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5 minutes

This work was supported by the Postgraduate Support Program (PROAP) through the Financial Assistance for Educational or Research Project, process number 88881.594204/2020-01, grant number 1359/2020. We thank the professors, technicians, and students of the Physiology, Metabolism, and Human Anatomy Laboratories of UNIOESTE (Universidade Estadual do Oeste do Paran&#225;, Cascavel, Brazil) for their technical and academic support throughout the project.

The protocol followed the guidelines for the care and use of laboratory animals established by the National Council for the Control of Animal Experimentation (CONCEA) and was approved by the Ethics Committee on Animal Experimentation (CEUA) of the State University of Western Paran&#225; (UNIOESTE), protocol number 25-23.
1. Preoperative preparation
<ol>
	<li>Prepare surgical instruments, gauze, cotton pads, and position the stereomicroscope (Figure 1).</li>
	<li>Clean the surgical table and equipment with 70% ethanol. Prepare sterile surgical instruments and perform all surgical procedures using aseptic technique.</li>
</ol>
2. Anesthesia
<ol>
	<li>Induce anesthesia with a mixture of ketamine (3.75 mL), xylazine (2.5 mL), and 0.9% sodium chloride (3.75 mL). Administer 0.2 mL of the mixture per 100 g of body weight intraperitoneally. Confirm anesthesia depth using a hind paw or tail pinch.</li>
	<li>If necessary, administer additional doses corresponding to one-third of the maximum dose to avoid overdose.</li>
	<li>Administer the prophylactic antibiotic enrofloxacin (20 mg/kg) and the analgesic tramadol hydrochloride (12 mg/kg) intraperitoneally.</li>
	<li>Measure rectal temperature and maintain body temperature at 37&#176;C (&#177; 2&#176;C) using a heating pad throughout the procedure.</li>
</ol>
3. Positioning
<ol>
	<li>Place the rat in lateral decubitus on a cotton pad on the microscope stage (Figure 1A). Support the head with a small soft roll and align it at 180&#176; to expose the left lateral surface of the skull. Secure the head with adhesive tape (Figure 2A).</li>
	<li>Apply lubricating eye drops and tape the eyelids closed.</li>
	<li>Recheck rectal temperature and adjust the heating source if needed.</li>
</ol>
4. Craniectomy
<ol>
	<li>Perform trichotomy between the lateral canthus and the tragus (approximately 1.6 cm &#215; 0.5 cm) using a mini trichotomizer or razor blade (Figure 1B).</li>
	<li>Disinfect the shaved area with 2% chlorhexidine scrub and then 0.2% aqueous chlorhexidine.</li>
	<li>Make a 1-cm skin and subcutaneous incision using straight scissors (Figure 1C), maintaining a 0.3-cm margin posterior to the lateral canthus and anterior to the tragus (Figure 2B). 
	&#8203;NOTE: Retract this layer with Adson forceps (Figure 1D) or hemostatic forceps.</li>
	<li>Identify and coagulate the middle temporal branch of the superficial temporal vessels using electrocautery (Figure 1E).</li>
	<li>Palpate the zygomatic arch using a freer elevator (Figure 1F) and incise the temporal muscle fascia longitudinally.</li>
	<li>Dissect the temporal muscle cranially and caudally to expose the temporal and frontal bones and part of the masseter muscle. Isolate the zygomatic arch longitudinally and remove an approximately 0.5-cm segment (Figure 2C).
	<ol>
		<li>Precautions:
		<ol>
			<li>Control bleeding from the anterior deep temporal branch of the maxillary vessels using compression or electrocautery.</li>
			<li>Avoid posterior oblique cuts to prevent damage to the temporomandibular joint.</li>
			<li>Avoid injury to the extraorbital lacrimal gland located beneath the arch.</li>
			<li>Avoid opening the orbital cavity.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Drill the lateral temporal bone immediately posterior to the zygomatic arch removal site (Figure 2D) using a 3.5-mm diameter round diamond-coated burr mounted on a low-speed rotary motor (Figure 1G). Intermittently irrigate with 0.9% saline to prevent thermal injury. Leave small bone fragments attached to the dura mater (Figure 2E) and remove them gently with Halstead forceps (Figure 1H). 
	&#8203;NOTE: A craniectomy using a smaller drill bit is an alternative but increases the risk of dural and brain injury.</li>
</ol>
5. MCA occlusion (Figure 3)
<ol>
	<li>Open the dura mater using a 6 mm &#215; 0.25 mm ultrafine needle. 
	NOTE: Avoid damaging adjacent cortical vessels.</li>
	<li>Identify the inferior cerebral vein (ICV) below the craniectomy window and the MCA crossing perpendicularly above it (Figure 3A,C). Identify the basal and thickest MCA branch located in the center of the craniectomy field.</li>
	<li>Pass a non-absorbable 8-0 polypropylene suture around the MCA using a fine needle holder (Figure 3B and Figure 1I). Tie three knots to occlude the vessel (Figure 3C). 
	NOTE: Other non-absorbable sutures such as nylon, cotton, or silk may be used.</li>
	<li>Confirm the absence of downstream blood flow. If an additional MCA branch is present, ligate it as well.</li>
	<li>Irrigate the cavity with saline and perform hemostasis with electrocautery or gentle compression, as needed. 
	&#8203;NOTE: Avoid coagulating brain tissue to prevent histological or neuroinflammatory artifacts.</li>
</ol>
6. Closure
<ol>
	<li>Reapproximate the dura mater when possible.</li>
	<li>Reapproximate the deep muscle layer and suture the temporal muscle fascia with absorbable 4-0 suture (Figure 3D).</li>
	<li>Close the subcutaneous and skin layers using absorbable 4-0 polyglactin 910 sutures in a continuous pattern. 
	NOTE: Avoid leaving exposed suture ends. Continuous suturing is recommended.</li>
	<li>Clean the surgical site with 0.2% aqueous chlorhexidine.</li>
	<li>Keep the animals warm until recovery from anesthesia. Monitor temperature before returning them to their cages. The total surgical duration, from skin incision to wound closure, typically ranges between 30 min and 45 min.</li>
	<li>Postoperatively, administer a second dose of tramadol hydrochloride (12 mg/kg, intraperitoneally) 12 h after surgery for postoperative analgesia. Administer a second prophylactic antibiotic dose the day after surgery.</li>
</ol>

