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Method Article
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 bor...
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 i...
Authors have no conflicts of interest to disclose. Authors have no affiliation with commercial names and trademarks mentioned in this work.
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.
Name | Company | Catalog Number | Comments |
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 |
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