We thank all our collaboration partners of the ImmunoStroke Consortia (FOR 2879, From immune cells to stroke recovery) for suggestions and discussions. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany&#39;s Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy - ID 390857198) and under the grants LI-2534/6-1, LI-2534/7-1 and LL-112/1-1.

The experiments reported in this video were conducted following the national guidelines for the use of experimental animals, and the protocols were approved by the German governmental committees (Regierung von Oberbayern, Munich, Germany). Ten-week-old male C57Bl/6J mice were used and housed under controlled temperature (22 &#177; 2 &#176;C), with a 12 h light-dark cycle period and access to pelleted food and water ad libitum.
1. Preparation of the material and instruments
<ol>
	<li>Connect the heat blanket to maintain the temperature of the operation area and the mouse body temperature during anesthesia at 37 &#176;C.</li>
	<li>Autoclave scissors and forceps, prepare 70% ethanol solution and keep available dexpanthenol eye ointment, several pieces of cotton, and 5-0 coated braided polyester suture ready for use. Prepare a 1 mL syringe with 0.9% saline solution (without needle) to keep the animal&#39;s incision site hydrated. Prepare the anesthesia gas (100% O2 + isoflurane).</li>
	<li>Prepare a holder for the laser Doppler probe by cutting the tip of a 10 &#181;L pipet tip (3-5 mm length). 
	NOTE: All instruments are sterilized using a hot bead sterilizer. Surfaces are also disinfected before and after surgery with a microbial disinfectant spray. Prior to surgery, the areas surrounding the head and chest of mice are disinfected with a wound disinfection spray.</li>
</ol>
2. Preparation of the laser Doppler
<ol>
	<li>Inject analgesia to the mouse 30 min before the surgery (4 mg/kg Carprofen and 0,1 mg/kg Buprenorphine, intraperitoneally).</li>
	<li>Anesthetize the mouse by placing it in the induction chamber with an isoflurane flow rate of 4% until the cessation of spontaneous body movement and vibrissae.</li>
	<li>Place the mouse in a prone position in the operation area with its nose in the anesthesia mask. Maintain isoflurane concentration at 4% for another minute, then reduce it and keep it at 2%.</li>
	<li>Set the associated feedback-controlled heating pad for maintaining the mouse body temperature at 37 &#176;C, and gently insert the rectal probe to monitor the temperature throughout the surgical procedures.</li>
	<li>Apply dexpanthenol eye ointment on both eyes.</li>
	<li>Disinfect the skin and hair surrounding the left eye and ear with 70% ethanol.</li>
	<li>Cut the scalp between the left ear and the eye (1 cm long) to expose the skull bone.</li>
	<li>Cut and retire the temporal muscle to visualize the MCA beneath the skull.</li>
	<li>Fix with glue the outer part of the tip holding the laser Doppler probe/fiber on top of the left MCA with glue. Then, glue the skin to close the wound around the tip holder. Apply 2-3 drops of hardener glue to speed up the process. Make sure that the laser Doppler fiber is not glued and can be easily removed from the tip holder at any time.</li>
</ol>
3. Transient MCAo model (occlusion)
<ol>
	<li>Turn the mouse into the supine position. Put the snout into the anesthesia cone and fix the paws with tape.</li>
	<li>Disinfect the skin and hair surrounding the chest and make a 2-cm-long midline incision in the neck.</li>
	<li>Use forceps to pull the skin and the&#160;submandibular glands apart. Use retractors to hold the sternomastoid muscle,&#160;expose the surgical field and find the left common carotid artery (CCA). Dissect the CCA free from connective tissue and surrounding nerves (without harming the vagal nerve) and perform a transient ligation before the bifurcation.</li>
	<li>Dissect the external carotid artery (ECA) and tie a permanent knot at the most distal visible part. Place another suture under the ECA, close to the bifurcation, and prepare a loose knot to be used later.</li>
	<li>Dissect the internal carotid artery (ICA) and place a microvascular clip on it, 5 mm over the bifurcation. Make sure not to damage the vagal nerve.</li>
	<li>Cut a small hole into the ECA between the tight and the loose ligations; be careful not to cut the entire ECA.</li>
	<li>Introduce the filament and advance it towards the CCA. Tighten the loose ligation in the ECA around the lumen to shortly&#160;secure the filament in that position and avoid bleeding when removing the microvascular clip.</li>
	<li>Remove the microvascular clip and insert the filament through the ICA until the origin of the MCA is reached by detecting a sharp reduction (&#62;80%) in the cerebral blood flow as measured by the laser Doppler. Fix the filament in this position by further tightening the knot around the ECA. 
	NOTE: When the filament goes toward the appropriate direction, it advances smoothly, and no resistance should be observed.</li>
	<li>Record laser Doppler values before and after filament insertion.</li>
	<li>Remove the retractor and relocate the sternomastoid muscle and the submandibular glands before suturing the wound. Remove the laser Doppler probe, and place the animal in a recovery chamber at 37 &#176;C for 1 h (until filament removal).</li>
</ol>
4. Transient MCAo model (Reperfusion)
<ol>
	<li>Anesthetize the mouse by placing it in the induction chamber with an isoflurane flow rate of 4% until the cessation of spontaneous body movement and vibrissae.</li>
	<li>Apply dexpanthenol eye ointment on both eyes.</li>
	<li>Place the mouse in a prone position in the operation area with its snout in the anesthesia mask. Maintain isoflurane concentration at 4% for another minute, then reduce it and keep it at 2%. Fix the animal&#180;s paws with tape.</li>
	<li>Insert the laser Doppler probe into the probe holder.</li>
	<li>Remove the wound suture, use forceps to pull the skin and the submandibular glands apart. Use retractors to gently pull the sternomastoid muscle and expose the surgical field.</li>
	<li>Loosen the ECA suture that tightens the filament, and gently pull the filament. Avoid damaging the silicone-rubber coating of the filament during the removal.</li>
	<li>Tightly tie the ECA suture.</li>
	<li>Confirm the increase in the cerebral blood flow in the laser Doppler device (&#62;80% of the initial value before reperfusion).</li>
	<li>Record laser Doppler values before and after filament removal.</li>
