(A-C) Confocal images showing the effects of parabiosis on proliferative (A), neural stem (B) and progenitor cells (C) in the SVZ of isochronic and heterochronic mice. Scale bar: 50μm. (D-E) Quantification of neural stem (D) and progenitor cell (E) populations of the above images (n=9 animals for each experimental group, *p<0.05, **p<0.01, ***p<0.001). Data shown as mean±S.E.M; statistical analysis by ANOVA.

(A) Representative images of olfactory bulbs showing newborn neurons in isochronic and heterochronic parabionts. Scale bar: 100μm. Circles in higher magnification inserts indicate BrdU⁺/NeuN⁺ double-positive cells. (B, C) Quantification of neurogenesis in the olfactory bulbs of old (B) and young (C) parabionts. (n=4, *p<0.05). (D) Measurement of the exploratory time during the olfactory sensitivity assay (n=3). Data shown as mean±S.E.M; statistical analysis by t-test. “n=” indicates the number of animals for each experimental group.

(A) Confocal images of the SVZ area showing the changes in vasculature after heterochronic parabiosis. Scale bar: 50μm. (B) Measurement of blood vessel volume in isochronic and heterochronic parabionts (n_old= 9, n_young=6). (C, D) Measurements of cerebral blood flow in the SVZ region of the parabionts: Iso-O versus Iso-Y (C) or Het-O (D) mice (n=4). (E) Perfusion MRI images of the brain. “V” indicates the ventricles. Data shown as mean±S.E.M; statistical analysis by ANOVA in (B) and t-test in (C,D); *p<0.05, **p<0.01, ***p<0.001. “n=” indicates the number of animals for each experimental group.

(A, B) Confocal images of coronal SVZ sections showing that 22-month old mice injected with rGDF11 for 4 weeks have enhanced vascularization (A) as well as increased Sox2⁺ neural stem cell populations (B) compared to control. (C) Measurement of blood vessel volume in rGDF11-treated and control mice (n=9). (D) Quantification of Sox2⁺ cells in the SVZ area (n=6); “n=” indicates the number of animals for each experimental group. (E, F) Quantification (E) and representative images (F) of the percentage of phospho-SMAD2/3⁺ cells in primary brain capillary endothelial cell cultures treated with either GDF11 (40ng/ml) or TFG-β (10ng/ml) in the presence of sodium orthovanadate used to inhibit phosphatase activity for 30 minutes (n=7). Scale bar: 100 μm. Data shown as mean±S.E.M; statistical analysis by t-test, between each experimental condition and the untreated control, *p<0.05, **p<0.01, ***p<0.001.