Journal
BMC NEUROSCIENCE
Volume 5, Issue -, Pages -Publisher
BIOMED CENTRAL LTD
DOI: 10.1186/1471-2202-5-34
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Funding
- NINDS NIH HHS [R01 NS042273, 1RO1NS042273] Funding Source: Medline
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Background: Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin. Results: Continuous exposure of wild-type cultures to 1-10 muM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC50 of 1-2 muM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 muM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR) staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect. Conclusions: These results suggest that HO-2 gene deletion protects neurons in mixed neuron-astrocyte cultures from heme-mediated oxidative injury. Selective inhibition of neuronal HO-2 may have a beneficial effect after CNS hemorrhage.
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