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Background/Purpose: Cholesterol homeostasis is vital for synaptogenesis and myelination during fetal and neonatal brain development. Both brain and plasma sterol levels have been shown to be associated with outcomes in adult stroke, and animal studies have suggested brain and plasma sterol changes in neonatal hypoxic-ischemic brain injury. MicroRNA-335 (miR-335) has been associated with brain sterol metabolism and our preliminary data showed that sterol metabolism is dysregulated in the brain after hypoxic-ischemic injury in a mouse model. This study sought to describe the effect of oxygen-glucose deprivation in vitro on individual brain cell populations and to validate the associations between sterol levels and miR-335 levels. Lastly, we assessed the feasibility of transfecting miR-335 mimics or miRNA antagonists (antagomiRs) in these brain cell cultures to set up future experiments to alter miR-335 levels for neuroprotection.

Methods: Primary cells were isolated from embryonic day 18 CD1 mouse brains. Cells were maintained in DMEM for mixed glial culture and Neurobasal media with B27 for neurons. Once astrocytes were confluent, microglia were separated from the mixed glial culture by shaking to provide pure cell populations. Each of the three cell types underwent oxygen and glucose deprivation (OGD: glucose/pyruvate/B27-free media, 1% O2, 5% CO2) for 4 hours followed by replacement of glucose/pyruvate/B27 and resumption of normoxia. At 24 hours after OGD, cells (n=12 wells OGD and 12 normoxia) were washed, counted, and sterols analyzed by LC-MS/MS, normalized to the number of cells/well. Additional cells underwent measurement of miR-335 expression using quantitative PCR and/or transfection with miR-335 mimic or antagomiR.

Results: Although lanosterol is increased after OGD in neurons, desmosterol and cholesterol levels were decreased. In microglia and astrocytes, cholesterol levels were lower than in neurons but increased after OGD. MiR-335 expression in neurons and astrocytes were inverse to cholesterol level changes, though this association was not seen in microglia. Lastly, red fluorescent protein-labelled miR-335 mimic was visualized in both neurons and astrocytes after transfection and miR-335 expression changes were seen after transfection in astrocytes.

Conclusions: Sterol levels are altered after OGD and may be associated with OGD changes. Transfection of miR-335 mimic and antagomiR is feasible and future studies using these tools will allow for better understanding of the effects of neonatal HIBI on sterol levels. This approach could allow for identification of targets to aid in developing therapeutics.

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Relationship between miR-335 and sterol levels after in vitro hypoxia-ischemia of primary brain cells

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