Protection from oxygen-glucose deprivation by neurosteroid treatment in primary neurons and oligodendrocytes.

Preterm birth results in an increased risk of neonatal brain injury and neurobehavioural disorders. Despite the seriousness of these adverse outcomes, there are currently no effective therapies to protect the vulnerable developing brain. We propose that neurosteroid replacement therapy may be a novel approach in reducing detrimental neurological outcomes following preterm birth. The use of guinea pig primary neuronal and oligodendrocyte cultures with relevance to late gestation allows insight into the mechanisms behind the effectiveness of these treatments. Primary neuronal and oligodendrocyte cultures were derived from fetal guinea pig frontal cortex brain tissue at gestational age 62 (GA62). Cell cultures were pre-treated with either etifoxine (5 µM) or zuranolone (1 µm) for 24 h prior to insult. Cells were then exposed to either oxygen-glucose deprivation (OGD; 0% O₂ and no glucose DMEM; preterm birth insult) or sham (standard cell culture conditions; 25 mM DMEM) for 2 h. Lactate dehydrogenase assay (LDH) was performed following OGD as a measure of cytotoxicity. Relative mRNA expression of key neuronal and oligodendrocyte markers, as well as neuronal receptors and transporters, were quantified using high throughput (Fluidigm) RT-PCR. OGD significantly increased cellular cytotoxicity in both neurons and oligodendrocytes. Additionally, key neuronal marker mRNA expression was reduced following OGD, and oligodendrocytes displayed arrested mRNA expression of key markers of lineage progression. Treatment with etifoxine restored a number of parameters back to control levels, whereas treatment with zuranolone provided a robust improvement in all parameters examined. This study has demonstrated the neuroprotective potential of neurosteroid replacement therapy in a model of hypoxia related to preterm birth. Neuroprotection appears to be mediated through glutamate reduction and increased brain derived neurotrophic factor (BDNF). Future work is warranted in examining these treatments in vivo, with the overall aim to suppress preterm associated brain damage and reduce long term outcomes for affected offspring.