neck region, as well as in the region between the right eye and right ear.</li>
	<li>Use epilating cream to clear the fur from the mouse&#8217;s body and disinfect the surgical site alternating scrbus with povidione-iodine and&#160;&#160;70% ethanol.</li>
	<li>Use iris scissors to cut a 1 cm-long midline incision at the neck.</li>
	<li>Use iris forceps to carefully dissect the CCA free from the vagus nerves without causing physical injury.</li>
	<li>Use 5-0 silk sutures to isolate the CCA.</li>
	<li>Make a 0.3 cm incision in the scalp at the midpoint between the right eye and right ear.</li>
	<li>Use microscissors to cut the temporalis muscle to expose the zygomatic and squamosal bone.</li>
	<li>Under a stereo dissecting microscope, use a microdrill to create a 2 mm-diameter hole directly over the right-side distal MCA.</li>
	<li>Ligate the trunk of the right-side distal MCA using a 10-0 suture.</li>
	<li>Occlude the right-side CCA using a nontraumatic aneurysm clip.</li>
	<li>After either 10 or 40 min of ischemia, remove the aneurysm clips and suture to restore blood flow to the MCA and CCA.</li>
	<li>Use a suture clip to seal the skin incision on the head.</li>
	<li>Seal the cervical skin incisions using a&#160;single suture followed by closing neck skin with suture or staple9.&#160;</li>
	<li>Subcutaneously inject buprenorphine (0.1 mg/kg) for pain relief9.</li>
	<li>Maintain the mouse&#8217;s body temperature at 36.5 &#177; 0.5 &#176;C on the heating pad until it has fully recovered from the anesthesia. Do not return the animal that has undergone surgery to the company of other animals until it has fully recovered. Do not leave the animal unattended until it regains sufficient consciousness.</li>
	<li>Place the mouse into the autoclaved cage so that it can freely access water and chow after it has fully recovered.</li>
</ol>
2. Staining with 2,3,5-triphenyltetrazolium chloride
<ol>
	<li>Anesthetize the mouse with 0.8% chloral hydrate via an intraperitoneal injection.</li>
	<li>Use operating scissors to decapitate the animal.</li>
	<li>Expose the skull by using iris scissors to make an incision in the skin of the head.</li>
	<li>Use operating scissors to cut the anterior of the frontal bone.</li>
	<li>Use iris scissors to cut the skull along the sagittal suture.</li>
	<li>Use a bone rongeur to push aside the frontal and parietal bone and expose the brain.</li>
	<li>Use iris forceps to dissect the brain.</li>
	<li>Use a mouse brain matrix and razor blades to obtain 2 mm coronal slices.</li>
	<li>Stain the brain slices for 10 min at 37 &#176;C with 2% 2,3,5-triphenyltetrazolium chloride (TTC) in 1x phosphate-buffered saline.</li>
	<li>Rinse the brain 2x with 10% formalin.</li>
	<li>Fix the brain in 10% formalin at room temperature for 24 h.&#160;</li>
</ol>
3. Measurement of infarct size
<ol>
	<li>Arrange the sections on a clean plastic slide and orient the sections from rostral to caudal.</li>
	<li>Scan the slide using a scanner. Place a metric ruler and make sure it is visible in the scanned image. Flip the slide over and scan the reverse side.</li>
	<li>Calculate the infarction area of each section using ImageJ software.
	<ol>
		<li>Open the image file and set up the scale for the image.</li>
		<li>Use freehand selection to select the infarct area.</li>
		<li>Use the regions of interest (ROI) manager to measure the area of interest.</li>
	</ol>
	</li>
	<li>Sum the infarction areas for each section and multiply the result by the section thickness to estimate the total infarction volume.</li>
</ol>
4. Statistical analysis
<ol>
	<li>Use GraphPad Prism 6 to determine the statistical significance with Student&#8217;s t-test. 
	NOTE: The error bars on the bar graphs represent standard errors of the mean (SEMs).</li>
	<li>Use G*Power 3.1 to calculate the appropriate sample size and perform a power analysis10.</li>
</ol>

