Proceedings of the 13th International Newborn Brain Conference: Fetal And/or Neonatal Brain Development, Both Normal and Abnormal.

BACKGROUND AND PURPOSE: White matter injury (WMI) is the most common brain injury leading to poor neurologic outcomes in premature infants.Sepsis and infl ammation are signifi cant risk factors and there are no treatment options available.Developing novel drug therapies for neonates is challenged by appropriate concerns for safety.Here, we identifi ed oxysterols in human maternal breast milk and explored their therapeutic potential in directing neural stem cells (NSCs) into the oligodendrocyte (OL) lineage via the Sonic Hedgehog (Shh) pathway in vitro.Further, we investigated whether breast milk-associated oxysterols can rescue neonatal WMI in vivo.METHODOLOGY: Oxysterols were identifi ed in breast milk using Liquid Chromatography/Electrospray Ionization/Tandem Mass Spectrometry.NSCs were cultured from the subventricular zone (SVZ) of mice and treated with oxysterols.Cells were analyzed by immunohistochemistry, western blot, fl ow cytometry, and scRNAseq, looking for markers of the OL lineage.Shh pathway activation was established by quantifying upregulation of target gene, Gli1, by western blot and RT-PCR.Gli-dependence was explored using pharmacological and genetic approaches.Neonatal sepsis leading to WMI was induced in mice on postnatal day 5, using an intraperitoneal stool injection.Mice then received either breast milk-associated 20-hydroxycholesterol (20HC) or vehicle.Stereology determined OL numbers in the periventricular white matter regions.Myelination was evaluated by performing g-ratios to determine myelin thickness.Motor function in mice was analyzed using the CatWalk gait analysis system.To lineage trace postnatal nestin+ SVZ NSCs in vivo, neonatal sepsis was induced in Nestin-CreERT2;R26r-TdTomato pups as above, and then treated with either vehicle or 20HC.