In vivo models have provided insight into the pathophysiology of stroke. However, most animal models of ischemia are extremely time-consuming to conduct and contain the variation inherent in using a whole animal as opposed to a single cell or slice of tissue. With the advent of new cellular technologies and genetic and molecular manipulation, there is a great opportunity for the development of in vitro systems to model stroke and improve the drug discovery pipeline.

Ace Therapeutics offers a comprehensive range of ischemic stroke in vitro assays to help our clients study study pathogenic mechanisms and drug discovery screening.

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Modeling Ischemic Stroke In Vitro

Chemical and Enzymatic Methods

The chemical methods use rotenone, antimycin, and sodium azide to inhibit the electron transport chain. The enzymatic methods used were the GOX/CAT system consisting of glucose oxidase (GOX) and catalase (CAT) and 2-deoxyglucose.

While these chemical and enzymatic methods are suitable for high-throughput applications due to their ease and quick responses, they may compromise relevance. For example, chemical hypoxia tends to generate more free radicals than true anoxia, which can affect the accuracy of the model in reflecting physiological conditions.

Oxygen-glucose Deprivation (OGD)

To this end, the atmosphere in the incubator with cultured cells or brain slices is exchanged. The normal O₂/CO₂ equilibrated medium is replaced by an N₂/CO₂ equilibrated medium in a hypoxic chamber with glucose omitted from the medium.

Compared to in vivo models, there is characteristically a need for a longer episode of energy deficiency to induce neuronal death. Typically, cell cultures are exposed to OGD for 1-24 h. In contrast to single-deprivation paradigms, 1 h exposure to OGD is already sufficient to induce widespread neuronal death.

Excitotoxicity

Experiments with excitotoxicity can be carried out using agonists of the glutamate receptor in vitro, like N-Methyl-D-aspartate (NMDA) or glutamate, independent of ischemic damage. That allows researchers to study which glutamate receptor subtypes are involved in excitotoxicity.

Cell-Based Assays

In vitro models cannot fully replicate the complex conditions of ischemic stroke due to the absence of intact blood vessels, blood flow, and leukocyte infiltration. However, they are valuable for investigating specific biochemical and molecular mechanisms associated with energy deficiency similar to ischemia. These models allow for direct study of critical control points and molecular pathways related to necrotic cell death, programmed cell death, and autophagy. Additionally, in vitro systems enable high-throughput analyses, which are crucial for testing new neuroprotective pharmaceuticals. The use of human or humanized cells in these models is becoming increasingly important for relevance to human conditions.

Caspase Activation

The principal executioners of apoptosis are caspases and activation (caspase 3, 6, 7 in particular) is a hallmark of programmed cell death. These caspases can be quantified to determine how much apoptotic signalling is triggered by OGD.

Lactate Dehydrogenase (LDH) Release

When the plasma membrane is damaged, lactate dehydrogenase (LDH) spills out into the extracellular fluid as a cell-damaging and necrosis marker. We can measure both cumulative cell damage and necrotic cell death by measuring the LDH in the culture medium after OGD.

Neurite Outgrowth Studies

Nerve synapse growth is critical for neuronal recovery and regeneration. Neuronal synapse extension and branching are assessed in neurons surviving after OGD to evaluate their ability to recover and form connections.

Cell Viability

Scientists can perform many different measurements (e.g., MTT or ATP tests) of cell viability following OGD. These tests assess how cells are being metabolized and functioning, which allows us to see just how many neurons remain functional after an ischemic event.

Mitochondrial Damage

Ischemic cell death is caused by mitochondrial dysfunction. Scientists can determine mitochondrial integrity with dyes or tests that evaluate mitochondrial membrane potential and reactive oxygen species (ROS).

Oxidative Stress

When the blood is hypoxic and low on glucose, it's going to be at higher oxidative stress, which accelerates cell damage. The degree of oxidative stress is measured through assays using markers like malondialdehyde (MDA) or detection of particular ROS.

Studies of Efflux Transporter Expression and Function in the Ischemic Carrier

In vitro OGD models can be used to study efflux transporter expression and function in the ischemic barrier, both in vivo and endothelial cell lines in coculture with astrocytes in vitro.

Fig.1. Factors defining the predictive value of in vitro ischemic stroke models. (Van, et al., 2022)

1. Van Breedam, E., & Ponsaerts, P. (2022). Promising Strategies for the Development of Advanced In Vitro Models with High Predictive Power in Ischaemic Stroke Research. International journal of molecular sciences, 23(13), 7140.