Figure 2: Heterochronic blood exchange reduces the proliferative potential of old neural stem cells.

The effects of isochronic and heterochronic blood exchange on SGZ neurogenesis were determined in animals from Fig. 1, with and without muscle injury. (a) Brains from YY, YO, OY and OO mice that had muscle injury were frozen and sectioned at 25 μm. Cryo-sections were immunostained for the proliferation marker Ki67 (red) and counterstained for nuclei (Hoechst, blue). Shown are representative images of the dentate gyrus (DG). Scale bar, 100 μm. (b) Proliferating (Ki67+/Hoechst+) cells in SGZ were quantified in serial 25-μm cryo-sections for each experimental cohort spanning the DG. Ki67+/Hoechst+ cells were clearly identifiable as seen in the enlarged inset image from a, outlined in white. Ki67+ SGZ cells decrease with age and also a decrease is seen in heterochronic young brains compared with the isochronic young controls. At the same time, there in no enhancement of SGZ cell proliferation occurring in heterochronic old brains as compared with the isochronic old controls. T-test **P<0.005. N=4, YY to YO (P=0.0034), OY (P=0.0002) and OO (P=0.000159), YO to OY (P=0.0047), and OO (P=0.0032). (c) Ki67 largely colocalized with Sox2 by immunodetection in of brains from YY, YO, OY and OO mice with and without the experimental muscle injury. Hoechst (blue) was used to label all nuclei. Representative image of YY cohort with muscle injury is shown. Scale bar, 100 μm. (d) Quantification of Ki67+/Sox2+/Hoechst+ cells per SGZ was performed for all blood exchange cohorts above; shown are the relative numbers compared with the in YY cohort without injury that is set to 100%. Similarly to SGZ Ki67+/Hoechst+ cells, the numbers of SGZ proliferating Sox2+ cells diminished with age and significantly decreased after exposure of young cells to old blood by a single procedure of exchange. Notably, neurogenesis was significantly attenuated in YO mice with muscle injury as compared with the uninjured animals of the same cohort (P=0.001). No significant positive effects on old Ki67+/Sox2+/Hoechst+ cells were detected with or without muscle injury. n=4, *P<0.05, **P<0.005.