Hemoglobin-Induced Cytotoxicity in Rat Cerebral Cortical Neurons

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.

Genetic Enhancement of Visual Learning by Activation of Protein Kinase C Pathways in Small Groups of Rat Cortical Neurons

“…Cultures of dissociated temporal cortex cells were prepared from embryonic day 17 Sprague Dawley rats (Dichter, 1978;Wang et al, 2002) and maintained in Neurobasal medium with B-27 supplement (NB medium) (lacks Glu, Gln, and GABA; Invitrogen, Carlsbad, CA) and 0.5 mM Gln (omitted during the release procedure). Cultures were treated with 10 M cytosine arabinoside (days 3-4) and infected (multiplicity of infection, 0.2) on day 8 or 9, and release of Glu (Feasey et al, 1986;Di Iorio et al, 1996) and GABA (Snodgrass et al, 1980) was measured 24 h later.…”

Thrombin and hemin as central factors in the mechanisms of intracerebral hemorrhage–induced secondary brain injury and as potential targets for intervention

“…103 Its mechanism of action occurs through oxidative stress and the activation of caspases, resulting in the injury of astrocytes, neurons, and microglia. 102,135 However, microglia that clear hemin have protective mechanisms that prevent cell death.…”

Effects of U83836E on nerve functions, hyperalgesia and oxidative stress in experimental diabetic neuropathy

“…Oxidative stress also activates down stream pathways such as poly(ADP-ribose) polymerase (PARP) and mitogen-activated protein kinases (MAPK) (Evans et al, 2002;Obrosova et al, 2004). Oxidative stress causes vascular impairment leading to endoneurial hypoxia resulting in impaired neural function (Wang et al, 2002;Yorek et al, 2004), reduced nerve conduction velocity and loss of neurotrophic support. Longterm oxidative stress can also mediate apoptosis of neurons and Schwann cells leading to nerve damage (Sekido et al, 2004).…”