<ol><li>Woodruff, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathophysiology%2C+treatment%2C+and+animal+and+cellular+models+of+human+ischemic+stroke%5BTitle%5D)+AND+%22Molecular+Neurodegeneration%22%5BJournal%5D)">Pathophysiology, treatment, and animal and cellular models of human ischemic stroke.</a> Molecular Neurodegeneration. 6 (1), 11(2011).</li>
<li>Chamorro, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+immunology+of+acute+stroke%5BTitle%5D)+AND+%22Nature+Reviews.+Neurology%22%5BJournal%5D)">The immunology of acute stroke.</a> Nature Reviews. Neurology. 8 (7), 401-410 (2012).</li>
<li>Engel, O., Kolodziej, S., Dirnagl, U., Prinz, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+stroke+in+mice+-+Middle+cerebral+artery+occlusion+with+the+filament+model%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Modeling stroke in mice - Middle cerebral artery occlusion with the filament model.</a> Journal of Visualized Experiments. (47), e2423(2011).</li>
<li>Lee, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin+15+blockade+protects+the+brain+from+cerebral+ischemia-reperfusion+injury%5BTitle%5D)+AND+%22Brain%2C+Behavior%2C+and+Immunity%22%5BJournal%5D)">Interleukin 15 blockade protects the brain from cerebral ischemia-reperfusion injury.</a> Brain, Behavior, and Immunity. 73, 562-570 (2018).</li>
<li>Barone, F. C., Knudsen, D. J., Nelson, A. H., Feuerstein, G. Z., Willette, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+strain+differences+in+susceptibility+to+cerebral+ischemia+are+related+to+cerebral+vascular+anatomy%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+and+Metabolism%3A+Official+Journal+of+the+International+Society+of+Cerebral+Blood+Flow+and+Metabolism%22%5BJournal%5D)">Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 13 (4), 683-692 (1993).</li>
<li>Kitagawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cerebral+ischemia+after+bilateral+carotid+artery+occlusion+and+intraluminal+suture+occlusion+in+mice%3A+evaluation+of+the+patency+of+the+posterior+communicating+artery%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+and+Metabolism%3A+Official+Journal+of+the+International+Society+of+Cerebral+Blood+Flow+and+Metabolism%22%5BJournal%5D)">Cerebral ischemia after bilateral carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 18 (5), 570-579 (1998).</li>
<li>Wellons, J. C. 3rd, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparison+of+strain-related+susceptibility+in+two+murine+recovery+models+of+global+cerebral+ischemia%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">A comparison of strain-related susceptibility in two murine recovery models of global cerebral ischemia.</a> Brain Research. 868 (1), 14-21 (2000).</li>
<li>Doyle, K. P., Fathali, N., Siddiqui, M. R., Buckwalter, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distal+hypoxic+stroke%3A+a+new+mouse+model+of+stroke+with+high+throughput%2C+low+variability+and+a+quantifiable+functional+deficit%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Distal hypoxic stroke: a new mouse model of stroke with high throughput, low variability and a quantifiable functional deficit.</a> Journal of Neuroscience Methods. 207 (1), 31-40 (2012).</li>
<li>Doyle, K. P., Buckwalter, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+model+of+permanent+focal+ischemia%3A+Distal+middle+cerebral+artery+occlusion%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">A mouse model of permanent focal ischemia: Distal middle cerebral artery occlusion.</a> Methods in Molecular Biology. , 103-110 (2014).</li>
<li>Wayman, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Performing+Permanent+Distal+Middle+Cerebral+with+Common+Carotid+Artery+Occlusion+in+Aged+Rats+to+Study+Cortical+Ischemia+with+Sustained+Disability%5BTitle%5D)+AND+%22Journal+Of+Visualized+Experiments%22%5BJournal%5D)">Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability.</a> Journal Of Visualized Experiments. (108), e53106(2016).</li>
<li>Noor, R., Wang, C. X., Shuaib, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+hyperthermia+on+infarct+volume+in+focal+embolic+model+of+cerebral+ischemia+in+rats%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">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/pubmed?term=((Long-term+hypothermia+reduces+infarct+volume+in+aged+rats+after+focal+ischemia%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">Long-term hypothermia reduces infarct volume in aged rats after focal ischemia.</a> Neuroscience Letters. 438 (2), 180-185 (2008).</li>
<li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rodent+models+of+focal+stroke%3A+size%2C+mechanism%2C+and+purpose%5BTitle%5D)+AND+%22NeuroRx%3A+The+Journal+of+the+American+Society+for+Experimental+NeuroTherapeutics%22%5BJournal%5D)">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx: The Journal of the American Society for Experimental NeuroTherapeutics. 2 (3), 396-409 (2005).</li>
<li>Lin, T. N., Te, J., Huang, H. C., Chi, S. I., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prolongation+and+enhancement+of+postischemic+c-fos+expression+after+fasting%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Prolongation and enhancement of postischemic c-fos expression after fasting.</a> Stroke. 28 (2), 412-418 (1997).</li>
<li>Glazier, S. S., O'Rourke, D. M., Graham, D. I., Welsh, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+ischemic+tolerance+following+brief+focal+ischemia+in+rat+brain%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+and+Metabolism%3A+Official+Journal+of+the+International+Society+of+Cerebral+Blood+Flow+and+Metabolism%22%5BJournal%5D)">Induction of ischemic tolerance following brief focal ischemia in rat brain.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 14 (4), 545-553 (1994).</li>
<li>Tachibana, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+Reperfusion+After+Brain+Ischemia+Has+Beneficial+Effects+Beyond+Rescuing+Neurons%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Early Reperfusion After Brain Ischemia Has Beneficial Effects Beyond Rescuing Neurons.</a> Stroke. 48 (8), 2222-2230 (2017).</li>
<li>Gan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ischemic+neurons+recruit+natural+killer+cells+that+accelerate+brain+infarction%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Ischemic neurons recruit natural killer cells that accelerate brain infarction.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (7), 2704-2709 (2014).</li>
<li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocyte-derived+interleukin-15+exacerbates+ischemic+brain+injury+via+propagation+of+cellular+immunity%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Astrocyte-derived interleukin-15 exacerbates ischemic brain injury via propagation of cellular immunity.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (3), E396-E405 (2017).</li>
<li>Wang, S., Zhang, H., Dai, X., Sealock, R., Faber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+architecture+underlying+variation+in+extent+and+remodeling+of+the+collateral+circulation%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Genetic architecture underlying variation in extent and remodeling of the collateral circulation.</a> Circulation Research. 107 (4), (2010).</li>
</ol>

