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. 2023 Feb 2;120(6):e2218915120. doi: 10.1073/pnas.2218915120

Fig. 1.

Fig. 1.

OPN deletion diminishes AD pathology and rescues cognitive deficits in 5XFAD mice. (A) Flow cytometric analysis of OPN expression in CD11c+ and CD11c− microglia from 5XFAD mice (n=6) at different disease stages. ****P < 0.0001 by two-way ANOVA with Bonferroni’s multiple comparisons test. (B) Flow cytometric analysis showing the increased percentage in CD11c+ OPN+ microglia from 5XFAD mice compared with age-matched WT mice during disease progression (n=5 to 6). ****P < 0.0001, **P < 0.01 by two-way ANOVA with Bonferroni’s multiple comparisons test. (C) Ex vivo analysis of microglial tumor necrosis factor-alpha (TNF-α) expression in WT, 5XFAD, and OPN-KO.5XFAD mice (n=6) during disease progression according to flow cytometry. *: WT vs. 5XFAD, ***P < 0.001, ****P < 0.0001; #: 5XFAD vs. OPN-KO.5XFAD, ####P < 0.0001, ns: not significant by two-way ANOVA with Bonferroni’s multiple comparisons test. (D) Representative immunofluorescent images of brain sections from 9-mo-old 5XFAD mice and OPN-KO.5XFAD mice stained with 6E10 (red) and Thioflavin-S (green) displaying different forms of Aβ plaques. Total plaques were defined as 6E10+. White arrows indicate diffuse plaques (6E10+ Thio-S−), while compact plaques (6E10+ Thio-S+) are indicated by yellow arrows. (Scale bar, 100 μm.) (E–G) Quantification of total area (6E10+), compact area (6E10+Thio-S+), and diffuse area (6E10+Thio-S−) of Aβ plaques in 3-, 6-, and 9-mo-old 5XFAD mice and OPN-KO.5XFAD mice (n= 54 fields from 3 mice/group). ***P < 0.001, **P < 0.01, *P < 0.05, ns: not significant by two-way ANOVA with Bonferroni’s multiple comparisons test. (H) The compactness index of Aβ plaques in 3-, 6-, and 9-mo-old 5XFAD mice and OPN-KO.5XFAD mice. The compactness index was calculated as 6E10+Thio-S+ area/ 6E10+ area (n =54 fields from three mice/group). **P < 0.01, ns: not significant by two-way ANOVA with Bonferroni’s multiple comparisons test. (I and J) Representative immunofluorescent images and quantification of dystrophic neurites (labeled with N terminus APP) per plaque (labeled with Thioflavin-S) in brain sections of 5XFAD and OPN-KO.5XFAD mice. The number of APP+ dystrophic neurites was quantified within 25 µm area of each plaque (n=30 to 40 plaques from three mice per group). ****P < 0.0001, ***P < 0.001, ns: not significant by two-way ANOVA with Bonferroni’s multiple comparisons test. (Scale bar, 200 μm.) (K) Spatial learning and memory (acquisition trials) and cognitive flexibility (reversal trials) were assessed by water T maze in 9-mo-old 5XFAD and OPN-KO.5XFAD mice. Age-matched WT mice were included as controls (n=3 to 5, #: WT vs. 5XFAD, #P < 0.05, ##P < 0.01, ###P < 0.001; *: 5XFAD vs. OPN-KO.5XFAD, **P < 0.01). Statistical analysis was performed by two-way ANOVA with Bonferroni’s multiple comparisons test. All data are presented as mean ± SEM.