This study was supported by the Grants from the Nature Science Foundation of Hubei Province (2022CFC057).

The experimental protocol was approved by the Institutional Animal Care and Use Committee of Jianghan University and was conducted in accordance with Experimental Animals Ethical Guidelines issued by the Center for Disease Control of China. Adult male C57BL/6J mice, 10 weeks old, weighted 24-26 g, were used in this protocol. All mice were housed under a 12-h light/dark cycle controlled environment with food and water ad libitum.
1. Preoperative preparation
NOTE: The key instruments and equipment required for this protocol are shown in Figure 1.
<ol>
	<li>Prior to the procedure, sterilize the surgical instruments by autoclaving.</li>
	<li>Administer meloxicam subcutaneously at a dose of 5 mg/kg for analgesia 60 min before surgery.</li>
	<li>Anesthetize the mouse with 5% isoflurane in an induction chamber.</li>
	<li>Wipe the heating surgery board with 75% ethanol and activate the heating switch with the temperature set to 37 &#176;C. Then, position the mouse in a left lateral position on the board, with the head positioned away from the surgeon and the tail facing towards the surgeon.</li>
	<li>Attach a mask to the mouse face that provides a constant supply of 2% isoflurane and 0.3 L/min oxygen for maintenance anesthesia. Apply the eye ointment on the cornea to protect them from drying during the procedure.</li>
	<li>Shave the fur from the left orbit to the left ear canal with a shaver. Use depilatory creams to remove the remaining fur for sterilization.</li>
	<li>Prepare the surgical area by applying povidone-iodine disinfectant and 75% ethanol three times.</li>
</ol>
2. Distal MCAO model
<ol>
	<li>Check if the mouse is deeply anesthetized by assessing the reflexes of the toe pinch.</li>
	<li>Make a vertical incision between the left orbit and the left ear canal using a sterile scalpel blade.</li>
	<li>Retract the skin&#39;s soft tissue with retractors and expose the temporal muscle.</li>
	<li>Use straight microforceps to separate the apical and dorsal segments of the temporal muscle from the skull.</li>
	<li>Identify the bifurcation of the MCA, which assumes a &#34;Y&#34; shape beneath the temporal bone with a bias towards the base of the skull (Figure 2A). 
	NOTE: If the bifurcation of MCA is not visually discernible (due to an anatomically normal variation), ascertain the most rostral vessel.</li>
	<li>Use an electric cranial drill to thin out the skull to make a bone window (4 mm in diameter) until the dura mater, with its translucent texture, becomes visible. 
	NOTE: Intermittently add drops of normal saline to the skull to prevent overheating during this process.</li>
	<li>Remove the dura mater above the artery with curved micro forceps to expose the MCA branch.</li>
	<li>Set the electrocautery to 7 W. Coagulate the artery using the electric coagulation forceps at the sites of proximal and distal to the bifurcation of MCA (Figure 2A). When the bifurcation is not visible due to an anatomical variation, perform the coagulation on the accurately identified MCA branch (as mentioned above) at two separate locations approximately 1 mm apart13.</li>
	<li>Monitor the blood flow of the left MCA cortical branch utilizing a laser speckle blood flow meter. 
	NOTE: Mice exhibiting a blood flow reduction of less than 40% of the baseline value following electrocoagulation were excluded from the study.</li>
	<li>Suture the muscle and the skin separately using 5-0 polyglycolic acid sutures. Apply diclofenac sodium gel and mupirocin ointment to the skin incision using a sterile cotton swab.</li>
	<li>Place the mouse in a recovery chamber. Monitor all mice until they are fully awake. Provide meloxicam (5 mg/kg, SC) for analgesia every 24 h&#160;up to 72 h.</li>
</ol>
3. Sham Operation
<ol>
	<li>Reproduce the same procedure as those mentioned above, excluding the process of arterial coagulation.</li>
</ol>
4. Behavioral tests
NOTE: Prior to the dMCAO, the mice underwent behavioral training twice daily for 3 days. On 3rd day post-dMCAO, move the mice to the behavioral testing room for a 2 h environmental adaptation before testing.
<ol>
	<li>Grip strength test14 
	&#8203;NOTE: The grip strength meter comprises a push-pull strain gauge and a metal horizontal bar.
	<ol>
		<li>Grasp the base of the mouse tail. Gradually lower the mouse toward the metal grip bar until it grips the horizontal bar with both forepaws (Figure 2B).</li>
		<li>Pull the mouse backward at a constant speed of about 2 cm/s and keep its body horizontal.</li>
		<li>Record the peak force in grams (g) when the mouse releases its forepaws from the bar.</li>
	</ol>
	</li>
	<li>Pole test15
	<ol>
		<li>Place the mouse on the top of a vertical wooden pole (50 cm high, 1 cm in diameter) with their head facing upwards, and allow them to climb down the pole (Figure 2B).</li>
		<li>Record the time taken by the mouse to turn around (T-turn) and the total time to climb down the pole (T-total) with a 60-s cut-off time.</li>
	</ol>
	</li>
	<li>Adhesive tape removal test16
	<ol>
		<li>Hold the mouse and attach a patch of adhesive tape (2 &#215; 2 mm) to the right forepaw of the mouse (Figure 2B).</li>
		<li>Put the mouse back into the rearing cage and allow it to move freely.</li>
		<li>Record the time taken by the mouse to remove adhesive with a limit of 60 s.</li>
	</ol>
	</li>
	<li>Cylinder test17
	<ol>
		<li>Place the mouse in an open-top, clear glass cylinder (diameter: 14 cm; height: 20 cm) and record its spontaneous standing exploratory behavior for 3 min using a camera (Figure 2B). 
		NOTE: During the experimental procedure, two mirrors were positioned alongside the cylinder to ensure optimal recording of forelimb movements.</li>
