This work was funded by the Croatian Science Foundation project BRADISCHEMIA (UIP-2017-05-8082); GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund and by the European Union through the European Regional development Fund under Grant Agreement No. KK.01.1.1.07.0071, project &#34;SineMozak. The work of doctoral students Rok Ister and Marta Pongrac has been fully supported by the &#34;Young researchers&#39; career development project - training of doctoral students&#34; of the Croatian Science Foundation funded by the European Union from the European Social Fund. The procedure was filmed using an Android smartphone mounted to a surgical microscope using a generic camera mount. Video materials were edited, and voiceover was recorded using the Wondershare Filmora video editor.

All animal handling and procedures were approved by the Ethics Licensing Committee of the University of Zagreb School of Medicine and the Ethics Committee for the protection of animals used for scientific purposes of the Ministry of Agriculture of the Republic of Croatia. Experimental procedures were conducted according to Croatian Animal Protection Act (NN 102/17, 32/19), Amendments to the Animal Protection Act (NN 37/13) and the Guidelines on the Protection of Animals Used for Scientific Purposes (NN 55/13) which are in line with the European Guide for the Care and Use of Laboratory Animals (Directive 2010/63/EU).
1. Preparation of the animal and the surgical site
<ol>
	<li>Place the animal in a knock-out box and induce anesthesia using 5% volume of isoflurane in an oxygen/nitrogen mixture (1:2 ratio).</li>
	<li>After visually inspecting the animal&#39;s consciousness status and breathing frequency, bring it to a laboratory scale and weigh it.</li>
	<li>Check the reflex by pinching the interdigital skin of a lower limb to ensure proper anesthesia depth.</li>
	<li>Position the animal on the heated operating table and bring its nose into the anesthesia mask. Set the initial operating table temperature to 36 &#176;C and adjust it to keep the animal&#39;s body temperature at 37 &#176;C (measured via a rectal probe introduced in later steps). Throughout all further steps, adjust the volume fraction of isoflurane to keep the respiratory rate of the animal at 100 breaths/min. 
	NOTE: All surfaces and materials in direct contact with the animal must be disinfected thoroughly before the procedure.</li>
	<li>Apply eye ointment to the animal&#39;s eyes to protect them from dehydration and isoflurane gas.</li>
	<li>Turn the animal on its back and extend its neck by placing a small pillow made of surgical tape beneath it.</li>
	<li>Secure the animal&#39;s limbs in place using surgical tape. Take care not to extend the forelimbs too much so as not&#160;to cause shoulder joint dislocation inadvertently.</li>
	<li>Lubricate the rectal probe using petroleum white jelly and insert it into the rectum for continuous body temperature measurement. Secure the probe in place together with the tail using surgical tape.</li>
	<li>Apply a preoperative injection of saline and buprenorphine (0.5 mg/kg) intraperitoneally to keep the animal well hydrated and under analgesia during the procedure.</li>
	<li>Shave the animal&#39;s fur in the neck region using a cordless animal clipper. Collect all shaven fur using pieces of surgical tape. Additionally, shave the region with a razor to make it completely free of fur.</li>
	<li>Choose an MCAO filament based on the measured animal&#39;s weight and/or age. Bring it out of its container and put it in a Petri dish in a visible place. Put a drop of saline on the silicone part of the filament to keep it clean and dust-free. 
	NOTE: The thickness of the filament&#39;s silicone coating should be chosen according to the manufacturer&#39;s official recommendations in the case of a 12-16-week-old mouse. Additionally, the length of the silicone coating should be carefully planned in advance, depending on the size of the irrigation area that is to be occluded (see Discussion).</li>
	<li>Place a clean surgical drape over the operating table.</li>
	<li>Apply a drop of Betadine to the animal&#39;s shaven skin. Use a cotton swab to rub the disinfectant into the skin in a circular fashion from the inside out. Afterward, do the same with an ethanol-soaked cotton swab. Repeat this step 3 times with a fresh pair of sterile cotton swabs for each repetition.</li>
	<li>Apply a dab of lidocaine gel to the disinfected region of the future incision site for local wound analgesia.</li>
</ol>
2. Ischemia induction surgery
<ol>
	<li>Make an initial incision on the disinfected skin using a scalpel. Keep the number of skin incision strokes at a minimum to make the surgical wound heal easier. 
	NOTE: All instruments must have been previously sterilized. The reader should refer to their institutional guidelines for using sterile or surgical gloves.</li>
	<li>Using type 7 and 5 forceps, tear away the superficial fascia and detach the salivary glands from the underlying tissue. 
	NOTE: Always advance with closed forceps and extend it while moving away to push the tissue away. This way, anatomical spaces can be traversed with minimal tissue trauma.</li>
