Sevoflurane protects rat mixed cerebrocortical neuronal-glial cell cultures against transient oxygen-glucose deprivation: involvement of glutamate uptake and reactive oxygen species.
BACKGROUND
The purpose of this study was to clarify the role of glutamate and reactive oxygen species in sevoflurane-mediated neuroprotection on an in vitro model of ischemia-reoxygenation.


METHODS
Mature mixed cerebrocortical neuronal-glial cell cultures, treated or not with increasing concentrations of sevoflurane, were exposed to 90 min combined oxygen-glucose deprivation (OGD) in an anaerobic chamber followed by reoxygenation. Cell death was quantified by lactate dehydrogenase release into the media and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium by mitochondrial succinate dehydrogenase. Extracellular concentrations of glutamate and glutamate uptake were assessed at the end of the ischemic injury by high-performance liquid chromatography and incorporation of L-[H]glutamate into cells, respectively. Free radical generation in cells was assessed 6 h after OGD during the reoxygenation period using 2',7'-dichlorofluorescin diacetate, which reacts with intracellular radicals to be converted to its fluorescent product, 2',7'-dichlorofluorescin, in cell cytosol.


RESULTS
Twenty-four hours after OGD, sevoflurane, in a concentration-dependent manner, significantly reduced lactate dehydrogenase release and increased cell viability. At the end of OGD, sevoflurane was able to reduce the OGD-induced decrease in glutamate uptake. This effect was impaired in the presence of threo-3-methyl glutamate, a specific inhibitor of the glial transporter GLT1. Sevoflurane counteracted the increase in extracellular level of glutamate during OGD and the generation of reactive oxygen species during reoxygenation.


CONCLUSIONS
Sevoflurane had a neuroprotective effect in this in vitro model of ischemia-reoxygenation. This beneficial effect may be explained, at least in part, by sevoflurane-induced antiexcitotoxic properties during OGD, probably depending on GLT1, and by sevoflurane-induced decrease of reactive oxygen species generation during reoxygenation.
Canas
Paula T
PT
Département d'Anesthésie Réanimation, Centre Hospitalier Universitaire Timone, Marseille, France.
Velly
Lionel J
LJ
Labrande
Christelle N
CN
Guillet
Benjamin A
BA
Sautou-Miranda
Valérie
V
Masmejean
Frédérique M
FM
Nieoullon
André L
AL
Gouin
François M
FM
Bruder
Nicolas J
NJ
Pisano
Pascale S
PS
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D018920:Coculture Techniques; D005947:Glucose / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D008738:Methyl Ethers / Q000494:pharmacology*; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D000077149:Sevoflurane
D008738:Methyl Ethers; D018696:Neuroprotective Agents; D017382:Reactive Oxygen Species; D000077149:Sevoflurane; D018698:Glutamic Acid; D005947:Glucose