	<li>Open the transient ligation before the bifurcation from the CCA.</li>
	<li>Remove the retractor, and relocate the sternomastoid muscle and the submandibular glands before suturing the wound. Place the animal in a recovery chamber at 37 &#176;C for 1 h to recover from anesthesia.</li>
	<li>After recovery, return the mice to their cages in a temperature-controlled room.</li>
	<li>Take care of the animals by adding wet food pellets and hydrogel in small Petri dishes on the cage floor until day 3 after the surgery.</li>
	<li>Inject analgesia every 12 h for 3 d after surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).</li>
</ol>
5. Sham operation
<ol>
	<li>Perform all procedures as described above, including the ligation of the arteries and the introduction of the filament (steps 1-3.7).</li>
	<li>Remove the filament immediately after its insertion. Then, place the animal in the recovery chamber for 1 h.</li>
	<li>Place the animal in the operation area again, and remove the transient ligation of the CCA to ensure complete cerebral blood flow restoration.</li>
	<li>Suture the wound, and place the animal in a recovery chamber at 37 &#176;C for 1 h to recover from anesthesia. After recovery, return the mice to their cages in a temperature-controlled room.</li>
	<li>Take care of the animals by adding wet food pellets and hydrogel in small Petri dishes on the cage floor until day 3 after surgery.</li>
	<li>Inject analgesia every 12 h for 3 d after surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).</li>
</ol>
6. Neuroscore
<ol>
	<li>Perform the Neuroscore always at the same time of the day, and use surgical clothes to maintain a &#34;neutral smell&#34; between individual surgeons.</li>
	<li>Let the mice rest for 30 min in the room with an &#34;open&#34; cage before the test.</li>
	<li>Observe each item in Table 1 and Table 2 for 30 s.</li>
</ol>
7. Intracardiac perfusion
<ol>
	<li>Prepare a 20 mL syringe containing phosphate-buffered saline (PBS)-heparin (2 U/mL) and place it 1 m above the bench to facilitate gravity-driven perfusion. (OPTIONAL: Perform intracardiac perfusion with 4% paraformaldehyde (PFA) using a 20 mL syringe containing 4% PFA in PBS, pH 7.4).</li>
	<li>Inject intrperitoneally 100 &#181;L of ketamine and xylazine (120 and 16&#160; mg/kg body weight, respectively). Wait 5 min and confirm the cessation of spontaneous body movement and vibrissae.</li>
	<li>Fix the animal in a supine position, and disinfect the abdominal body surface with 70% ethanol.</li>
	<li>Make a 3-cm-long incision into the abdomen; cut the diaphragm, the ribs, and sternum to visualize the heart completely.</li>
	<li>Make a small incision in the right atrium, and insert the perfusion cannula into the left ventricle.</li>
	<li>Perfuse with 20 mL of PBS-heparin.</li>
	<li>After perfusion, decapitate the animal and remove the brain.</li>
	<li>Freeze the brain on powdered dry ice and store at -80 &#176;C until further use.</li>
</ol>
8. Infarct volumetry
<ol>
	<li>For cryosectioning, use a cryostat to cut the brains into 20-&#181;m-thick sections every 400 &#181;m. Place the sections on slides, and store the slides at &#8722;80 &#176;C until use.</li>
	<li>Cresyl violet (CV) staining
	<ol>
		<li>Prepare the staining solution by stirring and heating (60 &#176;C) 0.5 g of CV acetate in 500 mL of H2O until the crystals are dissolved. After the solution has cooled, store it in a dark bottle. Reheat to 60 &#176;C and filter before every use.</li>
		<li>Let the slides dry at room temperature for 30 min. Immerse them in 95% ethanol for 15 min, in 70% ethanol for 1 min, and then in 50% ethanol for 1 min.</li>
		<li>Immerse the slides in distilled water for 2 min; refresh the distilled water and place the slides in the water for 1 min. Afterward, immerse the slides in the pre-heated staining solution for 10 min at 60 &#176;C. Wash the slides twice in distilled water for 1 min.</li>
		<li>Immerse the slides in 95% ethanol for 2 min. Place them in 100% ethanol for 5 min; refresh the 100% ethanol and place the slides again in the ethanol for 2 min. Afterward, cover the slides with a mounting medium.</li>
		<li>Analysis (Figure 4C)
		<ol>
			<li>Scan the slides and analyze the indirect infarct volume by the Swanson method12 to correct for edema by using the following&#160;equation: 
			(Ischemic area) = (ischemic region)-((ipsilateral hemisphere)-(contralateral hemisphere))</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Donnan, G. A., Fisher, M., Macleod, M., Davis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stroke%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Stroke.</a> Lancet. 371 (9624), 1612-1623 (2008).</li>
<li>O'Collins, V. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((1%2C026+experimental+treatments+in+acute+stroke%5BTitle%5D)+AND+%22Annals+of+Neurology%22%5BJournal%5D)">1,026 experimental treatments in acute stroke.</a> Annals of Neurology. 59 (3), 467-477 (2006).</li>
<li>Tureyen, K., Vemuganti, R., Sailor, K. A., Dempsey, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infarct+volume+quantification+in+mouse+focal+cerebral+ischemia%3A+a+comparison+of+triphenyltetrazolium+chloride+and+cresyl+violet+staining+techniques%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Infarct volume quantification in mouse focal cerebral ischemia: a comparison of triphenyltetrazolium chloride and cresyl violet staining techniques.</a> Journal of Neuroscience Methods. 139 (2), 203-207 (2004).</li>
<li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+rat+model+of+thrombotic+focal+cerebral+ischemia%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+and+Metabolism%22%5BJournal%5D)">A new rat model of thrombotic focal cerebral ischemia.</a> Journal of Cerebral Blood Flow and Metabolism. 17 (2), 123-135 (1997).</li>
<li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</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%22%5BJournal%5D)">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx. 2 (3), 396-409 (2005).</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%3A+JoVE%22%5BJournal%5D)">Modeling stroke in mice - middle cerebral artery occlusion with the filament model.</a> Journal of Visualized Experiments: JoVE. (47), e2423(2011).</li>
<li>Dirnagl, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+concerted+appeal+for+international+cooperation+in+preclinical+stroke+research%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">A concerted appeal for international cooperation in preclinical stroke research.</a> Stroke. 44 (6), 1754-1760 (2013).</li>
