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

Summary

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.

Abstract

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 “replication crisis”, detailed standardized models for all procedures are urgently needed. As an effort within the “ImmunoStroke” 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 “filament” stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Introduction

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, 80–85% 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. 

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 neocortex^3,4. Besides those, the most commonly used stroke model is probably the so-called “filament model,” 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 "filament model" 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 “filament model” 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 “filament model.” 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 far^2,8. A potential cause of the current “replication crisis,” 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 “ImmunoStroke” 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.

Protocol

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 ± 2 °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

1. Connect the heat blanket to maintain the temperature of the operation area and the mouse body temperature during anesthesia at 37 °C.
2. Autoclave scissors and forceps, prepare 70% ethanol solution and keep available dexpanthenol eye ointment, several pieces of cotton, and 5-0 coated braided polyester suture ready for use. Prepare a 1 mL syringe with 0.9% saline solution (without needle) to keep the animal's incision site hydrated. Prepare the anesthesia gas (100% O₂ + isoflurane).
3. Prepare a holder for the laser Doppler probe by cutting the tip of a 10 µL pipet tip (3-5 mm length).
   NOTE: All instruments are sterilized using a hot bead sterilizer. Surfaces are also disinfected before and after surgery with a microbial disinfectant spray. Prior to surgery, the areas surrounding the head and chest of mice are disinfected with a wound disinfection spray.

2. Preparation of the laser Doppler

1. Inject analgesia to the mouse 30 min before the surgery (4 mg/kg Carprofen and 0,1 mg/kg Buprenorphine, intraperitoneally).
2. Anesthetize the mouse by placing it in the induction chamber with an isoflurane flow rate of 4% until the cessation of spontaneous body movement and vibrissae.
3. Place the mouse in a prone position in the operation area with its nose in the anesthesia mask. Maintain isoflurane concentration at 4% for another minute, then reduce it and keep it at 2%.
4. Set the associated feedback-controlled heating pad for maintaining the mouse body temperature at 37 °C, and gently insert the rectal probe to monitor the temperature throughout the surgical procedures.
5. Apply dexpanthenol eye ointment on both eyes.
6. Disinfect the skin and hair surrounding the left eye and ear with 70% ethanol.
7. Cut the scalp between the left ear and the eye (1 cm long) to expose the skull bone.
8. Cut and retire the temporal muscle to visualize the MCA beneath the skull.
9. Fix with glue the outer part of the tip holding the laser Doppler probe/fiber on top of the left MCA with glue. Then, glue the skin to close the wound around the tip holder. Apply 2-3 drops of hardener glue to speed up the process. Make sure that the laser Doppler fiber is not glued and can be easily removed from the tip holder at any time.

3. Transient MCAo model (occlusion)

1. Turn the mouse into the supine position. Put the snout into the anesthesia cone and fix the paws with tape.
2. Disinfect the skin and hair surrounding the chest and make a 2-cm-long midline incision in the neck.
3. Use forceps to pull the skin and the submandibular glands apart. Use retractors to hold the sternomastoid muscle, expose the surgical field and find the left common carotid artery (CCA). Dissect the CCA free from connective tissue and surrounding nerves (without harming the vagal nerve) and perform a transient ligation before the bifurcation.
4. Dissect the external carotid artery (ECA) and tie a permanent knot at the most distal visible part. Place another suture under the ECA, close to the bifurcation, and prepare a loose knot to be used later.
5. Dissect the internal carotid artery (ICA) and place a microvascular clip on it, 5 mm over the bifurcation. Make sure not to damage the vagal nerve.
6. Cut a small hole into the ECA between the tight and the loose ligations; be careful not to cut the entire ECA.
7. Introduce the filament and advance it towards the CCA. Tighten the loose ligation in the ECA around the lumen to shortly secure the filament in that position and avoid bleeding when removing the microvascular clip.
8. Remove the microvascular clip and insert the filament through the ICA until the origin of the MCA is reached by detecting a sharp reduction (>80%) in the cerebral blood flow as measured by the laser Doppler. Fix the filament in this position by further tightening the knot around the ECA.
   NOTE: When the filament goes toward the appropriate direction, it advances smoothly, and no resistance should be observed.
9. Record laser Doppler values before and after filament insertion.
10. Remove the retractor and relocate the sternomastoid muscle and the submandibular glands before suturing the wound. Remove the laser Doppler probe, and place the animal in a recovery chamber at 37 °C for 1 h (until filament removal).

