Effect of Caffeine on Cell Death, Oxidative Stress, and Microglial Morphology in a Ferret Organotypic Brain Slice Model of Hypoxia-Ischemia.

Brain injury after hypoxia-ischemia (HI) is the leading cause of morbidity and mortality in term and near-term neonates worldwide. The ferret is a promising translational model to study HI due to its gyrified brain and white-to-gray matter ratio that more closely resembles humans compared to rodents. Caffeine, an adenosine A2A receptor (A2AR) antagonist, shows neuroprotective potential after HI, but its effects have not been fully characterized. We sought to evaluate caffeine's effect on neuronal cell death, cytotoxicity, and inflammatory and oxidative stress markers in a term-equivalent ferret organotypic brain slice model of HI. Slices were cultured for 72 h, exposed to two hours of oxygen-glucose deprivation (OGD), and randomized to OGD alone, OGD with caffeine (20 or 50 mg/L), or OGD with caffeine and an A2AR agonist. Healthy slices served as controls. Outcomes included global cell death, regional cell death, microglial morphology, and expression of inflammatory and oxidative stress genes (46-48 slices/group for cell death assays and 18 slices/group for imaging, balanced by sex). Caffeine 50 mg/L significantly reduced global cell death compared to OGD (p = 0.02), and this effect persisted despite co-administration of an A2AR agonist (p = 0.01), suggesting that protection was not primarily mediated through A2AR signaling. Caffeine also did not change regional pyknotic nuclei counts (p > 0.05). Caffeine altered microglial morphology, increasing the proportion of microglia with features characteristic of control conditions. OGD significantly increased expression of inflammatory and oxidative stress-related genes (p < 0.05) compared with control slices, whereas caffeine did not significantly alter gene expression. In summary, caffeine partially reversed global cell death after OGD and altered microglial morphology. Larger, higher-powered studies are needed to further investigate caffeine's effects on neonatal HI.