Canas Paula T, Velly Lionel J, Labrande Christelle N, Guillet Benjamin A, Sautou-Miranda Valérie, Masmejean Frédérique M, Nieoullon André L, Gouin François M, Bruder Nicolas J, Pisano Pascale S
Département d'Anesthésie Réanimation, Centre Hospitalier Universitaire Timone, Marseille, France.
Anesthesiology. 2006 Nov;105(5):990-8. doi: 10.1097/00000542-200611000-00021.
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.
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.
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.
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.
本研究的目的是在体外缺血 - 再灌注模型中阐明谷氨酸和活性氧在七氟醚介导的神经保护作用中的作用。
将成熟的混合大脑皮质神经元 - 胶质细胞培养物，用不同浓度的七氟醚处理或不处理，置于厌氧培养箱中进行90分钟的联合氧 - 葡萄糖剥夺（OGD），随后再灌注。通过测定培养基中乳酸脱氢酶的释放量来量化细胞死亡，通过线粒体琥珀酸脱氢酶将3 - [4,5 - 二甲基噻唑 - 2 - 基] - 2,5 - 二苯基四氮唑还原的程度来评估细胞活力。在缺血损伤结束时，分别通过高效液相色谱法和L - [H]谷氨酸掺入细胞的方法来评估细胞外谷氨酸浓度和谷氨酸摄取。在再灌注期间，使用二氯荧光素二乙酸酯评估OGD后6小时细胞内自由基的产生，二氯荧光素二乙酸酯与细胞内自由基反应，在细胞质中转化为其荧光产物二氯荧光素。
OGD后24小时，七氟醚以浓度依赖的方式显著降低乳酸脱氢酶的释放量并提高细胞活力。在OGD结束时，七氟醚能够减少OGD诱导的谷氨酸摄取减少。在存在苏式 - 3 - 甲基谷氨酸（一种胶质转运体GLT1的特异性抑制剂）的情况下，这种作用受到损害。七氟醚抵消了OGD期间细胞外谷氨酸水平的升高以及再灌注期间活性氧的产生。
在这个体外缺血 - 再灌注模型中，七氟醚具有神经保护作用。这种有益作用至少部分可以通过七氟醚在OGD期间诱导的抗兴奋毒性特性来解释，这可能依赖于GLT1，以及七氟醚在再灌注期间诱导的活性氧产生减少来解释。
