Hemoglobin-Induced Cytotoxicity in Rat Cerebral Cortical Neurons
- 1 July 2002
- journal article
- Published by Ovid Technologies (Wolters Kluwer Health) in Stroke
- Vol. 33 (7), 1882-1888
- https://doi.org/10.1161/01.str.0000020121.41527.5d
Abstract
Background and Purpose — Apoptotic-like pathways may contribute to brain cell death after intracerebral hemorrhage. In this study, we used a simplified in vitro model of hemoglobin neurotoxicity to map the caspase cascades involved and to document the role of oxidative stress. Methods — Primary neuronal cultures were obtained from rat cerebral cortex and exposed to hemoglobin to induce cell death. Cytotoxicity was assessed via measurements of mitochondrial viability (MTT assay) and lactate dehydrogenase (LDH assay). Activation of caspase-3, -8, and -9 was measured by Western blot and enzyme activity assays. Various caspase inhibitors (zVADfmk, zDEVDfmk, zIETDfmk, and zLEHDfmk) were tested for neuroprotective efficacy. The role of oxidative stress was assessed with the use of U83836E as a potent scavenger of free radicals. Results — Exposure of primary cortical neurons to hemoglobin induced a dose- and time-dependent cytotoxicity. Western blots showed upregulation of cleaved caspase-3. Enzyme assays showed an increase in caspase-9–like and caspase-3–like activity. However, caspase inhibition did not result in neuroprotection. In contrast, the free radical scavenger U83836E significantly reduced hemoglobin-induced neuronal death. Combination treatment with both U83836E and the broad spectrum caspase inhibitor zVADfmk did not yield additional protection. Conclusions — Upstream and downstream caspases were upregulated after hemoglobin-induced neurotoxicity in vitro, but only an antioxidant approach with a potent free radical scavenger significantly improved neuronal survival. These data suggest that in addition to the activation of caspase cascades, parallel pathways of oxidative stress may predominate in this model of hemoglobin neurotoxicity.Keywords
This publication has 37 references indexed in Scilit:
- Protective Effect of Heme Oxygenase-1 Gene Transfer against Oxyhemoglobin-Induced Endothelial DysfunctionJournal of Cerebral Blood Flow & Metabolism, 2001
- Neuronal, but Not Microglial, Accumulation of Extravasated Serum Proteins after Intracerebral Hemolysate Exposure is Accompanied by Cytochrome c Release and DNA FragmentationJournal of Cerebral Blood Flow & Metabolism, 2001
- Effect of FK-506 on Inflammation and Behavioral Outcome Following Intracerebral Hemorrhage in RatExperimental Neurology, 2001
- Multiple Molecular Penumbras after Focal Cerebral IschemiaJournal of Cerebral Blood Flow & Metabolism, 2000
- Evidence for Apoptosis After Intracerebral Hemorrhage in Rat StriatumJournal of Cerebral Blood Flow & Metabolism, 2000
- Anti-apoptotic oncogenes prevent caspase-dependent and independent commitment for cell deathCell Death & Differentiation, 1998
- Tissue Plasminogen Activator for Acute Ischemic StrokeNew England Journal of Medicine, 1995
- Neurotoxicity of hemoglobin in cortical cell cultureNeuroscience Letters, 1993
- Intracerebral hemorrhage more than twice as common as subarachnoid hemorrhageJournal of Neurosurgery, 1993