Authors: Brian Snyder, MD; Danielle Goulding; R. Vogel; Brandon Miller, MD, PhD (Lexington, KY)

Introduction: An unfortunate consequence of preterm birth is neonatal intraventricular hemorrhage. This extravascular hemorrhage is frequently associated with periventricular leukomalacia. Our group hypothesizes that neuroinflammation, in particular activated microglia/macrophages, mediates this ultimate white matter loss. Methods: Mixed cortical cultures were obtained from post natal day two rats and allowed to expand. Microglia were isolated and exposed to different concentrations of hemoglobin. Nine different inflammatory cytokines were measured by using a meso-scale detection ELISA. We then co-cultured oligodendrocyte progenitor cells with microglia to test the possibility of hemoglobin-induced microglial OPC death. Cells were stained with IB4, a microglial marker, and NG2, a marker of immature oligodendrocytes. An in vivo model of neonatal IVH using rat pups was used to measure cytokine production and oxidative stress within the whole brain. Results: Numerous proinflammatory cytokines including TNF-⍺ and CXCL1 are significantly elevated in microglia exposed to hemoglobin. The cytokine profile is consistent with a “M1” or pro-inflammatory macrophage response. This inflammatory profile in vitro mirrors the cytokine profile seen in our in vivo model of intraventricular hemorrhage. Oxidative stress is present throughout the brain after IVH, and occurs after the initial inflammatory response. Analysis of OPC survival in response to hemoglobin alone and hemoglobin-activated microglia is in progress. Conclusion: Our rodent model of neonatal IVH induces a proinflammatory cytokine profile that mirrors our in vivo model of intraventricular hemorrhage of prematurity. Upon analysis of our OPC survival data we will be able to deduce whether or not hemoglobin induced microglia or hemoglobin alone kills oligodendrocyte progenitor cells. This in vitro model of IVH-induced PVL may be useful for future mechanistic and therapeutic studies.