Blood exchange between mice

To develop a better-controlled experimental system where only blood is exchanged, we developed a small animal blood exchange device, which operates similarly to a previously published circulatory cell-scrubbing device16. It consists of a computer controlled microfluidic peristaltic pump circuit and computer controlled extracorporeal blood manipulation system (Supplementary Fig. 1 and Supplementary Movie 1). A major design constraint of small animal blood manipulation that we have addressed is the low volume of total blood that can be removed from a small animal at once. It is not prudent to remove more than 10% of an animal’s blood at once, and mice contain 5–8% (w/w) blood. This translates to ∼150 μl of blood that can be removed from a 30 g mouse. Small volume microfluidic blood manipulation systems exist for lab on a chip and other diagnostic applications17, however, our device is the first to allow for continuous blood flow as required for larger scale experimental applications in live mice (Supplementary Movie 1). In contrast to parabiosis, where joint circulation is established in ∼7–10 days through growth of skin capillaries, blood exchange is instantaneous and well controlled by the device. The procedure is less invasive than parabiosis as it does not involve as much invasive surgery, only the catheterization of a jugular vein (Supplementary Fig. 1). The exchanged blood is visualized in the tubing and the exchange volumes are easily measured.

Using this small animal device we have exchanged blood between 4 pairs of young to old mice, using 4 pairs of isochronic, young to young exchanges and 4 pairs of isochronic, old to old exchanges, as controls. Virtually 100% animal viability is maintained when two series of 15 exchanges of 150 μl of blood per series are performed over the course of 24 h, establishing a blood equilibrium similar to parabiosis between the pairs in a fraction of the time. This regiment was employed for the studies and will be referred to as a single procedure of blood exchange thereafter.

Young blood improves old muscle regeneration

One day after the blood exchange, tibiallis anterior (TA) hind leg muscles of all mice were injured by cardiotoxin (CTX) and 5 days later this muscle, as well as non-injured livers and brains were isolated postmortem. The efficiency of muscle regeneration was determined in a manner identical to the heterochronic parabiosis studies3,4. TA muscles were injected with CTX 1 (CTX, Sigma, 0.1 mg ml−1). Ten micrometre muscle cryo-sections were prepared from TA muscle, which was isolated at 5 days post CTX injury. These cryo-sections were analysed by haematoxylin and eosin (H&E), staining and by eMyHC immuno-detection followed by microscopy and quantification of the percent of de- novo small eMyHC+ myofibers with centrally located nuclei that robustly appear in young, but are less in the old injured muscle, which typically shows more inflammation and incipient fibrosis. As shown in Fig. 1a–c, a single procedure of heterochronic blood exchange significantly improved the regeneration of old muscle after experimental injury (both when assayed by H&E staining or eMyHC immunofluorescence), while there was no statistically significant decline in the robust regeneration of the young muscle. The numbers of de-novo myofibers were slightly higher for all cohorts when counted by the more robust eMyHC immunofluorescence method as compared with H&E, but the relative differences between the YY, YO, OY and OO cohorts remained virtually the same. We also examined de-novo fibre size (minimal Feret diameter, as published6,18), as expected the regenerating fibre size declined with age but interestingly was increased in old mice transfused with young blood, while remaining unchanged in young mice transfused with old blood (Supplementary Fig. 2). Importantly, the degree of fibrosis (a culprit of muscle aging) was also reduced by the young blood exchange and was not increased in the young mice transfused with old blood (Fig. 1d).

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 images15. 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. Full size image

Old blood inhibits young performance

To assay functional performance, the blood exchange studies were repeated without muscle injury, and a four-limb hanging test was applied to the isochronic and heterochronic cohorts 6 days after the blood exchange (for example, the same time frame when muscle repair was studied in the injured mice). In this test animal strength, endurance and learning are all measured: mice hang inverted from a 1 cm mesh screen over soft bedding, and the time until the mouse drops is recorded over three trials, and the maximal time multiplied by the weight is expressed as hanging index19. Interestingly, exchange with old blood markedly diminished the maximal hanging index of young animals (3 out of 4 mice) but there was no increase in this parameter for the old mice transfused with young blood (Fig. 1e). Of note, the initial hanging indices in training session were not significantly different between the young and old mice, but young animals transfused with young blood became statistically better than the old mice after the training session, while young mice transfused with old blood remained statistically undistinguishable from the old cohorts (Fig. 1e).

