Hemoglobin-induced cytotoxicity in rat cerebral cortical neurons: caspase activation and oxidative stress.
OBJECTIVE
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.
Wang
Xiaoying
X
Neuroprotection Research Laboratory, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Mass 02129, USA. wangxi@helix.mgh.harvard.edu
Mori
Tatsuro
T
Sumii
Toshihisa
T
Lo
Eng H
EH
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D006454:Hemoglobins / Q000633:toxicity*; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D018696:Neuroprotective Agents / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation
D000975:Antioxidants; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D006454:Hemoglobins; D018696:Neuroprotective Agents; D007770:L-Lactate Dehydrogenase; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases