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

The experiments reported in this video were conducted following the national guidelines for the use of experimental animals, and the protocols were approved by the German governmental committees (Regierung von Oberbayern, Munich, Germany). Ten-week-old male C57Bl/6J mice were used and housed under controlled temperature (22 &#177; 2 &#176;C), with a 12 h light-dark cycle period and access to pelleted food and water ad libitum.
1. Preparation of the material and instruments
<ol>
	<li...

<ol>
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The authors have no competing interests to disclose.

The present protocol describes an experimental stroke model based on the consensus agreement of a German multicenter research consortium (&#8220;ImmunoStroke&#8221;) to establish a standardized transient MCAo model. The transient MCAo model established by introducing a silicon-coated filament through the ECA to the origin of the MCA is one of the most widely used stroke models to achieve arterial reperfusion after a delimitated occlusion period. Therefore, this procedure can be considered a translationally relevant 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.

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

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

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

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

In the filament model described here, complete restoration of blood flow and it's recording, are requirements to ensure a producible influx among mice, especially in translational stroke studies. This model has two main advantages compared to other previously described stroke models. No craniotomy is required, and a complete re-perfusion of the blood vessel is achieved.The filament model is a complex surgical intervention involving the process manipulation of different arteries. The visualization and explanation of all these are small but important steps, might accelerate the learning period of neurosurgeons. Connect the heat blanket to maintain the temperature of the operation area and the mouse body temperature during anesthesia at 37 degrees Celsius.Prepare autoclave scissors and forceps, 70%ethanol solution and dexpanthenol eye ointment, and keep several pieces of cotton and Five O coated braided polyester suture, ready to use. Prepare a 0.9%saline solution in a one milliliter syringe without a needle to keep the incision site hydrated. Prepare a holder for the laser Doppler probe by cutting the tip of a 10 microliter pipette tip.Once the mouse is completely anesthetized, place it in a prone position in the operation area. Gently insert the rectal probe to monitor the temperature throughout the surgical procedures. After disinfecting the surgical site, cut this scalp between the left ear and the eye to expose the skull bone.Next, cut and retire the temporal muscle to visualize the multiple cerebral arteries or MCA beneath the skull. Fix the outer part of the tip holding the laser Doppler probe or fiber on top of the left MCA with glue and close the skin so that the skin is glued as well. Apply two to three drops of hardener glue to speed up the process.Make sure that the laser Doppler fiber is not glued and can be easily removed from the tip holder at any time. Turn the mouse to the supine position, then put the snout into the anesthesia cone and fix the paws with tape. Disinfect the skin and hair surrounding the chest and make a two centimeter long midline incision in the neck.Use forceps to pull the skin submandibular gland and sternomastoid muscle apart. Use retractors to expose the surgical field and find the left common carotid artery or CCA. Dissect the CCA free from connective tissue and surrounding nerves without harming the vagal nerve and perform a transient ligation before the bifurcation.Dissect the external carotid artery or ECA and tie a permanent knot at the most distal visible part. Place another suture under the ECA and close to the bifurcation, then prepare a loose knot to be used later. Dissect the internal carotid artery or ICA and place a microvascular clip on it about five millimeters over the bifurcation.Make sure not to damage the vagal nerve. Then cut a small hole into the ECA between the tight and the loose ligations, ensuring not to cut the entire ECA. Introduce the filament and advance it towards the CCA.Tighten the loose ligation in the ECA around the lumen to momentarily secure the filament in that position. Removing the microvascular clip and prevent bleeding. After removing the microvascular clip, insert the filament through the ICA until the origin of the MCA is reached.Fix the filament in this position by further tightening the knot around the ECA. Record laser Doppler values before and after filament insertion. Remove the retractor and relocate the sternomastoid muscle in the submandibular gland before suturing the wound.Remove the laser Doppler probe and place the animal in a recovery chamber at 37 degrees Celsius for what one hour. Place the mouse in a prone position in the operation area with its snout in the anesthesia mask, then fix the animal's paws with tape. Insert the laser Doppler probe into the probe holder.Remove the wound suture and use forceps to pull the skin, the submandibular gland and the sternomastoid muscle apart. Use retractors to expose the surgical field. Loosen the ECA suture that tightens the filament and gently pull the filament.Avoid damaging the silicone rubber coating of the filament during the removal. Record the laser Doppler values before and after filament removal. Tightly tie the ECA suture and confirm the increase in the cerebral blood flow in the laser Doppler device.Open the transient ligation before the bifurcation from the CCA to restore the complete blood flow. Remove the retractor and relocate the sternomastoid muscle in the submandibular gland before suturing the wound. Then, place the animal in a recovery chamber at 37 degrees Celsius for one hour to recover from anesthesia.After recovery return the mice to their cages in a temperature controlled room and take care of the animals by adding wet food pellets and hydrogel in small Petri dishes on the cage floor and inject analgesia every 12 hours until day three after the surgery. Once the blood flow in the CCA is restored after removing the filament, complete re-perfusion of the brain occurs, which is similar to the situation observed after successful mechanical thrombectomy in human patients. Stroke animals presented a significant change in the composite and focal neuro score, but not in the general neuro score when compared to sham animals.Infarct volume retrieved was also performed using cresol violet staining of coronal serial brain sections 24 hours after stroke induction. The lesion area includes the somatosensory and motor cortex, as well as sub-cortical structures, such as the striatum. The infarct volume mean was 61 to 62 millimeters square, representing 48%of the effected brain hemisphere.It is important to perform an adequate dissection of the CCA, ECA and RCA without causing damage to the adjacent tissue, especially the vagus nerve, to have a good visualization of all structures, and to be able to insert the filament and occlude the MCA origin.

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Research

JoVE Journal

Neuroscience

6.6K Views.  LMU Munich. In the filament model described here, complete restoration of blood flow and it's recording, are requirements to ensure a producible influx among mice, especially in translational stroke studies. This model has two main advantages compared to other previously described stroke models. No craniotomy is required, and a complete re-perfusion of the blood vessel is achieved.The filament model is a complex surgical intervention involving the process manipulation of different arteries. The vi...