These data extrapolate the findings obtained with heterochronic parabiosis4, and establish that the beneficial effects of young blood for the regeneration of old muscle take place right away and without the contribution of young organ systems or altered activity levels between the isochronic and heterochronic pairs. Moreover, while one exchange of young blood improves muscle regeneration in old animals, it does not improve the functional performance as measured by the hanging test, while in young animals the functional performance declines very rapidly after one exchange of old blood.

Hippocampal neurogenesis responds to old blood and injury

The efficiency of hippocampal neurogenesis was determined similarly to refs 8 and (ref. 3). Mouse brains were collected and sectioned at 25 μm using a cryostat. Sections were fixed in 4% paraformaldehyde and immunostained with antibodies to Ki67, using Hoechst co-stain to detect all nuclei. The numbers of Ki67+ proliferating subgranular zone (SGZ) cells were quantified throughout the entire dentate gyrus (DG) of the hippocampus, as in ref. 8, where based on co-immunodetection of Sox-2, these Ki67+ SGZ cells are virtually all-neural stem cells. As shown in Fig. 2 and Supplementary Fig. 3, based on either SGZ Ki67+ or SGZ Ki67+/Sox2+ cell numbers, one exchange of heterochronic blood severely decreased hippocampal neurogenesis in young mice, and surprisingly, there was no significant positive effect in the old mice that had been exchanged with young blood. Of note, these were the same old animals that showed improvement in muscle regeneration and hepatogenesis (see below).

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. Full size image

In the some of published heterochronic parabiosis work, muscle was injured in animals that were assayed for hippocampal neurogenesis3,4 and similar effects on neurogenesis were later seen in the absence of muscle injury11. We compared the influences of heterochronic blood exchange on neurogenesis in the presence and absence of muscle injury, to assay for potential changes in the brain that might be caused by additional stress and peripheral inflammation. While a statistically significant inhibition of young neurogenesis by old blood persisted, its magnitude was less in the absence of muscle injury (Fig. 2c). There was no enhancement of old neurogenesis by the young blood, with or without muscle injury (Fig. 2 and Supplementary Fig. 3). These data confirm the negative effects of the old systemic milieu on neurogenesis in young hippocampi and identify that such inhibition is very rapid and is uncoupled from influences of old organ systems, pheromones and changes in the environmental stimulation or exercise. Muscle injury after blood exchange might add to the magnitude of the negative effects of old blood on young neurogenesis, and even without muscle injury, young hippocampal neurogenesis quickly declines after one old blood exchange.

Liver responds to heterochronic blood and muscle injury

We next assayed the efficiency of ongoing hepatogenesis in mice that were and were not experimentally injured in their muscle, as in ref. 4 by co-immunodetection of a proliferation marker, Ki67 and a hepatocyte marker albumin, in 10 μm liver cryosections. As shown in Fig. 3a,b, the numbers of proliferating old hepatocytes were increased after a single procedure of heterochronic blood exchange, while the numbers of Ki67+albumin+ young hepatocytes declined on transfusion of old blood. The ongoing hepatogenesis in animals that did not experience muscle injury was much less prominent even in young mice, suggesting that hepatogenesis increases during muscle repair, but still the heterochronic effects of a single blood exchange were observed (Fig. 3a,b).

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. Full size image

As previously reported4 there were many fibrotic areas in old livers, which at times had proliferating clusters of small albumin negative cells (Fig. 3c,d). Such areas were not present in young livers, and very interestingly the numbers of these fibrotic proliferative clusters declined in the livers of old animals that were exchanged with young blood, regardless of whether muscle was or was not injured (Fig. 3c,d).

As another metric for improvement in liver health we assayed liver tissue adiposity by Oil Red staining on 10 μm cryosections from the above-described animals. Old livers were markedly more positive for Oil Red, as compared with young and interestingly, transfusion with young blood somewhat reduced old liver adiposity, while there was no significant increase in young liver adiposity (Fig. 3e,f and Supplementary Fig. 4). These results demonstrate that heterochronic blood exchange and heterochronic parabiosis yield similar enhancement of old hepatogenesis and decline of young hepatogenesis; and suggest that muscle damage enhances ongoing hepatogenesis in young mice. Additionally, the fibrotic regions and adiposity rapidly decline in old livers after the exposure to young blood. Such effects manifest after just a single procedure of blood exchange and in the absence of the influences from heterochronic organ systems.