The authors have nothing to disclose.

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 procedure. Damage to the MCA affects the level of reperfusion in the ischemic brain7. The MCA reperfusion status must be checked after the MCAO. The occlusion and restoration of blood flow to the MCA can be analyzed by using a laser Doppler. (3) The CCA should not bleed during the CCA isolation. (4) The vagus nerve must not be damaged during the CCA isolation because this could increase the infarct size and probability of mortality. (5) The body temperature of the mouse should be maintained at 36.5 &#177; 0.5 &#176;C. Hyperthermia increases the infarct size and probability of mortality11. Hypothermia reduces the infarct volume after cerebral ischemia12.
The significance of this MCAO/reperfusion model is that it can create highly reproducible cortical infarcts and behavioral deficits4. Compared with different MCAO models, such as the hypoxic ischemic (H/I) stroke model as described in a previous study8, this two-vessel occlusion model induces a relatively small variability in infarct volume (the coefficient of variation ranged from 0.11-0.17) (Table 1).Alternative stroke models, such as the intraluminal filament model, might result in an unpredictable infarct volume because of the uncertain status of the occlusion and reperfusion condition after surgery13. Compared with the three-vessel MCAO model (ligation of the right MCA and right and left CCAs)14, the proposed model involves the ligation of only two vessels (the right MCA and right CCA) to achieve cerebral ischemia. Consequently, a shorter surgery time is required than in the three-vessel MCAO model. The main limitation of this MCAO/reperfusion model is that it requires craniectomy to perform MCA ligation. One study indicated that craniotomy causes transcriptional changes in the brain15. Therefore, a sham control is required to determine the effects of MCAO/reperfusion on gene expression.
Neuronal loss in the cortical area is less when a longer reperfusion duration is employed. Studies have demonstrated that the MAP2-negative area is smaller after 7 days of reperfusion, compared with 2 days of reperfusion4,16. However, this recovery effect is unlikely in a brain with ischemia induced by an intraluminal monofilament model of MCAO17,18. In addition, the intraluminal monofilament model of MCAO can sustain the infarct size for at least 7 days.
B6 mice have extensive collateralization between the anterior cerebral artery and the MCA19. When we permanently truncated the MCA in the ischemic brain, we found that the infarct volume was not significantly different from the mice with a reperfused MCA at 24 h after the MCAO (Figure 4). Therefore, we suggest that the blood flow from the anterior cerebral artery collaterals might compensate for the ischemia effects of the MCA territory when the distal MCA is permanently occluded.
In this study, the two-vessel MCAO/reperfusion model created an ischemia-reperfusion injury and caused peripheral immune cells to infiltrate into the ischemic brain. This model can be employed to investigate the interplay between the brain and the immune system. In addition, it can be used to test potential neuroprotectants or drugs that modulate the immune response after cerebral ischemia-reperfusion.

