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Microglia turnover with aging and in an Alzheimer's model via long-term in vivo single-cell imaging

Abstract

To clarify the role of microglia in brain homeostasis and disease, an understanding of their maintenance, proliferation and turnover is essential. The lifespan of brain microglia, however, remains uncertain, and reflects confounding factors in earlier assessments that were largely indirect. We genetically labeled single resident microglia in living mice and then used multiphoton microscopy to monitor these cells over time. Under homeostatic conditions, we found that neocortical resident microglia were long-lived, with a median lifetime of well over 15 months; thus, approximately half of these cells survive the entire mouse lifespan. While proliferation of resident neocortical microglia under homeostatic conditions was low, microglial proliferation in a mouse model of Alzheimer's β-amyloidosis was increased threefold. The persistence of individual microglia throughout the mouse lifespan provides an explanation for how microglial priming early in life can induce lasting functional changes and how microglial senescence may contribute to age-related neurodegenerative diseases.

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Figure 1: Visualization and long-term in vivo imaging of individual microglia in the mouse brain.
Figure 2: Estimated average lifetime of a microglial cell in the mouse neocortex.
Figure 3: New microglia appear in close proximity to existing microglial cells.
Figure 4: Microglial turnover rate in the neocortex of a mouse model of cerebral β-amyloidosis.

References

  1. 1

    Crotti, A. & Ransohoff, R.M. Microglial physiology and pathophysiology: insights from genome-wide transcriptional profiling. Immunity 44, 505–515 (2016).

    CAS  Article  Google Scholar 

  2. 2

    Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010).

    CAS  Article  Google Scholar 

  3. 3

    Sheng, J., Ruedl, C. & Karjalainen, K. Most tissue-resident macrophages except microglia are derived from fetal hematopoietic stem cells. Immunity 43, 382–393 (2015).

    CAS  Article  Google Scholar 

  4. 4

    Ajami, B., Bennett, J.L., Krieger, C., Tetzlaff, W. & Rossi, F.M. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat. Neurosci. 10, 1538–1543 (2007).

    CAS  Article  Google Scholar 

  5. 5

    Hashimoto, D. et al. Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity 38, 792–804 (2013).

    CAS  Article  Google Scholar 

  6. 6

    Pelvig, D.P., Pakkenberg, H., Stark, A.K. & Pakkenberg, B. Neocortical glial cell numbers in human brains. Neurobiol. Aging 29, 1754–1762 (2008).

    CAS  Article  Google Scholar 

  7. 7

    Hefendehl, J.K. et al. Homeostatic and injury-induced microglia behavior in the aging brain. Aging Cell 13, 60–69 (2014).

    CAS  Article  Google Scholar 

  8. 8

    Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Prinz, M. & Priller, J. Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease. Nat. Rev. Neurosci. 15, 300–312 (2014).

    CAS  Article  Google Scholar 

  10. 10

    Ransohoff, R.M. How neuroinflammation contributes to neurodegeneration. Science 353, 777–783 (2016).

    CAS  Article  Google Scholar 

  11. 11

    Sierra, A. et al. The “big-bang” for modern glial biology: translation and comments on Pío del Río-Hortega 1919 series of papers on microglia. Glia 64, 1801–1840 (2016).

    Article  Google Scholar 

  12. 12

    Gómez-Nicola, D., Fransen, N.L., Suzzi, S. & Perry, V.H. Regulation of microglial proliferation during chronic neurodegeneration. J. Neurosci. 33, 2481–2493 (2013).

    Article  Google Scholar 

  13. 13

    Graeber, M.B. et al. The microglia/macrophage response in the neonatal rat facial nucleus following axotomy. Brain Res. 813, 241–253 (1998).

    CAS  Article  Google Scholar 

  14. 14

    Lawson, L.J., Perry, V.H. & Gordon, S. Turnover of resident microglia in the normal adult mouse brain. Neuroscience 48, 405–415 (1992).

