Metal Neurotoxicity assessment: A new imaging and analysis pipeline for primary neurons in co-cultures.

BACKGROUND

The toxicity of certain metals poses serious problems for the environment and human health. Their toxicity affects neurological functions. However, toxicity assays performed in multi-well plates are not well suited to primary neuronal co-cultures, which more closely resemble physiological conditions than monocultures of cell lines.

NEW METHOD: We have developed a novel methodological workflow to specifically measure the viability of primary neurons co-cultured with astrocytes, and have used this to measure the toxicity of cadmium (Cd), arsenic (As), lead (Pb), mercury (Hg), zinc (Zn), copper (Cu) and manganese (Mn).

RESULTS

This method enables the imaging of large numbers of primary hippocampal neurons (>20,000) directly on coverslips while excluding glial cells from the analysis. Data analysis was performed using a dedicated ImageJ macro and an R notebook script to facilitate the reproducibility and traceability of the results. Inhibitory concentrations IC10 and IC50 were calculated using three dose-response models (log-logistic, Weibull 1 and Weibull 2), and were used to rank the metals in order of toxicity: Cd > As > Pb > Hg > Zn > Cu > Mn.

COMPARISON WITH EXISTING

METHODS

Contrary to other protocol, this analysis pipeline allows the specific measurement of neurotoxic effect on large numbers of primary neurons co-cultured with glial cells.

CONCLUSION

This new methodological approach enables the precise and reproductible measurement of neurotoxicity in primary neuronal co-cultures.