This work was funded by the excellence cluster of the German research foundation &#8220;Munich Cluster for Systems Neurology (SyNergy)&#8221; and by the Daimler-Benz foundation to A.L.

Ethics statement
The experiments reported in this video were conducted in accordance with national guidelines for the use of experimental animals and the protocols were approved by the German governmental committees (Regierung von Oberbayern, Munich, Germany). 10 week old, male C57Bl/6J mice are used in this study. The animals were housed under controlled temperature (22 &#177; 2 &#176;C), with a 12 hr light-dark cycle period and access to pelleted food and water ad libitum.&#160;Analgesia and sedation protocols are described as approved by the local governmental committee but might differ from protocols used in other laboratories.
1. Preparation of the Material and Instruments
<ol>
	<li>Connect the heat blanket in order to maintain the operation area warm and maintain the mouse body temperature during anesthesia (37 &#176;C).</li>
	<li>Prepare autoclaved scissors, forceps and&#160;cottons, dexpanthenol eye ointment and suture material. Prepare a syringe with saline solution (without needle) to maintain the operation area hydrated. Prepare the anesthesia gas (70% N2O + 30% O2 + isoflurane).</li>
	<li>Inject analgesics intraperitoneally: Metamizol 200 mg/kg, Carprofen 4 mg/kg and Buprenorphin 0.1 mg/kg.</li>
	<li>Place the mouse into the induction chamber with an isoflurane flow rate of 4% to anesthetize it until spontaneous movement of body and vibrissae stops.</li>
	<li>Transfer the mouse in lateral position with its nose into the anesthesia mask and maintain isoflurane concentration at 4% for approximately another minute, then reduce and it at approx.1.5% to maintain appropriate anesthesia.</li>
	<li>Apply dexpanthenol eye ointment on both eyes.&#160;</li>
</ol>
2. Distal MCAO Model
<ol>
	<li>Make a 1 cm skin incision between the ear and eye using little operation scissors&#160;after aseptical preparation of the surgical site using skin disinfectant.</li>
	<li>Separate the skin and localize the temporal muscle.</li>
	<li>Select in the high frequency generator the coagulation function, bipolar mode, select 12 W and connect the electrocoagulation forceps with the cable.</li>
	<li>Add a drop of saline and use the forceps to detach the temporal muscle from the skull in its apical and dorsal part, thereby, making a muscle flap without totally removing the muscle.</li>
	<li>Identify the MCA below the transparent skull, in the rostral part of the temporal area, dorsal to the retro-orbital sinus (Figure 2A). If the MCA bifurcation is not visible (due to an anatomical normal variation) identify the vessel most rostral.</li>
	<li>Add some saline on the skull and thin out the bone with the drill right above the MCA branch until it has a thin and translucent texture (Figure 2B).</li>
	<li>Carefully withdraw the bone above the artery with a very thin forceps.</li>
	<li>Select bipolar mode in the high frequency generator at 7 W. Coagulate the artery with the electrocoagulation forceps proximal and distal to the bifurcation (Figure 2C). When the bifurcation is not visible due to an anatomical variant, coagulate the correctly identified MCA branch (see above) at two sites of approx. 1mm distance. It is not necessary to grasp the artery with the forceps for coagulation, touching the artery carefully with the forceps on both sides from above is sufficient and induces less mechanical damage.</li>
	<li>Wait 30 sec and gently touch the artery with a blunted forceps to check for any blood flow due to spontaneous recanalization. In case of recanalization repeat the electrocoagulation once.</li>
	<li>Relocate the temporal muscle to its position, covering the burr hole.</li>
	<li>Suture the wound and place the animal in a nursing box at 32 &#176;C to recover from anesthesia and return it to the cage. In general it takes 5-10 min for the animal to recover from anesthesia.</li>
	<li>Inject postoperative analgesia (i.p.) after 24 hr and then daily until the fifth postoperative day: Carprofen 4 mg/kg.</li>
</ol>
3. Sham Operation
Perform all procedures identically to the operation described above &#8211; including thinning of the skull and its removal &#8211; except for not coagulating the exposed artery.
4. Cylinder Test13
<ol>
	<li>Place the animal in a transparent acrylic glass cylinder (diameter: 8 cm; height: 25 cm) in front of two mirrors and videotape for 5 min. Adjust the camera centrally in front of the two mirrors and the cylinder to obtain an optimal video (Figure 3A).</li>
	<li>For assessment of independent forelimb use, score (1) contact of the cylinder wall with one forelimb during full rear and (2) landing with only one forelimb on floor after full rear. Count at least 20 contacts for one forelimb using slow motion or frame-by-frame function of the Video Lan Client (VLC) freeware software (<a href="http://www.videolan.org/vlc">http://www.videolan.org/vlc</a>).</li>
	<li>For baseline analysis before surgery: perform the test twice per mouse, with a 1 hr break between trials. Forelimb use is expressed as a ratio of right/left-sided, independent forelimb use.</li>
	<li>After the MCA coagulation: perform the test again twice per mouse, with a 1 hr break between trials as indicated above.</li>
</ol>
5. Perfusion
<ol>
	<li>Anaesthetize the animals (e.g., by ketamine and xylazine 120/16 mg/kg body weight, respectively).</li>
	<li>Fix the animal in a supine position and open the abdominal cavity with a median cut. Remove rips and sternum. Make a small incision in the right atrium. Insert a perfusion cannula in the left ventricle and slowly perfuse with 20 ml of saline.</li>
	<li>Decapitate the animal, open the calvaria and gently detach the brain from the base of the skull.</li>
	<li>Freeze the brains in -40 &#176;C cold isopentane for 10 min. Afterwards remove the brains from the isopentane and store until further analysis at -80 &#176;C.</li>
</ol>
6. Infarct Volumetry
<ol>
	<li>Cryosectioning: Cut the brains serially on a cryostat to 20 &#181;m thick sections every 400 &#181;m on slides and store the slides at -20 &#176;C.</li>
	<li>Cresyl violet (CV) staining:
	<ol>
		<li>Prepare the staining solution: Mix 0.5 g of CV acetate in 500 ml H2O. Stir and heat (60 &#176;C) until crystals are dissolved. Let the solution cool and store in a dark bottle. Reheat to 60 &#176;C and filter before every use.</li>
		<li>Dry the slides at RT for 30 min. Then place them in 95% ethanol for 15 min and then in 70% ethanol for 1 min and afterwards in 50% ethanol for 1 min.</li>
		<li>Place the slides in distilled water for 2 min, refresh distilled water and place them in for 1 min. Afterwards place the slides in staining solution (60 &#176;C) for 10 min and wash them twice in distilled water for 1 min.</li>
		<li>Place the slides in 95% ethanol for 2 min. Then place them into 100% ethanol for 5 min, refresh the 100% ethanol and place them in for 2 min. Afterwards cover the slides with mounting medium.</li>
		<li>Analysis: 
		Scan the slides and analyze the indirect infarct volume by the Swanson method14 to correct for edema: 
		(Ischemic area) = (Cortex area of contralateral side) &#8211; (Non-ischemic cortex area of ipsilateral side) (Figure 4A).</li>
	</ol>
	</li>
</ol>