	<li>Place the wire retractor in its initial position while ensuring the salivary glands don&#39;t interfere with subsequent steps.</li>
	<li>Remove the deep neck fascia using Type 7 forceps and detach sternocleidomastoid muscles from the carotid region to reposition the retractor.</li>
	<li>Reposition the retractor to reach under the sternocleidomastoid muscles to enable access to the carotid region.</li>
	<li>Resect the omohyoid muscle to allow for a clear visual and easier approach to the carotid region.</li>
	<li>Free the carotid artery and its branches from the carotid vein and the vagus nerve, which are held together in the carotid fascia. 
	NOTE: This fascia must be removed carefully to avoid injuring the vagus nerve or causing any significant bleeding.
	<ol>
		<li>Pinch the carotid fascia on the lateral side of the carotid triad and gently pull it laterally to visually identify the artery, nerve, vein, and all the surrounding blood vessel branches.</li>
		<li>While pulling on the fascia laterally, using Type 5 forceps,&#160;separate the CCA from the adjacent nerve and vein up to the branching point and the superior thyroid vein, which crosses over the CCA.</li>
		<li>Completely detach the lower part of CCA from underlying tissue and fascia, using curved Type 7 forceps, making sure it is ready for clamping in future steps.</li>
		<li>Proceeding cranially in the region above the CCA branching point, tear open the carotid fascia using two forceps.</li>
		<li>Similarly, pull the loose end of the carotid fascia medially and laterally to precisely detach the ECA and internal carotid artery (ICA) from the fascia and surrounding tissue, respectively.</li>
		<li>Set the ECA and ICA free from the underlying tissue using curved Type 7 forceps.</li>
	</ol>
	</li>
	<li>With the CCA carefully prepared with its two respective branches, raise the ECA using curved type 7 forceps and clamp it with type N0 self-closing forceps.</li>
	<li>Lower the type N0 self-closing forceps carefully on the operating table. 
	NOTE: To reposition the clamping point of ECA, it is sometimes necessary to release the grip slightly and push the ECA branching point caudally. At this point, it&#39;s important to ensure that more than 1 mm of ECA remains between the ECA clamping and the branching point.</li>
	<li>With the ECA clamped and held securely with type N0 tweezers, introduce two braided silk threads behind the ECA. 
	NOTE: The cranial thread will make for the ligation knot and must be placed cranially from the clamping site.</li>
	<li>Tie down the cranial thread completely, as it is permanent, and clip the excess thread away using scissors.</li>
	<li>Tie the caudal thread into a loose securing knot. 
	NOTE: It must be put close to the ECA branching point and kept loose for the MCAO filament to pass through.</li>
	<li>Using vascular microclips, clamp the CCA and the ICA shut to prevent bleeding after the arteriotomy. 
	NOTE: It is crucial to ensure that the microclip applied to the CCA has a strong enough grip as the CCA is a pulsating high-throughput artery, and failure to do so can result in profuse bleeding and death of the animal in the later steps.</li>
	<li>Using micro-spring scissors, make a small arteriotomy right beneath the ECA clamping site. 
	NOTE: The arteriotomy needs to be large enough for the silicon part of the filament to go in but not larger than half of the ECA circumference in order not to compromise the tension kept by the type N0 forceps.</li>
	<li>With an angle matching that of the ECA, insert the filament into the arteriotomy site in a proximal direction to the CCA, passing through the loose securing knot at the branching site of the ECA and sticking into the lumen of the CCA with the filament.</li>
	<li>Tighten down the securing knot around the silicon part of the filament. 
	NOTE: Great care is needed at this point as the filament tends to slip out of the ECA halfway through the last maneuver, and putting it back in at that point is very hard.</li>
	<li>Carefully open the ICA micro clip and remove it.</li>
	<li>With the filament partially inserted into ECA and secured down, make a complete arteriotomy thereby setting the ECA stump loose with the MCAO filament inside. At this point, release the type N0 self-closing forceps and remove it.</li>
	<li>With one set of type 5 tweezers, firmly pinch the ECA stump and raise it slightly. While holding onto the partially inserted filament with another forceps, lower and gently pull the ECA stump to orient the inner end of the filament towards the ICA. Never pinch the silicone part of the filament.</li>
	<li>With forceps holding onto the filament, slowly advance the filament through the ICA towards the circle of Wills. 
	NOTE: The first branching point of the ICA is also the only one where the operator can miss the desired MCA branching point. Therefore, a slight lateral angle of advancement is necessary to guide the filament to the MCA branching point. With the slight lateral angle of advancement, the filament curves away from the pterygopalatine artery (PPA) and makes the advancing end more likely to enter the circle of Willis (Figure 1).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66720/66720fig01.jpg" /> 
