Hemorrhage accompanies most significant brain trauma, and is the primary event in about 15% of strokes. A growing body of evidence suggests that hemoglobin (Hb) release from lysing erythrocytes may contribute to oxidative injury to tissue surrounding a hematoma. Prior studies have demonstrated that Hb toxicity is mediated by transfer of its heme moieties to nearby cells, which is favored by its oxidation to methemoglobin. Hemin, the oxidized form of heme, is then catabolized by the heme oxygenase enzymes to iron, carbon monoxide, and biliverdin. The cytoprotective effects of the latter two products may be overwhelmed if iron sequestration is inadequate. However, the iron detoxification strategies of neurons and astrocytes remain largely undefined. Preliminary experiments suggest that neurons export extracellular iron, where it must be sequestered to prevent oxidative membrane injury;astrocytes induce ferritin, consistent with intracellular storage. The goal of this project is to define the role of ferritin and transferrin in mitigating heme-mediated injury to neurons and astrocytes, and to devise a strategy to optimize this process. Our experimental aims are as follows: 1) Specifically increase expression of H or L-ferritin subunits in cultured neurons and astrocytes by adenoviral transfer of their genes. Alternatively, culture astrocytes and neurons from iron regulatory protein (IRP) knockout mice, which have markedly increased cell ferritin. Determine the effect of ferritin gene transfer or IRP knockdown on culture reactive oxygen species (ROS) formation, protein oxidation, and cell viability after Hb or hemin exposure. 2) Treat cultured neurons and astrocytes with [55Fe]hemin. Quantify subsequent 55Fe release into the culture medium or deposited in cell ferritin. Assess the effect of apotransferrin and holotransferrin on cell vulnerability to heme-mediated injury. 3) Stereo- tactically inject blood into the striata of hypotransferrinemic mice or non-mutant littermates. Alternatively, increase ferritin expression in blood-injected mice by adenoviral transfer of ferritin genes or shRNA to IRP's. At 72 and 144 hours, determine the effect on cell viability, protein and lipid oxidation, and axonal injury in surrounding tissue. The information gained in this project may lead to new treatments for victims of hemorrhagic stroke and head trauma. The ultimate goal is to reduce brain injury in tissue surrounding a blood clot, and to thereby improve the likelihood of survival and return to an independent, productive life.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS042273-08
Application #
7846098
Study Section
Brain Injury and Neurovascular Pathologies Study Section (BINP)
Program Officer
Jacobs, Tom P
Project Start
2001-07-01
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2012-05-31
Support Year
8
Fiscal Year
2010
Total Cost
$302,104
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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