12209 Validation Of A Novel Placenta Explant Protocol For Studying Circadian Rhythms In The Mouse Placenta In Early, Mid And Late Pregnancy

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Abstract Disclosure: J.S. Lee: None. A.M. Yaw: None. H.M. Hoffmann: None. Introduction: The placenta serves as an interface facilitating the exchange of essential nutrients and oxygen between the mother and fetus. Preeclampsia, a complication of pregnancy driven by placental dysfunction, is one of the leading causes of maternal, fetal, and neonatal morbidity and mortality. While deregulation of the genes driving circadian rhythm has been associated with preeclampsia, the role of circadian rhythms in the placenta largely remains unknown. To address this gap of knowledge, our goal was to establish and validate an organotypic ex vivo preparation to study placenta circadian rhythms at gestation day (GD) 10, GD14, and GD18 in the mouse. Methods: To establish an organotypic placenta culture GD10, GD14, and GD18 placental explants, we sectioned the placenta into 350μm sections using a vibratome. The explants were prepared from the validated circadian Per2::Luciferase reporter mouse, allowing automated circadian rhythms recording of the explants in a LumiCycle. We compared explant success rate and rhythm quality in 2x2mm section vs whole slice explants at GD14. Using hematoxylin and eosin (H&E) staining we validated the explants to be decidua which is strictly of maternal origin, labyrinth which is strictly of fetal origin, and the junctional zone which consists of binucleated cells fused of maternal and fetal cells. Results: Through H&E staining, we confirmed the decidua is enriched with neutrophils, the junctional zone contains spongiotrophoblasts and glycogen trophoblasts, and the labyrinth has red blood cells. Both explant types were enriched with >80% of cells from the targeted region. We next established a protocol to record Per2::Luciferase rhythms for <6 days. The fragility of the GD10 placentas required the whole explant protocol, whereas the GD18 2x2mm sectioned explants produced strong rhythms with a high success rate. At GD14, the whole explants resulted in stronger rhythms with higher amplitude and higher goodness of fit compared to the 2x2mm explants. Conclusion: We found that depending on GD recording success differs. Specifically, the whole placental explant provides a more robust and reliable model for studying circadian rhythms in GD10 and GD14, whereas 2x2mm explants are successful at GD18. This novel placental preparation protocol will be valuable in studying disease- and drug-induced changes to circadian rhythms in a placenta layer-specific manner in the mouse. Presentation: 6/1/2024

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