Abstract
It is well known that activated microglia is involved in the pathology of central nervous system injury caused by toxic substances. In these circumstances, microglia are thought to change their shape, which may be an indicator of toxicity of toxic substances to the central nervous system. Therefore, in this study, we attempted to establish a method to quantitatively evaluate the morphology of microglia and examine neurotoxicity and neurodevelopmental toxicity. To efficiently evaluate the change of microglia shape in the whole brain in vivo, transgenic zebrafish larvae expressing fluorescent proteins in neurons and microglia were exposed to ethanol, which is well known for its neurotoxicity. Whole-brain live imaging was performed with multiphoton laser scanning microscope. The obtained images were processed and analyzed using Image J. Three-dimensional data of microglia present in specific regions of the zebrafish brain was obtained. For quantitative assessment of microglia shape, projections of each microglia were evaluated using Image J shape descriptors. As a result, it was found that circularity or solidity of microglia in the optic tectum of zebrafish larvae treated with 2% ethanol for 24 hours increased and approached 1.0 compared to the control group. Circularity approaches 1.0 when the shape is nearly circular, and solidity approaches 1.0 when the outer contour is nearly flat; therefore, it was thought that acute exposure to ethanol caused the increase in the proportion of ameboid microglia, which is near-circular and less-branched in the optic tectum. This result indicates that microglial morphological changes caused by neurotoxic substances could be evaluated quantitatively. We are currently testing various neurotoxic substances by similar experiments.
