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

Abstract

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 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, EGSH, 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 EGSH to be determined. EGSH becomes more reducing during OGD and more oxidizing during RP in mitochondria while changes in cytoplasm are not significant compared with controls.

Differences in Reperfusion-Induced Mitochondrial Oxidative Stress and Cell Death Between Hippocampal CA1 and CA3 Subfields Are Due to the Mitochondrial Thioredoxin System

“…We have specifically designed our investigations to minimize the likelihood that transcription/translation are responsible for the observed effects. We improved the OGD-RP procedure to switch solutions rapidly (70) so that ROS changes could be measured within a few minutes. We restricted our attention to pyramidal cells, but did not isolate them, thus allowing the natural interaction between glia and neurons to occur.…”

“…pCMV-tdTomato (Clontech) and pCS2+ (Addgene) were kindly provided by Zachary P. Wills (University of Pittsburgh). pMito-tdTomato was constructed in the Deiters laboratory (University of Pittsburgh) (70). The antibodies and dye used in WBs and IF are listed in Supplementary Data.…”

“…Measurement of OxD P and OxD G during OGD-RP with/without AF All fluorescence images in this work were acquired on a Leica TCS SP5II broadband confocal microscope. We followed the protocol established in our previous work (70). OHSCs were collected from a postnatal 7-day-old rat and cultured 3 days before transfection with plasmids for H 2 O 2 or GSH probes.…”

Genetically Encoded Tools for Research of Cell Signaling and Metabolism under Brain Hypoxia

“…However, many fewer studies have attempted to exploit genetically encoded sensors for the real-time evaluation of alterations in cell signaling and metabolism in in vitro models of brain hypoxic injury. For instance, the responses of the glutathione redox system in neuronal cytoplasm and mitochondria during the course of oxygen-glucose deprivation and reperfusion in organotypic brain slices (an in vitro model of the stroke) were recorded by using the genetically encoded fusion biosensor Grx1-roGFP2 [276]. Under precisely controlled treatment conditions within a sample, the fluorescence signal from the biosensor was accurately calibrated using a signal from the co-transfected fluorescent protein tdTomato as an internal standard.…”

“…The latter allowed for the quantitative determination of the reduction potential of the glutathione system in the neuronal cytoplasm and mitochondria during the course of oxygen-glucose deprivation and reperfusion. Using their improved protocol, the authors were able to define time dependence of the redox state of the glutathione system in this in vitro model of the stroke [276]. Moreover, they demonstrated that the mitochondrial reduction potential became more reducing during oxygen-glucose deprivation and more oxidizing during reperfusion, while the cytoplasmic reduction potential did not change significantly.…”