		<li>Wipe the cylinder with 75% ethanol to eliminate residual olfactory cues before testing the subsequent mouse.</li>
		<li>Reduce the playback speed of the video to 1/5 of the actual speed, and count the number of wall touches with the left (L) or right forepaw (R) or simultaneous use of both forepaws (B). Calculate the usage rate of the right forepaw as (L-R)/(L + R + B) &#215; 100%.</li>
	</ol>
	</li>
</ol>
5. Perfusion and sample preparation
<ol>
	<li>Euthanize the mouse by peritoneal injection overdose of pentobarbital (300 mg/kg). Position the mouse supine and immobilize the limbs.</li>
	<li>Make a midline incision on the skin, starting from the mid-abdominal cavity and extending towards the upper chest region.</li>
	<li>Open the chest using surgical scissors and fold the xiphoid upwards using a hemostat. Carefully remove the lung and the diaphragm to expose the heart.</li>
	<li>Insert the perfusion needle tip into the left ventricle and puncture the right atrium with scissors. Perfuse the heart with 20 mL of normal saline via the left ventricle.</li>
	<li>Decapitate the mouse and cut open the scalp along the midline between the eyes. Cut the bone along both sides, starting from the skull base and opening up to the eye socket.</li>
	<li>Lift the calvarium and gently detach the brain from the base of the skull by using a forceps. 
	NOTE: Brains from half of the mice were subjected to 2,3,5-triphenyl tetrazolium chloride (TTC) staining, while brains from the other half underwent histological examination.</li>
	<li>Soak the brains in 4% paraformaldehyde solution overnight for follow-up histological analysis.</li>
</ol>
6. Identification of infarction volume
<ol>
	<li>Place the brain in a -20 &#176;C freezer compartment of the refrigerator for 20 min.</li>
	<li>Position the brain into the 1 mm brain matrix and slice the brain into coronary sections with a thickness of 2 mm by using microtome blades.</li>
	<li>Transfer the brain sections to a 24-well culture plate and add 2% TTC to cover the brain tissue. Put the 24-well culture plate in a constant temperature oven for 30 min with the temperature set at 37 &#176;C.</li>
	<li>Carefully lift out the brain sections and fix them in 4% paraformaldehyde solution for 15 min.</li>
	<li>Perform optical scanning of these brain sections with a resolution of 1200 x 1200 dpi by a scanister.</li>
	<li>Measure the cerebral infarct volume by summing the infarct area of each section (area of the right hemisphere minus uninjured area of the left hemisphere) using Image J software (https://imagej.net/software/imagej/index)18. Calculate the infarct volume percentage as infarct volume/volume of the right hemisphere &#215; 100%.</li>
</ol>
7. Histopathological and immunofluorescent staining analysis
<ol>
	<li>Take the brain samples from the paraformaldehyde solution and flush them with running water for 1 h.</li>
	<li>Transfer the brain samples into the automation-tissue-dehydrating machine. Launch the preset program for dehydration, vitrification, and wax immersion. Embed the dehydrated brain samples in paraffin and slice them into 5-&#181;m thick sections by a microtome.</li>
	<li>Hematoxylin and eosin (H&#38;E) staining
	<ol>
		<li>Dewax the sections with xylene 3 times for 8 min each.</li>
		<li>Rehydratethe sections by successively immersing in gradient ethanol (100%, 95%, 90%, 80%, 70%) and distilled water for 5 min each time.</li>
		<li>Stain the sections with the hematoxylin solution for 5 min.</li>
		<li>Rinse the sections with distilled water to wash the residue hematoxylin, then dip them in 5% hydrochloric acid alcohol for a 5-s differentiation. Wash the sections with distilled water for 2 min.</li>
		<li>Stain the sections with eosin solution for 30 s and rinse off excess staining fluid using distilled water.</li>
		<li>Immerse the sections in 90% ethanol, 95% ethanol, 100% ethanol, and xylene (2 times) successively for 5 min each time. Mount the sections with a neutral balsam mounting medium.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining
	<ol>
		<li>Dewax the sections with xylene 3 times for 8 min each time.</li>
		<li>Rehydrate the sections by successively immersing in gradient ethanol (100%, 95%, 90%, 80%, 70%) and distilled water for 5 min each time.</li>
		<li>Preheat the citrate antigen retrieval solution to boil in the microwave. Immerse the sections in the antigen retrieval solution. Reheat the antigen retrieval buffer with low power (20 W) for 20 min.</li>
		<li>Turn off the microwave and let the antigen retrieval buffer cool to room temperature (RT).</li>
		<li>Wash the sections thrice with 0.1 mM phosphate buffered saline (PBS) for 5 min each time.</li>
		<li>Incubate the sections with a blocking solution (5% bovine serum albumin) at RT for 1 h to block the non-specific binding of immunoglobulin.</li>
		<li>Incubate the sections with rabbit anti-NeuN antibody (1: 300), rabbit anti-Iba-1 antibody (1: 500) and mouse anti-GFAP antibody (1: 500) at 4&#730;C overnight.</li>
		<li>Wash the sections thrice with 0.1 mM PBS for 5 min each time.</li>
		<li>Incubate the sections with goat anti-rabbit Alexa 594-conjugated IgG (1:500) or goat anti-mouse Alexa 488-conjugated IgG (1:500) for 1 h at RT.</li>
		<li>Wash the sections with 0.1 mM PBS and mount them with an antifade mounting media containing 4&#39;,6-diamidino-2-phenylindole (DAPI).</li>
		<li>Take images of three microscopic fields (300 &#181;m &#215; 300 &#181;m) within the ischemic penumbra using a fluorescent microscope at 594 nm and 488 nm, respectively.</li>
		<li>Using the ImageJ software (https://imagej.net/software/imagej/index), estimate the positively stained cell density by averaging counts in ischemic penumbra19.</li>
	</ol>
	</li>
</ol>