B2M and TGF-beta as mechanisms are tissue specific

To start looking into the molecular mechanisms that are responsible for these rapid influences of circulation on tissue repair and maintenance, we studied the levels of B2M. B2M is the invariant chain of MHC class I that becomes elevated with inflammation and based on current reports is over-pronounced in old muscle and brain, as compared with young8,14,20. B2M levels were assayed by immunofluorescence in tissue cryosections (Fig. 4a,b) and by western blotting (Supplementary Fig. 5) in the young and old mice that underwent isochronic versus heterochronic blood exchange as described above. For tissues derived from mice injured with CTX in their TAs, the immunofluorescence on muscle and brain tissue cryosections demonstrated that exchange with old blood rapidly (within 6 days), elevated the B2M levels in young muscle located outside of the CTX injury, and in the SGZ of the young hippocampus, (Fig. 4a,b). Interestingly, the B2M remained high in the old hippocampi of the heterochronically exchanged animals (Fig. 4a,b). Furthermore, for muscle these age-specific differences in B2M were less pronounced between YY and YO cohorts and were undetectable between the OO and OY cohorts when immunofluorescence was performed at the sites of CTX injury—muscle regeneration (Supplementary Fig. 5) in agreement with the previous findings that inflammation overlaps in space and time with muscle repair and that some degree of transient inflammation is needed for successful myogenesis7,21.

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. Full size image

Western blotting confirmed the results obtained by the immunofluorescence and demonstrated that B2M levels were increased with age in muscle and in brains, while there was no detectable age-specific increase of B2M in livers (Supplementary Fig. 5 and Supplementary Fig. 9). The regional tissue differences in B2M levels are not resolvable by the western analyses, thus the differences between the cohorts were less drastic, but in general agreement with those seen by the immunofluorescence.

The age-elevated increase of B2M was less noticeable in the muscle and brains of the animals that did not experience experimental muscle injury; for livers there was again no detectable age-specific change (Supplementary Fig. 5). By immunofluorescence, the regional (DG) age-specific difference in B2M persisted in brains of young versus old mice that did not experience muscle injury; and no significant modulation of B2M were detected between YY versus YO or OO versus OY cohorts (Supplementary Fig. 5).

The simplest explanation to these changes in tissue B2M would be an age-imposed increase in circulating B2M, which was suggested by the earlier reports8,14,20. Thus, we performed B2M western blotting analysis for circulating B2M in five young and five old blood serum samples. Interestingly, while there was a mild trend (∼10%) for the age-specific increase in systemic B2M, it was without statistical significance (Fig. 4c). And similar results were obtained with and without muscle injury (Supplementary Fig. 6 and Supplementary Fig. 9). Therefore, it is unlikely that systemic B2M accumulates in young tissues after heterochronic exchange, because there is no statistically significant age-imposed increase of B2M in circulation, but rapid and significant changes in B2M manifest as the result of heterochronic blood exchange and moreover, in specific regions of muscle (outside of injury) and brain (DG).

Transforming growth factor beta1 (TGF- beta1) becomes up regulated with age systemically and locally, and experimental attenuation of the age-increased TGF-beta/pSmad3 reduces B2M in muscle and brain1,8,22. However, while TGF-beta1 was expectedly elevated with age (myostatin and follistatin remained similar), a single procedure of heterochronic blood exchange did not significantly change the TGF-beta1/pSmad3 intensity in either young or old muscle (Supplementary Fig. 7). These data suggest that other than TGF-beta1/pSmad3 determinant(s) must account for the induction of B2M by old blood in young animals.

These results demonstrate an age-specific increase of B2M in muscle, brain, but not liver and blood serum, and establish that exchange of young mice with old blood elevates B2M in muscle and brain in a regional manner; however, no decline of B2M is observed in hippocampi or the entire brain of old mice exchanged with young blood. Moreover, the tissue increase of B2M after heterochronic blood exchange does not seem to be caused by age-elevated systemic B2M or TGF-beta1.