Fig. 2. Short-term effects of young sEV injection on memory ability, endurance performance and senescent phenotypes of aged mice.
Aged male mice (21 months) were intravenously injected with 200 μl of PBS or young sEVs (1.80 μg of total protein per microliter, from 2-month-old male mice) seven times over 2 weeks, and then the two groups of aged mice were either assessed by a series of behavioral paradigms to determine memory ability and endurance performance or assessed by multiple senescence biomarkers to determine senescent phenotypes. Young male mice (2 months) were simultaneously injected with PBS to serve as a control group. a, The escape latency of each group in the training phase of Morris water maze test (n = 6). Blue and red asterisks indicate statistically significant differences between PBS → Young versus PBS → Aged and between Young sEV → Aged versus PBS → Aged, respectively. b,c, Time spent in the target quadrant and the number of platform crossings by each group in the probe trial of Morris water maze test (n = 6). d, Freezing levels of each group in the contextual fear conditioning test (n = 6). e, Running time to exhaustion for each group in the treadmill running test (n = 10). f, Representative images of SA-β-gal staining in tissue sections. Scale bar, 100 μm. g, Western blot analysis of p21 and p16 protein levels in various tissues. Representative western blots are shown. h, Immunohistochemistry staining of Ki67 in the hippocampus sections. Representative immunohistochemistry staining images are shown. Scale bar, 100 μm. Ki-67-positive staining is identified by the presence of brown nuclear staining (green arrows) in cells. i–j, Relative ROS and AGE levels in various tissues (n = 4). k, Representative images of aldehyde fuchsine staining for lipofuscin in tissue sections. Aldehyde fuchsine-positive staining appears dark purple in the sections. Scale bar, 100 μm. Each experiment was independently repeated four times with similar results in f and k. Significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test in a–e, i and j. *P < 0.05, **P < 0.01 and ***P < 0.005.