Figure 1: An illustration of middle cerebral artery occlusion filament advancement past the pterygopalatine artery branching point. Proper filament advancement (on the left) is achieved by orienting the MCAO filament in such a way that it backs against the lateral wall of the ICA and curves away from the PPA branching point. Failure to do so (on the right) can result in filament entering the PPA and not causing stroke. In the latter case, the filament won&#39;t be able to advance as far as it should and the surgeon should withdraw the filament until its end becomes visible at the ICA branching point and begin to advance the filament again. Green-colored arteries represent posterior communicating arteries (PcomA), mostly non-patent in mice. Created with BioRender.com. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66720/66720fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="21">
	<li>Slowly advance the MCAO filament through the circle of Willis until a sudden increase in resistance is felt. 
	NOTE: At this point, it is important to observe the remaining length of the filament. The filament should proceed effortlessly at least 7 mm from the ICA branching point in an adult mouse.</li>
	<li>Tighten the securing knot to ensure the filament is not displaced during ischemia.</li>
	<li>Remove the CCA clip and take note of the time. 
	NOTE: This marks the beginning of the ischemia onset.</li>
	<li>Pack the loose end of the MCAO filament in the groove between the carotid triangle and the ipsilateral sternocleidomastoid muscle so it doesn&#39;t protrude out of the surgical wound.</li>
	<li>Remove the wire retractor and bring the edges of the surgical wound closer to each other. Let the surgical site settle for a few seconds for the tissue to return to its anatomical position.</li>
	<li>Close the surgical wound using tape wound closures to facilitate faster reopening of the wound after the ischemia period. 
	NOTE: If a researcher wishes to let the animal awaken during the ischemia period, suturing the wound is recommended to prevent untimely reopening of the wound.</li>
</ol>
3. Ischemia period
<ol>
	<li>Validate the success of the surgery using MRI or some other quantitative perfusion measuring method. 
	NOTE: In case of doing an MRI scan, perfusion-weighted imaging (PWI) should show critical ischemia in the wide MCA region and diffusion-weighted imaging (DWI) should outline the site of reduced apparent diffusion coefficient (ADC) due to cell edema.</li>
	<li>Place the animal into a clean cage until the ischemia period ends.</li>
</ol>
4. Filament withdrawal surgery
<ol>
	<li>Roughly 5 min before the allotted ischemia time ends, position the animal and secure it on the operating table again.</li>
	<li>Remove the tape wound closures and reopen the surgical wound with the wire retractor.</li>
	<li>Free the loose end of the MCAO filament from surrounding tissue and the ends of the securing knot thread.</li>
	<li>With type N0 forceps, pinch and pull the edge of the ECA stump ventrally to put tension on it. Carefully lower the pinching forceps down, similar to the first surgical procedure. 
	NOTE: The operator must avoid pinching the securing knot, as that will make the knot unable to untie.</li>
	<li>Clamp the CCA again with a microvascular clip.</li>
	<li>Using type 5 forceps, loosen up the securing knot to enable filament withdrawal. 
	NOTE: The best way to start loosening the knot is to keep pinching it until it separates into individual parts and then push the thread ends toward the knot.</li>
	<li>Slowly withdraw the MCAO filament until the silicon part starts to stick out of the ECA stump. Tighten down the securing knot slightly to prepare for complete filament withdrawal.</li>
	<li>When close to the silicon end of the filament, tighten the securing knot so that it pushes out the MCAO filament and closes the ECA stump in a single maneuver. Tighten the knot completely after the filament slips out.</li>
	<li>Open and remove the pinching forceps together with the CCA clip. 
	NOTE: This marks the end of the ischemia period and the start of the reperfusion process.</li>
	<li>Remove the wire retractor and bring the edges of the surgical wound together again.</li>
	<li>Suture down the surgical wound starting from the periphery, closing the wound completely without any underlying tissue visible. 
	NOTE: The number of sutures required depends on the size of the surgical wound.</li>
	<li>Clean and disinfect the surgical site using rubbing alcohol wipes.</li>
</ol>
5. Postsurgical care
<ol>
	<li>Place the animal in a clean cage and allow it to awake spontaneously from anesthesia.</li>
	<li>Place pelleted food on the bottom of the cage for easier reach. Soften the food pellets using drinking water. Fill a small Petri dish with drinking water and bring it to the floor of the cage as well.</li>
	<li>In the next 48 h, inject 0.25 mL saline with buprenorphine (0.5 mg/kg) every 24 h intraperitoneally.</li>
</ol>