4. Transient MCAo model (Reperfusion)

1. Anesthetize the mouse by placing it in the induction chamber with an isoflurane flow rate of 4% until the cessation of spontaneous body movement and vibrissae.
2. Apply dexpanthenol eye ointment on both eyes.
3. Place the mouse in a prone position in the operation area with its snout in the anesthesia mask. Maintain isoflurane concentration at 4% for another minute, then reduce it and keep it at 2%. Fix the animal´s paws with tape.
4. Insert the laser Doppler probe into the probe holder.
5. Remove the wound suture, use forceps to pull the skin and the submandibular glands apart. Use retractors to gently pull the sternomastoid muscle and expose the surgical field.
6. 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.
7. Tightly tie the ECA suture.
8. Confirm the increase in the cerebral blood flow in the laser Doppler device (>80% of the initial value before reperfusion).
9. Record laser Doppler values before and after filament removal.
10. Open the transient ligation before the bifurcation from the CCA.
11. Remove the retractor, and relocate the sternomastoid muscle and the submandibular glands before suturing the wound. Place the animal in a recovery chamber at 37 °C for 1 h to recover from anesthesia.
12. After recovery, return the mice to their cages in a temperature-controlled room.
13. Take care of the animals by adding wet food pellets and hydrogel in small Petri dishes on the cage floor until day 3 after the surgery.
14. Inject analgesia every 12 h for 3 d after surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).

5. Sham operation

1. Perform all procedures as described above, including the ligation of the arteries and the introduction of the filament (steps 1-3.7).
2. Remove the filament immediately after its insertion. Then, place the animal in the recovery chamber for 1 h.
3. Place the animal in the operation area again, and remove the transient ligation of the CCA to ensure complete cerebral blood flow restoration.
4. Suture the wound, and place the animal in a recovery chamber at 37 °C for 1 h to recover from anesthesia. After recovery, return the mice to their cages in a temperature-controlled room.
5. Take care of the animals by adding wet food pellets and hydrogel in small Petri dishes on the cage floor until day 3 after surgery.
6. Inject analgesia every 12 h for 3 d after surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).

6. Neuroscore

1. Perform the Neuroscore always at the same time of the day, and use surgical clothes to maintain a "neutral smell" between individual surgeons.
2. Let the mice rest for 30 min in the room with an "open" cage before the test.
3. Observe each item in Table 1 and Table 2 for 30 s.

7. Intracardiac perfusion

1. Prepare a 20 mL syringe containing phosphate-buffered saline (PBS)-heparin (2 U/mL) and place it 1 m above the bench to facilitate gravity-driven perfusion. (OPTIONAL: Perform intracardiac perfusion with 4% paraformaldehyde (PFA) using a 20 mL syringe containing 4% PFA in PBS, pH 7.4).
2. Inject intrperitoneally 100 µL of ketamine and xylazine (120 and 16  mg/kg body weight, respectively). Wait 5 min and confirm the cessation of spontaneous body movement and vibrissae.
3. Fix the animal in a supine position, and disinfect the abdominal body surface with 70% ethanol.
4. Make a 3-cm-long incision into the abdomen; cut the diaphragm, the ribs, and sternum to visualize the heart completely.
5. Make a small incision in the right atrium, and insert the perfusion cannula into the left ventricle.
6. Perfuse with 20 mL of PBS-heparin.
7. After perfusion, decapitate the animal and remove the brain.
8. Freeze the brain on powdered dry ice and store at -80 °C until further use.

8. Infarct volumetry

1. For cryosectioning, use a cryostat to cut the brains into 20-µm-thick sections every 400 µm. Place the sections on slides, and store the slides at −80 °C until use.
2. Cresyl violet (CV) staining
   1. Prepare the staining solution by stirring and heating (60 °C) 0.5 g of CV acetate in 500 mL of H₂O until the crystals are dissolved. After the solution has cooled, store it in a dark bottle. Reheat to 60 °C and filter before every use.
   2. Let the slides dry at room temperature for 30 min. Immerse them in 95% ethanol for 15 min, in 70% ethanol for 1 min, and then in 50% ethanol for 1 min.
   3. Immerse the slides in distilled water for 2 min; refresh the distilled water and place the slides in the water for 1 min. Afterward, immerse the slides in the pre-heated staining solution for 10 min at 60 °C. Wash the slides twice in distilled water for 1 min.
   4. Immerse the slides in 95% ethanol for 2 min. Place them in 100% ethanol for 5 min; refresh the 100% ethanol and place the slides again in the ethanol for 2 min. Afterward, cover the slides with a mounting medium.
   5. Analysis (Figure 4C)
      1. Scan the slides and analyze the indirect infarct volume by the Swanson method¹² to correct for edema by using the following equation:
         (Ischemic area) = (ischemic region)-((ipsilateral hemisphere)-(contralateral hemisphere))

