Multi-modal screening for synergistic neuroprotection of extremely preterm brain injury

Preterm brain injury affects both white and grey matter, including altered cortical development and gyrification, with associated neurodevelopmental sequelae such as cerebral palsy and learning deficits. The preterm brain also displays regionally heterogeneous responses to both injury and treatment, supporting the need for drug combinations to provide global neuroprotection. We developed an extremely preterm-equivalent organotypic whole hemisphere (OWH) slice culture injury model using the gyrencephalic ferret brain to probe treatment mechanisms of promising therapeutic agents and their combination. Regional and global responses to injury and treatment were assessed by cell death quantification, machine learning-augmented morphological microglia assessments, and digital transcriptomics. Using two promising therapeutic agents, azithromycin (Az) and erythropoietin (Epo), we show minimal neuroprotection by either therapy alone, but evidence of synergistic neuroprotection by Az*Epo both globally and regionally. This effect of Az*Epo involved emergent augmentation of transcriptomic responses to injury related to neurogenesis and neuroplasticity and downregulation of transcripts involved in cytokine production, inflammation, and cell death. This study supports the use of the ferret OWH slice culture model to provide a powerful high-throughput platform to examine combinations of therapeutics for extremely preterm brain injury.