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 at 48 h after the MCAO/reperfusion (Figure 2). A bulk of peripheral immune cells (CD45high cells) also infiltrated the ischemic brain (ipsilateral hemisphere) after the cerebral ischemia-reperfusion (Figure 3). In addition, we compared this two-vessel occlusion model with the MCAO model and found that the infarct volumes of these two models were not significantly different (Figure 4). The mortality rate was low (&#60;5%) in the two-vessel occlusion mouse model of cerebral ischemia-reperfusion. We excluded mice from further analyses if excessive bleeding had occurred during the surgery. When the surgical procedures were correctly followed, the rate of animal exclusion due to excessive bleeding from craniectomy or MCA was less than 15%. Occlusion of the right MCA or CCA alone did not cause infarction.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59078/59078fig1.jpg" /> 
Figure 1: Infarct volume and neuronal loss are positively correlated with the length of the vessel occlusion. (A) Representative TTC stains of brain slices from mice, 24 h after the MCAO/reperfusion. The duration of the MCAO was 10 or 40 min. Data shown are representative of three independent experiments. (B) Quantification of infarct volume. The error bars represent SEMs; n = 8; *p &#60; 0.05. (C) The MAP2 expression in B6 brains at 24 h after MCAO/reperfusion was determined using immunohistochemistry. MAP2-negative areas are enclosed by a dashed line in the representative image of MAP2 staining of the brain section. (D) Quantification of the MAP2 negative area.&#160;MAP2-negative area (%) = ipsilateral MAP2-negative area / contralateral hemisphere x 100; n = 3; *p &#60; 0.05.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59078/59078fig2.jpg" /> 
Figure 2: Locomotor activity decreased after cerebral ischemia-reperfusion. (A) Locomotor activity was analyzed 48 h after the MCAO/reperfusion. The duration of the MCAO was 40 min. The data were recorded for 60 min in an open-field assay. Mice&#39;s tracking distances were analyzed using CleverSys TopScan 1.0. The sham control group was comprised of mice that had undergone the surgery without the occlusion of the MCA or CCA. (B) Quantification of the distance moved by the sham and the MCAO/reperfusion mice. The data are presented as mean &#177; SEM; n = 7; *p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59078/59078fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59078/59078fig3.jpg" /> 
Figure 3: Peripheral immune cells infiltrate into the ischemic hemisphere after cerebral ischemia-reperfusion. (A)&#160;Brain-in&#64257;ltrating immune cells (CD45high cells) in the ipsilateral and contralateral hemisphere, at 24 h after the MCAO/reperfusion, were analyzed by &#64258;ow cytometry. The isolation of brain-infiltrating immune cells has been described in a previous study4. The duration of the MCAO was 40 min. (B) The quantification of brain-infiltrating immune cells in the ipsilateral and contralateral hemisphere, at 24 h after the MCAO/reperfusion. The data are presented as mean &#177; SEM; n = 4; *p &#60; 0.05.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59078/59078fig4.jpg" /> 
Figure 4: The infarct volume is not different between MCAO- and MCAO/reperfusion-induced injuries. (A) Representative TTC stains of brain slices from mice, 24 h after the MCAO. In the MCAO experimental group, the right MCA was permanently truncated using a vessel cauterizer, whereas the right CCA was transiently ligated for 40 min. In the MCAO/reperfusion (MCAO/Rep) experimental group, the procedure was as described in section 1 of the protocol. The duration of the MCAO was 40 min. (B) Quantification of infarct volume. The data are presented as mean &#177; SEM; n = 7.
<img alt="Table 1" src="/files/ftp_upload/59078/59078table1.jpg" /> 
Table 1: Comparison of infarct volume and variability from different experiments. The infarct volume was determined at 24 h after the MCAO/reperfusion from three independent experiments. The duration of the MCAO was 40 min. SD = standard deviation; n = number of mice used per experiment.

