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 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.
Stroke is a devastating disease that affects approximately 15 million people worldwide annually, according to the WHO. Around one-third of patients succumb to the condition, while another third experience permanent disability. Stroke is a complex pathology involving various cell types, such as neural and peripheral immune cells, vasculature, and systemic responses1. The intricate network of reactions triggered by stroke at the systems level cannot be currently replicated using in vitro models. Thus, experimental animal models are essential to delve into the disease's mechanisms and to develop and test new therapies. Currently, early tissue reperfusion is the only approved intervention, either through thrombolysis with tissue-type plasminogen activator (tPA) or endovascular thrombectomy1.
Occlusions of the middle cerebral artery (MCA) are frequent in stroke patients. Consequently, rodent models of transient MCA occlusion (tMCAo) were initially developed in rats2,3,4. Nowadays, genetically modified mice are the most commonly used animals in experimental stroke models. In this study, we describe a minimally invasive model of intraluminal tMCAo in mice. The approach is performed via the carotid artery at the neck level, without craniectomy.
The duration of the occlusion period is a critical factor that determines the extent of the ischemic lesion. Even short occlusions of 10 min can cause selective neuronal death without an apparent infarction, while longer occlusions, typically lasting 30 to 60 min, result in some degree of cerebral infarction. Unlike the proximal and distal branches of the MCA that supply the cortex and have collaterals, the lenticulo-striatal arteries providing blood to the striatum lack collaterals5. As a consequence, there is a greater reduction of blood flow in the striatum than in the cortex after tMCAo. Thus, occlusions of 30 min or less generally affect the striatum but not the cortex, whereas longer occlusions, from 45 min onwards, often generate an ischemic lesion in the entire MCA territory, including the striatum and dorsolateral cortex.
To ensure the well-being of the mice, we administer analgesics prior to the procedure and use anesthesia during surgery. Nevertheless, anesthesia can potentially introduce artificial alterations in the physiology of the mouse and affect some outcome measures6. The surgical intervention, when performed by experienced personnel, usually lasts about 15 min for inducing MCAo. Subsequently, the total time under anesthesia depends on the occlusion period. For experiments where minimizing anesthesia is crucial, an alternative step in the procedure involves discontinuing anesthesia during the occlusion period and limiting it only to the surgical steps for inserting and withdrawing the filament occluding the MCA. This approach reduces the duration of anesthesia and minimizes its potential artifactual effects on the experimental model7,8. Therefore, the method of inducing transient focal ischemia is presented by intraluminal occlusion of the MCA with two variants: with the mouse anesthetized during the entire occlusion period or with the mouse awake during this period. In either case, a sham surgery should be performed in parallel with the intervention carried out on the ischemic mice. Additionally, data on outcome assessment is provided as measured by behavioral tests and MRI at various time points after reperfusion. Finally, the main factors to consider when implementing the experimental procedure are discussed.
Animal work was conducted following the Catalan and Spanish laws (Real Decreto 53/2013) and the European Directives, with approval of the ethical committee (Comité Ètic d'Experimentació Animal, CEEA) of the University of Barcelona, and the local regulatory bodies of the Generalitat de Catalunya. Studies are reported in compliance with the ARRIVE guidelines. This procedure is designed to be performed in adult mice, starting at 8 weeks of age, with no age limit. Examples of the surgical procedure developed in C57BL/6 mice of 10-12 weeks of age are provided here. Anatomical differences depending on mouse strain should be considered.
1. Animal preparation
2. Cerebral blood flow (CBF) assessment with laser Doppler flowmetry (LDF)
3. Transient middle cerebral artery occlusion (tMCAo)
4. Post-operative care
There are different approaches to evaluate the outcome of the tMCAo procedure. In vivo neuroimaging methods (MRI) and behavioral testing are utilized here.
Mice develop ischemic lesions in the brain, mainly affecting the territory supplied by the MCA ipsilateral to the occlusion, such as the striatum and dorsolateral cortex. Several methods exist to determine the extent of the lesion, including 2,3,5-triphenyltetrazolium chloride (TTC) tissue staining, histological staining (hematoxylin/eosin, thionine acetate), and in vivo neuroimaging modalities like MRI. MRI has been chosen here due to its non-invasive nature and the ability to use the same tissue for other studies, providing a comprehensive assessment of the lesion in each mouse. Additionally, MRI allows for repeated measurements in the same animals, increasing the reproducibility of results and often reducing the number of animals required for a study.
