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.
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.
Stroke ranks as the second leading cause of death and the third leading cause of death and disability combined1. By 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'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 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'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.
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
2. Ischemia induction surgery
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'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. Please click here to view a larger version of this figure.
3. Ischemia period
4. Filament withdrawal surgery
5. Postsurgical care
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's core, accompanied by a significant decrease in the lesion'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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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'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. Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
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 "SineMozak. The work of doctoral students Rok Ister and Marta Pongrac has been fully supported by the "Young researchers' career development project – training of doctoral students" 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.
Betadine cutaneous solution 10g/100ml | Alkaloid Skopje | N/A | |
Braided silk suture | Fine Science Tools | 18020-60 | |
Dafilon suture 5/0 DS16 | B. Braun | C0936154 | |
Dolokain 20 mg/g gel | Jadran-Galenski Laboratorij | N/A | |
Dumont #5 forceps | Fine Science Tools | 11251-20 | 2 pieces |
Dumont #7 forceps | Fine Science Tools | 11271-30 | |
Dumont N0 self-closing forceps | Fine Science Tools | 11480-11 | |
Durapore Surgical Tape 1,25cm x 9,1m | 3M | 7100057169 | |
Durapore Surgical Tape 2,5cm x 9,1m | 3M | 7100057168 | |
External thermostat | Petnap | 1012536 | |
Halsey needle holders | Fine Science Tools | 12500-12 | |
Hot bead sterilizer | Fine Science Tools | 18000-50 | |
Iris scissors | Fine Science Tools | 14060-10 | |
Isoflurane USP | Piramal critical care | N/A | |
Laser Doppler Monitor | Moor | MOORVMS-LDF2 | |
Metal Pet Heat Pad | Petnap | 1012525 | |
Micro Vannas spring scissors | Fine Science Tools | 15000-00 | |
Mini-colibri retractor | Fine Science Tools | 17000-01 | |
Recugel eye ointment | Bausch&Lomb | N/A | |
S&T B-1 vessel micro clamp | Fine Science Tools | 00396-01 | 2 pieces |
S&T micro clamp applying forceps | Fine Science Tools | 00071-14 | |
Schwartz micro serrefines | Fine Science Tools | 18052-01 | |
Stemi DV4 Spot stereo microscope | Zeiss | 000000-1018-453 | |
Steri-Strip Reinforced Adhesive Skin Closures 3 mm x 75 mm | 3M | 7100236545 | |
Straight tissue forceps | Fine Science Tools | 11023-10 | |
SZX Stand Arm | Olympus | SZ2-STS | |
Tec III 300 series calibrated vaporizer | Vaporizer Sales and Service inc. | N/A | |
Universal Stand Type 2 | Olympus | SZ2-STU2 | |
VetFlo Six Channel Anesthesia Stand | Kent Scientific | VetFlo-1225 | Modified for O2/N2 mixing |