<li>McNutt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Journals+unite+for+reproducibility%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Journals unite for reproducibility.</a> Science. 346 (6210), 679(2014).</li>
<li>Llovera, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Results+of+a+preclinical+randomized+controlled+multicenter+trial+%28pRCT%29%3A+Anti-CD49d+treatment+for+acute+brain+ischemia%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Results of a preclinical randomized controlled multicenter trial (pRCT): Anti-CD49d treatment for acute brain ischemia.</a> Science Translational Medicine. 7 (299), (2015).</li>
<li>Llovera, G., Liesz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+next+step+in+translational+research%3A+lessons+learned+from+the+first+preclinical+randomized+controlled+trial%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">The next step in translational research: lessons learned from the first preclinical randomized controlled trial.</a> Journal of Neurochemistry. 139, Suppl 2 271-279 (2016).</li>
<li>Swanson, G. M., Satariano, E. R., Satariano, W. A., Threatt, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Racial+differences+in+the+early+detection+of+breast+cancer+in+metropolitan+Detroit%2C+1978+to+1987%5BTitle%5D)+AND+%22Cancer%22%5BJournal%5D)">Racial differences in the early detection of breast cancer in metropolitan Detroit, 1978 to 1987.</a> Cancer. 66 (6), 1297-1301 (1990).</li>
<li>Lourbopoulos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inadequate+food+and+water+intake+determine+mortality+following+stroke+in+mice%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+and+Metabolism%22%5BJournal%5D)">Inadequate food and water intake determine mortality following stroke in mice.</a> Journal of Cerebral Blood Flow and Metabolism. 37 (6), 2084-2097 (2017).</li>
<li>Clark, W. M., Lessov, N. S., Dixon, M. P., Eckenstein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monofilament+intraluminal+middle+cerebral+artery+occlusion+in+the+mouse%5BTitle%5D)+AND+%22Neurological+Research%22%5BJournal%5D)">Monofilament intraluminal middle cerebral artery occlusion in the mouse.</a> Neurological Research. 19 (6), 641-648 (1997).</li>
<li>Jackman, K., Kunz, A., Iadecola, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+focal+cerebral+ischemia+in+vivo%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Modeling focal cerebral ischemia in vivo.</a> Methods in Molecular Biology. 793, 195-209 (2011).</li>
<li>Kitano, H., Kirsch, J. R., Hurn, P. D., Murphy, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhalational+anesthetics+as+neuroprotectants+or+chemical+preconditioning+agents+in+ischemic+brain%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+and+Metabolism%22%5BJournal%5D)">Inhalational anesthetics as neuroprotectants or chemical preconditioning agents in ischemic brain.</a> Journal of Cerebral Blood Flow and Metabolism. 27 (6), 1108-1128 (2007).</li>
<li>Rousselet, E., Kriz, J., Seidah, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+model+of+intraluminal+MCAO%3A+cerebral+infarct+evaluation+by+cresyl+violet+staining%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Mouse model of intraluminal MCAO: cerebral infarct evaluation by cresyl violet staining.</a> Journal of Visualized Experiments: JoVE. (69), e4038(2012).</li>
<li>Rha, J. H., Saver, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+impact+of+recanalization+on+ischemic+stroke+outcome%3A+a+meta-analysis%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">The impact of recanalization on ischemic stroke outcome: a meta-analysis.</a> Stroke. 38 (3), 967-973 (2007).</li>
<li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+filament+occlusion+of+the+middle+cerebral+artery+in+rats%3A+does+the+reperfusion+method+matter+24+hours+after+perfusion%5BTitle%5D)+AND+%22BMC+Neuroscience%22%5BJournal%5D)">Transient filament occlusion of the middle cerebral artery in rats: does the reperfusion method matter 24 hours after perfusion.</a> BMC Neuroscience. 13, 154(2012).</li>
<li>Sommer, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ischemic+stroke%3A+experimental+models+and+reality%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">Ischemic stroke: experimental models and reality.</a> Acta Neuropathologica. 133 (2), 245-261 (2017).</li>
<li>Jones, B. J., Roberts, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rotarod+suitable+for+quantitative+measurements+of+motor+incoordination+in+naive+mice%5BTitle%5D)+AND+%22Naunyn-Schmiedebergs+Archiv+f%C3%BCr+Experimentelle+Pathologie+und+Pharmakologie%22%5BJournal%5D)">A rotarod suitable for quantitative measurements of motor incoordination in naive mice.</a> Naunyn-Schmiedebergs Archiv für Experimentelle Pathologie und Pharmakologie. 259 (2), 211(1968).</li>
<li>Bouet, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+adhesive+removal+test%3A+a+sensitive+method+to+assess+sensorimotor+deficits+in+mice%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">The adhesive removal test: a sensitive method to assess sensorimotor deficits in mice.</a> Nature Protocols. 4 (10), 1560-1564 (2009).</li>
<li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+test+for+detecting+long-term+sensorimotor+dysfunction+in+the+mouse+after+focal+cerebral+ischemia%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">A test for detecting long-term sensorimotor dysfunction in the mouse after focal cerebral ischemia.</a> Journal of Neuroscience Methods. 117 (2), 207-214 (2002).</li>
<li>Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CNS+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke%2C+cortical+ablation%2C+parkinsonism+and+spinal+cord+injury%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.</a> Neuropharmacology. 39 (5), 777-787 (2000).</li>
<li>Roth, S., Yang, J., Cramer, J., Malik, R., Liesz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+cytokine-induced+sickness+behavior+after+ischemic+stroke+by+an+optimized+behavioral+assessment+battery%5BTitle%5D)+AND+%22Brain%2C+Behavior%2C+and+Immunity%22%5BJournal%5D)">Detection of cytokine-induced sickness behavior after ischemic stroke by an optimized behavioral assessment battery.</a> Brain, Behavior, and Immunity. 91, 668-672 (2021).</li>
</ol>

