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IN VITRO SCREENING OF DEVELOPMENTAL NEUROTOXICANTS IN RAT PRIMARY CORTICAL NEURONS USING HIGH CONTENT IMAGE
Citation:
Harrill, J., B. ROBINETTE, T. M. FREUDENRICH, AND W. R. MUNDY. IN VITRO SCREENING OF DEVELOPMENTAL NEUROTOXICANTS IN RAT PRIMARY CORTICAL NEURONS USING HIGH CONTENT IMAGE. Presented at 27th International Neurotoxicology Conference, Apex, NC, November 01 - 02, 2011.
Impact/Purpose:
The present work describes the design and implementation of an in vitro protocol which quantifies chemical effects on the processes of neuritogenesis, neuronal maturation and synapse formation
Description:
There is a need for more efficient and cost-effective methods for identifying, characterizing and prioritizing chemicals which may result in developmental neurotoxicity. One approach is to utilize in vitro test systems which recapitulate the critical processes of nervous system development as screening tools. The present work describes the design and implementation of an in vitro protocol which quantifies chemical effects on the processes of neuritogenesis, neuronal maturation and synapse formation. These processes occur sequentially in dissociated primary rat cortical cultures prepared in a 96well format. Cultures were exposed to test chemicals, fixed, labeled using fluorescent immunocytochemistry, and analyzed using high-content image analysis: a combination of high-throughput automated image acquisition and morphometric analysis. Chemical exposure periods in vitro were selected which correspond the period in which each process occurs in untreated cultures. The concentration-response of 24 chemicals that are known developmental neurotoxicants in vivo (positives) and 6 chemicals that are generally regarded as safe (negatives) were tested for effects on each neurodevelopmental process at concentrations ranging from 0.001 to 100 uM. In each assay, cell number was analyzed in parallel with morphometric measurements to delineate effects on a neurodevelopmental process from general cytotoxicity. The concentration which caused a 30% decrease from control (i.e. E30) was then compared between endpoints and across assays to assess assay sensitivity and specificity. Overall, 10/24 positive chemicals produced effects on neuritogenesis in a short term exposure (first 24 hours in vitro). A larger number of positive chemicals produced effects in the neuronal maturation (19/24) and synaptogenesis (14/24) assays. In many instances, effects on neuronal morphology were observed at noncytotoxic concentrations. Only 1 of 6 negative chemicals had any effect. These data, when combined with analyses of other critical neurodevelopmental processes (e.g. proliferation, differentiation, migration) could be used to construct a scheme which prioritizes chemicals for further testing for developmental neurotoxicity. This abstract does not necessarily reflect USEPA policy.