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Cell-free oxidized hemoglobin drives reactive oxygen species production and pro-inflammation in an immature primary rat mixed glial cell culture.

  • Agyemang, Alex Adusei1
  • Kvist, Suvi Vallius1
  • Brinkman, Nathan2
  • Gentinetta, Thomas3
  • Illa, Miriam4
  • Ortenlöf, Niklas1
  • Holmqvist, Bo5
  • Ley, David1
  • Gram, Magnus6
  • 1 Lund University, Department of Clinical Sciences Lund, Pediatrics, Lund, Sweden. , (Sweden)
  • 2 R&D, CSL Behring, Kankakee, IL, USA.
  • 3 Research Bern, CSL Behring, Bern, Switzerland. , (Switzerland)
  • 4 Fetal i+D Fetal Medicine Research Center, BCNatal-Barcelona Center for Maternal-Fetal and Neonatal Medicine (Hospital Clínic and Hospital Sant Joan de Deu), Institut Clínic de Ginecologia, Obstetricia i Neonatologia, Universitat de Barcelona, Barcelona, Spain. , (Spain)
  • 5 ImaGene-iT AB, Medicon Village, Lund, Sweden. , (Sweden)
  • 6 Lund University, Department of Clinical Sciences Lund, Pediatrics, Lund, Sweden. [email protected] , (Sweden)
Published Article
Journal of Neuroinflammation
Springer (Biomed Central Ltd.)
Publication Date
Feb 11, 2021
DOI: 10.1186/s12974-020-02052-4
PMID: 33573677


Germinal matrix intraventricular hemorrhage (GM-IVH) is associated with deposition of redox active cell-free hemoglobin (Hb), derived from hemorrhagic cerebrospinal fluid (CSF), in the cerebrum and cerebellum. In a recent study, using a preterm rabbit pup model of IVH, intraventricularly administered haptoglobin (Hp), a cell-free Hb scavenger, partially reversed the damaging effects observed following IVH. Together, this suggests that cell-free Hb is central in the pathophysiology of the injury to the immature brain following GM-IVH. An increased understanding of the causal pathways and metabolites involved in eliciting the damaging response following hemorrhage is essential for the continued development and implementation of neuroprotective treatments of GM-IVH in preterm infant. We exposed immature primary rat mixed glial cells to hemorrhagic CSF obtained from preterm human infants with IVH (containing a mixture of Hb-metabolites) or to a range of pure Hb-metabolites, incl. oxidized Hb (mainly metHb with iron in Fe3+), oxyHb (mainly Fe2+), or low equivalents of heme, with or without co-administration with human Hp (a mixture of isotype 2-2/2-1). Following exposure, cellular response, reactive oxygen species (ROS) generation, secretion and expression of pro-inflammatory cytokines and oxidative markers were evaluated. Exposure of the glial cells to hemorrhagic CSF as well as oxidized Hb, but not oxyHb, resulted in a significantly increased rate of ROS production that positively correlated with the rate of production of pro-inflammatory and oxidative markers. Congruently, exposure to oxidized Hb caused a disintegration of the polygonal cytoskeletal structure of the glial cells in addition to upregulation of F-actin proteins in microglial cells. Co-administration of Hp partially reversed the damaging response of hemorrhagic CSF and oxidized Hb. Exposure of mixed glial cells to oxidized Hb initiates a pro-inflammatory and oxidative response with cytoskeletal disintegration. Early administration of Hp, aiming to minimize the spontaneous autoxidation of cell-free oxyHb and liberation of heme, may provide a therapeutic benefit in preterm infant with GM-IVH.

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