The authors declare no competing interests.

The MCAO model in rats has been used since 197516,17&#160;as an experimental paradigm for cerebral ischemia, and has been refined over the years, demonstrating efficacy in inducing neurological damage8,22,23,24,25,26,27,<s...

The ischemic penumbra surrounding the infarcted area in ischemic stroke (IS) is capable of recovery if treatments are administered in a timely manner, aiming to prevent or mitigate motor and memory impairments1. However, therapies such as chemical thrombolysis and mechanical thrombectomy remain poorly accessible in clinical practice for a large segment of the population2, supporting the need for experimental models to investigate more widely available treatment strategies. Animal studies are indispensable due to their reproducibility and the ability to analyze collateral circulation3, with rats being widely used in IS models4. Owing to its close correlation with human IS5 and its vessel diameter, the middle cerebral artery (MCA) is the primary target vessel in experimental studies of arterial occlusion6,7,8.
One of the most widely used animal models to induce cerebral ischemia is filament-based intraluminal occlusion followed by reperfusion9,10. Nevertheless, the variability associated with the precise positioning of the filament within the MCA, inadvertent occlusion of adjacent vessels of the circle of Willis, collateral effects due to external carotid artery (ECA) ligation required for the procedure, and the duration of occlusion all contribute to conflicting results in meta-analyses11,12.
In the clinical setting, most patients suffering from IS do not achieve cerebral reperfusion and do not exhibit hypothalamic damage or sequelae associated with ECA occlusion, highlighting the need for experimental approaches that induce selective ischemia without subsequent reperfusion. An alternative is permanent MCA occlusion (MCAO) performed via craniectomy. With direct visualization of the MCA, selective occlusion prevents damage associated with ECA ligation and injury to the anterior choroidal, hypothalamic, and posterior cerebral arteries13, resulting in an ischemic effect more closely resembling that observed in lacunar infarction.
Recent studies indicate that animal models of IS should exhibit a closer correlation between the ischemic area and unfavorable clinical outcomes14,15. Developing technical alternatives that enable targeted induction of ischemia with minimal complications interfering with the results appears to be the most suitable strategy. Within this context, the aim of this protocol was to establish a reproducible method for performing craniectomy followed by permanent and selective MCAO in rats, with the purpose of standardizing the model and reducing technical challenges, for use in studies of motor and spatial memory deficits. This approach was adapted from classical studies16,17,18,19&#160;that demonstrated high survival rates, allowing long-term evaluations.
Using the inferior cerebral vein (ICV) as an anatomical landmark, this protocol seeks to demystify the technical difficulty historically cited as the major limitation of this approach, enabling the investigation of selective cerebral ischemia in both acute and chronic phases. Motor function and spatial memory were assessed to verify the deficits induced by the proposed lesion model, in addition to histological analysis of ischemic brain regions.