The authors have no competing interests to disclose.

The present protocol describes an experimental stroke model based on the consensus agreement of a German multicenter research consortium (&#8220;ImmunoStroke&#8221;) to establish a standardized transient MCAo model. The transient MCAo model established by introducing a silicon-coated filament through the ECA to the origin of the MCA is one of the most widely used stroke models to achieve arterial reperfusion after a delimitated occlusion period. Therefore, this procedure can be considered a translationally relevant stroke model.
The &#8220;filament model&#8221; presented in the video has some advantages compared to other previously described stroke models, such as not requiring craniotomy and achieving complete reperfusion of transiently the occluded vessel. However, the complexity of the surgical intervention could be considered as a limitation, as it includes invasive surgery and a precise manipulation of the different arteries in close proximity to the trachea and the vagal nerve. The long exposure of the animal to anesthetics could also be a critical factor to consider, as the impact of anesthetics on neuroprotection and stroke outcome has already been well documented16. Lastly, despite the complexity of this surgical procedure, it can be completed in approx. 20 min when performed by a trained surgeon.
In contrast to the previously described &#8220;filament&#8221; stroke protocols17, the method described here also allows the measurement of cerebral blood flow during the occlusion and reperfusion phases. Blood flow monitoring during reperfusion could be an important parameter to prevent stroke reperfusion injury18, which is known to cause deleterious consequences in patients undergoing pharmacologic or endovascular interventions for recanalization of the thrombosed vessels. Despite the discrepancies between the consequences of cerebral blood flow restoration after MCAo19, the variability of blood flow restoration after stroke may influence the pathophysiological and biochemical events in the brain, as well as the infarct volume and the neurological deficits of&#160; stroke mice20. Therefore, in this model, complete restoration of blood flow and its recording are requirements to ensure reproducible infarcts among mice, especially in translational stroke studies.
The overall mortality during the surgical procedure is less than 5% and is mainly caused by anesthetic complications, bleedings, or sacrifice due to predefined exclusion criteria. This stroke model presents, however, a moderate mortality rate within the first 24&#8211;48 h after stroke induction, which could increase the number of animals needed per experiment to achieve an adequate cohort of stroke mice. In terms of infarct volume, this model induces large infarcts, with lesions encompassing up to 50% of the hemisphere. It also produces brain edema, affecting different brain regions, including cortical and subcortical regions.
To achieve a low variability and high reproducibility of the stroke model, several exclusion criteria should be taken into account, including: 1) operation time &#62; 20 min; 2) &#62;20% of blood flow reduction when CCA is ligated (step 3.3); 3) blood flow reduction during occlusion &#60; 80% of the initial pre-occlusion value; and 4) blood flow increase 10 min after reperfusion rate &#60;80% compared to the pre-reperfusion value. For an experienced and trained surgeon, no animals are excluded due to the operation time criterion. However, 10&#8211;15% of animals show a 20% reduction in blood flow upon CCA&#160; ligation, and 5&#8211;10% show no adequate reduction or increase in blood flow during occlusion or reperfusion, respectively. Therefore, the success rate after excluding animals based on these criteria is approximately 75&#8211;85%.&#160;
In addition, animals are examined daily after MCAo (body weight, temperature, and basic physiological behavior) to control for sickness, pain, or discomfort behavior. In addition to this general care, several tests have been developed for specific behavioral analysis after focal brain ischemia, despite all known tests to evaluate sensorimotor dysfunction, such as the Rotarod test21, Sticky label test22, Corner test23, or the Cylinder test24. Here, animals selected for the establishment of this stroke model were evaluated for focal and general deficits, since the filament model also induces cytokine-sickness behavior independent of focal (sensory or motor) deficits25. Taken together, the &#8220;filament&#8221; stroke model described here is a valuable model for basic and translational stroke research. This model is proposed as a standardized stroke model to be used to harmonize stroke models across laboratories.

