Background

With a global prevalence of more than 101.5 million, stroke is a leading cause of mortality and chronic disability worldwide (Benjamin et al., 2019; Diseases and Injuries, 2020). Though some strokes are caused when a vessel ruptures (i.e., hemorrhagic stroke), ischemic strokes account for 87% of clinical cases and occur following vascular occlusion, either by local arterial thrombus formation (e.g., cerebral atherosclerotic stroke) or a traveling embolic clot (e.g., cardioembolism) (Virani et al., 2021). Acute interventions to restore perfusion and halt neuronal death include pharmacological thrombolysis [i.e., intravenous recombinant tissue plasminogen activator (rtPA)] and mechanical endovascular thrombectomy (Pierot et al., 2018; Powers et al., 2018; Adams and Nudo, 2013), though clinical exclusion criteria restrict eligibility to just 20% of patients (National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995; Hacke et al., 2008; Saver et al., 2009; Smith and Furlan, 2016; Yaghi et al., 2017; Evans et al., 2017; Albers et al., 2018; Nogueira et al., 2018; Powers et al., 2018; Mokin et al., 2018).

The necessity for more inclusive therapeutic options underscores the significance of animal models, which have long been used to study disease pathology, identify molecular mechanisms of injury, and test treatment paradigms (Narayan et al., 2021). While large animal models (i.e., pigs, dogs, non-human primates) serve a key role, given their higher order resemblance to the human condition (Kaiser and West, 2020), small animals are most commonly used in preclinical stroke research. Factors specifically favoring murine models (i.e., mice and rats) include lower cost, reliable physiological responses, procedural reproducibility, available imaging modalities, a variety of functional and behavioral tests, and options for genetic manipulation (Fluri et al., 2015; Hermann et al., 2019).

The majority of clinical strokes occur in regions supplied by the middle cerebral artery (MCA), a terminal branch of the internal carotid artery (ICA) that supplies two-thirds of the lateral cortex, the infra-lateral surface of the anterior brain, and many deep subcortical structures (e.g., internal capsule and basal ganglia) (Nogles and Galuska, 2021). Numerous preclinical approaches to experimental MCA occlusion (MCAO) have been described, including direct suturing or electrocoagulation of the MCA (transcranial MCAO), intraluminal placement of a monofilament (intraluminal MCAO), delivery of synthetic (micro- or macro-sphere embolisation) or autologous clots (thromboembolisation), transcranial vessel illumination following photosensitive dye injection (photothrombosis), and stereotactic injection of a potent vasoconstrictor (Endothelin-1) (Fluri et al., 2015; Hermann et al., 2019; Kumar et al., 2016). Of these methods, intraluminal MCAO is the most common technique applied for ischemic stroke induction in murine models.

First introduced into mice in 1993 by Chan et al. (1993), the intraluminal MCAO procedure induces a proximal large vessel occlusion in mice, through placement of a coated monofilament tip at the MCA origin. The external carotid artery (ECA) is permanently ligated, after which the partially-coated monofilament is inserted and coursed proximally to the common carotid artery (CCA) bifurcation, before being introduced into the ICA. The coated tip of the monofilament is advanced to the origin of the MCA and secured for the duration of the occlusion. Reduction of blood flow into the MCA is confirmed using Laser Doppler Flowmetry (LDF) (40-70% reduction). Adjustable variables that alter stroke severity and distribution include monofilament composition (e.g., nylon) and diameter (e.g., 3-0), monofilament tip coating material (e.g., silicone), insertion length of the monofilament (e.g., 8 mm), duration of surgery (e.g., 45 min), and duration of occlusion (e.g., 30 min) (Engel et al., 2011; Fluri et al., 2015; Lee et al., 2014). Though this model allows for permanent occlusion of the MCA, transient MCAO (tMCAO) is achieved by retracting the monofilament tip to allow for reperfusion, and is thus more clinically relevant. Additional advantages of this model are its reproducibility, regarding both infarct volume and cognitive/functional deficits, and the fact that a craniotomy is not required. tMCAO also produces an ischemic core and penumbral region, which facilitates the study of neuronal death, neuroinflammation, and blood-brain-barrier breakdown, as well as testing of therapies to rescue the penumbra.

Our group has long used the intraluminal tMCAO model of ischemic stroke induction (Khanna et al., 2017; Rink et al., 2017; Sen et al., 2017; Balch et al., 2021; Lemmerman et al., 2021). Limitations to this model include larger infarction compared to human strokes and compromised hypothalamic regulation of body temperature, which is not typically present in humans and complicates survivability of murine models (Carmichael, 2005). Additionally, distal ligation of the ECA removes blood flow to masticatory muscles, which can further reduce eating in an animal facing significant weight loss (Dittmar et al., 2003). Despite these limitations, MCAO is well-established and widely accepted for murine ischemic stroke induction in preclinical research (Fluri et al., 2015). In conjunction with a dedicated small animal surgeon, our group has spent over seven years refining the tMCAO approach, to address the limitations related to food/water intake, weight loss, and mortality. Our refined protocol for surgery and post-operative care yields successful MCA-territory infarction, while enhancing reproducibility and animal survivability. Here, we provide a detailed protocol for surgical induction of unilateral transient ischemic stroke using our refined intraluminal thread model of transient unilateral MCAO.