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Authors have no conflicts of interest to disclose. Authors have no affiliation with commercial names and trademarks mentioned in this work.

MCAO is a highly demanding procedure for the operator and a debilitating one for the animal. For this reason, it is of utmost importance for researchers to have a standard operating procedure that minimizes stroke severity, reduces procedural failure, and improves the well-being of the animal post-procedure. This MCAO protocol highlights some of the key aspects of consideration when conducting this procedure on a mouse. 
The choice of the MCAO filament influences the size and location of the induced stroke lesion and therefore is a critical step in this procedure11. Silicon-coated filaments, such as those pioneered by Doccol, have made a big leap in lowering the incidence of filament-induced hemorrhages12. However, silicon-coated filaments come in a large variety of sizes and lengths, posing a challenge for new researchers to select appropriate ones for MCAO in experimental animals. While filament manufacturers usually provide guidelines for choosing the right filament diameter for a given animal weight, the length of the silicon coating is considered to be of the operator's personal preference and should be chosen only on the basis of the operator's skill. In this work, we advocate for minimizing the silicon length to ensure the induced stroke lesion only encompasses the MCA irrigation area thus improving reproducibility. Figure 4 illustrates that the posterior cerebral artery (PCA), the first intracranial ICA branch, supplies the posterior regions of the brain, including parts of the hippocampus. Recent micro-CT studies on mice have identified additional three smaller branches arising from ICA before the branching point of the MCA13. The delicate and variable hypothalamic artery (HTA), ventral tegmental artery (VTA) and anterior choroid artery (AChA), in their branching order, contribute significantly to intra-variant and intra-subject variability8. Moreover, micro-CT studies have shown that, in mice, the average distance from MCA and PCA branching points measures around 1.7 mm, considerably shorter than the silicon coating length of most manufactured filaments14. Since the only feedback an operator receives when advancing the filament is a sudden increase in resistance when the filament reaches the MCA branching point, the trailing length of the filament's silicon coating is what induces ischemia to the MCA and, depending on the length of the silicon coating, to the AChA, VTA, HTA and even PCA, introducing variability contrary to the intended MCAO model. Thus, to maintain reproducibility and postoperative animal well-being, the aim should solely be MCA occlusion. Doccol offers MCAO filaments with silicon coatings ranging from 1 to 2 mm, yet our experience indicates that using 1-2 mm coatings often results in a sham operation without causing stroke. Intraoperative MRI scans revealed that the filament advances up to 2 mm beyond the MCA branching point into the anterior cerebral artery (ACA) before providing critical resistive feedback to the operator. For this reason, we highly suggest use of silicon coatings ranging from 2 to 3 mm in length, which reduces hippocampal involvement in stroke lesions compared to longer silicon coatings on MCAO filaments. 
Second critical step in this procedure is the correct placement of the MCAO filament in order to achieve complete occlusion of the MCA. Mistakenly entering the first ICA branch, the pterygopalatine artery (PPA), is a common issue for the operator since there is hardly any feedback that MCAO filament has taken a wrong turn during its advancement. For this reason, we recommend a slight lateral angle of advancement so that the silicon-coated tip of the filament curves away from the branching point of PPA, making it more likely to enter the intracranial portion of ICA. (Figure 1) As stated in the protocol section, it is important to observe the trailing filament's length to conclude if the placement of the filament is adequate. In a case where the filament can't be inserted for at least 7mm from the ICA branching point, the operator should retract the filament and repeat. Any unnecessary force applied during the filament insertion can result in a hemorrhage and a failed procedure. For this matter, a continuous laser-doppler perfusion measurement can be used to confirm correct filament placement. 
Ensuring unimpeded blood flow from the CCA during the ischemic period is another critical step in this protocol. Contrary to most illustrations depicting a complete Willis circle in a mouse, studies have shown that the posterior communicating artery (PcomA) is most often not bilaterally patent in mice and cannot reliably supply the ipsilateral side of the Willis circle if the CCA remains closed during the ischemia period15,16. In those cases, even if a researcher carefully selects the correct length of filament's silicon coating, stroke will envelop the whole hemisphere or only the MCA region, depending on PcomA patency, giving rise to the unaccounted variance of stroke sizes. For this reason, we have modified the procedure to involve the instruction step where the operator removes the CCA clip right after setting the occluding MCAO filament. These modifications could increase the precision and reproducibility of the surgery and reduce the heterogeneity of the results at the same time. Standardization of the methods would further improve the success rate and comparability, as well as the translative potential of the results17.
The greatest limitation of the proposed technique is the fact that ECA remains ligated after the procedure, which can potentially worsen the animal's status during the recovery18. So far, we've been unsuccessful with efforts to repair the ECA while keeping the success rate of the procedure high enough. This remains a potential point for future improvements in the method. Furthermore, minimizing the time in which the CCA remains clamped is a challenge, but it is essential to prevent the recruitment of non-MCA regions in stroke. Since this could provide a source of inter- and intra-operator variance, it is advisable to measure the duration of CCA clamping, potentially enabling researchers to statistically account for at least a fraction of the observed total variance in stroke sizes. On the other hand, operator skill and experience play a crucial role in reducing the time of CCA ligature and overall procedure duration11.
The presented method is significant because it enables researchers to selectively occlude the MCA, thus making the onset of reperfusion sudden and highly controllable. The method mimics endovascular thrombectomy and its subsequent reperfusion injury more accurately than currently available methods, providing details for a reproducible and clinically relevant animal ischemia/reperfusion injury model.