Stroke is one of the most common causes of death and disability worldwide. Although there are mainly two distinct forms of stroke, ischemic and hemorrhagic, 80&#8211;85% of all stroke cases are ischemic1. Currently, only two treatments are available for patients with ischemic stroke: pharmacological treatment with recombinant tissue plasminogen activator (rtPA) or mechanical thrombectomy. However, due to the narrow therapeutic time window and multiple exclusion criteria, only a select number of patients can benefit from these specific treatment options. Over the last two decades, preclinical and translational stroke research has focused on the study of neuroprotective approaches. However, all compounds that reached clinical trials have so far shown no improvements for the patient2.&#160;
Since in vitro models cannot accurately reproduce all brain interactions and pathophysiological mechanisms of stroke, animal models are crucial for preclinical stroke research. However, mimicking all aspects of human ischemic stroke in a single animal model is not feasible, as ischemic stroke is a highly complex and heterogeneous disease. For this reason, different ischemic stroke models have been developed over time in different species. Photothrombosis of cerebral arterioles or permanent distal occlusion of the middle cerebral artery (MCA) are commonly used models that induce small and locally defined lesions in the neocortex3,4. Besides those, the most commonly used stroke model is probably the so-called &#8220;filament model,&#8221; in which a transient occlusion of MCA is achieved. This model consists of a transient introduction of a suture filament to the origin of the MCA, leading to an abrupt reduction of the cerebral blood flow and the subsequent large infarction of subcortical and cortical brain regions5. Although most stroke models mimic MCA occlusions 6, the &#34;filament model&#34; allows precise delimitation of the ischemic time. Reperfusion by filament removal mimics the human clinical scenario of cerebral blood flow restoration after spontaneous or therapeutic (rtPA or mechanical thrombectomy) clot lysis. To date, different modifications of this &#8220;filament model&#8221; have been described. In the most common approach, first described by Longa et al. in 19895, a silicon-coated filament is introduced via the common carotid artery (CCA) to the origin of the MCA7. Although it is a widely used approach, this model does not allow complete restoration of the blood flow during reperfusion, as the CCA is permanently ligated after removal of the filament.
Over the past decade, an increasing number of research groups have been interested in modeling stroke in mice using this &#8220;filament model.&#8221; However, the considerable variability of this model and the lack of standardization of the procedures are some of the reasons for the high variability and poor reproducibility of the experimental results and scientific findings reported so far2,8. A potential cause of the current &#8220;replication crisis,&#8221; referring to the low reproducibility among research laboratories, is the non-comparable stroke infarct volumes between research groups using the same experimental methodology9. In fact, after conducting the first preclinical randomized controlled multicenter trial study10, we were able to confirm that the lack of sufficient standardization of this experimental stroke model and the subsequent outcome parameters were the main reasons for the failure of reproducibility in preclinical studies between independent laboratories11. These drastic differences in the resulting infarct sizes, despite using the same stroke model, justifiably pose not only a threat to confirmatory research, but also for scientific collaborations due to the lack of robust and reproducible models.
In light of these challenges, we aimed to develop and describe in detail the procedure for a standardized transient MCAo model as used for the collaborative research efforts within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/). This consortium aims to understand the brain-immune interactions underlying the mechanistic principles of stroke recovery. In addition, histological and related functional methods for stroke outcome analysis are presented. All methods are based on established standard operating procedures used in all research laboratories of the ImmunoStroke consortium.