Representative Results

The model described here is a modification of the commonly used "filament" stroke model, which consists of introducing a silicon-coated filament through the ECA to transiently block the origin of the MCA (Figure 1). After removing the filament, only the blood flow in the ECA is permanently ceased, allowing complete recanalization of the CCA and ICA. This allows an adequate reperfusion of the brain (Figure 2), similar to the situation observed after successful pharmacological thrombolysis or mechanical thrombectomy in human patients. Moreover, this work also describes a method for measuring the cerebral blood flow during both occlusion and reperfusion procedures by fixing a cannula connected to the laser Doppler probe at the skull over the MCA territory.

The overall mortality rate of the surgical procedure is <5% when performed by a trained surgeon. At early time points after MCAo, animals generally present severe postural and movement deficits, general weakness, and loss in body weight¹³. These severe deficits are transient, and the animals show improved activity after approximately 1 week; thus, the deficits are more specific for focal neurological symptoms.

Behavioral deficits after MCA occlusion were assessed by the composite Neuroscore¹⁴; general and focal deficits were measured 24 h and 3 d after surgery. The general Neuroscore integrates 5 items (Table 1), including the evaluation of the fur, ears, eyes, posture, and spontaneous activity, with a maximum score of 18. The focal Neuroscore comprises 7 items (Table 2), including the evaluation of body symmetry, gait, climbing, circling behavior, forelimb symmetry, compulsory cycling, and whiskers response, with a maximum score of 28. The composite scale ranges from 0 (no deficits) to 46 (severe impairments). Stroke animals presented a significant change in the composite and focal Neuroscore, but not in the general Neuroscore, when compared to sham animals (Figure 3).

Infarct volumetry was also performed using Cresyl Violet staining of coronal serial brain sections 24 h after stroke induction. The infarct volume mean was 61.69 mm³, representing 48% of the affected brain hemisphere (Figure 4). When performed by a trained surgeon, the overall variability of this stroke model is low, with a coefficient of variation of <6%. The lesion area includes the somatosensory and motor cortex as well as subcortical structures such as the striatum (Figure 4).

Figure 1: Scheme for the access and intraluminal MCA occlusion. The filament (dotted line) is inserted between the proximal and distal suture knots in the ECA and advanced along the ICA until it reaches the origin of the MCA (see inset). Once in place, the ECA is ligated with a suture to fix the filament. Abbreviations: ACA = anterior cerebral artery; BA = basilar artery; CCA = common carotid artery; ECA = external carotid artery; ICA = internal carotid artery; MCA = middle cerebral artery; PCA = posterior communicating artery; PTG = pterygopalatine artery. This figure has been modified from Jackman et al.¹⁵. Please click here to view a larger version of this figure.

Figure 2: Blood flow during occlusion and reperfusion. Blood flow is registered before and after filament insertion and before and after filament removal. A reduction in the blood flow was observed during the occlusion and the restoration of the blood flow during the reperfusion. Every color represents one animal. Abbreviations: MCA = middle cerebral artery; CBF = cerebral blood flow; A.U. = arbitrary units. Please click here to view a larger version of this figure.

Figure 3: Neuroscore for functional deficits after tMCAo. (A) Total, (B) focal, and (C) general Neuroscore before and 24 h and 3 d after tMCAo. Open bars: sham; Black bars: tMCAo. n=10 per group. *p < 0.05. Abbreviations: tMCAo = transient middle cerebral artery occlusion; BL = before tMCAo. Please click here to view a larger version of this figure.