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

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The authors have no conflicting interests to disclose.

In the present protocol of the craniotomy electrocoagulation dMCAO model, the surgical procedures are conducted with minimal invasiveness, wherein only a portion of the temporalis muscle is separated to mitigate the adverse effects on masticatory function. The mice all recovered well after the procedure, with no observed instances of feeding difficulties. The MCA can be easily discerned in the temporal bone of the mouse, thereby facilitating precise identification of suitable craniotomy locations. This dMCAO model-induce...

Stroke is a common acute cerebrovascular disease characterized by high incidences of disability and fatality1. Of all stroke cases, nearly 80% belong to ischemic stroke2. Up to now, intravenous thrombolysis remains one of a limited number of productive approaches for the treatment of acute ischemic stroke. However, the effectiveness of thrombolytic treatment is restricted by the narrow effective time window and the occurrence of hemorrhagic transformation3. In the long-term rehabilitation phase following an ischemic stroke, a considerable number of patients are likely to experience durable neurological dysfunctions4. Further investigation is urgently required to unravel the underlying pathophysiological mechanisms of ischemic stroke, as well as to facilitate the development of novel therapeutic strategies targeting ischemic stroke. The establishment of a dependable and replicable model of ischemic stroke is crucial for basic research as well as subsequent translational research in the field of ischemic stroke.
In 1981, Tamura et al. developed a focal cerebral ischemia model by employing transcranial electrocoagulation at the proximal site of the middle cerebral artery (MCA)5. Since then, numerous researchers have utilized various methodologies such as ligation, compression, or clipping to induce distal MCA occlusion (dMCAO) for establishing transient or permanent ischemic stroke models6,7,8. Compared to the filament model, the dMCAO model exhibits notable advantages such as smaller infarct size and higher survival rate, rendering it more suitable for investigating long-term functional recovery subsequent to ischemic stroke9. In addition, the dMCAO model demonstrates a higher survival rate in aged rodents compared to the filament model, making it an advantageous tool for investigating ischemic stroke in elderly and comorbid animal models10. The photothrombotic (PT) stroke model has been demonstrated to possess the characteristics of less surgical invasiveness and a significantly low mortality rate. However, the PT model exhibits a greater degree of cellular necrosis and tissue edema compared to the dMCAO model, leading to the absence of collateral circulation11. Furthermore, it is noteworthy that the ischemic lesions observed in the PT model predominantly arise from microvascular occlusion, which differs substantially from the cerebral ischemia induced by large vessel embolism in the dMCAO model12.
In this paper, we present the methodology for inducing the murine dMCAO model by coagulating the distal MCA via small bone window craniotomy. Additionally, we conducted histological examinations and behavioral evaluations to comprehensively characterize the ischemic insults and stroke outcomes in this experimental model. We aim to acquaint researchers with this model and facilitate further investigations into the pathologic mechanisms of ischemic stroke.