<table><tbody><tr><td>45&amp;deg; ramp</td><td>H&amp;amp;S Kunststofftechnik</td><td></td><td>height: 18 cm</td></tr><tr><td>5/0 threat</td><td>Pearsalls</td><td>10C103000</td><td></td></tr><tr><td>5 mL Syringe</td><td>Braun</td><td></td><td></td></tr><tr><td>Acetic Acid</td><td>Sigma Life Science</td><td>695092</td><td></td></tr><tr><td>Anesthesia system for isoflurane</td><td>Drager</td><td></td><td></td></tr><tr><td>Bepanthen pomade</td><td>Bayer</td><td></td><td></td></tr><tr><td>C57Bl/6J mice</td><td>Charles River</td><td>000664</td><td></td></tr><tr><td>Clamp</td><td>FST</td><td>12500-12</td><td></td></tr><tr><td>Clip</td><td>FST</td><td>18055-04</td><td></td></tr><tr><td>Clip holder</td><td>FST</td><td>18057-14</td><td></td></tr><tr><td>Cotons</td><td>NOBA Verbondmitel Danz</td><td>974116</td><td></td></tr><tr><td>Cresyl violet</td><td>Sigma Life Science</td><td>C5042-10G</td><td></td></tr><tr><td>Cryostat</td><td>Thermo Scientific CryoStarNX70</td><td></td><td></td></tr><tr><td>Ethanol 70%</td><td>CLN Chemikalien Laborbedorf</td><td>521005</td><td></td></tr><tr><td>Ethanol 96%</td><td>CLN Chemikalien Laborbedorf</td><td>522078</td><td></td></tr><tr><td>Ethanol 99%</td><td>CLN Chemikalien Laborbedorf</td><td>ETO-5000-99-1</td><td></td></tr><tr><td>Filaments</td><td>Doccol</td><td>602112PK5Re</td><td></td></tr><tr><td>Fine 45 angled forceps</td><td>FST</td><td>11251-35</td><td></td></tr><tr><td>Fine forceps</td><td>FST</td><td>11252-23</td><td></td></tr><tr><td>Fine Scissors</td><td>FST</td><td>14094-11</td><td></td></tr><tr><td>Glue</td><td>Orechseln</td><td>BSI-112</td><td></td></tr><tr><td>Hardener Glue</td><td>Drechseln &amp;amp; Mehr</td><td>BSI-151</td><td></td></tr><tr><td>Heating blanket</td><td>FHC DC Temperature Controller</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Abbot</td><td>B506</td><td></td></tr><tr><td>Isopentane</td><td>Fluka</td><td>59070</td><td></td></tr><tr><td>Ketamine</td><td>Inresa Arzneimittel GmbH</td><td></td><td></td></tr><tr><td>Laser Doppler</td><td>Perimed</td><td>PF 5010 LDPM, Periflux System 5000</td><td></td></tr><tr><td>Laser Doppler probe</td><td>Perimed</td><td>91-00123</td><td></td></tr><tr><td>Phosphate Buffered Saline pH: 7.4</td><td>Apotheke Innestadt Uni Munchen</td><td>P32799</td><td></td></tr><tr><td>Recovery chamber</td><td>Mediheat</td><td></td><td></td></tr><tr><td>Roti-Histokit mounting medium</td><td>Roth</td><td>6638.1</td><td></td></tr><tr><td>Saline solution</td><td>Braun</td><td>131321</td><td></td></tr><tr><td>Scalpel</td><td>Feather</td><td>02.001.30.011</td><td></td></tr><tr><td>Silicon-coated filaments</td><td>Doccol</td><td>602112PK5Re</td><td></td></tr><tr><td>Stereomicropscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>Superfrost Plus Slides</td><td>Thermo Scientific</td><td>J1800AMNZ</td><td></td></tr><tr><td>Vannas Spring Scissors</td><td>FST</td><td>15000-00</td><td></td></tr><tr><td>Xylacine</td><td>Albrecht</td><td></td><td></td></tr></tbody></table>

modeling stroke in mice: transient middle cerebral artery occlusion via the external carotid artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

