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 published⁴, 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.