The same anesthesia protocol with isoflurane (induction 5%, maintenance 1.5%) was used in the MRI sessions. For lesion volume assessment, a fast T2-weighted sequence (T2w turbo RARE fast spin-echo)9 was used to minimize the time the animal is anesthetized, which is important when longitudinal studies with MRI acquisitions at different times are to be performed in the same mice. This procedure allows the evaluation of changes in the lesion over time in the same animals, and it is very useful when applied for neuroprotection studies or to test drug efficacy, among others. Image experiments were conducted on a 7T horizontal animal scanner. The technical specifications of the anatomical sequence (may differ depending on the magnetic field strength): T2_TurboRARE; 22 coronal sections; 0.5 mm thick; echo time (TE) = 33 ms; repetition time (TR) = 2336.39 ms. 2 averages. Flip angle, 90°; field of view (FOV) = 20 mm x 20mm, with a matrix size of 256 x 256. Figure 2A shows a representative example of MR images of lesion evolution in the same mouse, assessed at 40 min, 6 h, 24 h, and 48 h after reperfusion. Progression of the lesion volume takes hours to approximately two days to complete. Quantification of the lesion volume shows this evolution over time (Figure 2B).
A variety of neurological scales have been described to assess the neurological impairment caused by ischemic insult. We suggest using neuroscore tests that have been extensively described in previous manuscripts. For instance, the test reported in detail by Orsini et al. (2012)10 is recommended.
A wide variety of behavioral tests are available, mainly to detect differences in motor and sensory function impairment. For this purpose, the grip strength test and the corner test were used. The grip strength test is used to evaluate motor function. Forelimbs strength is measured with a Grip Strength Meter connected to a digital force transducer (see Table of Materials). Mouse holds on to a horizontal bar with both forepaws while gently pulling it backwards through the tail. The maximum strength of the grip prior to the forepaws release is noted. Five trials per animal are performed, and the main value is calculated after excluding the maximum and the minimum values. The corner test is used to detect unilateral abnormalities of sensory and motor functions. The apparatus consists of a corner with two boards (30 cm × 20 cm × 1 cm) attached with an angle of 30° and a small opening at the end. The mouse is placed halfway facing the corner. When the mouse enters deep into the corner, both sides of the vibrissae are stimulated together. The mouse then turns back to face the open end. A total of 10 trials are performed per animal, and the chosen sides are noted. 50% left and right turns are expected under physiological conditions, whereas a right preference is expected in mice with the right MCAo. A trial is considered valid when a complete turn is achieved or when the mouse turns its head ≥ 90º. Results are shown as the percentage of right (ipsilateral) turns.
The representative results showing the loss of strength exhibited by the mice 24h after tMCAo measured by the grip strength test are presented (Figure 3A), as well as their preference to turn to the side ipsilateral to the lesion when stimulated in the corner test (Figure 3B). Performing behavioral tests on the same day of the surgery may be less precise since some parameters could be altered due to the proximity of the anesthesia and the post-operative period.
Figure 1: Schematic representation of the vascular tree of the neck (right side). (A) The image shows the main arteries (Common Carotid Artery-CCA, External Carotid Artery-ECA, Internal Carotid Artery-ICA) and the different branches (Pterygopalatine artery Pt; Occipital artery Occ; Superior thyroid artery St; Maxillar and lingual arteries Max/Lin). (B) The first steps of the surgical procedure, with the CCA ligated by suture, the ICA circulation is interrupted by a vascular clamp, and the monofilament is introduced via the ECA. (C) Reorientation of the ECA to push the monofilament to the occlusion zone. Please click here to view a larger version of this figure.
Figure 2: Representative MR images. (A) T2-w images of the same mouse at different time points after reperfusion shows the evolution of the lesion in the acute phase. The area affected by the infarct increases rapidly over the first hours and experiences little variation thereafter. (B) Evolution of lesion volume in the acute phase after MCAo. Each bar represents the mean ± SD of percentage (%) of lesion volume. Lesion volume increases significantly during the first 24 h after reperfusion (*p = 0.0182; **p = 0.0088; 1-way ANOVA/ Kruskal-Wallis test). Please click here to view a larger version of this figure.
Figure 3: Behavioural tests before (basal) and 24 h after tMCAo (n = 16 mice). (A) Grip strength test shows the maximal (Max.) strength per mouse. (B) Corner test shows the percentage (%) of right turns. Graphs show box and whiskers (minimum to maximum values) per group, and points correspond with individual mice (****p < 0.0001; Wilcoxon matched-pairs signed rank test). Please click here to view a larger version of this figure.
The intraluminal tMCAo procedure is the most commonly used model of focal brain ischemia with reperfusion in basic research. Currently, mice are the preferred animal model due to the availability of genetically modified strains. However, it's essential to acknowledge that genetically modified mice and their genetic backgrounds can impact brain vascularization. The presence of collateral circulation and anastomoses between different arterial territories can significantly influence the outcomes of experimental procedures11.
