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Oxygen Glucose Deprivation Model

Stroke is a devastating disease accounting for 5.5 million deaths annually worldwide. Despite significant preclinical and clinical investigations, with more than 1000 candidate neuroprotective stroke drugs investigated and nearly 200 clinical trials, no effective therapy other than tissue plasminogen activator (TPA) has been approved. The appropriate use of animal models and clinical trial design will undoubtedly improve the odds of identifying effective therapeutics.

Ischemic conditions can either be modeled by removal of oxygen and glucose or via chemical or enzymatic inhibition of metabolism. Oxygen–glucose deprivation (OGD) model is the most frequent used models in ischemia studies.

Creative Bioarray has enriched experience in OGD model establishing. We build ischemia-like conditions by replacing the normal O2/CO2 equilibrated medium with N2/CO2 equilibrated medium and maintaining cells in a hypoxic chamber. While oxygen deprivation is usually used in combination with glucose deprivation, a number of in vitro studies have demonstrated hypoxia alone causes dramatic alterations in endothelial cell (EC) actin cytoskeleton and tight junction protein localization in Blood Brain Barrier models.

Creative Bioarray combines oxygen and glucose deprivation, causes primary neurons to undergo acute cell body swelling followed by apoptotic and excitotoxic necrotic cell death. In OGD reperfusion experiments, neuronal degeneration occurs over several hours, despite return to standard culture conditions, as is consistent with observations of in vivo I/R injury. OGD in primary mouse cortical neurons is also associated with a large increase in extracellular glutamate concentration consistent with excitotoxic effects in vivo.

Multiple models can be selected in Creative Bioarray for ischemia studies when building OGD model.


4-1-11 Oxygen Glucose Deprivation Model-1.jpg

Fig. 1 Ischemia and normoxia protocols of OGD treatment on PC12 cells


Brain slices

We utilize a thin slice of brain tissue (usually 300-400 µm), allowing for in-depth probing of neuronal circuitry. The slice is perfused with artificial CSF (aCSF), allowing rodent slices to be maintained for up to 12h. Removing glucose and replacing oxygen with nitrogen in this solution provides global OGD, causing neuronal and astrocytic depolarization within 10min in rat cortical slices.

Advantages

  • Different vulnerabilities of neuronal cell types can be assessed.

  • Reaching focal neuronal effects of ischemia separated from cerebrovascular influences by perfusing OGD aCSF over a brain slice in a focal stream, while the remainder of the slice is perfused with normal aCFS.

Organotypic cell culture

Existing in the realm between brain slice and primary cell culture, these ex vivo cultures are obtained from different anatomical regions of the brain, usually taken from neonatal animals, and allowed to mature in vitro. This method maintains structural organization however many cultures experience synaptic rearrangement due to lack of extrinsic afferent and efferent signals in vitro. These cultures have increasingly been used to investigate neuronal cell death, myelination, synapse plasticity and potential stroke therapies in OGD models.


4-1-11 Oxygen Glucose Deprivation Model-2.jpg

Fig. 2 Distinct neuronal vulnerability after 30 min-OGD in hippocampal organotypic culture


Primary cells

The particular benefit of primary cells is evident in brain endothelial cell (BEC) primary cultures, where trans-endothelial electrical resistance (TEER) is comparatively high compared to cell lines. Primary glial cell cultures are some of the most commonly used in vitro model for neurobiological studies and the molecular properties and differentiation of these cells in culture reflect their in vivo phenotype well.

Cell lines

Creative Bioarray provides ready supply of human cells suitable for use in high-throughput screening assays. Immortalized human microglia cells have been produced and established from primary human embryonic microglia and displayed a wide variety of inflammatory responses typical of the primary cell. We also provide the human teratoma-derived NT2 cell line as a promising investigational tool from which neuronal cells, astrocytes, and oligodendrocytes can be derived.


4-1-11 Oxygen Glucose Deprivation Model-3.jpg

Fig.3 Growth pattern post 24-h of re-oxygenation in 6-h OGD received vs. normoxic PC 12 cells under the influence of various concentrations of glucose


Stem cells

Embryonic stem cells in principle allow for unlimited quantities of every cell type in vitro, and numerous techniques have been established for the generation of lineage-restricted neural progenitor cells followed by their specific differentiation into neurons, astrocytes or oligodendrocytes. Ethical issues limit the use of human embryonic stem cells, however, reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides an attractive alternative. Protocols for iPSC cell differentiation to neurons are improving to give near 100% purity of neurons.

iPSCs have been utilized to model a number of diseases for drug screening, and also been used in modeling neuronal disease. The particular benefit of these cells is in producing patient specific cells that can aid in modeling neurological disorders with significant genetic contributions, such as Multiple Sclerosis (MS). While stroke is a multifactorial disease, mendelian stroke syndromes do exist, such as Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leucoencephalopathy (CADASIL). The use of iPSCs from such patients may aid a further understanding of stroke pathophysiology.

References

  1. Voytenko LP, et al. Hippocampal GABAergic interneurons coexpressing alpha7-nicotinic receptors and connexin-36 are able to improve neuronal viability under oxygen-glucose deprivation. Brain Res. 2015; 1616: 134–145.

  2. Holloway PM, Gavins FNE. Modeling Ischemic Stroke In Vitro: The Status Quo and Future Perspectives. Stroke. 2016; 47: 561–569.

  3. Singh G, et al. Oxygen glucose deprivation model of cerebral stroke in PC-12 cells: glucose as a limiting factor. Toxicology mechanisms and methods. 2009, 19: 154-160.

  4. Tabakman R, et al. Neuroprotection by NGF in the PC12 in vitro OGD model: Involvement of mitogen-activated protein kinases and gene expression. Ann N Y Acad Sci. 2005; 1053: 84–96.


For research use only.

Related Section

  • Xenopus Oocyte Screening Model
  • Acute Isolated Cardiomyocytes
  • Acute Dissociated Neurons
  • Primary Cultured Neurons
  • Cultured Neuronal Cell Lines
  • iPSC-derived Cardiomyocytes/Neurons
  • Acute/Cultured Organotypic Brain Slices
  • 3D Cell Culture
  • iPSC-derived Neurons
  • Isolation and culture of neural stem/progenitor cells

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