Development of an In Vitro Phenotypic Assay for Screening Chronic Pain Therapeutics

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Dorsal root ganglia (DRG) are a collection of first order sensory neurons involved in the perception of innocuous as well as noxious stimuli, and the conduction of corresponding signals to the spinal cord. Upon suprathreshold stimulation, sensory fibers from the DRG are depolarized, transmitting signals originating in the periphery in the form of all-or-nothing action potentials to be processed by the central nervous system. The DRG play a critical role in the manifestation of chronic pain, a disorder affecting 100 million people throughout the US. Enhanced DRG excitability is a hallmark feature of chronic pain, which can be induced by exposure to cytokines in vivo and modeled in vitro. Currently, there is a critical technology void in our ability to screen for potential chronic pain therapeutics. Phenotypic screening approaches that focus on cell behavior endpoints such as excitability have been previously limited to single cell measurements (e.g., patch clamp) or extracellular recording from cultured embryonic tissue. Furthermore, unlike the in vivo scenario, these in vitro approaches do not spatially segregate axonal processes from cell bodies and, therefore, increasingly rely on cell soma responses and do not allow selective axonal manipulation. Our novel approach will leverage a combination of adult mouse DRG neurons cultured on multi-well microelectrode arrays (MEAs) with specially fabricated microchannels to allow long-term monitoring of phenotypic activity from spatially and chemically segregated DRG cell bodies and axons. To date, the only related prior effort has focused on embryonic derived DRG cultures, which require neurotrophic factors to maintain viability in culture. These factors (e.g., nerve growth factor) are known to play a role in inflammatory pain by causing sensitization through the increased expression and trafficking of transient receptor potential cation channel subfamily V member 1 (TRPV1). Therefore, there is a need for alternative, more physiologically relevant in vitro models for the study of sensitization of DRG neurons. Firstly, we developed and described methodologies for the culture of spontaneously active adult DRG sensory neurons on MEAs. The spontaneous and stimulus evoked activity is characterized over chronic time periods in vitro which are consistent with pharmacological interventions in the context of a screening paradigm. Additionally, we demonstrate that the observed activity can be modulated with inflammatory cytokine interleukin6 (IL-6), giving rise to persistent hyperexcitability in vitro. As a next step, to enable translation as a high-content screening (HCS) platform an assay quality indicator, such as the Z’-factor was determined. A robust version, which leverages the insensitivity of parameters such as the median and median average deviation in the context of extracellular electrophysiological recordings from DRG neurons is developed. Additionally, a hit detection methodology is assessed based on putative compounds to assess our ability to identify true hits in the assay. Lastly, using a PDMSbased microfluidic culture system, we illustrate the utility of compartmentalized cultures for the study of the axonal microenvironment independent of the cell body

Spinal ganglia, Chronic pain, Medical screening, Array processors, Nerves, Peripheral
©2020 Rahul Atmaramani. All rights reserved.