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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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.

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			<td height="21" style="height:21px;width:281px;">Camera Canon Eos 500D</td>
			<td style="width:224px;">Canon</td>
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			<td style="width:324px;">optics: 18-55 mm; 30 fps; 640x480 video function</td>
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			<td height="21" style="height:21px;width:281px;">Mirror</td>
			<td style="width:224px;">Kristallform</td>
			<td style="width:119px;">2677089</td>
			<td>30x30 cm&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Transparent acrylic glass cylinder</td>
			<td style="width:224px;">H&#38;S Kunststofftechnik</td>
			<td style="width:119px;"></td>
			<td>diameter: 8 cm,&#160; height: 25 cm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Heating blanket</td>
			<td>FHC DC Temperature Controller</td>
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			<td></td>
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			<td height="21" style="height:21px;width:281px;">Phosphate Buffered Saline PH: 7,4</td>
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			<td height="21" style="height:21px;width:281px;">Ethanol 96%</td>
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			<td height="21" style="height:21px;">Ethanol 99%</td>
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			<td height="21" style="height:21px;width:281px;">Roti-Histokit mounting medium</td>
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			<td height="21" style="height:21px;width:281px;">C57Bl/6J mice</td>
			<td style="width:224px;">Charles River</td>
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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 &#177; 0.326; p &#60; 0.05) and 3 days (1.36 &#177; 0.17; p &#60; 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 &#177; 0.29; p &#60; 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="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51729/51729fig1highres.jpg" width="500" /> 
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="Figure 2" fo:content-width="3in" src="/files/ftp_upload/51729/51729fig2highres.jpg" width="400" /> 
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="Figure 3" fo:content-width="4in" src="/files/ftp_upload/51729/51729fig3highres.jpg" width="500" /> 
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.&#160;(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 &#60; 0.05 between indicated time point and baseline (control) value.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51729/51729fig4highres.jpg" width="500" /> 
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.&#160;(C) Representative cresyl violet stained coronal brain sections every 400 &#181;m 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/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 ...

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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 ...

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

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

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

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

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

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 ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...