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<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, 396-409 (2005).</li>
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<li>Swanson, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+semiautomated+method+for+measuring+brain+infarct+volume%5BTitle%5D)+AND+%22J.+Cereb.+Blood+Flow+Metab%22%5BJournal%5D)">A semiautomated method for measuring brain infarct volume.</a> J. Cereb. Blood Flow Metab. 10, 290-293 (1990).</li>
<li>Kitano, H., et al. <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+%22J.+Cereb.+Blood+Flow+Metab%22%5BJournal%5D)">Inhalational anesthetics as neuroprotectants or chemical preconditioning agents in ischemic brain.</a> J. Cereb. Blood Flow Metab. 27, 1108-1128 (2007).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+intravenous+recombinant+tissue+plasminogen+activator+on+ischemic+stroke+lesion+size+measured+by+computed+tomography.+NINDS%3B+The+National+Institute+of+Neurological+Disorders+and+Stroke+%28NINDS%29+rt-PA+Stroke+Study+Group%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Effect of intravenous recombinant tissue plasminogen activator on ischemic stroke lesion size measured by computed tomography. NINDS; The National Institute of Neurological Disorders and Stroke (NINDS) rt-PA Stroke Study Group.</a> Stroke. 31, 2912-2919 (2000).</li>
<li>Sowell, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+cortical+change+across+the+human+life+span%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">Mapping cortical change across the human life span.</a> Nat. Neurosci. 6, 309-315 (2003).</li>
<li>Brott, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurements+of+acute+cerebral+infarction%3A+lesion+size+by+computed+tomography%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Measurements of acute cerebral infarction: lesion size by computed tomography.</a> Stroke. 20, 871-875 (1989).</li>
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<li>Liesz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Boosting+regulatory+T+cells+limits+neuroinflammation+in+permanent+cortical+stroke%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Boosting regulatory T cells limits neuroinflammation in permanent cortical stroke.</a> J. Neurosci. 33 (44), 17350-17362 (2013).</li>
<li>Liesz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+lymphocyte+trafficking+shields+the+brain+against+deleterious+neuroinflammation+after+stroke%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Inhibition of lymphocyte trafficking shields the brain against deleterious neuroinflammation after stroke.</a> Brain. 134, 704-720 (2011).</li>
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<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+%22Nat.+Protoc%22%5BJournal%5D)">The adhesive removal test: a sensitive method to assess sensorimotor deficits in mice.</a> Nat. Protoc. 4, 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+%22J.+Neurosci.+Methods%22%5BJournal%5D)">A test for detecting long-term sensorimotor dysfunction in the mouse after focal cerebral ischemia.</a> J. Neurosci. Methods. 117, 207-214 (2002).</li>
<li>Orset, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+model+of+in+situ+thromboembolic+stroke+and+reperfusion%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Mouse model of in situ thromboembolic stroke and reperfusion.</a> Stroke. 38, 2771-2778 (2007).</li>
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</ol>