<table><tbody><tr><td>2,3,5-Triphenyltetrazolium&lt;br/&gt; Chloride (TTC)</td><td>Sigma-Aldrich</td><td>108380</td><td>Dye for TTC staining</td></tr><tr><td>24-well culture plate</td><td>Corning (USA)</td><td>CLS3527</td><td>Vessel for TTC staining</td></tr><tr><td>4% paraformaldehyde</td><td>Wuhan Servicebio Technology&lt;br/&gt; Co., Ltd.</td><td>G1101</td><td>Tissue fixation</td></tr><tr><td>5% bovine serum albumin</td><td>Wuhan BOSTER Bio Co., Ltd.</td><td>AR004</td><td>Non-specific antigen blocking</td></tr><tr><td>5-0 Polyglycolic acid suture</td><td>Jinhuan Medical Co., Ltd</td><td>KCR531</td><td>Material for surgery</td></tr><tr><td>Anesthesia machine</td><td>Midmark Corporation</td><td>VMR</td><td>Anesthetized animal</td></tr><tr><td>Antifade mounting medium</td><td>Beyotime Biotech</td><td>P0131</td><td>Seal for IF staining</td></tr><tr><td>Automation-tissue-dehydrating&amp;nbsp;&lt;br/&gt; machine</td><td>Leica Biosystems (Germany)</td><td>TP1020</td><td>Dehydrate tissue</td></tr><tr><td>Depilatory cream</td><td>Veet (France)</td><td>20220328</td><td>Material for surgery</td></tr><tr><td>Diclofenac&amp;nbsp;sodium&amp;nbsp;gel</td><td>Wuhan Ma Yinglong Pharmaceutical&lt;br/&gt; &amp;nbsp;Co., Ltd.</td><td>H10950214</td><td>Analgesia for animal</td></tr><tr><td>Drill tip (0.8 mm)</td><td>Rwd Life Science Co., Ltd.</td><td></td><td>Equipment for surgery</td></tr><tr><td>Eosin staining solution</td><td>Wuhan Servicebio Technology&lt;br/&gt; Co., Ltd.</td><td>G1001</td><td>Dye for H&amp;amp;E staining</td></tr><tr><td>Eye ointment</td><td>Guangzhou Pharmaceutical Co., Ltd</td><td>H44023098</td><td>Material for surgery</td></tr><tr><td>Fluorescence microscope</td><td>Olympus (Japan)</td><td>BX51</td><td>Image acquisition</td></tr><tr><td>GFAP Mouse monoclonal antibody</td><td>Cell Signaling Technology Inc.&lt;br/&gt; (Danvers, MA, USA)</td><td>3670</td><td>Primary antibody for IF staining</td></tr><tr><td>Goat anti-mouse Alexa&lt;br/&gt; 488-conjugated IgG</td><td>Cell Signaling Technology Inc.&lt;br/&gt; (Danvers, MA, USA)</td><td>4408</td><td>Second antibody for IF staining</td></tr><tr><td>Goat anti-rabbit Alexa&lt;br/&gt; 594-conjugated IgG</td><td>Cell Signaling Technology Inc.&lt;br/&gt; (Danvers, MA, USA)</td><td>8889</td><td>Second antibody for IF staining</td></tr><tr><td>Grip strength meter</td><td>Shanghai Xinruan Information Technology Co., Ltd.</td><td>XR501</td><td>Equipment for behavioral test</td></tr><tr><td>Hematoxylin staining solution</td><td>Wuhan Servicebio Technology&lt;br/&gt; Co., Ltd.</td><td>G1004</td><td>Dye for H&amp;amp;E staining</td></tr><tr><td>Iba1 Rabbit monoclonal antibody</td><td>Abcam</td><td>ab178846</td><td>Primary antibody for IF staining</td></tr><tr><td>Isoflurane</td><td>Rwd Life Science Co., Ltd.</td><td>R510-22-10</td><td>Anesthetized animal</td></tr><tr><td>Laser doppler blood flow meter</td><td>Moor Instruments (UK)</td><td>moorVMS</td><td>Blood flow monitoring</td></tr><tr><td>Meloxicam</td><td>Boehringer-Ingelheim</td><td>J20160020</td><td>Analgesia for animal</td></tr><tr><td>Microdrill</td><td>Rwd Life Science Co., Ltd.</td><td>78001</td><td>Equipment for surgery</td></tr><tr><td>Microsurgical instruments set</td><td>Rwd Life Science Co., Ltd.</td><td>SP0009-R</td><td>Equipment for surgery</td></tr><tr><td>Microtome</td><td>Thermo Fisher Scientific (USA)</td><td>HM325</td><td>Tissue section production</td></tr><tr><td>Microtome blade</td><td>Leica Biosystems (Germany)</td><td>819</td><td>Tissue section production</td></tr><tr><td>Monopolar electrocoagulation generator</td><td>Spring Scenery Medical Instrument&lt;br/&gt; Co., Ltd.</td><td>CZ0001</td><td>Equipment for surgery</td></tr><tr><td>Mupirocin ointment</td><td>Tianjin Smith Kline &amp;amp; French&lt;br/&gt; Laboratories Ltd.</td><td>H10930064</td><td>Anti-infection for animal</td></tr><tr><td>NeuN Rabbit monoclonal antibody</td><td>Cell Signaling Technology Inc.&lt;br/&gt; (Danvers, MA, USA)</td><td>24307</td><td>Primary antibody for IF staining</td></tr><tr><td>Neutral balsam</td><td>Absin Bioscience</td><td>abs9177</td><td>Seal for H&amp;amp;E staining</td></tr><tr><td>Paraffin embedding center</td><td>Thermo Fisher Scientific (USA)</td><td>EC 350</td><td>Produce paraffin blocks</td></tr><tr><td>Pentobarbital sodium</td><td>Sigma-Aldrich</td><td>P3761</td><td>Euthanized animal</td></tr><tr><td>Phosphate buffered saline</td><td>Shanghai Beyotime Biotech Co., Ltd</td><td>C0221A</td><td>Rinsing for tissue section</td></tr><tr><td>Shaver</td><td>Shenzhen Codos Electrical Appliances&lt;br/&gt; Co.,Ltd.</td><td>CP-9200</td><td>Equipment for surgery</td></tr><tr><td>Sodium citrate solution</td><td>Shanghai Beyotime Biotech Co., Ltd.</td><td>P0083</td><td>Antigen retrieval for IF staining</td></tr></tbody></table>

