Disruption of AQP4 polarity in the retina of AD patient, 5×FAD mice, and aged mice. (A) Immunofluorescence images of the retina and optic nerve sections from 10-mo-old 5×FAD mice, showing staining of 6E10 (gray) for Aβ, IB4 (red) for blood vessels, GFAP (green) for astrocytes, and AQP4 (purple) for AQP4. The circular dashed outline indicated the location of the optic nerve head, where the expression of AQP4 was absent. (B) Representative images of immunofluorescence staining for 6E10, GFAP, and AQP4 in cross-sections of the optic nerve from WT and 5×FAD mice. (C) Representative immunofluorescence images of 6E10, GFAP, AQP4, and IB4 in the retina of WT, 5×FAD, and aged mice (18 mo). (D) Statistical analysis of AQP4 fluorescence intensity along blood vessels (yellow dashed lines), indicating more dispersed AQP4 localization in 5×FAD mice and aged mice in comparison to WT mice. (E) Box plot of the polarization index, where the polarization index was the peak fluorescence value of blood vessels minus the baseline fluorescence value, all values were normalized to the peak value (n = 6). (F and G) Representative immunofluorescence images of GFAP and AQP4 in the optic nerve and retina of AD donors and healthy controls. Dashed lines and arrowheads indicated the location of blood vessels (identified through AQP4 channel Z-axis imaging), revealing abnormal distribution of AQP4 in the optic nerve and retina of AD patients, indicative of polarity disruption. Representative of two independent experiments. All data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Tukey’s post hoc test (E).