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. 2016 Nov 22;7:13363.
doi: 10.1038/ncomms13363.

A Single Heterochronic Blood Exchange Reveals Rapid Inhibition of Multiple Tissues by Old Blood

Free PMC article

A Single Heterochronic Blood Exchange Reveals Rapid Inhibition of Multiple Tissues by Old Blood

Justin Rebo et al. Nat Commun. .
Free PMC article


Heterochronic parabiosis rejuvenates the performance of old tissue stem cells at some expense to the young, but whether this is through shared circulation or shared organs is unclear. Here we show that heterochronic blood exchange between young and old mice without sharing other organs, affects tissues within a few days, and leads to different outcomes than heterochronic parabiosis. Investigating muscle, liver and brain hippocampus, in the presence or absence of muscle injury, we find that, in many cases, the inhibitory effects of old blood are more pronounced than the benefits of young, and that peripheral tissue injury compounds the negative effects. We also explore mechanistic explanations, including the role of B2M and TGF-beta. We conclude that, compared with heterochronic parabiosis, heterochronic blood exchange in small animals is less invasive and enables better-controlled studies with more immediate translation to therapies for humans.


Figure 1
Figure 1. Heterochronic blood exchange effects on muscle regeneration and performance.
One day after blood exchange mice were injured by intramuscular injections of CTX into TA. Five days after injury, TA muscles were isolated, cryo-sectioned and analysed. (a) TA muscles from young mice receiving young blood (YY), young mice receiving old blood (YO), old mice receiving young blood (OY) and old mice receiving old blood (OO) were analysed by haematoxylin and eosin (H&E) staining and immunofluorescence with anti-eMyHC antibody. Representative images show an injury site and nascent de-novo formed eMyHC+ myofibers which are smaller in size with central nuclei than uninjured myofibers. Scale bar, 50 μm for H&E panel and 25 μm for immunofluorescence panel. (b,c) Regeneration indices ±s.e.m. were quantified from H&E images (b) and eMyHC images (c) by counting the number of nascent de-novo formed myofibers and dividing by the total number of nuclei present at the injury/regeneration site. By H&E: *P<0.05 N=4 per group. Significant students t test differences exist between YO and OY (P=0.045), YY and OY (P=0.043), YY and OO (P=0.0004), YO and OO (P=0.0042) and between OY and OO (P=0.015). By eMyHC: *P<0.05, N=4 per group; OY and OO P=0.041, YY and OO P=0.00009, and YO to OO P=0.001. (d) Fibrotic/inflammatory indexes were quantified as total injury area minus regenerated myofiber area, per injury site, using the H&E images. T-test **P<0.005, n=3–4 per group. Muscle from old to old isochronic exchange had diminished regenerative capacity and more fibrosis, as compared with muscle from young to young isochronic exchange. Heterochronic blood exchange significantly improved regeneration of old muscle after experimental injury and reduced fibrosis, but no significant decline in young muscle regeneration was seen. (e) A four-limb hanging test was conducted with isochronically and heterochronically transfused mice that were not injured, before and at 6 days after the blood exchange. Maximal hanging time was multiplied by body weight (hang index). T-test n=4–8, P=0.01 YY post transfusion compared with O training, and YO, OY, OO post-transfusion performance. Y to O training and YO, OY and OO were NS=not statistically different.
Figure 2
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.
Figure 3
Figure 3. Heterochronic blood exchange effects on hepatogenesis and liver fibrosis and adiposity.
(a) Livers from YY, YO, OY and OO mice with and without experimental muscle injury as above were cryo-sectioned at 10 μm and immuno-stained for Ki67 (red), hepatocyte marker albumin (green) and Hoechst (blue). Representative images show YY livers with and without injury. Scale bar, 50 μm. (B&C. Quantification of hepatocyte proliferation was by counting the average number of Ki67+,abumin+,Hoechst+ cells per 10 μm section from multiple sections of each blood exchange cohort. (b) Old hepatocyte showed increased proliferation and young hepatocytes showed less proliferation with heterochronic blood as compared with isochronic blood exchanges in animals with injured muscle (t test P=0.00028). (c) This trend continues without muscle injury, but the total numbers of proliferating hepatocytes decline by twofold, (P=0.02411). *P<0.05; **P<0.005; n=3–5. (d) As previously published, there were fibrotic clusters exclusively in the old livers of small Ki67+ve, albumin negative Ki67+ cells. Scale bar, 50 μm, × 40 magnification. (e,f) Fibrotic index was calculated as the average number of albumin negative proliferative cell clusters per four 10 μm sections. The fibrotic index diminished in old mice exchanged with young blood with muscle injury (e) (t test P=0.048 N=4, *P<0.05) or without (f) (t test P=0.00776. N=3; **P<0.005). (g) Liver adiposity was assayed by Oil Red in 10 μm cryosections. Shown are representative images acquired at × 20 magnification. (h). Liver adiposity (red) was quantified by Image J, dramatically increased with age and was attenuated by young blood in old mice (t test N=3, P=0.022), while adiposity remained unchanged in young mice that were transfused with the old blood (see Supplementary Figure 4). Shown are means±s.e.m. for all histograms.
Figure 4
Figure 4. Levels of B2M in young muscle and brain correlate positively with the heterochronicity of blood exchange.
(a) Muscle cryosections of 10 μm and 25 μm brain-SGZ cryosections of isochronically and heterochronically apheresed mice (that had experimental muscle injury) were immuno-stained for B2M and counter-stained for Hoechst to label all nuclei. Representative images were acquired at the sites of muscle injury (Mu in) outside the injury-repair (Mu out) and at the hippocampi-DG areas (brain DG), scale bar is 50 μm for muscle and liver, and 100 μm for brain. (b) Pixel density of B2M was quantified using Image J from serial cryosections represented in a; and shown are the means and standard errors. In muscle: ***,**P<0.005. Significant differences were observed between YY and YO (P=0.004), OY and OO (0.001), YO and OY (P=0.0007), and YY and OO (P=0.006), N=5–7 per group. In brain: ****P<0.00005. Significant differences were observed between YY and YO (P=0.00001), and YY and OO (P=0.004). (c) Western SDS–polyacrylamide gel electrophoresis was used to analyse B2M levels in one microlitre of cell-free blood serum from 5 young (Y) and 5 old (O) mice. ECL images were quantified by ImageJ and expressed as background-corrected pixel volume. N=5. P=0.5. B2M becomes increased with age in muscle and brain but it is not elevated in old blood serum as compared with young. After heterochronic blood exchange B2M is increased by old blood in young muscle and decreased by young blood in old muscle (regionally, outside of the injury site). B2M is also increased in young hippocampi-DG after exchange with old blood, but B2M is not diminished in the old DG after young blood exchange. Shown are means±s.e.m. for all histograms.

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