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. 2021 May 4;11:9520. doi: 10.1038/s41598-021-89086-7

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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MAT treatment protected RGCs from apoptosis. Neuroprotective effects of MAT were evaluated by counting RGCs immune-labeled with Brn3a antibody and estimating the number of RGC deaths through TUNEL. (A) RGCs in the both sides of temporal retina were examined by immunofluorescent double staining by anti-Brn3a (red) and TUNEL (green), and all cells were co-stained with DAPI (blue). ONL, outer nuclear layer; INL: inner nuclear layer; GCL, ganglion cell layer. Scale bars = 100 µm. (B) Quantitative analysis for the numbers of apoptotic RGCs (TUNEL⁺ Brn3a⁺DAPI⁺). (C) Scatter plots between optic nerve inflammation scores and numbers of Brn3a⁺TUNEL⁺ cells showing significant positive correlation. Numbers of Brn3a + TUNEL + cells were compared between EAE + Vehicle and EAE + MAT groups using generalized estimating equation (GEE) models with optic nerve inflammation scores as a covariate to adjust for within-subject inter-eye correlations. Subsequently, an adjusted P value was used for multiple comparison according to the Bonferroni correction methods. ***P < 0.001, comparison with the naive group. ^###P < 0.001, comparisons between vehicle- and MAT-treated groups.