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. 2020 Jan 17;10:614. doi: 10.1038/s41598-019-57234-9

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© The Author(s) 2020

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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Secondary blood-perfusion insufficiency induced the apoptosis of neuron and glial (in vivo/in vitro). (A) Example of images (each representative of 6 zebrafishes) depicting endothelial cells (green) of cerebral capillaries (left) and spontaneous fluorescence (white) of NADPH (right) in 0 and 10 Gy group at 4-day post radiation. Scale bars, 100 µm. (B) Example of images (each representative of 6 zebrafishes) depicting intracranial injection of hypoxia probe (green) and neurons (red) of HuC:mCherry zebrafish in 0 and 10 Gy group at 4-day post radiation. The boxed areas indicated the changes in severity of hypoxia and changes in the number of neurons between 0 and 10 Gy groups. Scale bars, 100 µm. (C) In vivo, glial cells (GFAP:mCherry zebrafish lines) and neurons (HuC:mCherry zebrafish lines) were gated for apoptosis staining in control and 10 Gy group at 4-day post radiation (n = 6 zebrafishes per group). (D) Example of images (each representative of 6 visions) depicting vitro 3-D culture of cerebral endothelial cells in 0 and 10 Gy groups at the fluorescence channel (left) and bright field (right). The arrowheads and boxes indicate the apoptosis of endothelial cells in 10 Gy group compared with 0 Gy group. (E) Flow analysis of percentage of endothelial cells in vitro 3-D culture at 4-day post radiation among 0, 5 and 10 Gy groups. (F) Example of images (each representative of 6 visions) depicting vitro 3-D culture of neurons (white) in 0 and 10 Gy groups at 4-day post radiation. (G) Flow analysis of percentage of neurons in vitro 3-D culture at 4-day post radiation among 0, 5 and 10 Gy groups. (H) Neurons (HuC:mCherry zebrafish lines) in vitro 3-D culture were gated for apoptosis staining in 0 and 10 Gy group at 4-day post radiation (n = 6 zebrafishes per group). Statistical analysis in (E,G) was performed using t-test: **P < 0.05, ***P < 0.01. Data represent the mean ± s.e.m.