Delayed Treatment of Hemoglobin Neurotoxicity

Abstract

Hemoglobin is an oxidative neurotoxin that may contribute to cell injury after CNS trauma and hemorrhagic stroke. Prior studies have demonstrated that concomitant treatment with iron-chelating antioxidants prevents its neurotoxicity. However, the efficacy of these agents when applied hours after hemoglobin has not been determined, and is the subject of the present investigation. Consistent with prior observations, an increase in reactive oxygen species generation, detected by 2',7'-dichlorofluorescin oxidation, was observed when mixed neuronal/astrocyte cultures prepared from mouse cortex were exposed to hemoglobin alone. However, this oxidative stress developed slowly. A significant increase in the dichlorofluorescein signal compared with control, untreated cultures was not observed until four hours after addition of hemoglobin, and was followed by loss of membrane integrity and propidium iodide staining. Treating cultures with the 21-aminosteroid U74500A or the ferric iron chelator deferoxamine four hours after initiating hemoglobin treatment markedly attenuated reactive oxygen species production within 2 h. Continuous exposure to 5 micro M hemoglobin for 24 h resulted in death of about three-quarters of neurons, without injuring astrocytes. Most neuronal loss was prevented by concomitant treatment with U74500A; its effect was not significantly attenuated if treatment was delayed for 2-4 h, and it still prevented over half of neuronal death if treatment was delayed for 8 h. Similar neuroprotection was produced by delayed treatment with deferoxamine or the lipid-soluble iron chelator phenanthroline. None of these agents had any effect on neuronal death when added to cultures 12 h after hemoglobin. These results suggest that hemoglobin is a potent but slowly-acting neurotoxin. The delayed onset of hemoglobin neurotoxicity may make it an attractive target for therapeutic intervention.

Deferoxamine Mesylate

“…Regan and Rogers showed that delayed treatment with deferoxamine markedly attenuates the production of reactive oxygen species and neuronal death induced by adding hemoglobin to mixed neuronal and astrocyte cell cultures 10 and that deferoxamine attenuates the effects of hemoglobin, which potentiates the neurotoxicity of glutamate agonists in murine cortical cultures. 3 Deferoxamine also reduces the production of reactive oxygen species and cell death induced by hemin in human neuron-like cells 4 and pheochromocytoma and neuroblastoma cell lines.…”

Traumatic injury to the immature brain: Inflammation, oxidative injury, and iron-mediated damage as potential therapeutic targets

“…It attenuates oxidative stress in cultured neurons and astrocytes, likely a result of reducing the amount of iron available to generate free radicals. 133 It may also confer added protection to the injured immature brain by upregulating hypoxia-inducible factor-1, which promotes cell survival via the activation of molecules such as erythropoetin and vascular endothelial growth factor. 134 This later phenomenon is supported in a recent in vitro study of ischemic injury where the beneficial effects of deferoxamine in reducing hippocampal neuronal death were diminished in the presence of an antisense oligonucleotide specific for hypoxia-inducible factor-1alpha.…”

Astrocyte Overexpression of Heme Oxygenase-1 Improves Outcome After Intracerebral Hemorrhage

“…8, 9 This effect is mitigated by protein or low molecular weight iron chelators, resulting in net neuroprotection that may be mediated by the other reaction products. 3, 10, 11 Astrocytes, which are relatively resistant to acute iron toxicity due to ferritin induction, 12 are protected from hemin by both HO-1 and HO-2 in the absence of exogenous chelators. 13, 14 The effect of HO expression in any model may therefore depend on the cell population at greatest risk and the iron binding capacity of the cellular microenvironment.…”