Hemorrhage complicates many traumatic injuries to the CNS and about 20% of strokes. Over subsequent hours, erythrocytes lyse and release their contents into the extravascular space. The most abundant protein released is hemoglobin (Hb). A growing body of experimental evidence suggests that the oxidative toxicity of extracellular Hb contributes to the pathogenesis of hemorrhagic CNS injury. Moreover, because of its prolonged time course, Hb toxicity may be an ideal target for therapeutic intervention. Further insight into the cellular mechanisms and prevention of this toxicity therefore seems desirable. ? ? Cultured neurons are highly vulnerable to Hb, but astrocytes are resistant via a mechanism that requires protein synthesis. Preliminary experiments suggest that this discrepancy may be explained in part by the effects of two inducible antioxidants: heme oxygenase (HO)- and ferritin. The former is rapidly induced by Hb and may facilitate synthesis of L-rich ferritin in astrocytes. In contrast, Hb decreases the expression of L-rich ferritin in neurons; iron released as a product of heme breakdown may then be toxic. ? ? This project will address the role of HO and ferritin in cell culture and in vivo models. Overexpression of HO-1 will be accomplished in glial, neuronal, or mixed cultures via gene transfer; the relationship between activity, heme-mediated reactive oxygen species formation, and cell death will be established. Cellular vulnerability to Hb or hemin will then be compared in cultures prepared from wild-type, HO-1 knockout, and HO-2 knockout mice. Using antibodies that specifically recognize H- or L-ferritin, the subunit content of ferritin will be assessed at baseline and in response to Hb in these cultures. Expression of HasA, which binds to and may facilitate heme iron uptake, will also be determined. H and L-rich ferritin heteropolymers will be constructed from recombinant H or L-ferritin. Neuronal and glial uptake of these heteropolymers via receptor-mediated endocytosis will allow investigation of the effect of the H:L ratio on cellular vulnerability to heme-mediated injury. Finally, the putamen of wild type, HO-1 or HO-2 knockout, and transgenic mice that overexpress HO-1 will be injected with Hb, or with collagenase to induce an endogenous hemorrhage. Surrounding neuronal loss, DNA cleavage, and caspase-3 activation will then be quantified at defined time points 12-96 hours after injection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS042273-01A1
Application #
6474840
Study Section
Special Emphasis Panel (ZRG1-BDCN-1 (25))
Program Officer
Jacobs, Tom P
Project Start
2002-12-01
Project End
2006-11-30
Budget Start
2002-12-01
Budget End
2003-11-30
Support Year
1
Fiscal Year
2003
Total Cost
$298,300
Indirect Cost
Name
Thomas Jefferson University
Department
Emergency Medicine
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Chen-Roetling, Jing; Liu, Wenpei; Regan, Raymond F (2012) Hemopexin decreases hemin accumulation and catabolism by neural cells. Neurochem Int 60:488-94
Chen-Roetling, Jing; Sinanan, Jesse; Regan, Raymond F (2012) Effect of iron chelators on methemoglobin and thrombin preconditioning. Transl Stroke Res 3:452-9
Chen, Lifen; Zhang, Xuefeng; Chen-Roetling, Jing et al. (2011) Increased striatal injury and behavioral deficits after intracerebral hemorrhage in hemopexin knockout mice. J Neurosurg 114:1159-67
Chen-Roetling, Jing; Chen, Lifen; Regan, Raymond F (2011) Apotransferrin protects cortical neurons from hemoglobin toxicity. Neuropharmacology 60:423-31
Chen-Roetling, Jing; Liu, Wenpei; Regan, Raymond F (2011) Iron accumulation and neurotoxicity in cortical cultures treated with holotransferrin. Free Radic Biol Med 51:1966-74
Jaremko, Kellie M; Chen-Roetling, Jing; Chen, Lifen et al. (2010) Accelerated hemolysis and neurotoxicity in neuron-glia-blood clot co-cultures. J Neurochem 114:1063-73
Chen, Mai; Awe, Olatilewa O; Chen-Roetling, Jing et al. (2010) Iron regulatory protein-2 knockout increases perihematomal ferritin expression and cell viability after intracerebral hemorrhage. Brain Res 1337:95-103
Chen-Roetling, Jing; Chen, Lifen; Regan, Raymond F (2009) Minocycline attenuates iron neurotoxicity in cortical cell cultures. Biochem Biophys Res Commun 386:322-6
Li, Zhi; Chen-Roetling, Jing; Regan, Raymond F (2009) Increasing expression of H- or L-ferritin protects cortical astrocytes from hemin toxicity. Free Radic Res 43:613-21
Regan, Raymond F; Chen, Mai; Li, Zhi et al. (2008) Neurons lacking iron regulatory protein-2 are highly resistant to the toxicity of hemoglobin. Neurobiol Dis 31:242-9

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