The authors have no competing interests to disclose.

The present protocol describes the experimental stroke model of distal, permanent MCAO by transcranial electrocoagulation &#8211; the so-called &#8220;coagulation model&#8221;. This model has meanwhile become one of the most frequently used animal models in experimental stroke research12. Compared to other focal brain ischemia models, the coagulation model as presented in this video has the advantage of a very short operation time of approximately 10 min when performed by a trained scientist. Hence, brief anesthesia times can be achieved in this model, which is a favorable feature of an experimental stroke model because the impact of anesthetics on neuroprotection and stroke outcome is well-known15. Moreover, as previously described by Carmichael et al.11, we corroborate that the resulting infarct volume and localization after distal permanent MCA coagulation corresponds to ischemic brain lesions in the majority of human strokes in proportion to brain size. Human stroke is mainly small in size with lesions of approximately 5-15% of the hemisphere, based on previous population studies and clinical imaging trials16-18, in contrast to extensive stroke lesions with compressive brain edema which occur in less than 10% of clinical strokes19. Thereby, stroke lesions in the MCA territory of around 12% of the hemisphere achieved by the presented model can be regarded as a translationally relevant stroke volume. However, it has to be taken into account that different mouse strains or used anesthesia protocols might affect the resulting lesion volume20.
Mortality during the observation period after stroke induction in this model is virtually absent. The overall mortality of less than 5% consists mainly of deaths during operation because of anesthesiological complications or sacrifice because reaching of exclusion criteria. In order to warrant the low variability of this model and its excellent reproducibility, we suggest the following exclusion criteria: 1) Any subarachnoid hemorrhage during the operation. 2) Operation time longer than 15 min. 3) Recanalization of the MCA after two attempts for electrocoagulation with only transient MCAO. Moreover, animals need to be examined daily after MCAO (basic physiological behavior, fur appearance and body weight) to control for pain, discomfort or sickness behavior.
Several measures might be implemented for analysis of stroke outcome like laser speckle measurement, magnetic resonance imaging, behavioral tests or histological analysis. In this protocol we provide exemplary methods for behavior analysis and infarct volume analysis. Several test for behavioral analysis after focal brain ischemia have been developed and used in experimental stroke research. Suitable tests for sensorimotor dysfunction previously used by our group in this stroke model21,22 were the Rotarod test23, Sticky label test24, Corner test25 and the Cylinder test13, which is demonstrated in this video. The cylinder test consistently depicts motor asymmetry in the acute phase after distal permanent MCAO and also detects consecutive regain of motor function.
Despite the obvious advantages, some limitations of this stroke model have to be taken into account. First, a trepanation of the skull is needed in order to coagulate the artery, thereby producing a potential access for peri-operational infections of the brain, although bacterial infections of the surgical wound, temporal muscle or the brain itself have never been detected by ourselves or documented by others using this model. Moreover, mechanical damage to the cortex during preparation and coagulation cannot be excluded but can be limited by careful drilling and removal of the skull, constant humidification of the surgical site and minimal necessary electrocoagulation (see exclusion criteria). Although the course of the MCA as depicted in Figure 2 is found in the majority of C57Bl/6 mice, we describe in the protocol how to proceed in normal variations of the vessel course to minimize model variability. Furthermore, we suggest the use of multiple (3 in case of bifurcation; 2 without bifurcation of the MCA) occlusion sites to minimize the risk of partial recanalization of the MCA, which in our experience is an important factor for the variability of this model.
In terms of behavioral tests, only minor behavioral deficits can be detected in the above-mentioned behavioral tests and functional regeneration can be observed within the first week after stroke. Thus, more advanced test systems with higher sensitivity and qualitative test parameters like the skilled reaching test27 might be more suitable to detect long-term functional outcome in this model.
Finally, due to the permanent coagulation of the MCA no reperfusion can be obtained, which is a feature observed in a substantial percentage of stroke patients due to spontaneous clot lysis or therapy28. However, a previously described thromboembolic stroke model26 provides the option for a complimentary stroke model with reperfusion of cortical brain ischemia. Taken together, the high reproducibility, the possible long-term observation due to minimal mortality and the comparable relative infarct volume and localization in respect to human stroke distinguish the &#8220;coagulation model&#8221; as a valuable model for basic and translational stroke research.