Figure 4: Volumetric infarct analysis and infarct outcome 24 h after tMCAo. (A) Representative cresyl violet-stained coronal brain sections every 400 µm at 24 h after tMCAo. Dashed lines demarcate the lesion area. (B) Analysis of infarct volume of 10 brains (each dot representing one individual brain) 24 h after tMCAo. The horizontal red line represents the mean (61.69 mm³), error bars indicate standard deviation (3.78 mm³). (C) Representative image for infarct volume calculation from a cresyl violet coronal section. Blue = Contralateral hemisphere; Red = Ipsilateral hemisphere; Pale striped area = Ischemic region. Please click here to view a larger version of this figure.

	Time-point of scoring		score
General Neuroscore	Hair	0. Hair neat and clean
		1. Localized piloerection and dirty hair in 2 body parts (nose and eyes)
		2. Piloerection and dirty hair in >2 body parts
	Ears (mouse on an open bench top)	0. Normal (ears are stretched laterally and behind, they react by straightening up following noise)
		1. Stretched laterally but not behind (one or both), they react to noise
		2. Same as 1. NO Reaction to noise.
	Eyes (mouse on OBT)	0. Open, clean and quickly follow the surrounding environment
		1. Open and characterized by aqueous mucus. Slowly follow the surrounding environment
		2. Open and characterized by dark mucus
		3. Ellipsoidal shaped and characterized by dark mucus
		4. Closed
	Posture (place the mouse on the palm and swing gently)	0. The mouse stands in the upright position with the back parallel to the palm. During swing, it stands rapidly.
		1. The mouse stands humpbacked. During the swing, it flattens the body to gain stability.
		2. The head or part of the trunk lies on the palm.
		3. The mouse lies on one side, barely able to recover the upright position.
		4. The mouse lies in a prone position, not able to recover the upright position.
	Spontaneos activity (mouse on OBT)	0.The mouse is alert and explores actively.
		1.The mouse seems alert, but it is calm and sluggish.
		2.The mouse explores intermittently and sluggishly.
		3.The mouse is somnolent and numb, few movements on-the-spot.
		4.No spontaneous movements
	Total score for general scoring
	(normal=0 max=18)

Table 1: General Neuroscore. Animals received between 0 and 4 points, depending on the severity, for each of the five general deficits measured. The scores on the different areas are then added to provide a total general score ranging from 0 to 18. This table has been modified from Clark et al.¹⁴ . Abbreviation: OBT = open benchtop.

	Time-point of scoring		score
Focal Neuroscore	Body symmetry (mouse on OBT, observe the nose-tail line)	0. Normal (Body: normal posture, trunk elevated from the bench, with fore and hindlimbs leaning beneath the body. Tail: straight)
		1. Slight asymmetry (Body: leans on one side with fore and hindlimbs leaning beneath the body. Tail: slightly bent)
		2. Moderate asymmetry (Body: leans on one side with fore and hindlimbs stretched out. Tail: slightly bent)
		3. Prominent asymmetry (Body: bent, on one side lies on the OBT. Tail: bent)
		4. Extreme asymmetry (Body: highly bent, on one side constantly lies on the OBT. Tail: highly bent)
	Gait (mouse on OBT. Observed undisturbed)	0. Normal (gait is flexible, symmetric and quick)
		1. Stiff, inflexible (humpbacked walk, slower than normal mouse)
		2. Limping, with asymmetric movements
		3. Trembling, drifting, falling
		4. Does not walk spontaneously (when stimulated by gently pushing the mouse walks no longer than 3 steps)
	Climbing (mouse on a 45o surface. Place the mouse in the center of the gripping surface)	0. Normal (mouse climbs quickly)
		1. Climbs with strain, limb weakness present
		2. Holds onto slope, does not slip or climb
		3. Slides down slope, unsuccessful effort to prevent fail
		4. Slides immediately, no effort to prevent fail
	Circling behavior (mouse on OBT, free observation)	0. Absent circling behavior
		1. Predominantly one-side turns
		2. Circles to one side, although not constantly
		3. Circles constantly to one side
		4. Pivoting, swaying, or no movement
	Forelimb symmetry (mouse suspended by tail)	0. Normal
		1. Light asymmetry: mild flexion of contralateral forelimb
		2. Marked asymmetry: marked flexion of contralateral limb, the body slightly bends on the ipsilateral side
		3. Prominent asymmetry: contralateral forelimb adheres to the trunk
		4. Slight asymmetry, no body/limb movement
	Compulsory circling (forelimbs on bench, hindlimbs suspended by the tail: it reveals the presence of the contralateral limb palsy)	0. Absent. Normal extension of both forelimbs
		1. Tendency to turn to one side (the mouse extends both forelimbs, but starts to turn preferably to one side)
		2. Circles to one side (the mouse turns towards one side with a slower movement compared to healthy mice)
		3. Pivots to one side sluggishly (the mouse turns towards one side failing to perform a complete circle)
		4. Does not advance (the front part of the trunk lies on the bench, slow and brief movements)
	Whisker response (mouse on the OBT)	0. Normal
		1. Light asymmetry (the mouse withdraws slowly when stimulated on the contralateral side)
		2. Prominent asymmetry (no response when stimulated to the contralateral side)
		3. Absent response contralaterally, slow response when stimulated ipsilaterally
		4. Absent response bilaterally
	Total score for focal deficits
	(normal=0 max=28)

