Haptoglobin and Hemopexin Redirect Heme-Driven Oxidative Stress and Neurotoxicity in Organotypic Brain Slices

Hemorrhagic stroke triggers secondary brain injury through the red blood cell toxins hemoglobin (Hb) and heme, which fuel iron-driven lipid peroxidation and neuronal injury. We sought to use organotypic brain-slice cultures to dissect how the high-affinity scavenger proteins haptoglobin (Hp) and hemopexin (Hpx) modulate this cascade. By day 7 of culture, slices remained structurally intact, metabolically active, and responsive to oxidative stress, enabling precise toxin exposure studies. Isotopic Fe tracing revealed that upon cell-free Hb and heme exposures, heme-iron accumulated in brain slices and heightened lipid peroxidation. In contrast, Hpx neutralized heme, nearly abolishing iron deposition, while Hp partially reduced Hb-driven iron accumulation. Both scavengers attenuated lipid peroxidation and reduced neuronal cell death. Transcriptomic profiling revealed that free toxins increased oxidative stress and neuroinflammatory activation markers, whereas Hpx suppressed the expression of heme-induced genes. Remarkably, HbHp complexes triggered a strong Nrf2-centered adaptive program that enhanced iron metabolism and glutathione synthesis. Integrating five readouts─iron accumulation, lipid peroxidation, neuronal cell death, heme-stress transcripts, and Nrf2/metabolic transcripts─via bootstrap-based principal component analysis yielded two orthogonal axes. An oxidative toxicity axis (PC1) captured iron-driven reactive oxygen species and cell death, while a metabolic adaptation axis (PC2) reflected Nrf2-mediated reprogramming. Free toxins clustered at the toxic extreme on PC1, and heme-Hpx aligned near baseline. HbHp shifted slices upward on PC2, reducing neuronal loss through safe adaptation. These findings establish that Hpx neutralizes free heme, whereas Hp stabilizes Hb and elicits cytoprotective gene expression, offering a rational, dual-scavenger strategy to mitigate secondary brain injury in hemorrhagic stroke.