Purification and Characterization of Mature Murine and Human Satellite Glial Cells

University Of Cincinnati, Cincinnati OH

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Abstract

More than 100 million Americans suffer from chronic pain or severe pain. However, current pain treatments are inefficient and accompanied by undesirable side effects. Both inflammation and satellite glial cells (SGCs) in sensory ganglia are common features of chronic pain such as headache, temporomandibular joint disorders, neuropathic and inflammatory pain, and they may represent new therapeutic targets. However, there is a lack of adequate tools for the direct interrogation of SGCs from adult individuals and their interactions with inflammation. In this proposal we have established a new and transformative tool for the study of adult mouse and human SGCs by immunopanning, which consists in passing a cell suspension over several dishes coated with specific antibodies to deplete unwanted cell types, and a final antibody-coated dish being used to select the cell type of interest. Immunopanning is relatively simple, inexpensive, gentle on cells, and can produce very high yields of purified cells for transcriptional, biochemical and pharmacological analyses. Our long-term goal is to better understand how maladaptive perturbations between neurons, glial and immune cells can participate in pain and how they can be targeted for the development of more effective and safer therapeutic approaches. The main objective of this application is to establish a reliable protocol for the characterization and understanding of the inflammatory pathophysiology of adult murine and human SGCs. Our central hypothesis is that acutely isolated SGCs from adult mice and humans exhibit a unique molecular profile that can be modulated by inflammation to display either pathogenic or protective functions. This hypothesis will be tested by pursuing the following specific aims: (1) Identification of a unique transcriptional signature in murine and human SGCs; and (2) Characterization of murine and human SGC responses to inflammation. These aims will be accomplished by immunopanning and RNA sequencing in combination with bioinformatics, immunohistochemical, calcium imaging, in situ hybridization and real-time RT-PCR approaches. The proposed work is innovative because it will challenge conventional concepts that are biased toward exclusive neuronal mechanisms and therapies of pain and provide, for the first time, a comprehensive, unbiased analysis of the pattern of gene expression and functional characterization of mouse and human SGCs in health and disease conditions. The outcomes of these investigations will be (1) the establishment of a simple and relatively inexpensive procedure to isolate a large, and highly enriched population of mature SGCs, (2) the identification of the molecular repertoire of mouse and human SGCs, (3) the determination of the functions of inflammation and SGCs in a mouse model of chronic pain, and (4) the establishment of an in vitro model to study these functions in mouse and human SGCs. The application is particularly significant because these sets of tools and databases should greatly benefit the pain research field and accelerate the development of new therapeutics to efficiently suppress chronic pain.