Figure 3. Neurogenesis of aged mice is enhanced by one procedure of neutral blood exchange. (A) Immunofluorescence was performed to assay for proliferative Ki67-positive cells in the subgranular zone (SGZ) of the dentate gyrus (DG). Representative images of Ki67(red)+/Hoechst (blue)+ cells in the DG are shown for OO and ONBE mice. (B) Quantification of the number of Ki67+/Hoechst+ cells per SGZ of the DG (extrapolated from serial sections that span the entire hippocampus). ONBE mice have a ~8-fold increase in the number of these cells when compared to OO (****p-value = 0.0000145). The number of these proliferating neural precursor cells in the SGZ of YY mice is not significantly different from that of ONBE mice (N.S. p-value = 0.15235). A trend for ~44% increase in YNBE mice as compared to the YY mice, is not statistically significant (N.S. = 0.20123). Isotype-matched IgG negative control confirms low non-specific fluorescence. N=4 for YY and OO, N=6 for YNBE and N=7 for ONBE. Scale bar is 50-micron. Representative images for YY versus YNBE cohorts are shown in Supplementary Figure 6. These data demonstrate that hippocampal neurogenesis improves in old mice after just one NBE, e.g. without young blood or its fractions, and that young mice do not decline in this parameter when their blood plasma is diluted through the NBE.