Stroke is the third most common cause of death and one the main reasons of acquired adult disability in developed countries1. Approximately 80% of this acute neurological disease is caused by brain ischemia resulting from an obstruction of cerebral blood flow while about 15% are caused by an intracerebral hemorrhage2. Despite ongoing research, intravenous administration of tissue plasminogen activator is the only approved pharmacological treatment for ischemic stroke so far and only available to a minority of stroke patients due to the short approved time window of 4.5 hr after stroke onset3,4. As there are no in vitro models, which can properly model the complex interactions between the brain, vasculature and systemic pathophysiological mechanisms during stroke, animal models are essential for preclinical stroke research.
Therefore, several ischemic stroke models have been developed in a variety of species. One of the most commonly used stroke models is the &#8220;filament model&#8221; where a suture filament is transiently introduced into the&#160;internal carotid artery&#160;and forwarded until the tip occludes the origin of the&#160;Middle Cerebral Artery (MCA), resulting in a stop of blood flow and subsequent brain infarction&#160;of subcortical and in case of prolonged occlusion also cortical regions5,6. In photothrombotic models of ischemic stroke, a photochemical occlusion of the irradiated cortical vessel is achieved after injection of a photosensitizer resulting in small, locally circumscribed lesions7. The permanent occlusion of the MCA distal of the lenticulostriatal arteries can be achieved by a ligation of the artery, its transient compression or by permanent coagulation8,9. The resulting infarct in this model predominantly affects the neocortex10 because the occlusion of the MCA in this model is distal to the lenticulostriatal arteries, which supply the basal ganglia.
Since the majority of human stroke lesions are located in the territory of the middle cerebral artery, all of the common stroke models resemble occlusions of the MCA or one of its branches11. The MCA is one of the major arteries that provides blood supply to the brain; it arises from the internal carotid artery, routes along the lateral sulcus&#160;where it then branches and projects to the basal ganglia and the lateral surfaces of the frontal, parietal and temporal lobes, including the primary motor and sensory cortex. The right and left MCA are connected to the&#160;anterior cerebral arteries and the&#160;posterior communicating arteries, which connect to the&#160;posterior cerebral arteries, creating the Circle of Willis (Figure 1).
As previously reported by Carmichael et al.11, infarcts modeled by the distal Middle Cerebral Artery Occlusion (MCAO) model in mice encompass about 10-15% of the hemisphere, thereby mimicking a majority of human stroke lesions which are located in the cortical MCA territory11,12. In 1981, Tamura et al. described a permanent, transcranial MCAO coagulation model in rats 8. However, the model described by Tamura involved a proximal occlusion of the MCA in order to circumvent the more distal bifurcation of the artery. Thus, the original &#8220;Tamura model&#8221; induces not only cortical but also striatal lesions, similar to the lesions obtained by the &#8220;filament model&#8221;6. Here, we describe the permanent distal MCAO model by transcranial electrocoagulation in mice. Additionally, we report related histological and functional methods to analyze the stroke outcome in this model. All methods are based on standard operating procedures developed and used in our laboratories.