<table><tbody><tr><td>Adson toothed forceps</td><td>ABC stainless</td><td>L47-P4-164</td><td>Other options may be available from different companies</td></tr><tr><td>Castroviejo needle holder</td><td>Golgran</td><td>135-3</td><td>Other options may be available from different companies</td></tr><tr><td>Electrocautery</td><td>Chunguang medical</td><td>128</td><td>Other options may be available from different companies</td></tr><tr><td>Freer elevator</td><td>ABC stainless</td><td>47P4-819</td><td>Other options may be available from different companies</td></tr><tr><td>Halsted curved</td><td>ABC stainless</td><td>AI1055</td><td>Other options may be available from different companies</td></tr><tr><td>Micro drill</td><td>Nail drill</td><td>ZS-710</td><td>Other options may be available from different companies</td></tr><tr><td>Mini trichotomizer</td><td>Kemei</td><td>KM-666</td><td>Other options may be available from different companies</td></tr><tr><td>Prolene</td><td>Ethicon</td><td>&amp;nbsp;8730H</td><td>8-0 size suture</td></tr><tr><td>Stereomicroscope</td><td>Alltion</td><td>ASM-112</td><td>Other options may be available from different companies</td></tr><tr><td>Straight scissor</td><td>ABC stainless</td><td>L23-W4</td><td>Other options may be available from different companies</td></tr><tr><td>Surgical compresse</td><td>Cremer</td><td>158595</td><td>Other options may be available from different companies</td></tr><tr><td>Surgical gauze</td><td>Cremer</td><td>198669</td><td>Other options may be available from different companies</td></tr><tr><td>Thermometer</td><td>G-Tech</td><td>DMT-2027</td><td>Other options may be available from different companies</td></tr><tr><td>Vicryl</td><td>Ethicon</td><td>&amp;nbsp;VCP346H</td><td>4-0 size suture</td></tr></tbody></table>

selective and permanent occlusion of the middle cerebral artery in rats: an experimental approach for studying motor and spatial memory deficits

Selective and Permanent Occlusion of the Middle Cerebral Artery in Rats: An Experimental Approach for Studying Motor and Spatial Memory Deficits

With major clinical relevance, ischemic stroke (IS) demands experimental approaches that enable the development of novel adjuvant therapies. Due to the similarity of the cerebral vascular anatomy to humans, rats are widely used as experimental models of IS. However, there remains a need for methods capable of generating an ischemic environment that more closely resembles that observed in humans, employing an approach that prioritizes selective arterial occlusion. In this context, the aim of this study was to establish a reproducible method for performing a craniectomy followed by permanent and selective occlusion of the middle cerebral artery (MCA) in rats, to standardize the model and reduce technical challenges for application in studies of motor and spatial memory deficits. Wistar rats (N = 28) were equally divided into two groups, one undergoing craniectomy alone (SHAM) and the other craniectomy followed by selective MCA occlusion (MCAO). On the seventh and eighth postoperative days, the postural test and the object location recognition memory test were performed, respectively. After euthanasia, brain specimens were collected, and Nissl-stained histological sections were prepared. To demonstrate the feasibility and effectiveness of the method, a detailed surgical protocol was developed, including preoperative preparation, anesthesia, positioning, craniectomy, MCAO, and closure. The applied method demonstrated deficits in the MCAO group, as evidenced by the postural test (p = 0.015) and the object location recognition memory test (p &#60; 0.001). Histological analysis confirmed the selectivity of the approach, with injury involving hippocampal-associated areas such as the perirhinal, entorhinal, and piriform cortices, in addition to the external capsule, while deeper brain structures were preserved. This protocol proved to be a reproducible and effective method for inducing selective MCA ischemia in rats and represents a valid and technically accessible tool for experimental studies investigating pathophysiological and therapeutic mechanisms in ischemic stroke.

A total of 28 male Wistar rats (10–12 weeks old, mean weight 349 g) were randomly and equally divided into two groups: MCAO (submitted to the complete protocol described) and SHAM (submitted only to the craniectomy stage). In this study, 3 animals were excluded intraoperatively and replaced during surgery due to venous bleeding (2 MCAO) or minimal cortical injury during craniectomy (1 SHAM). One additional MCAO animal died on postoperative day 5. The overall mortality rate of the model was 3.6%. The final analyzed sample...

