In Vitro and In Vivo Models of Ischemic Stroke

At a glance

    For the past 20 years, the pathophysiology of stroke has come a long way thanks to in vivo and in vitro models of neurotoxicity and ischemia. All of this increased understanding has spurred the invention of many neuroprotective therapeutics, but these need to be models of cerebral ischemia that can predict human consequences.

    Ace Therapeutics has extensive experience in stroke modeling and offers a wide range of high-quality and customized animal and in vitro models of ischemic stroke designed for mechanistic studies and for testing new thrombolytic and neuroprotective strategies.

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    In Vitro Ischemia Models

    In vitro models were created to fill the gap between animal models for ischemia that are time-consuming and variable. Scientists have turned their efforts to developing in vitro test systems that can assess neuroprotective effects in vivo. We generally conduct our experiments using primary cell cultures of neurons or tissue slices and most commonly in the cortex, hippocampus or cerebellum.

    Different types of insults are used to induce damage in these in vitro systems, primarily modeling neurotoxicity rather than ischemia. However, a notable exception is the combination of hypoxia and glucose deprivation, which aims to mimic the effects of impaired blood supply and more closely simulate ischemic conditions.

    Table 1. In vitro ischemia models

    Cellular platform Physiological relevance Structural complexity modelled Possible application to stroke research
    Primary cells High Artificial arrangement required Oxygen‐glucose deprivation (OGD), chemical ischemia, excitotoxicity BBB models, thrombosis and leukocyte responses
    Cell lines Low Artificial arrangement required OGD, chemical ischemia, excitotoxicity BBB models
    Brain slice High To a high degree OGD, chemical ischemia, excitotoxicity
    Organotypic cell culture High To some degree OGD, chemical ischemia, excitotoxicity
    Embryonic stem cells Potentially High Artificial arrangement required OGD, chemical ischemia, excitotoxicity, BBB models
    iPSCs Potentially High Artificial arrangement required OGD, chemical ischemia, excitotoxicity, BBB models

    Modeling the BBB in stroke

    Blood-brain barrier (BBB) compromise is a critical pathological process in stroke, disrupting brain homeostasis. Various in vitro models have been developed to study ischemic damage and potential therapeutic interventions for BBB breakdown. These models typically utilize a two-layer or transwell cell culture system, enabling the measurement of permeability to molecules like sucrose or transendothelial electrical resistance (TEER) across the cell monolayer. Recently, commercially available 3D co-culture models have also been introduced, enhancing the study of BBB dynamics in a more physiologically relevant context.

    There are several different in vitro models of the ischemic BBB, which differ regarding the cell types used (primary cells mainly isolated from mouse, rat, or bovine brain or immortalized cell lines), the cell configuration (monoculture of brain capillary endothelial cells (BCECs) or cocultured with other brain cell types such as glial cells, astrocytes, pericytes, neurons, in contact or not), and the protocol applied to mimic the ischemic event (nutrients, oxygen levels used, and time points).

    The choice of cell type and configuration is critical to the experimental results. Primary brain endothelial cell lines grown in co-culture with astrocytes has the benefit of mimicking astrocyte-endothelial interactions, leading to less open endothelial cell junctions for vectorial drug delivery experiments. Monocultures of immortalised cell lines, like hCMEC/D3, are typically more permeable, but do not reproduce the in vivo BBB exactly, although hCMEC/D3 cells are human, and so the findings translate to humans easily.

    Fig. 1. Currently used blood-brain barrier models.Fig.1. Currently used BBB models. (Holloway, et al., 2016)

    Animal Models of Ischemic Stroke

    Models of cerebral ischemia are typically divided into two categories: global and focal. Global models simulate the deficits associated with cardiac arrest and cardiac bypass surgery, resulting in selective necrosis in vulnerable brain regions.

    Table 2. Global ischemia models

    Experimental models Advantages Disadvantages Animals
    Four-vessel occlusion (4VO) model Easy to prepare; high reproducibility; low incidence of seizures Two-stage surgical procedure; permanent occlusion vertebral arteries; high mortality Rats, mice, rabbits, dogs, pigs
    Two-vessel occlusion (2VO) model One-stage surgical procedure; controllable recirculation; lower mortality Poor reproducibility; strains-depended Rats, mice, rabbits, cats, dogs, sheep, pigs
    Complete global brain ischemia Close to human condition of cardiac arrest and resuscitation Extracerebral complications; complicated procedure; poor survival rate and coma Rats, rabbits, cats, dogs, pigs, sheep
    Ventricular fibrillation cardiac arrest Relatively easy to induce Affects all organs of the body, not just the brain Rats, rabbits, cats, dogs, pigs, sheep
    Aorta/vena cava occlusion models Simple surgery, rapid for screening purposes; delayed and selective neurodegeneration; reliable measurement of damage Small animals, so difficulties of undertaking physiological measurements; variable outcome due to variations in cerebral circulation Dogs and pigs

    In contrast, focal models are more relevant for studying acute ischemic stroke, as they produce brain infarction. Despite their utility, it is important to note that no single model has been proven to reliably predict drug efficacy in humans.

    Table 3. Focal ischemia models

    Experimental models Advantages Disadvantages Animals
    Endovascular filament occlusion Localization of the infarct (mostly MCAO), penumbra, blood-brain barrier injury, inflammatory processes and cell death pathways (Permanent and transient ischemia); no craniectomy Tremendous variations; spontaneous hyperthermia; not suitable for thrombolysis Rats, mice, monkeys
    Transcranial occlusion Penumbra, blood-brain barrier injury, inflammatory processes and cell death pathways Destroy dura; intracranial infection; one-sided blindness Rats, mice, cats, sheep, pigs, monkeys
    Photothrombotic stroke Reproducibility; easy manipulation; less trauma; long-term survival Lack of penumbra; poor responses to rt-PA Rats, mice
    Endothelin-1 model Infarcts of variable sizes in nearly any brain region; Subcortical stroke
    Recovery and plasticity mechanisms in chronic stroke
    Affected by anesthetics; neural transmission/modulation Rats, monkeys
    Thromboembolic clot models Thromboembolic infarcts; Transient ischemia with unpredictable time point of lysis of the embolus; Possibility to test thrombolytic therapies Poor reproducibility; spontaneous recirculation Rats, rabbits, dogs
    Artificial spheres occlusion Microspheres induce graded infarcts; reproductivity of macrosphere embolization Poor reproducibility of microspheres models; not suitable for transient occlusion and thrombolysis Rats, rabbits, monkeys
    References
    1. Holloway, P. M., & Gavins, F. N. (2016). Modeling ischemic stroke in vitro: status quo and future perspectives. Stroke47(2), 561-569.
    2. Tajiri, N., et al. (2013). In vivo animal stroke models: a rationale for rodent and non-human primate models.Translational stroke research4, 308-321.
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