<table><tbody><tr><td>Camera Canon Eos 500D</td><td>Canon</td><td></td><td>Optics: 18-55 mm; 30 fps; 640 x 480 video function</td></tr><tr><td>Mirror</td><td>Kristallform</td><td>2677089</td><td>30 x 30 cm&amp;nbsp;</td></tr><tr><td>Transparent acrylic glass cylinder</td><td>H&amp;#38;S Kunststofftechnik</td><td></td><td>Diameter: 8 cm,&amp;nbsp; height: 25 cm</td></tr><tr><td>Heating blanket</td><td>FHC DC Temperature Controller</td><td></td><td></td></tr><tr><td>Fine Scissors</td><td>FST</td><td>15000-00</td><td></td></tr><tr><td>Mayo Scissors</td><td>FST</td><td>1410-15</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>11616-15</td><td></td></tr><tr><td>Cottons</td><td>NOBA Verbondmitel Danz</td><td>974116</td><td></td></tr><tr><td>Saline solution</td><td>Braun</td><td>131321</td><td></td></tr><tr><td>Bepanthen pommade</td><td>Bayer</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Abbot</td><td>B506</td><td></td></tr><tr><td>Anesthesia system for isoflurane</td><td>Drager</td><td></td><td></td></tr><tr><td>Stereomikroskop&amp;nbsp;</td><td>Zeiss</td><td>Stemi DV4</td><td></td></tr><tr><td>Electrosurgical device</td><td>ERBETOM ICC 80/50 HF-Chirurgieger&amp;auml;t</td><td></td><td></td></tr><tr><td>Drill</td><td>Proxxon</td><td>D-34343</td><td></td></tr><tr><td>Ketamine</td><td>Inresa Arzneimittel GmbH</td><td></td><td></td></tr><tr><td>Xylacine</td><td>Albrecht</td><td></td><td></td></tr><tr><td>5ml Syringe&amp;nbsp;</td><td>Braun</td><td></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>Isopentane</td><td>Fluka</td><td>59070</td><td></td></tr><tr><td>Cryostat</td><td>Thermo Scientific CryoStarNX70</td><td></td><td></td></tr><tr><td>Superfrost Plus Slides</td><td>Thermo Scientific</td><td>J1800AMNZ</td><td></td></tr><tr><td>Cresyl violet</td><td>Sigma Life Science</td><td>C5042-10G</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma Life Science</td><td>695092</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>Roti-Histokit mounting medium</td><td>Roth</td><td>6638.1</td><td></td></tr><tr><td>C57Bl/6J mice</td><td>Charles River</td><td>000664</td><td></td></tr></tbody></table>

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.