induction of acute ischemic stroke in mice using the distal middle artery occlusion technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

The key instruments used to perform the dMCAO are the microsurgical instruments set, the isoflurane vaporizer, and the monopolar microsurgical electrocoagulation generator shown in Figure 1. The experimental procedure of this study is illustrated in Figure 2.&#160;In brief, a small bone window craniotomy was employed to expose the distal MCA, which was subsequently coagulated to induce permanent focal cerebral ischemia in C57BL/6 mice. Furthermore, the ischemic ...

Watch this Scientific Journal Video about Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique at JoVE.com

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

induction-acute-ischemic-stroke-mice-using-distal-middle-artery

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JoVE Journal

Neuroscience

1.8K Views.  Jianghan University. In het huidige onderzoek streven we ernaar een uitgebreid en gedetailleerd protocol te bieden voor het vaststellen van een occlusiemodel voor de middelste hersenslagader bij C57BL/6J-muizen met behulp van transcraniële elektrocoagulatie en we evalueerden het daaropvolgende neurologische gedrag en histopathologische kenmerken. De middelste hersenslagader of MCA wordt erkend als de primaire plaats van ischemische beroerte bij mensen. De MCA-occlusiemethoden, waaronder techniek, fotochemische i...

Video: Auteur in de schijnwerpers: het opzetten van een betrouwbaar distaal MCA-occlusiemodel bij muizen voor onderzoek naar beroertes

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 ...

Medicine

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 ...

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.

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 ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

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

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

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

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

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

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

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

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

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

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

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

A Model of Transient Cerebral Ischemia by Occlusion of Middle Cerebral Artery

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Author Spotlight: Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

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

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

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