Essential tremor: A three-dimensional neurosphere in vitro model to assess the neurotoxicity of harmane

Abstract

Objectives: To use a novel in vitro model of three-dimensional (3D) neurosphere cultures to assess neurotoxic or neuroprotective effects with harmane as a model compound. Methods: A reproducible model of 3D spheroids was developed from embryonic mouse cortical neurons, using molded agarose micro-wells; this method seems particularly practical as it is customizable and widely available and does not require specific cell treatments or assay components different from 2D cultures, allowing for the easy transposition of routine protocols. To assess the neurotoxic effects of harmane, a resazurin assay was performed to measure cell viability, and a highly sensitive fluorometric method, based on the oxidation of dichlorodihydrofluorescein, was applied to measure eventually induced reactive oxygen species (ROS) after exposure to harmane at increasing concentrations of 50 100, and 250 μm. Results: Hydrogel microwells facilitated the assembly of spheroids containing neurons and glial cells into a complex 3D structure and prevented the agglomeration of spheroids. Exposure to harmane induced cytotoxicity in 3D neural spheroids, which was correlated with harmane concentrations, with a 27% reduction in viability at 250 μm. Harmane that did not induce significant levels of oxidative stress was detected for all tested concentrations. Conclusion: This 3D neurosphere model mimics a neuronal microenvironment, allowing a fine study of neurodegenerative disorders and the effects of chemicals on the brain. This model opens novel opportunities, not only from a pathogenetic point of view but also from a therapeutic perspective.

Keywords

• 3D spheroid
• Toxicity
• Neurotoxicity
• Harmane
• Essential tremor