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Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening
Citation:
RADIO, N., T. M. FREUDENRICH, B. ROBINETTE, K. M. CROFTON, AND W. R. MUNDY. Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening. NEUROTOXICOLOGY AND TERATOLOGY. Elsevier Science Ltd, New York, NY, 32(1):25-35, (2010).
Impact/Purpose:
In vitro models may be useful for the rapid toxicological screening of large numbers of chemicals for their potential to produce toxicity. Such screening could facilitate prioritization of resources needed for in vivo toxicity testing towards those chemicals most likely to result in adverse health effects. Cell cultures derived from nervous system tissue can be used in automated, high content screening assays to examine the chemical effects on neuronal outgrowth. However, it is not clear whether cell lines or primary cultures have the same sensitivity to chemicals. In the present study, chemical effects on neurite outgrowth were compared between the PC12 cell line and primary cultures of rat cerebellar granule cells. Neurite outgrowth was quantified using a high content screening protocol which allowed for automated image acquisition and analysis of neurite length for cells grown in 96-well microplates. Following optimization of culture conditions for both PC12 cells and cerebellar granule cells, chemical-induced changes in neurite outgrowth was evaluated using a set of pharmacologic and environmental compounds previously shown to affect this process. Chemicals with little evidence of effects on neurite outgrowth were also examined. The results demonstrate that the choice of model system can influence the ability to detect chemical-induced changes in neurite outgrowth. Primary cultures were more sensitive in predicting the effects of the positive control chemicals.
Description:
Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process underlying nervous system development that can be quantified using automated microscopy and image analysis (high content analysis). The present study characterized and compared the PC-12 cell line (NS-1) and primary cultures of cerebellar granular cells (CGC), as models for assessing chemical effects on neurite outgrowth. High content analysis of neurite outgrowth was performed using the Cellomics ArrayScan VTi automated epifluorescent imaging system to acquire and analyze images of β-tubulin immunostained cells in 96-well plates. Cell viability was assessed using the CellTiter-Glo® assay. Culture of NS-1 or CGC in nerve growth factor or serum respectively, rapidly induced neurite outgrowth that increased over four days in vitro. Seven compounds previously shown to affect neurite outgrowth in vitro were tested in both models for changes in total neurite length and cell viability. In NS-1 cells, four chemicals (PKC inhibitor Bis-I, MEK inhibitor U0126, trans-retinoic acid, methylmercury) inhibited neurite outgrowth, while lead, amphetamine and valproic acid had no effect. In CGC, five chemicals inhibited neurite outgrowth (Bis-I, U0126, lead, methylmercury, amphetamine), while trans-retinoic acid decreased cell viability but not neurite outgrowth. Valproic acid was without effect. The sensitivity of the two models was chemical specific: NS-1 cells were more sensitive to Bis-I, methylmercury and trans-retinoic acid, while CGC were more sensitive to U0126, lead, and amphetamine. For every chemical (except trans-Retinoic acid), neurite outgrowth was equal to or more sensitive than cell viability. In comparison, out of seven chemicals without prior evidence for effects on neurite outgrowth, only one decreased neurite outgrowth (diphenhydramine in CGC). These findings demonstrate that the effects of chemicals on neurite outgrowth may be cell type specific.
