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. 2021 Apr 21;11:8647. doi: 10.1038/s41598-021-88118-6

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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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Modulation by the kappa opioid receptor agonist nalfurafine of the hemangiogenesis and lymphangiogenesis in neovascularized cornea. (a) Slit-lamp images showing corneas at 3 and 14 days after neovascularization induction in vivo (magnification × 25) (b) Neovascularization scores in the corneas 14 days after neovascularization induction (mean ± standard error of the mean, n = 15 per group; statistical significance is indicated as follows: *P = 0.034, **P = 0.007; two-way ANOVA followed by the post hoc Bonferroni test). (c) Representative images showing CD31 staining of the whole cornea and the processed images. (d) The fractions of the total cornea area covered with blood vessels (CD31⁺) were significantly reduced in corneas that were treated with nalfurafine once or twice a day compared to that in control, untreated corneas on day 14 post neovascularization induction (mean ± standard error of the mean, n = 5, statistical significance is indicated as follows: *P = 0.033 and ***P < 0.001 vs. control). (e) Relative mRNA expression levels of Pecam1, encoding CD31, were significantly decreased in corneas that were treated with nalfurafine twice a day compared to that in control, untreated corneas on day 14 post neovascularization induction (mean ± standard error of the mean, n = 3, statistical significance is indicated as follows: ***P ≤ 0.001 vs. control). (f) Images showing lymphatic vessel endothelial hyaluronan receptor1 (LYVE1) staining of the whole cornea and the processed images. (g) The fractions of the cornea area covered with lymphatic vessels were significantly reduced in corneas that were treated with nalfurafine once or twice a day compared with the control, untreated corneas on day 14 post neovascularization induction (mean ± standard error of the mean, n = 3, **P < 0.01, ***P < 0.001 vs. control, respectively). (h) Relative Lyve1 mRNA expression was significantly decreased in corneas that were treated with nalfurafine twice a day compared to that in control, untreated corneas on day 14 post neovascularization induction (mean ± standard error of the mean, n = 3, statistical significance is indicated as follows: *P = 0.016, vs. control). ns no significant difference.