Name: modeling stroke in mice: transient middle cerebral artery occlusion via the external carotid artery
Uploaded: 2021-05-24T14:00:00
Description: Watch this Scientific Journal Video about Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery at JoVE.com

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

The experiments reported in this video were conducted following the national guidelines for the use of experimental animals, and the protocols were approved by the German governmental committees (Regierung von Oberbayern, Munich, Germany). Ten-week-old male C57Bl/6J mice were used and housed under controlled temperature (22 &#177; 2 &#176;C), with a 12 h light-dark cycle period and access to pelleted food and water ad libitum.
1. Preparation of the material and instruments
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The present protocol describes an experimental stroke model based on the consensus agreement of a German multicenter research consortium (&#8220;ImmunoStroke&#8221;) to establish a standardized transient MCAo model. The transient MCAo model established by introducing a silicon-coated filament through the ECA to the origin of the MCA is one of the most widely used stroke models to achieve arterial reperfusion after a delimitated occlusion period. Therefore, this procedure can be considered a translationally relevant strok...

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

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			<td>45&#176; ramp</td>
			<td>H&#38;S Kunststofftechnik</td>
			<td></td>
			<td>height: 18 cm</td>
		</tr>
		<tr>
			<td>5/0 threat</td>
			<td>Pearsalls</td>
			<td>10C103000</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Syringe</td>
			<td>Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma Life Science</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia system for isoflurane</td>
			<td>Drager</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bepanthen pomade</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57Bl/6J mice</td>
			<td>Charles River</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp</td>
			<td>FST</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Clip</td>
			<td>FST</td>
			<td>18055-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Clip holder</td>
			<td>FST</td>
			<td>18057-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotons</td>
			<td>NOBA Verbondmitel Danz</td>
			<td>974116</td>
			<td></td>
		</tr>
		<tr>
			<td>Cresyl violet</td>
			<td>Sigma Life Science</td>
			<td>C5042-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Thermo Scientific CryoStarNX70</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>CLN Chemikalien Laborbedorf</td>
			<td>521005</td>
			<td></td>
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		<tr>
			<td>Ethanol 96%</td>
			<td>CLN Chemikalien Laborbedorf</td>
			<td>522078</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 99%</td>
			<td>CLN Chemikalien Laborbedorf</td>
			<td>ETO-5000-99-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Filaments</td>
			<td>Doccol</td>
			<td>602112PK5Re</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine 45 angled forceps</td>
			<td>FST</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>FST</td>
			<td>11252-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>FST</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>Orechseln</td>
			<td>BSI-112</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardener Glue</td>
			<td>Drechseln &#38; Mehr</td>
			<td>BSI-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating blanket</td>
			<td>FHC DC Temperature Controller</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbot</td>
			<td>B506</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopentane</td>
			<td>Fluka</td>
			<td>59070</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Inresa Arzneimittel GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Doppler</td>
			<td>Perimed</td>
			<td>PF 5010 LDPM, Periflux System 5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Doppler probe</td>
			<td>Perimed</td>
			<td>91-00123</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline pH: 7.4</td>
			<td>Apotheke Innestadt Uni Munchen</td>
			<td>P32799</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery chamber</td>
			<td>Mediheat</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Roti-Histokit mounting medium</td>
			<td>Roth</td>
			<td>6638.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td>Braun</td>
			<td>131321</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>02.001.30.011</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon-coated filaments</td>
			<td>Doccol</td>
			<td>602112PK5Re</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicropscope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Slides</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>FST</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylacine</td>
			<td>Albrecht</td>
			<td></td>
			<td></td>
		</tr>
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modeling stroke in mice: transient middle cerebral artery occlusion via the external carotid artery

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

The model described here is a modification of the commonly used &#34;filament&#34; stroke model, which consists of introducing a silicon-coated filament through the ECA to transiently block the origin of the MCA (Figure 1). After removing the filament, only the blood flow in the ECA is permanently ceased, allowing complete recanalization of the CCA and ICA. This allows an adequate reperfusion of the brain (Figure 2), similar to the situation observed after succe...

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

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

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

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

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

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

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

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

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

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, ...

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

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

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

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

Innovative Techniques and Future Directions in Stroke Research (Video) | JoVE

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

Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model (Video) | JoVE

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...