The model described here is a modification of the commonly used &#34;filament&#34; stroke model, which consists of introducing a silicon-coated filament through the ECA to transiently block the origin of the MCA (Figure 1). After removing the filament, only the blood flow in the ECA is permanently ceased, allowing complete recanalization of the CCA and ICA. This allows an adequate reperfusion of the brain (Figure 2), similar to the situation observed after successful pharmacological thrombolysis or mechanical thrombectomy in human patients. Moreover, this work also describes a method for measuring the cerebral blood flow during both occlusion and reperfusion procedures by fixing a cannula connected to the laser Doppler probe at the skull over the MCA territory.
The overall mortality rate of the surgical procedure is &#60;5% when performed by a trained surgeon. At early time points after MCAo, animals generally present severe postural and movement deficits, general weakness, and loss in body weight13. These severe deficits are transient, and the animals show improved activity after approximately 1 week; thus, the deficits are more specific for focal neurological symptoms.
Behavioral deficits after MCA occlusion were assessed by the composite Neuroscore14; general and focal deficits were measured 24 h and 3 d after surgery. The general Neuroscore integrates 5 items (Table 1), including the evaluation of the fur, ears, eyes, posture, and spontaneous activity, with a maximum score of 18. The focal Neuroscore comprises 7 items (Table 2), including the evaluation of body symmetry, gait, climbing, circling behavior, forelimb symmetry, compulsory cycling, and whiskers response, with a maximum score of 28. The composite scale ranges from 0 (no deficits) to 46 (severe impairments). Stroke animals presented a significant change in the composite and focal Neuroscore, but not in the general Neuroscore, when compared to sham animals (Figure 3).
Infarct volumetry was also performed using Cresyl Violet staining of coronal serial brain sections 24 h after stroke induction. The infarct volume mean was 61.69 mm3, representing 48% of the affected brain hemisphere (Figure 4). When performed by a trained surgeon, the overall variability of this stroke model is low, with a coefficient of variation of &#60;6%. The lesion area includes the somatosensory and motor cortex as well as subcortical structures such as the striatum (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62573/62573fig01.jpg" style="font-size: 14px;" />
Figure 1: Scheme for the access and intraluminal MCA occlusion. The filament (dotted line) is inserted between the proximal and distal suture knots in the ECA and advanced along the ICA until it reaches the origin of the MCA (see inset). Once in place, the ECA is ligated with a suture to fix the filament. Abbreviations: ACA = anterior cerebral artery; BA = basilar artery; CCA = common carotid artery; ECA = external carotid artery; ICA = internal carotid artery; MCA = middle cerebral artery; PCA = posterior communicating artery; PTG = pterygopalatine artery. This figure has been modified from Jackman et al.15. <a href="https://www.jove.com/files/ftp_upload/62573/62573fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62573/62573fig02.jpg" /> 
Figure 2: Blood flow during occlusion and reperfusion. Blood flow is registered before and after filament insertion and before and after filament removal. A reduction in the blood flow was observed during the occlusion and the restoration of the blood flow during the reperfusion. Every color represents one animal. Abbreviations: MCA = middle cerebral artery; CBF = cerebral blood flow; A.U. = arbitrary units. <a href="https://www.jove.com/files/ftp_upload/62573/62573fig02large.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/62573/62573fig03.jpg" /> 
Figure 3: Neuroscore for functional deficits after tMCAo. (A)&#160;Total, (B)&#160;focal, and (C)&#160;general Neuroscore before and 24 h and 3 d after tMCAo. Open bars: sham; Black bars: tMCAo. n=10 per group. *p &#60; 0.05. Abbreviations: tMCAo = transient middle cerebral artery occlusion; BL = before tMCAo. <a href="https://www.jove.com/files/ftp_upload/62573/62573fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62573/62573fig04.jpg" /> 
Figure 4: Volumetric infarct analysis and infarct outcome 24 h after tMCAo. (A) Representative cresyl violet-stained coronal brain sections every 400 &#181;m at 24 h after tMCAo. Dashed lines demarcate the lesion area. (B)&#160;Analysis of infarct volume of 10 brains (each dot representing one individual brain) 24 h after tMCAo. The horizontal red line represents the mean (61.69 mm3), error bars indicate standard deviation (3.78 mm3). (C)&#160;Representative image for infarct volume calculation from a cresyl violet coronal section. Blue = Contralateral hemisphere; Red = Ipsilateral hemisphere; Pale striped area = Ischemic region. <a href="https://www.jove.com/files/ftp_upload/62573/62573fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Time-point of scoring</td>
			<td>score</td>
		</tr>
		<tr>
			<td rowspan="23">General Neuroscore</td>
			<td rowspan="3">Hair</td>
			<td>0. Hair neat and clean</td>
			<td></td>
		</tr>
		<tr>
			<td>1. Localized piloerection and dirty hair in 2 body parts (nose and eyes)</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Piloerection and dirty hair in &#62;2 body parts</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Ears (mouse on an open bench top)</td>
			<td>0. Normal (ears are stretched laterally and behind, they react by straightening up following noise)</td>
			<td></td>
		</tr>
		<tr>
			<td>1. Stretched laterally but not behind (one or both), they react to noise</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Same as 1. NO Reaction to noise.</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Eyes (mouse on OBT)</td>
			<td>0. Open, clean and quickly follow the surrounding environment</td>
			<td></td>
		</tr>
		<tr>
			<td>1. Open and characterized by aqueous mucus. Slowly follow the surrounding environment</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Open and characterized by dark mucus</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Ellipsoidal shaped and characterized by dark mucus</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Closed</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Posture (place the mouse on the palm and swing gently)</td>
			<td>0. The mouse stands in the upright position with the back parallel to the palm. During swing, it stands rapidly.</td>
			<td></td>
		</tr>
		<tr>
			<td>1. The mouse stands humpbacked. During the swing, it flattens the body to gain stability.</td>
			<td></td>
		</tr>
		<tr>
			<td>2. The head or part of the trunk lies on the palm.</td>
			<td></td>
		</tr>
		<tr>
			<td>3. The mouse lies on one side, barely able to recover the upright position.</td>
			<td></td>
		</tr>
		<tr>
			<td>4. The mouse lies in a prone position, not able to recover the upright position.</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Spontaneos activity (mouse on OBT)</td>
			<td>0.The mouse is alert and explores actively.</td>
			<td></td>
		</tr>
		<tr>
			<td>1.The mouse seems alert, but it is calm and sluggish.</td>
			<td></td>
		</tr>
		<tr>
			<td>2.The mouse explores intermittently and sluggishly.</td>
			<td></td>
		</tr>
		<tr>
			<td>3.The mouse is somnolent and numb, few movements on-the-spot.</td>
			<td></td>
		</tr>
		<tr>
			<td>4.No spontaneous movements</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Total score for general scoring</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td colspan="2">(normal=0 max=18)</td>
		</tr>
	</tbody>
</table>
Table 1: General Neuroscore. Animals received between 0 and 4 points, depending on the severity, for each of the five general deficits measured. The scores on the different areas are then added to provide a total general score ranging from 0 to 18. This table has been modified from Clark et al.14 . Abbreviation: OBT = open benchtop.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Time-point of scoring</td>
			<td>score</td>
		</tr>
		<tr>
			<td rowspan="37">Focal Neuroscore</td>
			<td rowspan="5">Body symmetry (mouse on OBT, observe the nose-tail line)</td>
			<td>0. Normal (Body: normal posture, trunk elevated from the bench, with fore and hindlimbs leaning beneath the body. Tail: straight)</td>
			<td></td>
		</tr>
		<tr>
			<td>1. Slight asymmetry (Body: leans on one side with fore and hindlimbs leaning beneath the body. Tail: slightly bent)</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Moderate asymmetry (Body: leans on one side with fore and hindlimbs stretched out. Tail: slightly bent)</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Prominent asymmetry (Body: bent, on one side lies on the OBT. Tail: bent)</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Extreme asymmetry (Body: highly bent, on one side constantly lies on the OBT. Tail: highly bent)</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Gait (mouse on OBT. Observed undisturbed)</td>
			<td>0. Normal (gait is flexible, symmetric and quick)</td>
			<td></td>
		</tr>
		<tr>
			<td>1. Stiff, inflexible (humpbacked walk, slower than normal mouse)</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Limping, with asymmetric movements</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Trembling, drifting, falling</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Does not walk spontaneously (when stimulated by gently pushing the mouse walks no longer than 3 steps)</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Climbing (mouse on a 45o surface. Place the mouse in the center of the gripping surface)</td>
			<td>0. Normal (mouse climbs quickly)</td>
			<td></td>
		</tr>
		<tr>
			<td>1. Climbs with strain, limb weakness present</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Holds onto slope, does not slip or climb</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Slides down slope, unsuccessful effort to prevent fail</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Slides immediately, no effort to prevent fail</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Circling behavior (mouse on OBT, free observation)</td>
			<td>0. Absent circling behavior</td>
			<td></td>
		</tr>
		<tr>
			<td>1. Predominantly one-side turns</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Circles to one side, although not constantly</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Circles constantly to one side</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Pivoting, swaying, or no movement</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Forelimb symmetry (mouse suspended by tail)</td>
			<td>0. Normal</td>
			<td></td>
		</tr>
		<tr>
			<td>1. Light asymmetry: mild flexion of contralateral forelimb</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Marked asymmetry: marked flexion of contralateral limb, the body slightly bends on the ipsilateral side</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Prominent asymmetry: contralateral forelimb adheres to the trunk</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Slight asymmetry, no body/limb movement</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Compulsory circling (forelimbs on bench, hindlimbs suspended by the tail: it reveals the presence of the contralateral limb palsy)</td>
			<td>0. Absent. Normal extension of both forelimbs</td>
			<td></td>
		</tr>
		<tr>
			<td>1. Tendency to turn to one side (the mouse extends both forelimbs, but starts to turn preferably to one side)</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Circles to one side (the mouse turns towards one side with a slower movement compared to healthy mice)</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Pivots to one side sluggishly (the mouse turns towards one side failing to perform a complete circle)</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Does not advance (the front part of the trunk lies on the bench, slow and brief movements)</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Whisker response (mouse on the OBT)</td>
			<td>0. Normal</td>
			<td></td>
		</tr>
		<tr>
			<td>1. Light asymmetry (the mouse withdraws slowly when stimulated on the contralateral side)</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Prominent asymmetry (no response when stimulated to the contralateral side)</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Absent response contralaterally, slow response when stimulated ipsilaterally</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Absent response bilaterally</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Total score for focal deficits</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td colspan="2">(normal=0 max=28)</td>
		</tr>
	</tbody>
</table>
Table 2: Focal Neuroscore. Animals received between 0 and 4 points depending on the severity for each of the seven general deficits measured. The scores on the different areas are then added to provide a total focal score ranging from 0 to 28. This table has been modified from Clark et al.14 . Abbreviation: OBT = open benchtop.

