Vulnerability of glial cells to hydrogen peroxide in cultured hippocampal slices.
Massive production of free radicals (FR) has been associated with a variety of pathological conditions in the central nervous system (CNS). We have used the FR generating compound hydrogen peroxide (H2O2) in organotypic hippocampal slice cultures to model oxidative injury in the brain. Necrotic cell death was monitored for up to 48 h using propidium iodide (PI) and confocal microscopy. A 1 h exposure to H2O2 (0.5-2.5 mM) caused a dose-dependent, and region specific cell death in hippocampal slice cultures. Glial cells demonstrated a high degree of vulnerability to H2O2. During the initial 3 h post-injury period, regions of the slice where glial cell bodies predominated showed massive cell death. The majority of neurons in the pyramidal layers were spared, though at later time points they appeared damaged as well. Carboxy-dichlorofluorescein imaging revealed a corresponding early increase in ROS generation in glial cells compared to pyramidal neurons. Immunohistochemistry of PI labeled slices identified astrocytes as the cells most sensitive to H2O2 toxicity. In dissociated cell cultures of hippocampal astrocytes and neurons, astrocytes also exhibited a significantly higher sensitivity to H2O2 than neurons. Hydrogen peroxide-induced cytotoxicity in all regions of the hippocampal slice culture was significantly attenuated by pre-treatment with antioxidants (alpha-tocopherol and glutathione), and was not prevented by blockade of Ca2+ influx, or NMDA channel activation. Cyclosporin A, an inhibitor of mitochondrial permeability transition, reduced cytotoxicity in glial areas by more than 50%, while in the CA2-CA3 pyramidal layers a much smaller, but still significant, attenuation of cytotoxicity was observed. Our results suggest that mitochondria are primary targets of H2O2 toxicity, particularly in astrocytes.
Feeney
Chris J
CJ
Institute of Medical Science, University of Toronto, Ontario, Canada.
Frantseva
Marina V
MV
Carlen
Peter L
PL
Pennefather
Peter S
PS
Shulyakova
Natalya
N
Shniffer
Chloe
C
Mills
Linda R
LR
D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000494:pharmacology; D002545:Brain Ischemia / Q000378:metabolism*; D016923:Cell Death; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004734:Energy Metabolism / Q000187:drug effects; D005456:Fluorescent Dyes; D005609:Free Radicals / Q000378:metabolism*; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006861:Hydrogen Peroxide / Q000633:toxicity; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism; D009336:Necrosis / Q000378:metabolism; D009410:Nerve Degeneration / Q000139:chemically induced / Q000378:metabolism*; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009924:Organ Culture Techniques; D018384:Oxidative Stress; D017966:Pyramidal Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D051381:Rats; D013997:Time Factors
D000975:Antioxidants; D005456:Fluorescent Dyes; D005609:Free Radicals; D016572:Cyclosporine; D006861:Hydrogen Peroxide