Fig. 4
Microglia-specific p16ink4a downregulation protects against deterioration of spatial memory and learning in the 5XFAD mouse model of Alzheimer’s disease and promotes the division of microglia, which interferes with cellular senescence. A Schematic of the experimental design for PLGA NP injection into 5XFAD mice. i.t.: intrathecal. B Distance and velocity graphs from the Barnes maze test for mice injected with p16ink4a siRNA NPs or scrambled siRNA NPs. Data are expressed as mean ± SEM (n = 7) of 3 trials and were analyzed by 2-way ANOVA followed by the Bonferroni test for multiple comparisons. n.s.: not significant. C Schematic of the experimental design for the Barnes maze test. D Representative movement paths of mice in the probe test of the Barnes maze test 24 h after the last training day. E The time taken for mice to reach the target in the Barnes maze test over 5 training days. Data are expressed as mean ± SEM (n = 8) and were analyzed by 2-way ANOVA followed by the Bonferroni test for multiple comparisons. ****p < 0.001 F Representative searching tracks of mice injected with p16ink4a NPs or scrambled siRNA NPs in the radial maze test. G In both cortex and hippocampus, Aβ and Iba1 immunostaining paralleled a decrease in the area covered by Aβ plaques in mice injected with p16ink4a siRNA NPs. Scale bar: 100 µm. Data are expressed as mean ± SEM (n = 7) and were analyzed by 1-way ANOVA followed by the Tukey test for multiple comparisons. ****p < 0.001 H The surface area of microglia and the number of microglia around the plaque were measured. White arrows indicate masses of 3 or more microglia around the plaque. ****p < 0.001, versus scrambled siRNA NPs; unpaired Student’s t test. I ELISA of cortex lysate showed decreased of Aβ1-42 of mice infected with PLGA NPs. Each protein lysate samples from cortex were measured based on 50 μg by bradford assay. ****p < 0.001, versus scrambled siRNA NPs; unpaired Student’s t test. J Western blot of p-Rb, Rb, cyclin D1, and cyclin B1 expression in cortex tissue of mice injected with PLGA NPs. β-Actin (ACTB) was used as a protein loading control. K Quantification of the protein levels in (J) after injection of p16ink4a siRNA PLGA NPs in 5XFAD mice brain tissue. **p < 0.01 and ****p < 0.001, versus scrambled siRNA PLGA NPs; unpaired Student’s t test