Stroke ranks as the second leading cause of death and the third leading cause of death and disability combined1. By&#160;its cause, stroke can be ischemic or hemorrhagic, with ischemic stroke being significantly more prevalent in clinical practice. Ischemic stroke arises from a blockage in an artery supplying blood to brain tissue, leading to ischemia, cell death, and inflammation. Since the advent of reperfusion therapies such as thrombolysis and mechanical thrombectomy, a great advancement has been made in the treatment of stroke. However, all reperfusion therapies carry the risk of exacerbating the patient&#39;s condition by causing what is commonly referred to as reperfusion injury2. The exact mechanism of reperfusion injury remains unclear, and it is up to preclinical studies to identify potential causes and preventive measures. For that purpose, developing a pertinent animal model for reperfusion injury that follows ischemic stroke becomes crucial.
Middle cerebral artery occlusion (MCAO) is the most commonly used animal model for studying ischemic stroke. It is predominantly conducted in rodents and has many different variants described in scientific literature so far3,4. The two main types, Koizumi and Longa, known as common carotid artery (CCA) and external carotid artery (ECA) variants, technically differ by the arteriotomy site for filament insertion5,6. In our recent article, by in vivo monitoring of vascular perfusion, we showed that only the Longa method can be truly considered a brain ischemia/reperfusion model7. The procedure involves inserting the filament into the ECA, advancing it through the ICA, and securing it at the branching point of the middle cerebral artery (MCA) to induce brain tissue ischemia. Following a predetermined period of ischemia, withdrawal of the filament permits reperfusion, simulating transient brain ischemia. After the stroke onset, the primary outcome variable used in research is most often the volume of the infarcted lesion, which can be measured either using ex vivo histology or in vivo brain scans. Challenges in MCAO models revolve around low reproducibility attributed to inter-variant, inter-operator, and inter-subject variances, with the latter posing a significant limitation in preclinical stroke research4.
Moreover, infarcted regions following MCAO in rodents are massive relative to the size of the rodent brain. In addition, hippocampal posterior regions of the brain often get recruited into the infarct volume despite those regions being primarily dependent on blood flow from the posterior cerebral artery (PCA), and not MCA8. As in both Koizumi and Longa methods described in the literature, the CCA is kept ligated during the ischemia period due to the incomplete patency&#160;of the Willis circle in mice, leading to ischemia induction in a much wider region than intended5,6,9. Even in methods where the CCA is reopened or repaired after the ischemia period, the usual 30-60 min of ischemia results in irreversible tissue injury in non-MCA regions10. Furthermore, contrary to expectations, previous research showed the length of the silicon coating of a filament has no impact on the lesion size11. However, the choice of the filament&#39;s silicon coating length was addressed solely in models with ligated CCA during the occlusion period.
The goal of this method was to modify the Longa MCAO method in mice to enable uninterrupted blood flow from the CCA during the ischemia period, thereby increasing the selectivity of MCAO, as well as ensuring complete reperfusion of the infarcted region after the procedure. These modifications would greatly benefit longitudinal studies researching ischemia-reperfusion injury in mice by lowering the mortality rate and reducing the inter-subject variance.

