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

A redox-sensitive Grx1-roGFP2 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 glucose 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-roGFP2. To mimic the conditions of acute stroke, we developed an optimized superfiision system with the capability of rapid and reproducible exchange of the solution bathing the OHSCs. Measurements of pO(2) as a function of tissue depth show that in the region containing the transfected cells, the pO(2) is well-controlled. We also found that the pO(2) changes on the same time scale as changes in intracranial pressure, cerebral blood flow, and pO(2) during acute stroke. Determining the reduction potential, E-GSH, from the ratiometric fluorescence signal requires an absolute intensity measurement during calibration of the Grx1-roGFP2. Using the signal from cotransfected tdTomato as an internal standard during calibration improves quantitative measurements of Grx1-roGFP2 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.