Ischemic Stroke Model based In Vitro Assay Service

Ischemic stroke (IS) is one of the global diseases that seriously threaten human life, which seriously endangers the health of individuals and imposes a great burden on the society and economy. IS usually occurs when cerebral blood vessels are blocked. Brain injury of post-stroke are closely associated with neuronal death, oxidative stress, neuroinflammation, lactate accumulation, mitochondrial dysfunction, autophagy and blood-brain barrier (BBB) disruption.

Creative Biolabs offers a variety of approaches in vitro models and assays to assist you in uncovering innovative therapeutic targets for the treatment and improvement of IS.

Available Assay Models

• Neural Cell Culture

In addition to PC12 cell line and SH-SY5Y cell line, primary rodent-derived neuron culture and neuron-astrocyte (or microglia) co-culture are widely used for in vitro IS research. Oxygen–glucose deprivation (OGD) and oxygen-glucose deprivation/Reperfusion (OGD/R) are most common and most physiologically relevant methods to induce IS.

• BBB Models

BBB integrity will be disrupted by IS, which leads to subsequent secondary damage. We are professional to conduct BBB model with human CMEC/D3 cell line, human primary cerebral microvascular endothelial cells in co-culture with human primary or iPSC-derived astrocytes, pericytes and neurons to study drug screening and permeability studies.

• Organotypic Slice Culture

Organotypic slices are obtained from animal brains and are able to be cultured in vitro for a few weeks. Benefits of organotypic slices lies in retaining the tissue structures and functions during IS research.

• Cerebral Organoids

Human cerebral organoids have unique advantages as they closely mimic the complex structure of the human brain, with diverse cell types and neural connections. With human cerebral organoids, establishing an in vitro humanized IS model of OGD to test anti-stroke drugs is the most promising application.

Available In Vitro Assays

• Cell Viability Assay

After conducting IS model by utilizing OGD/OGDR procedure in cell models, we can perform MTT assay, CCK8 assay, LDH assay, Calcein/ Annexin V / Caspase 3/7of apoptosis assay and TUNEL assay to evaluate antioxidant and neuroprotective effects.

Fig.1 Retinal precursor cells R28 induced by OGD/R demonstrate features of pyroptosis, apoptosis, and necroptosis.^{1, 8}

• Neuroinflammation Assay

With OGD/R treated single microglia culture or co-cultures of neuron and microglia, we are able to detect proinflammatory cytokines or other inflammatory markers by evaluation of protein and/or gene expression levels, which will help figure out if inhibiting neuroinflammation with drug treatment could potentially reducing neuronal death and protect brain from IS injury.

Fig.2 Representative fluorescence image of Iba1 to assess effects of Sirt1 on microglial activation and inflammation from cultured N9 after OGD/R injury.^{2, 7}

• Oxidative Stress Assay

Reactive oxygen species (ROS) during cerebral IS leads to oxidative stress (OS), which more likely cause cell death and cascade development of brain injury. We offer ROS assays, antioxidant assays, lipid peroxidation assays, protein oxidation / nitration assays and DNA / RNA damage and repair assays to explore the interaction between OS and cell death after stroke.

Fig.3 CPI-1189 inhibits OGD/R-induced oxidative injury in SH-SY5Y cells.³

• BBB Permeability Assay

Utilizing different kinds of BBB models, we provide many options to learnt BBB permeability and function in IS by measuring trans-endothelial electrical resistance (TEER) and detecting the permeability of paracellular flux markers after the treatment of different combinations and concentrations of any compounds of your interest. Also, we can analysis the medium samples by RT-qPCR and WB.

Fig.4 Changes on cell viability, permeability, and occludin in bEnd.3 cell line after OGD.^{4, 8}

• Lactate Metabolism Assay

New research indicates that both exogenous lactate and endogenous lactate produced by astrocytes have neuroprotective effects. We can carry out a series of experiments to help you explore the possibility of lactic acid metabolism becoming therapeutic approaches in IS.

Fig.5 Lactate is neuroprotective in human iPSC-derived neurons exposed to hypoxia.^{5, 7}

• Mitochondrial Dysfunction Assay

Significant mitochondrial dysfunction induced by middle cerebral artery obstruction (MCAO) or OGD is reported to contribute to neuronal death after IS and is a potential and attractive therapeutic strategy. We offer comprehensive assays to explore the effects of drug treatment on mitochondrial dysfunction, such as observing mitochondrial morphology with transmission electron microscopy, determining mitochondrial membrane potential (MMP) with JC-1 fluorescent probe, detecting ROS level through flow cytometry (FC), measuring levels of malondialdehyde (MDA) and superoxide dismutase (SOD) with ELISA and other mitochondrial toxicity assays.

Fig.6 MMP results determined using the JC-1 assay show the effects of RSV on mitochondrial function in cortical neurons exposed to OGD.^{6, 7}

• Autophagy Assay

Autophagy is an intracellular degradation pathway. Atter IS, it is activated in diverse cell types such as astrocytes, neurons and brain microvascular cells. We can quantify the LC3-II protein with WB, observe GFP-labeled autophagosomes with Fluorescence microscopy, observe autophagosome particles inside the cells Scanning Electron Microscopy and stain acidic particles in the cells for FC to explore autophagic activity, which will provide new perspectives for treatment of IS in the future.

Fig.7 Activation of autophagy in brain endothelial cells after OGD injury.^{4, 8}

• Neurite Outgrowth Assay

IS causes severe neuronal damage in the brain. Neurite outgrowth plays a key role in repairing and reestablish neural connections. We can evaluate the effects of compounds on neurite outgrowth by staining neurons with MAP2 or TUJ1 antibody and then analyzing the neuronal body quantification, neurite quantification, neurite node quantification, as well as neurite length.

Fig.8 Representative fluorescence image of TUJ1 to show the effect of RSV on neurite outgrowth in cultured cortical neurons after OGD.^{6, 7}

Creative Biolabs is committed to advance your project with our comprehensive portfolio and services. If you'd like to screen compounds of interest to improve poststroke recovery, please feel free to contact us.

References

1. Yan, Wei-Tao et al. "PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons." Neural Regen Res. 2023;18(2):357-363.
2. Liao, H et al. "Sirt1 regulates microglial activation and inflammation following oxygen-glucose deprivation/reoxygenation injury by targeting the Shh/Gli-1 signaling pathway." Mol Biol Rep. 2023, 50(4):3317-3327.
3. Li, Yong-Jun et al. "CPI-1189 protects neuronal cells from oxygen glucose deprivation/re-oxygenation-induced oxidative injury and cell death." Aging (Albany NY). 2021, 13(5):6712-6723. Distributed under Open Access License CC BY 3.0. The original image was modified.
4. Kim, Kyeong-A et al. "Autophagy-mediated occludin degradation contributes to blood-brain barrier disruption during ischemia in bEnd.3 brain endothelial cells and rat ischemic stroke models." Fluids Barriers CNS. 2020, 17(1):21.
5. Cerina, M et al. Neuroprotective role of lactate in a human in vitro model of the ischemic penumbra. Sci Rep. 2024, 14:7973.
6. He, Weiliang et al. "Rosuvastatin Improves Neurite Outgrowth of Cortical Neurons against Oxygen-Glucose Deprivation via Notch1-mediated Mitochondrial Biogenesis and Functional Improvement." Front Cell Neurosci. 2018, 12:6.
7. Distributed under Open Access License CC BY 4.0. The original image was modified.
8. Distributed under Open Access License CC BY 4.0 without modification.