Table 2: Focal Neuroscore. Animals received between 0 and 4 points depending on the severity for each of the seven general deficits measured. The scores on the different areas are then added to provide a total focal score ranging from 0 to 28. This table has been modified from Clark et al.¹⁴ . Abbreviation: OBT = open benchtop.

Discussion

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

The “filament model” presented in the video has some advantages compared to other previously described stroke models, such as not requiring craniotomy and achieving complete reperfusion of transiently the occluded vessel. However, the complexity of the surgical intervention could be considered as a limitation, as it includes invasive surgery and a precise manipulation of the different arteries in close proximity to the trachea and the vagal nerve. The long exposure of the animal to anesthetics could also be a critical factor to consider, as the impact of anesthetics on neuroprotection and stroke outcome has already been well documented¹⁶. Lastly, despite the complexity of this surgical procedure, it can be completed in approx. 20 min when performed by a trained surgeon.

In contrast to the previously described “filament” stroke protocols¹⁷, the method described here also allows the measurement of cerebral blood flow during the occlusion and reperfusion phases. Blood flow monitoring during reperfusion could be an important parameter to prevent stroke reperfusion injury¹⁸, which is known to cause deleterious consequences in patients undergoing pharmacologic or endovascular interventions for recanalization of the thrombosed vessels. Despite the discrepancies between the consequences of cerebral blood flow restoration after MCAo¹⁹, the variability of blood flow restoration after stroke may influence the pathophysiological and biochemical events in the brain, as well as the infarct volume and the neurological deficits of  stroke mice²⁰. Therefore, in this model, complete restoration of blood flow and its recording are requirements to ensure reproducible infarcts among mice, especially in translational stroke studies.

The overall mortality during the surgical procedure is less than 5% and is mainly caused by anesthetic complications, bleedings, or sacrifice due to predefined exclusion criteria. This stroke model presents, however, a moderate mortality rate within the first 24–48 h after stroke induction, which could increase the number of animals needed per experiment to achieve an adequate cohort of stroke mice. In terms of infarct volume, this model induces large infarcts, with lesions encompassing up to 50% of the hemisphere. It also produces brain edema, affecting different brain regions, including cortical and subcortical regions.

To achieve a low variability and high reproducibility of the stroke model, several exclusion criteria should be taken into account, including: 1) operation time > 20 min; 2) >20% of blood flow reduction when CCA is ligated (step 3.3); 3) blood flow reduction during occlusion < 80% of the initial pre-occlusion value; and 4) blood flow increase 10 min after reperfusion rate <80% compared to the pre-reperfusion value. For an experienced and trained surgeon, no animals are excluded due to the operation time criterion. However, 10–15% of animals show a 20% reduction in blood flow upon CCA  ligation, and 5–10% show no adequate reduction or increase in blood flow during occlusion or reperfusion, respectively. Therefore, the success rate after excluding animals based on these criteria is approximately 75–85%. 

In addition, animals are examined daily after MCAo (body weight, temperature, and basic physiological behavior) to control for sickness, pain, or discomfort behavior. In addition to this general care, several tests have been developed for specific behavioral analysis after focal brain ischemia, despite all known tests to evaluate sensorimotor dysfunction, such as the Rotarod test²¹, Sticky label test²², Corner test²³, or the Cylinder test²⁴. Here, animals selected for the establishment of this stroke model were evaluated for focal and general deficits, since the filament model also induces cytokine-sickness behavior independent of focal (sensory or motor) deficits²⁵. Taken together, the “filament” stroke model described here is a valuable model for basic and translational stroke research. This model is proposed as a standardized stroke model to be used to harmonize stroke models across laboratories.

Materials

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