Watch this Scientific Journal Video about Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery at JoVE.com

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

modeling-stroke-mice-transient-middle-cerebral-artery-occlusion-via

Universidad Científica del Sur

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Neuroscience

7.0K Views.  LMU Munich. Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Video: Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

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 ...

Medicine

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 ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

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. ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

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 ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

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 ...

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 ...

Middle Cerebral Artery Occlusion in Rats with Preserved Vascular Anatomy

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have significantly contributed to our understanding of its pathological mechanisms and the development of potential treatments. Presently, there are two common methods involving filament (endovascular suture) techniques to induce animal models of cerebral ischemia. However, these methods have inherent limitations, such as reduced blood perfusion to the brain, damage to the external carotid artery system, impaired food and/or water intake, and sensory dysfunction of the face. This article introduces a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. In this study, the common carotid artery (CCA) of Sprague-Dawley rats was exposed, and an incision was made. A filament was then inserted through the incision into the internal carotid artery to occlude the middle cerebral artery. After 1.5 h of induced ischemia, the occluding filament was fully removed from both the internal carotid artery and the CCA. The incision in the CCA was subsequently sutured using 11-0 microsurgical sutures under a microscope (magnification 4x). Through the utilization of microsurgical techniques to repair the CCA, this study successfully developed a unique method to induce an ischemic stroke model in rats while preserving the anatomical integrity of cerebral blood vessels.

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.