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 ...

two-vessel-occlusion-mouse-model-of-cerebral-ischemia-reperfusion

Nanyang Technological University

Research

JoVE Journal

Medicine

11.7K Views.  Taipei Medical University. Dit twee-vaans occlusie muis model van cerebrale ischemie-reperfusie kan de pathofysiologie van een beroerte te onderzoeken. Dit model zal leiden tot een stabiel infarct volume van de bulk van de hersenen infiltreren immuuncellen in de ischemische hersenen en het gedragstekort na cerebrale ischemie-reperfusie. In dit model wordt een infarct veroorzaakt in de corticale gebieden en is het sterftecijfer laag.Omdat de ligatie MCAO reperfusie model vereist craniotomie om de grootte van dist...

Video: Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

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.

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 ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

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

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 

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 ...

Neuroscience

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

Optimization of the Longa Middle Cerebral Artery Occlusion Method for Complete Reperfusion

The middle cerebral artery occlusion model serves as the primary animal model for studying ischemic stroke. Despite being used in research for over three decades, its standardization remains inadequate. Predominantly conducted on rats and mice, the procedure poses challenges due to mice's smaller and more fragile nature. Unlike the Koizumi common carotid artery method, the Longa external carotid artery is the sole intraluminal filament stroke model ensuring complete reperfusion post-ischemia. This aspect holds critical significance for studies investigating reperfusion phenomena. The surgical modifications demonstrated in this article ensure continuous blood flow from the common carotid artery throughout the ischemic phase and after the reperfusion onset. The goal of these modifications is to selectively occlude the middle cerebral artery by keeping the perfusion uninterrupted in branches proximal to the middle cerebral artery during the ischemia period. Furthermore, the onset of reperfusion is sudden and can be precisely controlled, thereby modeling endovascular thrombectomy in human medicine more accurately. Our aim in presenting this comprehensive video article is to ease the training of new surgeons and promote the standardization of surgical procedures within the scientific community.

This protocol presents a step-by-step guide for researchers to perform the middle cerebral artery occlusion procedure on mice using the modified Longa external carotid artery method. Modifications presented in this article aim to increase the accuracy of middle cerebral artery occlusion and ensure complete reperfusion.

The middle cerebral artery occlusion model serves as the primary animal model for studying ischemic stroke. Despite being used in research for over three ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

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