Browsing by Author "Atmaramani, Rahul"
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Item Development of an In Vitro Phenotypic Assay for Screening Chronic Pain Therapeutics(2020-05) Atmaramani, Rahul; Pancrazio, JosephDorsal 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 bodyItem Emerging Neurotechnology for Antinoceptive Mechanisms and Therapeutics Discovery(Elsevier Advanced Technology, 2018-11-13) Black, Bryan J.; Atmaramani, Rahul; Plagens, Sarah; Campbell, Zachary T.; Dussor, Gregory; Price, Theodore J.; Pancrazio, Joseph J.; 0000-0002-3768-6996 (Campbell, ZT); 0000-0002-6971-6221 (Price, TJ); 0000-0001-8276-3690 (Pancrazio, JJ); Black, Bryan J.; Atmaramani, Rahul; Plagens, Sarah; Campbell, Zachary T.; Dussor, Gregory; Price, Theodore J.; Pancrazio, Joseph J.The tolerance, abuse, and potential exacerbation associated with classical chronic pain medications such as opioids creates a need for alternative therapeutics. Phenotypic screening provides a complementary approach to traditional target-based drug discovery. Profiling cellular phenotypes enables quantification of physiologically relevant traits central to a disease pathology without prior identification of a specific drug target. For complex disorders such as chronic pain, which likely involves many molecular targets, this approach may identify novel treatments. Sensory neurons, termed nociceptors, are derived from dorsal root ganglia (DRG) and can undergo changes in membrane excitability during chronic pain. In this review, we describe phenotypic screening paradigms that make use of nociceptor electrophysiology. The purpose of this paper is to review the bioelectrical behavior of DRG neurons, signaling complexity in sensory neurons, various sensory neuron models, assays for bioelectrical behavior, and emerging efforts to leverage microfabrication and microfluidics for assay development. We discuss limitations and advantages of these various approaches and offer perspectives on opportunities for future development.Item Spontaneous and Evoked Activity from Murine Ventral Horn Cultures on Microelectrode Arrays(Frontiers Media SA, 2018-06-01) Black, Bryan J.; Atmaramani, Rahul; Pancrazio, Joseph J.; 0000-0001-8276-3690 (Pancrazio, JJ); Black, Bryan J.; Atmaramani, Rahul; Pancrazio, Joseph J.Motor neurons are the site of action for several neurological disorders and paralytic toxins, with cell bodies located in the ventral horn (VH) of the spinal cord along with interneurons and support cells. Microelectrode arrays (MEAs) have emerged as a high content assay platform for mechanistic studies and drug discovery. Here, we explored the spontaneous and evoked electrical activity of VH cultures derived from embryonic mouse spinal cord on multi-well plates of MEAs. Primary VH cultures from embryonic day 15-16 mice were characterized by expression of choline acetyltransferase (ChAT) by immunocytochemistry. Well resolved, all-or-nothing spontaneous spikes with profiles consistent with extracellular action potentials were observed after 3 days in vitro, persisting with consistent firing rates until at least day in vitro 19. The majority of the spontaneous activity consisted of tonic firing interspersed with coordinated bursting across the network. After 5 days in vitro, spike activity was readily evoked by voltage pulses where a minimum amplitude and duration required for excitation was 300 mV and 100 μs/phase, respectively. We characterized the sensitivity of spontaneous and evoked activity to a host of pharmacological agents including AP5, CNQX, strychnine, omega-agatoxin IVA, and botulinum neurotoxin serotype A (BoNT/A). These experiments revealed sensitivity of the cultured VH to both agonist and antagonist compounds in a manner consistent with mature tissue derived from slices. In the case of BoNT/A, we also demonstrated intoxication persistence over an 18-day period, followed by partial intoxication recovery induced by N- and P/Q-type calcium channel agonist GV-58. In total, our findings suggest that VH cultures on multi-well MEA plates may represent a moderate throughput, high content assay for performing mechanistic studies and for screening potential therapeutics pertaining to paralytic toxins and neurological disorders.Item The Effect of Microfluidic Geometry on Myoblast Migration(MDPI AG) Atmaramani, Rahul; Black, Brian J.; Lam, Kevin H.; Sheth, Vinit M.; Pancrazio, Joseph J.; Schmidtke, David W.; Alsmadi, Nesreen Zoghoul; 0000-0002-9325-547X (Atmaramani, R); 0000-0001-8276-3690 (Pancrazio, JJ); 0000-0001-6404-318X (Schmidtke, DW); Atmaramani, Rahul; Black, Brian J.; Lam, Kevin H.; Sheth, Vinit M.; Pancrazio, Joseph J.; Schmidtke, David W.; Alsmadi, Nesreen ZoghoulIn vitro systems comprised of wells interconnected by microchannels have emerged as a platform for the study of cell migration or multicellular models. In the present study, we systematically evaluated the effect of microchannel width on spontaneous myoblast migration across these microchannels-from the proximal to the distal chamber. Myoblast migration was examined in microfluidic devices with varying microchannel widths of 1.5-20 µm, and in chips with uniform microchannel widths over time spans that are relevant for myoblast-to-myofiber differentiation in vitro. We found that the likelihood of spontaneous myoblast migration was microchannel width dependent and that a width of 3 µm was necessary to limit spontaneous migration below 5% of cells in the seeded well after 48 h. These results inform the future design of Polydimethylsiloxane (PDMS) microchannel-based co-culture platforms as well as future in vitro studies of myoblast migration. © 2019 by the authors.