title: Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation.
abstract: BACKGROUND
  Cerebral intraventricular hemorrhage (IVH) is a major cause of severe neurodevelopmental impairment in preterm infants. To date, no therapy is available that prevents infants from developing serious neurological disability following IVH. Thus, to develop treatment strategies for IVH, it is essential to characterize the initial sequence of molecular events that leads to brain damage. In this study, we investigated extracellular hemoglobin (Hb) as a causal initiator of inflammation in preterm IVH.
  
  
  METHODS
  Using a preterm rabbit pup model, we investigated the molecular mechanisms and events following IVH. We also characterized the concentrations of cell-free Hb metabolites and pro-inflammatory mediators in the cerebrospinal fluid (CSF) of preterm human infants and rabbit pups. Finally, Hb metabolites were evaluated as causal initiators of inflammation in primary rabbit astrocyte cell cultures.
  
  
  RESULTS
  Following IVH in preterm rabbit pups, the intraventricular CSF concentration of cell-free methemoglobin (metHb) increased from 24 to 72 hours and was strongly correlated with the concentration of TNFα at 72 hours (r2 = 0.896, P <0.001). Also, the mRNA expression of TNFα, IL-1β, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining). Furthermore, exposure of primary rabbit astrocyte cell cultures to metHb caused a dose-dependent increase in TNFα mRNA and protein levels, which was not observed following exposure to oxyhemoglobin (oxyHb) or hemin. Finally, a positive correlation (r2 = 0.237, P <0.03) between metHb and TNFα concentrations was observed in the CSF of preterm human infants following IVH.
  
  
  CONCLUSIONS
  Following preterm IVH, increased metHb formation in the intraventricular space induces expression of pro-inflammatory cytokines. Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH. Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.
authors.affiliation: Division of Infection Medicine, Lund University, S-221 84 Lund, Sweden. magnus.gram@med.lu.se
authors.forename: Magnus
authors.initials: M
authors.lastname: Gram
authors.forename: Snjolaug
authors.initials: S
authors.lastname: Sveinsdottir
authors.forename: Karsten
authors.initials: K
authors.lastname: Ruscher
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Hansson
authors.forename: Magnus
authors.initials: M
authors.lastname: Cinthio
authors.forename: Bo
authors.initials: B
authors.lastname: Akerström
authors.forename: David
authors.initials: D
authors.lastname: Ley
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002552:Cerebral Ventricles / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006427:Hemin / Q000134:cerebrospinal fluid / Q000378:metabolism; D006454:Hemoglobins / Q000633:toxicity*; D006801:Humans; D007231:Infant, Newborn; D007234:Infant, Premature; D007249:Inflammation / Q000139:chemically induced* / Q000473:pathology; D020300:Intracranial Hemorrhages / Q000378:metabolism* / Q000473:pathology; D008706:Methemoglobin / Q000134:cerebrospinal fluid / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D010108:Oxyhemoglobins / Q000134:cerebrospinal fluid / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000134:cerebrospinal fluid / Q000378:metabolism
chemical_list: D006454:Hemoglobins; D009419:Nerve Tissue Proteins; D010108:Oxyhemoglobins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D006427:Hemin; D008706:Methemoglobin
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