Due to the short anesthesia time and moderate brain damage, approximately 10 min after transfer to their cages all the animals were awake, freely moving in the cage and interacting with littermates. The mortality during the MCAO surgery was less than 5%, mainly as a result of accidental subarachnoid hemorrhage or incorrect anesthesia. Mortality during the 7 day observation time after stroke induction occurs only very rarely in approximately 1-2% of animals. In the operation series of 10 animals for this report all of the animals survived the operation and the 7 day observation period, none of them had to be excluded due to exclusion criteria.
Behavioral deficits after MCA coagulation were assessed by the cylinder test13 analyzing forepaw use asymmetry. In this test, the ratio of independent left and right forepaw use is measured at the indicated time points after stroke induction and compared to baseline values obtained 24 hr before MCAO (Figure 3B). The animals presented a significant change in limb use asymmetry for combined wall exploration in the cylinder test 24 hr (1.72 ± 0.326; p &lt; 0.05) and 3 days (1.36 ± 0.17; p &lt; 0.05) after MCAO. Although the ratio improved during the 1 week observation time, motor asymmetry was still significant 7 days after MCAO (1.35 ± 0.29; p &lt; 0.05) compared to baseline values.
We performed infarct volumetry using cresyl violet stained serial coronal brain sections 7 days after stroke induction (Figure 4B). Mean infarct volume was 15.4 mm3, thereby representing 12% of one brain hemisphere (Figure 4C). The variability of this stroke model is exceptionally low with a standard deviation of approximately 10%. The lesion area encompasses the somatosensory and motor cortex with only minor affection of subcortical structures. Moreover, localization of the infarct area is highly predictable with only minimal variability as shown in the schematic distribution diagram (Figure 4D).
<img alt="Circle of Willis diagram showing major cerebral arteries and distal MCA occlusion site." fo:content-width="5in" src="/files/ftp_upload/51729/51729fig1highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Schematic representation of the Circle of Willis. The arterial Circle of Willis is formed by the middle cerebral arteries (MCA) and the anterior cerebral arteries (ACA) which branch from the internal carotid artery (ICA), as well as by the posterior cerebral arteries (PCA) and the posterior communicating arteries (PComA). The MCA runs into the lateral sulcus where it branches to the cerebral cortex. The dominant MCA branch supplying the major part of the motor cortex and part of the somatosensory cortex is permanently occluded by the demonstrated model (AcomA = Anterior communicating artery; BA =Basilar artery; SuCA =Superior cerebellar artery).
<img alt="Mouse brain electrode application; brain stimulation method; neuroscience experimental diagram." fo:content-width="3in" src="/files/ftp_upload/51729/51729fig2highres.jpg" width="400" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Transcranial view after removal of the temporal muscle and schematic view of MCA occlusion. (A) After removing the temporal muscle the cortical arteries can be viewed through the partially translucent mouse skull (in 8-12 week old mice). The dominant MCA branch can be identified in the rostral part of the temporal view as well as further cortical arteries branching from the MCA and the PCA in the caudal part. (B) Schematic view on the dominant MCA branch in its predominant variation with a bifurcation on the lateral temporal cortex after drilling a burr hole and removing the skull. (C) Black squares represent the MCA coagulation sites at the proximal and distal sides of the bifurcation.
<img alt="Mouse behavioral test setup and bar graph comparing forelimb use ratios across time points." fo:content-width="4in" src="/files/ftp_upload/51729/51729fig3highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Analysis of behavioral deficits. (A) Cylinder test set-up: the mouse is placed into a vertical cylinder and mirrors are placed behind in order to register all of the movements using a video camera. (B) Forelimb use asymmetry was analyzed using the cylinder test. Left/right ratios of independent forelimb use were calculated 24 hr before MCAO and at the indicated time points after stroke induction. N = 10, *p &lt; 0.05 between indicated time point and baseline (control) value.
<img alt="Mouse brain infarction analysis, MCAO experiment, infarct volume graph, Bregma level slices diagram." fo:content-width="5in" src="/files/ftp_upload/51729/51729fig4highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Volumetric infarct analysis and infarct outcome after distal MCAO. (A) Representative image of a cresyl violet stained coronal brain section 7 days after MCAO. The yellow line is indicating selection of the contralateral (right) cortex and the red line is selecting the non-infarcted (stained) cortex of the ipsilateral brain. The pale area in the left hemisphere depicts the infarcted tissue area. (B) Infarct volume analysis of 10 brains (each dot representing one individual brain) 24 hr, 3 days and 7 days after distal MCA coagulation. The horizontal red line represents the mean, error bars indicate standard deviation. (C) Representative cresyl violet stained coronal brain sections every 400 µm at 7 days after MCA coagulation. (D) Schematic distribution of the infarcted brain tissue 7 days after MCAO. Each slide depicts the accumulated information of infarct distribution (color coded as indicated) at the given section in relation to the bregma (modified image from: Liesz A et al., Brain, 2011; by permission of Oxford University Press). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51729/51729fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery at JoVE.com

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

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 ...

modeling-stroke-mice-permanent-coagulation-distal-middle-cerebral

Nanyang Technological University

Research

JoVE Journal

Medicine

37.0K Views.  University Hospital Munich.  Verschillende muismodellen van midden cerebrale slagader occlusie (MCAO) worden veel gebruikt in experimenteel onderzoek hersenen. Hier tonen we het model van transcraniële permanente distale MCAO die consistent zijn corticale infarct van een omvang die overeenkomt met die door de meerderheid van de menselijke ischemische beroertes schade veroorzaakt. 

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

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 ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

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

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

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

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

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, ...

Neuroscience

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.

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 ...

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 ...

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

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