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. 2021 Jan 6;9:6. doi: 10.1186/s40478-020-01102-5

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© The Author(s) 2021

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Locally reactivated microglia and macrogliosis in the retinas of aged App^NL-G-F mice. a, b Quantification of microglial density (a) and soma area (b) on Iba1-labeled retinal wholemounts of 3- to 18-month-old App^NL-G-F and WT mice reveals no differences between genotypes when analyzed for the entire retina. An effect of aging was observed for microglia density (Two-way ANOVA, F_4,33 = 2.976, p = 0.0334); n = 3–6. c Morphometric analysis of plaque-associated microglia versus microglia distant from Aβ plaques in 18-month-old App^NL-G-F x CX₃CR-1^GFP/+ retinas reveals that plaque-associated microglia have a larger soma, which is indicative of their activation. Unpaired two-tailed t-test (t₆₃ = 2.614, p = 0.0112); n = 2–40 cells from 6 mice. d–g Representative images of immunostaining for Aβ (6E10) on retinal wholemounts of 18-month-old CX3CR-1GFP/⁺ (d–e) and App^NL-G-F x CX₃CR-1^GFP/+ mice (f–g), illustrating that green fluorescent microglia surrounding Aβ plaques display morphological alterations typical for reactive microglia, with thicker and less ramified processes and a larger soma. h Counting the number of GFAP⁺ radial fibers on retinal sections of App^NL-G-F and WT mice shows that macrogliosis increases with age, and significantly differs between genotypes at 18 months. Two-way ANOVA with Sidak’s multiple comparisons test (F_4,50 = 34.02 for the effect of age, F_1,50 = 4.411 for the effect of genotype, p = 0.0432); n = 5–8. i–j Representative images of a GFAP immunostaining on retinal cross-sections of 18-month-old WT (i) and App^NL-G-F (j) mice. k Quantification of the S100B immunopositive area on retinal sections of 18-month-old App^NL-G-F and WT mice shows astrogliosis in the App^NL-G-F retina. Unpaired two-tailed t-test (t₁₂ = 3.358, p = 0.0057); n = 7. (l-m) Representative images of the S100B immunostaining on WT (l) and App^NL-G-F (m) retinas. Scalebar (d–g): 20 µm, scalebar (i, j, l, m): 50 µm. Data are shown as mean ± SEM. GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer