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/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.

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			<td>Stemi DV4 Spot stereo microscope</td>
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			<td>Tec III 300 series calibrated vaporizer</td>
			<td>Vaporizer Sales and Service inc.</td>
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			<td>Modified for O2/N2 mixing</td>
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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/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/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/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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

609 Views.  University of Zagreb School of Medicine. 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.

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

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

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

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.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

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

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

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

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

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

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

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

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 ...

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

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.

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.

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