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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1,026+experimental+treatments+in+acute+stroke.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infarct+volume+quantification+in+mouse+focal+cerebral+ischemia:+a+comparison+of+triphenyltetrazolium+chloride+and+cresyl+violet+staining+techniques.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+rat+model+of+thrombotic+focal+cerebral+ischemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+stroke:+size,+mechanism,+and+purpose.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+stroke+in+mice+-+middle+cerebral+artery+occlusion+with+the+filament+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+concerted+appeal+for+international+cooperation+in+preclinical+stroke+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journals+unite+for+reproducibility.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+of+a+preclinical+randomized+controlled+multicenter+trial+(pRCT):+Anti-CD49d+treatment+for+acute+brain+ischemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+next+step+in+translational+research:+lessons+learned+from+the+first+preclinical+randomized+controlled+trial.">The next step in translational research: lessons learned from the first preclinical randomized controlled trial.</a> Journal of Neurochemistry. 139, 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial+differences+in+the+early+detection+of+breast+cancer+in+metropolitan+Detroit,+1978+to+1987.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inadequate+food+and+water+intake+determine+mortality+following+stroke+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monofilament+intraluminal+middle+cerebral+artery+occlusion+in+the+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+focal+cerebral+ischemia+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhalational+anesthetics+as+neuroprotectants+or+chemical+preconditioning+agents+in+ischemic+brain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+intraluminal+MCAO:+cerebral+infarct+evaluation+by+cresyl+violet+staining.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+recanalization+on+ischemic+stroke+outcome:+a+meta-analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+filament+occlusion+of+the+middle+cerebral+artery+in+rats:+does+the+reperfusion+method+matter+24+hours+after+perfusion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+stroke:+experimental+models+and+reality.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rotarod+suitable+for+quantitative+measurements+of+motor+incoordination+in+naive+mice.">A rotarod suitable for quantitative measurements of motor incoordination in naive mice.</a> Naunyn-Schmiedebergs Archiv f&#252;r Experimentelle Pathologie und Pharmakologie. 259 (2), 211 (1968).</li><li>Bouet, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adhesive+removal+test:+a+sensitive+method+to+assess+sensorimotor+deficits+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+test+for+detecting+long-term+sensorimotor+dysfunction+in+the+mouse+after+focal+cerebral+ischemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke,+cortical+ablation,+parkinsonism+and+spinal+cord+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+cytokine-induced+sickness+behavior+after+ischemic+stroke+by+an+optimized+behavioral+assessment+battery.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>45&#176; ramp</td>
			<td>H&#38;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 &#38; 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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.9K 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

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

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. ...

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

Summary

Explore More Videos

Chapters in this video

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

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