Edmands Scott D, Hall Adam C
Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts, USA.
Anesthesiology. 2009 Mar;110(3):538-47. doi: 10.1097/ALN.0b013e3181974bba.
Pretreatment with inhaled anesthetics, including isoflurane, can induce long-lasting cellular protection against ischemia-derived toxicity in multiple tissues, including brain tissue. Metal-regulatory genes, metallothioneins-I/II (MT-I/II), have been shown to protect against oxidative damage in multiple tissues. Furthermore, MT have been found to be differentially regulated in response to isoflurane and ischemic preconditioning. In this study, we assess the role of MT-I/II in mediating isoflurane preconditioning in primary neuronal-glial cultures.
Primary mouse neuronal-glial cultures were preconditioned with isoflurane (3 h, 1.5%) 24-96 h before 3-h oxygen-glucose deprivation (OGD, ischemic model). After OGD, isoflurane protection and responsiveness of MT-I/II knockdown and knockout cultures to preconditioning were assessed by lactate dehydrogenase release. Immunoassays for microtubule associated protein 2 and glial fibrillary acidic protein determined neuronal-glial sensitivity to preconditioning. MT-I/II messenger RNA was assessed by quantitative reverse transcriptase-polymerase chain reaction. Cultures transfected with exogenous MT-I/II were analyzed for protection against OGD toxicity.
Isoflurane preconditioning reduced OGD-mediated toxicity by 11.6 +/- 7.9% at 24 h, with protection increasing to 37.5 +/- 2.5% at 72 h after preconditioning. Immunolabeling showed that neurons were more sensitive to OGD and more responsive to isoflurane preconditioning compared to glia. Quantitative reverse transcriptase-polymerase chain reaction showed MT-I/II messenger RNA were upregulated (approximately 2.5-fold) by isoflurane treatments. Also MT-I/II protein transfection significantly decreased OGD-mediated toxicity. Finally, knockdown and knockout of MT-I/II diminished and abolished isoflurane-mediated protection, respectively.
MT-I/II play an important role in isoflurane-mediated delayed preconditioning against OGD toxicity of neuronal and glial cells in vitro.
包括异氟烷在内的吸入性麻醉剂预处理，可在包括脑组织在内的多种组织中诱导对缺血衍生毒性的持久细胞保护作用。金属调节基因金属硫蛋白-I/II（MT-I/II）已被证明可保护多种组织免受氧化损伤。此外，已发现MT对异氟烷和缺血预处理有不同的调节作用。在本研究中，我们评估MT-I/II在原代神经元-神经胶质细胞培养中介导异氟烷预处理的作用。
原代小鼠神经元-神经胶质细胞培养物在3小时氧糖剥夺（OGD，缺血模型）前24 - 96小时用异氟烷（3小时，1.5%）进行预处理。OGD后，通过乳酸脱氢酶释放评估异氟烷保护作用以及MT-I/II敲低和敲除培养物对预处理的反应性。用微管相关蛋白2和胶质纤维酸性蛋白的免疫测定法确定神经元-神经胶质细胞对预处理的敏感性。通过定量逆转录-聚合酶链反应评估MT-I/II信使核糖核酸。分析转染外源性MT-I/II的培养物对OGD毒性的保护作用。
异氟烷预处理在24小时时将OGD介导的毒性降低了11.6±7.9%，预处理后72小时保护作用增加至37.5±2.5%。免疫标记显示，与神经胶质细胞相比，神经元对OGD更敏感，对异氟烷预处理反应更敏感。定量逆转录-聚合酶链反应显示，异氟烷处理使MT-I/II信使核糖核酸上调（约2.5倍）。此外，MT-I/II蛋白转染显著降低了OGD介导的毒性。最后，MT-I/II的敲低和敲除分别减弱和消除了异氟烷介导的保护作用。
MT-I/II在异氟烷介导的对体外神经元和神经胶质细胞OGD毒性的延迟预处理中起重要作用。