The objective of this study was to establish a reproducible method for performing a craniectomy followed by permanent, selective middle cerebral artery occlusion in rats for application in experimental studies of motor and spatial memory deficits.

With major clinical relevance, ischemic stroke (IS) demands experimental approaches that enable the development of novel adjuvant therapies. Due to the ...

A rat model of selective permanent MCA occlusion was developed to study motor and spatial memory deficits. Many stroke models are less selective, so this protocol allows direct vessel viewing and precise distal MCA occlusion. To begin, assemble the clean sterile surgical instruments around the microscope stage covered with a sterile cloth.Expose the left lateral side of the anesthetized Worcester rat placed in 180 degree lateral decubitus on the padded microscope stage with the head supported by a soft roll. Secure the head with adhesive tape. Apply lubricating eye drops.Tape the eyelids closed. Recheck the rectal temperature to ensure it remains within the ideal range of approximately 37 degrees Celsius. Adjust the heating source if needed.Perform trichotomy over a 1.6 by 0.5 centimeter region between the lateral canthus and the tragus using a mini trichotomizer or razor blade. Disinfect the shaved area with 2%chlorhexidine scrub, followed by 0.2%aqueous chlorhexidine. Use a sterile scalpel blade to make a one centimeter skin and subcutaneous incision, maintaining a 0.3 centimeter margin posterior to the lateral canthus and anterior to the tragus.Use hemostatic forceps to retract the skin and subcutaneous layer. After identifying the middle temporal branch of the superficial temporal vessels, coagulate the vessel with electrocautery. Using a freer elevator, palpate the zygomatic arch.Incise the temporal muscle fascia longitudinally. Dissect the temporal muscle cranially and caudally to expose the temporal and frontal bones and a part of the mass seeder muscle. Isolate the zygomatic arch longitudinally.Remove an approximately 0.5 centimeter segment of the zygomatic arch. Take appropriate precautions while performing the procedure. Use a round diamond-coated burr to drill the lateral temporal bone immediately posterior to the zygomatic arch removal site with intermittent 0.9%saline irrigation.During drilling, leave a thin residual bony layer attached to the dura mater. Use Halsted forceps to gently remove the remaining attached fragments. Use an ultrafine needle to open the dura mater.Identify the inferior cerebral vein, the middle cerebral artery, or MCA, and the basal and thickest MCA branch at the center. Use a fine needle holder to pass a non-absorbable 8.0 polypropylene suture around the MCA, and occlude the vessel by tying three knots around it. After confirming the absence of downstream blood flow, use the ultra-fine needle to check for additional MCA branches to ligate if present.Irrigate the cavity with saline. Perform hemostasis with electrocautery or gentle compression. After MCA occlusion and dural closure, reapproximate the deep muscle layer and suture the temporal muscle fascia using absorbable 4-O suture.Use absorbable 4-0 polyglactin 910 sutures to stitch the subcutaneous layer and the skin layer in a continuous pattern. Conduct postoperative evaluation across specific days. Motor function scores in the postural test on postoperative day seven were significantly higher in MCA occlusion animals compared to sham animals.MCA occlusion animals showed reduced exploration of the relocated object on postoperative day eight compared to sham animals. This protocol helps researchers study selective cortical ischemia and its effects on motor function and spatial memory. The main challenge is doing the craniectomy safely and identifying the middle cerebral artery correctly.Future studies can use this model to test treatments and study long-term functional and histological outcomes.

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Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

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.

Mouse Model of Middle Cerebral Artery Occlusion

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.

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

This video demonstrates the procedure of performing permanent distal middle cerebral artery occlusion, or MCAO, with the common carotid artery, or CCA ligation, in aged rats to model ischemic stroke. This blocks blood flow to the brain, leading to neuronal death and generating a model to study stroke mechanisms and potential treatments.

Inducing Ischemia via Middle Cerebral and Common Carotid Artery Occlusion in a Rat Model

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel.  This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

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.

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

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

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

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.

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

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

State University of Western Paraná.  The objective of this study was to establish a reproducible method for performing a craniectomy followed by permanent, selective middle cerebral artery occlusion in rats for application in experimental studies of motor and spatial memory deficits.

Selective and Permanent Occlusion of the Middle Cerebral Artery in Rats: An Experimental Approach for Studying Motor and Spatial Memory Deficits

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