Optimized Real-Time Monitoring of Glutathione Redox Status in Single Pyramidal Neurons in Organotypic Hippocampal Slices during Oxygen鈥揋lucose Deprivation and Reperfusion

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• 作者：Bocheng Yin ; Germ谩n Barrionuevo ; Stephen G. Weber
• 刊名：ACS Chemical Neuroscience
• 出版年：2015
• 出版时间：November 18, 2015
• 年：2015
• 卷：6
• 期：11
• 页码：1838-1848
• 全文大小：682K
• ISSN：1948-7193

文摘

A redox-sensitive Grx1鈥搑oGFP2 fusion protein was introduced by transfection into single pyramidal neurons in the CA1 subfield of organotypic hippocampal slice cultures (OHSCs). We assessed changes in the GSH system in neuronal cytoplasm and mitochondria during oxygen鈥揼lucose deprivation and reperfusion (OGD/RP), an in vitro model of stroke. Pyramidal cells in a narrow range of depths below the surface of the OHSC were transfected by gene gun or single-cell electroporation with cyto- or mito-Grx1鈥搑oGFP2. To mimic the conditions of acute stroke, we developed an optimized superfusion system with the capability of rapid and reproducible exchange of the solution bathing the OHSCs. Measurements of pO₂ as a function of tissue depth show that in the region containing the transfected cells, the pO₂ is well-controlled. We also found that the pO₂ changes on the same time scale as changes in intracranial pressure, cerebral blood flow, and pO₂ during acute stroke. Determining the reduction potential, E_GSH, from the ratiometric fluorescence signal requires an absolute intensity measurement during calibration of the Grx1鈥搑oGFP2. Using the signal from cotransfected tdTomato as an internal standard during calibration improves quantitative measurements of Grx1鈥搑oGFP2 redox status and allows E_GSH to be determined. E_GSH becomes more reducing during OGD and more oxidizing during RP in mitochondria while changes in cytoplasm are not significant compared with controls.