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. 2019 Oct 10;9:14554. doi: 10.1038/s41598-019-49815-5

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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ROS generated in the cochlear explant. Cellular oxidative stress within the cochlea explant was examined using a fluorogenic probe which could be oxidized by reactive oxygen species (ROS). On day 2, there were no detectable ROS signals (green) in the untreated explants (A), while fuzzy fluorescent signals were detected around the hair cell band region (marked as HC region) in the 2 mM arecoline-treated explants (B). (C) The ROS signals could be detected within 3 h when the arecoline concentration was increased to 10 mM, especially in the hair cells (arrows). Disruption and disappearance of hair bundles within the hair cell region could also be observed. The z-projection column demonstrates the summed-up signals of all the optical sections, which represents the total ROS signal in the explant. The z-section column is specifically for the section at the nuclei level. The images of 10 mM group showed that many ROS signals were detected at the nuclei, indicating the oxidative stress generated (C, lower panel). HC, hair cells; IHC, inner hair cells; OHC: outer hair cells. Bar = 80 μm (A,B) and 30 μm (C).