Background Paraquat (PQ) is a widely used herbicide. Its neurodevelopmental toxicity and relevant mechanism are not completely clear. Furthermore, few studies focus on the effects of PQ on neural stem cell differentiation.
Objective This experiment is conducted to study the effects of PQ on the differentiation of neural stem cells and explore the appropriate observation time point of neural stem cell differentiation outcome.
Methods The primary murine neural stem cells (mNSCs) were isolated from subventricular zone of neonatal C57BL/6 mice and cultured in vitro. The mNSCs were treated with different concentrations of PQ (0, 10, 20, and 40 μmol/L) for 24 h. Cell viability was detected by MTT assay; intracellular reactive oxygen species (ROS) was detected using DCFH-DA probe. After treatment with PQ for 24 h, the mNSCs were maintained in differentiation medium without PQ for 5 d, and observed for differentiation outcome under light microscope after 1, 3, and 5 d, respectively. Immunofluorescence was used to detect the effects of PQ on differentiated neurons and astrocytes.
Results When being treated with 40μmol/L PQ, the cell viability decreased to 86% of the control group, and the ROS level increased to 167% of the control group (P < 0.05). After 3d of differentiation, the cells showed obvious cell stratification. The immunofluorescence identification showed that the upper layer was mainly neuron cells. After 5 d of differentiation, the upper-layer cells displayed a distinct neurite-like structure; however, the number of cells was not visually different from that of the differentiation after 3 d. By immunofluorescence method, it was found that PQ decreased the proportions of neuron cells in a dose-dependent manner; moreover, the ratio of neurons to astrocytes also decreased, and the difference was statistically significant between the 20 μmol/L and the 40 μmol/L PQ groups (P < 0.05).
Conclusion PQ could inhibit the differentiation of primary mNSCs into neurons, which may be related to PQ-induced cellular oxidative stress. Five days could be a good time point to study mNSCs differentiation in vitro.
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