title: Deletion of the hemopexin or heme oxygenase-2 gene aggravates brain injury following stroma-free hemoglobin-induced intracerebral hemorrhage.
abstract: BACKGROUND
  Following intracerebral hemorrhage (ICH), red blood cells release massive amounts of toxic heme that causes local brain injury. Hemopexin (Hpx) has the highest binding affinity to heme and participates in its transport, while heme oxygenase 2 (HO2) is the rate-limiting enzyme for the degradation of heme. Microglia are the resident macrophages in the brain; however, the significance and role of HO2 and Hpx on microglial clearance of the toxic heme (iron-protoporphyrin IX) after ICH still remain understudied. Accordingly, we postulated that global deletion of constitutive HO2 or Hpx would lead to worsening of ICH outcomes.
  
  
  METHODS
  Intracerebral injection of stroma-free hemoglobin (SFHb) was used in our study to induce ICH. Hpx knockout (Hpx(-/-)) or HO2 knockout (HO2(-/-)) mice were injected with 10 μL of SFHb in the striatum. After injection, behavioral/functional tests were performed, along with anatomical analyses. Iron deposition and neuronal degeneration were depicted by Perls' and Fluoro-Jade B staining, respectively. Immunohistochemistry with anti-ionized calcium-binding adapter protein 1 (Iba1) was used to estimate activated microglial cells around the injured site.
  
  
  RESULTS
  This study shows that deleting Hpx or HO2 aggravated SFHb-induced brain injury. Compared to wild-type littermates, larger lesion volumes were observed in Hpx(-/-) and HO2(-/-) mice, which also bear more degenerating neurons in the peri-lesion area 24 h postinjection. Fewer Iba1-positive microglial cells were detected at the peri-lesion area in Hpx(-/-) and HO2(-/-) mice, interestingly, which is associated with markedly increased iron-positive microglial cells. Moreover, the Iba1-positive microglial cells increased from 24 to 72 h postinjection and were accompanied with improved neurologic deficits in Hpx(-/-) and HO2(-/-) mice. These results suggest that Iba1-positive microglial cells could engulf the extracellular SFHb and provide protective effects after ICH. We then treated cultured primary microglial cells with SFHb at low and high concentrations. The results show that microglial cells actively take up the extracellular SFHb. Of interest, we also found that iron overload in microglia significantly reduces the Iba1 expression level and resultantly inhibits microglial phagocytosis.
  
  
  CONCLUSIONS
  This study suggests that microglial cells contribute to hemoglobin-heme clearance after ICH; however, the resultant iron overloads in microglia appear to decrease Iba1 expression and to further inhibit microglial phagocytosis.
authors.affiliation: Department of Anesthesiology, Center for Translational Research in Neurodegenerative Disease, University of Florida College of Medicine, P.O. Box 100159, Gainesville, FL, 32610, USA. bma@anest.ufl.edu.
authors.forename: Bo
authors.initials: B
authors.lastname: Ma
authors.affiliation: Department of Anesthesiology, Center for Translational Research in Neurodegenerative Disease, University of Florida College of Medicine, P.O. Box 100159, Gainesville, FL, 32610, USA. jasonpday@ufl.edu.
authors.forename: Jason Patrick
authors.initials: JP
authors.lastname: Day
authors.affiliation: Department of Anesthesiology, Center for Translational Research in Neurodegenerative Disease, University of Florida College of Medicine, P.O. Box 100159, Gainesville, FL, 32610, USA. hfphilli@ufl.edu.
authors.forename: Harrison
authors.initials: H
authors.lastname: Phillips
authors.affiliation: Department of Anesthesiology, Center for Translational Research in Neurodegenerative Disease, University of Florida College of Medicine, P.O. Box 100159, Gainesville, FL, 32610, USA. bsloots3@ufl.edu.
authors.forename: Bryan
authors.initials: B
authors.lastname: Slootsky
authors.affiliation: Departments of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy. emanuela.tolosano@unito.it.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Tolosano
authors.affiliation: Department of Anesthesiology, Center for Translational Research in Neurodegenerative Disease, University of Florida College of Medicine, P.O. Box 100159, Gainesville, FL, 32610, USA. sdore@ufl.edu.|Departments of Neuroscience, Neurology, Psychiatry, Psychology and Pharmaceutics, University of Florida College of Medicine, Gainesville, FL, 32610, USA. sdore@ufl.edu.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Doré
mesh_terms: D042964:Acyl-CoA Dehydrogenase / Q000378:metabolism; D000818:Animals; D029681:Arabidopsis Proteins / Q000378:metabolism; D001930:Brain Injuries / Q000209:etiology* / Q000235:genetics*; D002478:Cells, Cultured; D002543:Cerebral Hemorrhage / Q000139:chemically induced / Q000150:complications* / Q000401:mortality; D004195:Disease Models, Animal; D005452:Fluoresceins / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000172:deficiency* / Q000235:genetics; D006454:Hemoglobins / Q000633:toxicity; D006466:Hemopexin / Q000172:deficiency* / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D017628:Microglia / Q000187:drug effects / Q000378:metabolism; D009043:Motor Activity / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009460:Neurologic Examination; D010587:Phagocytosis / Q000187:drug effects / Q000235:genetics; D013997:Time Factors
chemical_list: D029681:Arabidopsis Proteins; D005452:Fluoresceins; D006454:Hemoglobins; D009419:Nerve Tissue Proteins; C435731:fluoro jade; C016484:hemoglobin, stroma free; D006466:Hemopexin; D006419:Heme Oxygenase (Decyclizing); C105245:heme oxygenase-2; D042964:Acyl-CoA Dehydrogenase; C520627:IBR3 protein, Arabidopsis
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