<table><tbody><tr><td>Betadine cutaneous solution 10g/100ml</td><td>Alkaloid Skopje</td><td>N/A</td><td></td></tr><tr><td>Braided silk suture</td><td>Fine Science Tools</td><td>18020-60</td><td></td></tr><tr><td>Dafilon suture 5/0 DS16</td><td>B. Braun</td><td>C0936154</td><td></td></tr><tr><td>Dolokain 20 mg/g gel</td><td>Jadran-Galenski Laboratorij</td><td>N/A</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-20</td><td>2 pieces</td></tr><tr><td>Dumont #7 forceps</td><td>Fine Science Tools</td><td>11271-30</td><td></td></tr><tr><td>Dumont N0 self-closing forceps</td><td>Fine Science Tools</td><td>11480-11</td><td></td></tr><tr><td>Durapore Surgical Tape 1,25cm x 9,1m</td><td>3M</td><td>7100057169</td><td></td></tr><tr><td>Durapore Surgical Tape 2,5cm x 9,1m</td><td>3M</td><td>7100057168</td><td></td></tr><tr><td>External thermostat</td><td>Petnap</td><td>1012536</td><td></td></tr><tr><td>Halsey needle holders</td><td>Fine Science Tools</td><td>12500-12</td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools</td><td>18000-50</td><td></td></tr><tr><td>Iris scissors</td><td>Fine Science Tools</td><td>14060-10</td><td></td></tr><tr><td>Isoflurane USP</td><td>Piramal critical care</td><td>N/A</td><td></td></tr><tr><td>Laser Doppler Monitor</td><td>Moor</td><td>MOORVMS-LDF2</td><td></td></tr><tr><td>Metal Pet Heat Pad</td><td>Petnap</td><td>1012525</td><td></td></tr><tr><td>Micro Vannas spring scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>Mini-colibri retractor</td><td>Fine Science Tools</td><td>17000-01</td><td></td></tr><tr><td>Recugel eye ointment</td><td>Bausch&amp;amp;Lomb</td><td>N/A</td><td></td></tr><tr><td>S&amp;amp;T B-1 vessel micro clamp&amp;nbsp;</td><td>Fine Science Tools</td><td>00396-01</td><td>2 pieces</td></tr><tr><td>S&amp;amp;T micro clamp applying forceps</td><td>Fine Science Tools</td><td>00071-14</td><td></td></tr><tr><td>Schwartz micro serrefines</td><td>Fine Science Tools</td><td>18052-01</td><td></td></tr><tr><td>Stemi DV4 Spot stereo microscope</td><td>Zeiss</td><td>000000-1018-453</td><td></td></tr><tr><td>Steri-Strip Reinforced Adhesive Skin Closures 3 mm x 75 mm</td><td>3M</td><td>7100236545</td><td></td></tr><tr><td>Straight tissue forceps</td><td>Fine Science Tools</td><td>11023-10</td><td></td></tr><tr><td>SZX Stand Arm</td><td>Olympus</td><td>SZ2-STS</td><td></td></tr><tr><td>Tec III 300 series calibrated vaporizer</td><td>Vaporizer Sales and Service inc.</td><td>N/A</td><td></td></tr><tr><td>Universal Stand Type 2</td><td>Olympus</td><td>SZ2-STU2</td><td></td></tr><tr><td>VetFlo Six Channel Anesthesia Stand</td><td>Kent Scientific</td><td>VetFlo-1225</td><td>Modified for O2/N2 mixing</td></tr></tbody></table>

