Electroporation is a technique that creates transient pores in cell membranes. It is mostly used for transfection, and applied to suspensions of cells. Single-cell electroporation is also used for transfection but on single cells, typically in suspension. This project addresses the need to do analytical chemistry on single cells without sacrificing them. As single-cell electroporation creates transient ports in cell membranes, it is an excellent approach to obtaining samples of cytoplasmic contents. Cells taken out of their context, e.g. suspensions of naturally adherent cells may not be representative of their natural state, so the project focuses on adherent cells and tissues, not on suspended cells. We have recently found that adherent cells in culture are remarkably robust. Cells survive even after losing a significant fraction of the low-molecular weight solutes in the cytoplasm. We have also found that we can control single-cell electroporation conditions so that a desired fraction of the low-molecular weight solutes in the cytoplasm, e.g., 20%, diffuses through the transient pores. This observation provides the foundation for obtaining samples from single cells without killing them. In this project, we will develop significant tools for single-cell biochemical investigations. One tool will be able to perfuse single adherent cells with high spatial resolution and simultaneously electroporate the perfused cell. We can then learn in detail the mass transport rates for solutes entering or leaving single cells. Another method will be developed for making measurements on single cells in cultured hippocampal tissue. It will be applied to an important question related to stroke and similar incidents in which blood flow to a region of the brain is temporarily lost. We will establish this method for determining the status of the important glutathione redox system in a single neuron in a hippocampal culture. This includes obtaining cytoplasmic contents by electroporation and microfluidic-based derivatization, separation, and quantitation. We also will develop a means to diminish the astrocytes'ability to communicate with each other through gap junctions based on focal electroporation of siRNA for the protein that creates the gap junctions. We will test the hypothesis that solute transport between adjacent astrocytes is important for maintenance of neuronal glutathione levels following oxygen/glucose deprivation.
New tools for controlling and measuring the chemical composition of the intra- and extracellular space of single cells are required for understanding biochemical responses to injury, especially ischemia. Our approach to making measurements of the glutathione status of single cells has far-reaching implications not only for studying ischemia/reperfusion, but also in a number of widespread conditions, namely Alzheimer's and Parkinson's diseases, schizophrenia, and epilepsy. Making measurements on single cells in tissue cultures will lead to a clarification of the role of astrocytes on neuronal health in ischemia/reperfusion.
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