Cellular distribution of AQP4 in the retina and optic nerve. (A) UMAP cell type distribution of retinal cells in 10-mo-old WT and 5×FAD mice based on scRNA-seq data. (B) Identification of 12 cell groups by UMAP clustering. (C) Expression levels of Aqp4 in the cell clustering plot. (D) The dot plot illustrated the percentage and expression levels of Aqp4 across various cell populations. The data was obtained through single-cell analysis of the retinas from 5xFAD mice. (E and F) Representative immunofluorescence images of GFAP (green), AQP4 (purple), and IB4 (red) in the retina and optic nerve of 3-mo-old WT mice. (G) A schematic diagram depicting the relationship between astrocytes, AQP4, and vascular positioning, illustrating the localization of AQP4 on the endfeet of astrocytes, encompassing the blood vessels. (H) Bioluminescence imaging showing Aβ transportation fluorescence intensity in the brain and optic nerve of WT and AQP4 KO mice 30 min after injection. (I and J) Immunofluorescence staining of the optic nerve and eye sections of WT and AQP4 KO mice showing significantly reduced Aβ fluorescence intensity, with subregion statistical analysis in the intraocular, intraorbital, intracanalicular, and intracranial segments of the optic nerve (n = 4). (K–N) Representative confocal images and quantitative analysis showing the transportation of Aβ in the retinas and optic nerve of WT and AQP4 KO mice 30 min after injection (n = 8). Data are representative of two independent experiments. All data are presented as mean ± SEM. Statistical significance was evaluated using two-tailed unpaired t test (L and N), two-way ANOVA with post hoc Tukey test (J).