optimization of the longa middle cerebral artery occlusion method for complete reperfusion

The middle cerebral artery occlusion model serves as the primary animal model for studying ischemic stroke. Despite being used in research for over three decades, its standardization remains inadequate. Predominantly conducted on rats and mice, the procedure poses challenges due to mice's smaller and more fragile nature. Unlike the Koizumi common carotid artery method, the Longa external carotid artery is the sole intraluminal filament stroke model ensuring complete reperfusion post-ischemia. This aspect holds critical significance for studies investigating reperfusion phenomena. The surgical modifications demonstrated in this article ensure continuous blood flow from the common carotid artery throughout the ischemic phase and after the reperfusion onset. The goal of these modifications is to selectively occlude the middle cerebral artery by keeping the perfusion uninterrupted in branches proximal to the middle cerebral artery during the ischemia period. Furthermore, the onset of reperfusion is sudden and can be precisely controlled, thereby modeling endovascular thrombectomy in human medicine more accurately. Our aim in presenting this comprehensive video article is to ease the training of new surgeons and promote the standardization of surgical procedures within the scientific community.

Intraoperative or postoperative MRI scans, specifically perfusion-weighted imaging (PWI) and/or diffusion weighted imaging (DWI) scans (Figure 2) can offer definitive proof of a successful procedure. Intraoperative PWI shows critical ischemia in the ipsilateral MCA region thus confirming that the filament placement resulted in complete occlusion. Postoperative PWI of a successful procedure displays hyperperfusion at the lesion core, as well as some degree of reperfusion at the MCA region borders. Intraoperative DWI lesions correspond to the observed ischemic regions on the intraoperative PWI. Postoperative DWI lesions exhibit shrinkage toward the ischemic lesion&#39;s core, accompanied by a significant decrease in the lesion&#39;s apparent diffusion coefficient, rendering it darker than the intraoperative DWI lesions and the surrounding reperfused brain tissue. Standard T2 anatomical scans, both intraoperative and postoperative, typically show nothing noteworthy unless filament-induced hemorrhage occurs
(Figure 3). In such cases, PWI and DWI scans reveal incomplete or no ischemia.
The reperfusion process follows in the next 48 hours, and it is confirmed to have taken place on MRI scans done on the second day after the procedure (Figure 2, CBF on Day 2). PWI scans show complete reperfusion in the lesion site with slight hyperperfusion in the lesion core. DWI scans show multiple regions of lowered apparent diffusion coefficients due to brain edema formation. T2 anatomical scans indicate hyperintense regions of edema enveloping MCA regions, with no involvement of non-MCA regions such as the hippocampus.
Upon awakening, the animal exhibits clear signs of acute stroke. Common behavioral symptoms include increased muscle tone on the side of the lesion, causing the animal to flex its torso toward that direction. Circling and rotating in the ipsilateral direction are also clear signs of an acute stroke. Gait ataxia often suggests the involvement of the hippocampus in the ischemic lesion volume. However, this symptom may lack specificity in the hyperacute phase due to isoflurane anesthesia side effects. In the subsequent days, the animal should develop front limb flexion ipsilaterally and worsening of its postural status, accompanied by an absence of whisker reflex. Mortality rate peaks at around 3 days post-procedure due to brain edema. The chronic phase begins after the brain edema starts to resolve. During this time, the animal can present with urine retention. For this reason, it is important to inspect the animal for bladder stiffness as well as a firm cap on the urethral exit. Most of the time, this issue can be resolved by gently massaging the bladder to facilitate urination. Weight recovery signs usually appear within the initial 7 days post-stroke onset.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66720/66720fig02.jpg" /> 
