Transient Middle Cerebral Artery Occlusion Model of Stroke

Summary

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.

Abstract

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.

Introduction

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 rats^2,3,4. 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 model^7,8. 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.

Protocol

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

1. Before starting the surgical procedure, gather and sterilize all the required materials and tools. Set up the operating table with all the necessary surgical materials (listed in the Table of Materials).
2. Anesthetize the animal using isoflurane inhalation in a mixture of oxygen and nitrous oxide (30%/70%).
3. Administer buprenorphine (see Table of Materials) subcutaneously at a dosage of 0.05 mg/kg BW to provide analgesia and alleviate any pain and discomfort.
   NOTE: Analgesia is mandatory, but different protocols are accepted. Pain and discomfort signs must also be controlled during the first days after MCAo (see step 4). Apply corrective solutions when necessary.
4. Place the animal in an anesthesia induction box (see Table of Materials) with 5% isoflurane until it reaches a state of deep anesthesia (loss of reflex in paw puncture and ocular reflex).
5. Position the mouse on the operating table and decrease the level of isoflurane to 1.5%, delivered by face mask. Apply vet ointment to avoid eye dryness during the procedure.
6. Maintain body temperature at 37 ± 0.5 °C controlled by a rectal probe connected to a heating pad (see Table of Materials).
7. Shave the ventral part of the neck and the head (calvaria) with an electric razor. Carefully remove fur debris and disinfect the skin areas three times in circular movements with iodine-based disinfectant and 70% alcohol.
   .

2. Cerebral blood flow (CBF) assessment with laser Doppler flowmetry (LDF)

1. With scissors, make an incision on the skin of the head, in the direction of the sagittal suture, from the ears to the area between the eyes.
2. Retract the skin and remove the periosteum on the right side of the skull.
3. Find the coordinates (2.5 mm lateral from Bregma) and attach the Doppler holder (see Table of Materials) using cyanoacrylate. After the glue has dried, connect the Doppler probe and check for the correct signal readout.

3. Transient middle cerebral artery occlusion (tMCAo)

1. Turn the mouse over to the supine position, and fix it to the surgical table with medical tape.
2. Make a midline incision on the neck. Laterally pull back the skin and salivary glands using retractors (see Table of Materials) to expose the carotid territory.
3. Identify the vascular anatomy of the common carotid artery (CCA), the ICA, and the ECA, as well as the different arteries derived from them (maxillary and lingual, superior thyroid, occipital, and pterygopalatine) (Figure 1A).
4. Detach the main arteries from the adjacent connective tissue so that they can be handled.
   NOTE: Take special care not to damage the nerves, especially the vagus nerve, which runs parallel to the CCA.
5. Wrap a 6-0 silk suture (see Table of Materials) around the ECA at the maxillary/lingual bifurcation. Tightly secure a knot to permanently interrupt the circulation.
6. Pass a second suture around the same artery, between the first knot and the CCA bifurcation, and keep this knot loose.
7. Place a third thread around the CCA and tie a slip knot that can be easily untied.
   NOTE: This can also be carried out with a vascular clip, but the thread allows more movement and flexibility. At this stage it is possible to observe a first decrease in CBF in the LDF signal.
8. Place a vascular clip (see Table of Materials) interrupting blood circulation from the ICA.
9. Make a small incision in the ECA, close to the area where the tight knot is located.
10. Insert the monofilament until the thick coating has completely entered the arterial lumen.
11. Tighten the second knot to hold the monofilament inside the artery and prevent the pressure exerted by the blood from pushing it out (Figure 1B).
12. Remove the vascular clip from the ICA.
13. Cut the ECA below the first knot and rotate the stump to orient it in the direction of the ICA (Figure 1C).
14. Advance the monofilament via the ICA until the point where the MCA branches out.
    NOTE: The occlusion is reflected in an abrupt blood flow drop in the LDF readout. We consider a successful occlusion when the drop in CBF is greater than 70% from the basal value. If CBF measurement systems are not available, the point of occlusion can be noted by the resistance to advance, which in adult mice is usually about 11 mm from the bifurcation of the CCA.
    1. If anesthesia is continued during the occlusion period, monitor the mouse and keep it under constant observation for 45 min.
    2. In case the mouse is awakened during the occlusion period, suture the skin of the neck with several stitches. Without disconnecting the LDF probe, place the mouse in the temperature-controlled box, allowing recovery from anesthesia.
       NOTE: It is common for the mouse to exhibit spontaneous circling behavior during this period, indicative of successful occlusion.
    3. After 40 min, anesthetize the mouse again following the same anesthesia and disinfection procedures as indicated in points 1.4, 1.5 and 1.7. Place it back on the surgical table, and remove the stitches from the neck.
15. After 45 min of occlusion, loosen the knot holding the monofilament in place. Pull slowly and gently on the filament and check that tissue recanalization occurs.
16. Pull out the filament and tighten the knot to prevent blood loss.
17. Untie the CCA knot. Ensure that there is no arterial wall damage.
18. Remove the retractors and re-position the muscles, glands, and skin. Suture the skin (6-0) and apply disinfectant.
19. Disconnect the Doppler probe, and detach the holder. Suture and disinfect the skin of the head.
20. During the recovery period from anesthesia, leave the mouse in a cage provided with a heater to maintain the temperature. Keep it under constant observation until it is fully recovered from anesthesia. After recovery, the mouse can be returned to its cage.
    ​NOTE: Housing with social enrichment is highly recommended. However, never mix operated mice with non-operated mice in the same cage without any physical separation in order to prevent aggression.

4. Post-operative care

1. Periodically supervise the animals following the procedures and regulations established according to the local regulations. Provide analgesic treatment on the appropriate schedule to minimize pain after the surgery.
   NOTE: In the present study, the same analgesic was applied as at the beginning of the intervention (Buprenorphine 0.05mg/kg BW) at 6 h and 24 h after surgery.
2. Perform euthanasia when the supervision parameters indicate so, following the institutionally approved protocols.
3. Daily monitor the weight of the animals. Provide soft food to the animals during the first few days after surgery. In addition, hydrate them by subcutaneous injection of saline (200 µL) immediately after surgery and periodically thereafter if it is observed that the mouse does not hydrate on its own. Arrange food and water in a way that is easily accessible to the animal.
4. Once the in vivo study is completed, anesthetize the mice, euthanize them, and remove the brain tissue for further histopathological analysis (if needed).

Representative Results

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.

Discussion

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 reduction^5,12. 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 flowmetry^13,14. 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 inflammation^6,15. 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.

Materials

Name	Company	Catalog Number	Comments
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