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COMPARISON OF NEUROSCREEN-1 AND CEREBELLAR GRANULE CELL CULTURES FOR EVALUATING NEURITE OUTGROWTH USING THE ARRAYSCAN HIGH CONTENT ANALYSIS SYSTEM
Citation:
RADIO, N., T. M. FREUDENRICH, B. ROBINETTE, AND W. R. MUNDY. COMPARISON OF NEUROSCREEN-1 AND CEREBELLAR GRANULE CELL CULTURES FOR EVALUATING NEURITE OUTGROWTH USING THE ARRAYSCAN HIGH CONTENT ANALYSIS SYSTEM. Presented at Society of Toxicology, Seattle, WA, March 16 - 20, 2008.
Impact/Purpose:
This study compared the two in vitro neural cell types, PC-12 neuronal cell line (NS-1)cells, and primary neuronal cultures of cerebellar granular cells (CGC) as potential models for assessing chemical effects onscreening neurite outgrowth in a high-throughput format.
Description:
A major challenge facing the Environmental Protection Agency is the development of high-throughput screening assays amendable to resource-efficient developmental neurotoxicity for chemical screening and toxicity prioritization. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is a critical cellular process underlying the developing nervous system development that can be quantified using high content analysis technology. This study compared the two in vitro neural cell types, PC-12 neuronal cell line (NS-1)cells, and primary neuronal cultures of cerebellar granular cells (CGC) as potential models for assessing chemical effects onscreening neurite outgrowth in a high-throughput format. High content analysis of neurite outgrowth was performed using the Cellomics ArrayScan VTi automated epifluorescent imaging system to acquire and analyze images of â-tubulin immunostained cells in 96-well plates. METHODS? Incubation in NS-1 or CGC in NGF or serum respectively, induced neurite outgrowthneurite outgrowth within 24 hours and continued to elongate over several days in vitro. ….. A series of optimization assays were performed to enhance the assay sensitivity to chemical-mediated outgrowth inhibition. Differentiation time course analysis indicated a linear increase in neurite outgrowth in both models Identification of potential pharmacological controls to be utilized as internal plate controls were evaluated using tThe MEK inhibitor U0126 (at concentrations that were not cytotoxic. 15 – 30 uM??) and the PKC inhibitor Bis-1 (0.3 – 10 uM), known positive controls, Both compounds specifically inhibited neurite outgrowth in both NS-1 and CGC. Neurite outgrowth inhibition was further evaluated using eight chemicals that are known a ddevelopmental neurotoxicants in vivocity training set.. Cells were exposed for either 4 (NS-1) or 2 (CGC) days to 1 nM - 100 uM. COMPARE RESULTS IN TWO MODELS IN MORE DETAILExposure to methylmercury or cadmium reduced neurite outgrowth in both models. Inhibition of neurite outgrowth with trans-retinoic acid and dexamethasone was more pronounced in NS-1 cells than CGC. In contrast, lead and amphetamine inhibited neurite outgrowth only in CGC. These results suggest differences in sensitivities of in vitro models to chemical effects on neurite outgrowth. Future research will evaluate additional developmental processes, including proliferation and synaptogenesis, for screening using high content analysis.to form an in vitro screening battery. The development of such high-throughput screening tests will greatly enhance the Environmental Protection Agency’s efficiency of risk assessment for the thousands of chemicals requiring developmental neurotoxicity characterization.