Figure 2: Representative example of a successful MCAO with complete reperfusion confirmed with MRI scans. CBF, ADC, and T2w stand for cerebral blood flow maps, apparent diffusion coefficient maps and T2-weighted anatomical scans, respectively. IntraOP, postop, and D02 signify 3 different time points of imaging after ischemia onset, after reperfusion onset, and on the second day after MCAO, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66720/66720fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66720/66720fig03.jpg" /> 
Figure 3: Representative MRI scans of a filament-induced hemorrhage during the MCAO procedure. CBF, ADC and T2w stand for cerebral blood flow maps, apparent diffusion coefficient maps and T2-weighted anatomical scans, respectively. IntraOP, postOP, and D02 signify 3 different time points of imaging: after ischemia onset, after reperfusion onset, and on the second day after MCAO, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66720/66720fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66720/66720fig04.jpg" /> 
Figure 4: An illustration of proper filament placement at the middle cerebral artery branch. In most cases, the silicon part of the filament (colored white) gets lodged in the proximal part of the anterior cerebral artery (ACA) about 2mm further than the MCA branching point. Normally the ACA brain region doesn&#39;t get affected in mice as ACA joins with the contralateral one into a singular azygos ACA. Affected arteries are shown in grey. On the left, CCA is left patent during the ischemia period, thus supplying blood to all ICA branches up to the MCA which is occluded with the silicon part of the filament. On the right, the filament is positioned identically, except the CCA ligation is kept during the ischemia period. Due to PcomA (colored green) not being patent in most cases, if the CCA ligation is kept, the stroke non-selectively envelops the majority of the ipsilateral hemisphere via MCA, AchA, VTA, HTA and PCA. In those cases, signs of acute infarction of the eye (leukocoria and anisocoria) can also be observed due to blocked PPA. Created with BioRender.com. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66720/66720fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol presents a step-by-step guide for researchers to perform the middle cerebral artery occlusion procedure on mice using the modified Longa external carotid artery method. Modifications presented in this article aim to increase the accuracy of middle cerebral artery occlusion and ensure complete reperfusion.

Optimization of the Longa Middle Cerebral Artery Occlusion Method for Complete Reperfusion

The middle cerebral artery occlusion model serves as the primary animal model for studying ischemic stroke. Despite being used in research for over three ...

optimization-longa-middle-cerebral-artery-occlusion-method-for

Nanyang Technological University

Research

JoVE Journal

Neuroscience

771 Views.  University of Zagreb School of Medicine. Welkom bij onze videogidsen over voorbijgaande occlusie van de middelste hersenslagader bij muizen. Mijn naam is Rok Ister, en dit experiment vond plaats in het Laboratory for Regenerative Neuroscience van de University of Zagreb School of Medicine. Transient middle cerebrale artery-methode, of kortweg tMCAO, is het meest gebruikte diermodel van ischemische beroerte.Het wordt voornamelijk gedaan bij knaagdieren, zoals ratten en muizen. Twee belangrijke...

Video: Optimalisatie van de Longa occlusiemethode van de middelste cerebrale slagader voor volledige reperfusie

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 ...

Medicine

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

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.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

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

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

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.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

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 ...

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

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

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.

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

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 ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Optimization of the Longa Middle Cerebral Artery Occlusion Method for Complete Reperfusion

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