When conducting this procedure, certain crucial points must be considered. Injuries can occur outside the territory of the MCA, affecting areas like the hippocampus, thalamus, or hypothalamus, usually due to occlusion of the posterior communicating artery. Additionally, a small percentage of mice may not display apparent infarction despite a seemingly successful surgical procedure.
Several variables require monitoring during the procedure. The development of brain lesions depends directly on the severity of the cerebral blood flow (CBF) drop and the duration of this reduction5,12. To track CBF during the surgical process and assess flow changes during occlusion and after reperfusion, it is highly recommended to use systems like LDF (Laser Doppler Flowmetry) or Laser Speckle flowmetry13,14. The duration of occlusion also influences the extent of the lesion, with occlusions lasting 30 min or less primarily affecting the striatum and occlusions longer than 45 min, also affecting the cortex regions supplied by the MCA. Considering the multiple variability factors, it's crucial to establish inclusion/exclusion criteria before the study commences and to report them.
Furthermore, other factors like blood pressure, body temperature, and blood glucose can significantly affect stroke outcomes. Maintaining mice under anesthesia during occlusion may impact parameters such as blood pressure, synaptic excitability, or inflammation6,15. An alternative option is to awaken the animals during occlusion.
Anesthesia can influence blood pressure, which in turn affects the size of the infarct15. Maintaining proper body temperature is essential due to the well-documented effects of hypothermia and hyperthermia on cerebral ischemia16. Additionally, hyperglycemia has been shown to increase ischemic damage17. Moreover, age and gender are factors that must be considered when designing experiments and analyzing results.
Instead of being seen as a drawback, the multiplicity of factors should be viewed as an advantage, but it is crucial to record variables and consider variability when calculating the sample size. Failures in translating results from experimental research to clinical practice can be attributed, in part, to under-powered experimental groups and the use of animal models that do not adequately represent pathological conditions in humans. Typically, young, healthy, mostly male mice are used in experimental models, but these can be augmented to investigate mice with comorbidities such as hypertension, hyperglycemia, or hypercholesterolemia, as well as different age groups and sexes.
The authors have nothing to disclose.
Study supported by grant PID2020-113202RB-I00 funded by Ministerio de Ciencia e Innovación (MCIN)/Agencia Estatal de Investigación (AEI), Gobierno de España/10.13039/501100011033 and "European Regional Development Fund (ERDF). A way of making Europe". NCC and MAR had predoctoral fellowships (PRE2021-099481 and PRE2018-085737, respectively) funded by MCIN/AEI/ 10.13039/501100011033 and by "European Social Fund (ESF) Investing in your future". We thank Francisca Ruiz-Jaén and Leonardo Márquez-Kisinousky for their technical support. We acknowledge the support of the MRI imaging facility of Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS). The Centres de Recerca de Catalunya (CERCA) Program of Generalitat de Catalunya supports IDIBAPS.
6/0 suture | Arago | Vascular ligatures | |
6/0 suture with curved needle | Arago | Skin sutures | |
9 mg/mL Saline | Fresenius Kabi | CN616003 EC | For hydration |
Anaesthesia system | SurgiVet | ||
Blunt retractors, 1 mm wide | Fine Science Tools | 18200-09 | |
Buprenorfine | Buprex | For pain relief | |
Clamp applying forceps | Fine Science Tools | S&T CAF4 | |
Dumont mini forceps | Fine Science Tools | M3S 11200-10 | |
Forceps | Fine Science Tools | 91106-12 | |
Glue | Loctite | To stick LDF probe to the skull | |
Grip Strength Meter | IITC Life Science Inc. | #2200 | |
Isoflurane | B-Braun | CN571105.8 | |
LDF Perimed | Perimed | Periflux System 5000 | |
LDF Probe Holders | Perimed | PH 07-4 | |
Medical tape | |||
MRI magnet | Bruker BioSpin, Ettlingen, Germany | BioSpec 70/30 horizontal animal scanner | |
Needle Holder with Suture Cutter | Fine Science Tools | 12002-14 | |
Nylon filament | Doccol | 701912PK5Re | |
Recovery cage with heating pad | |||
Sirgical scissors | Fine Science Tools | 91401-12 | |
Small vessel cauterizer kit | Fine Science Tools | 18000-00 | |
Stereomicroscope and cold light | Leica | M60 | |
Suture tying forceps | Fine Science Tools | 18025-10 | |
Thermostat, rectal probe and mouse pad | Letica Science Instruments | LE 13206 | |
Vannas spring scissors (4mm cutting edge) | Fine Science Tools | 15019-10 | |
Vascular clamps | Fine Science Tools | 00396-01 |