    CAS  Article  Google Scholar 

  15. 15

    Askew, K. et al. Coupled proliferation and apoptosis maintain the rapid turnover of microglia in the adult brain. Cell Rep. 18, 391–405 (2017).

    CAS  Article  Google Scholar 

  16. 16

    Tay, T.L. et al. A new fate mapping system reveals context-dependent random or clonal expansion of microglia. Nat. Neurosci. 20, 793–803 (2017).

    CAS  Article  Google Scholar 

  17. 17

    Nowakowski, R.S. & Hayes, N.L. New neurons: extraordinary evidence or extraordinary conclusion? Science 288, 771 (2000).

    CAS  Article  Google Scholar 

  18. 18

    Rakic, P. Neurogenesis in adult primates. Prog. Brain Res. 138, 3–14 (2002).

    CAS  Article  Google Scholar 

  19. 19

    Knuesel, I. et al. Maternal immune activation and abnormal brain development across CNS disorders. Nat. Rev. Neurol. 10, 643–660 (2014).

    CAS  Article  Google Scholar 

  20. 20

    Perry, V.H. & Holmes, C. Microglial priming in neurodegenerative disease. Nat. Rev. Neurol. 10, 217–224 (2014).

    CAS  Article  Google Scholar 

  21. 21

    Heppner, F.L., Ransohoff, R.M. & Becher, B. Immune attack: the role of inflammation in Alzheimer disease. Nat. Rev. Neurosci. 16, 358–372 (2015).

    CAS  Article  Google Scholar 

  22. 22

    Streit, W.J., Xue, Q.S., Tischer, J. & Bechmann, I. Microglial pathology. Acta Neuropathol. Commun. 2, 142 (2014).

    Article  Google Scholar 

  23. 23

    Hefendehl, J.K. et al. Repeatable target localization for long-term in vivo imaging of mice with 2-photon microscopy. J. Neurosci. Methods 205, 357–363 (2012).

    Article  Google Scholar 

  24. 24

    Radde, R. et al. Aβ42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology. EMBO Rep. 7, 940–946 (2006).

    CAS  Article  Google Scholar 

  25. 25

    Jucker, M. & Ingram, D.K. Murine models of brain aging and age-related neurodegenerative diseases. Behav. Brain Res. 85, 1–26 (1997).

    CAS  Article  Google Scholar 

  26. 26

    Bruttger, J. et al. Genetic cell ablation reveals clusters of local self-renewing microglia in the mammalian central nervous system. Immunity 43, 92–106 (2015).

    CAS  Article  Google Scholar 

  27. 27

    Elmore, M.R. et al. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron 82, 380–397 (2014).

    CAS  Article  Google Scholar 

  28. 28

    Grabert, K. et al. Microglial brain region-dependent diversity and selective regional sensitivities to aging. Nat. Neurosci. 19, 504–516 (2016).

    CAS  Article  Google Scholar 

  29. 29

    Moore, D.L. & Jessberger, S. Creating age asymmetry: consequences of inheriting damaged goods in mammalian cells. Trends Cell Biol. 27, 82–92 (2017).

    Article  Google Scholar 

  30. 30

    Streit, W.J., Sammons, N.W., Kuhns, A.J. & Sparks, D.L. Dystrophic microglia in the aging human brain. Glia 45, 208–212 (2004).

    Article  Google Scholar 

  31. 31

    Frisén, J. Neurogenesis and gliogenesis in nervous system plasticity and repair. Annu. Rev. Cell Dev. Biol. 32, 127–141 (2016).

    Article  Google Scholar 

  32. 32

    Hoeijmakers, L., Heinen, Y., van Dam, A.M., Lucassen, P.J. & Korosi, A. Microglial priming and Alzheimer's disease: a possible role for (early) immune challenges and epigenetics? Front. Hum. Neurosci. 10, 398 (2016).

    Article  Google Scholar 

  33. 33

    Muzumdar, M.D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

    CAS  Article  Google Scholar 

  34. 34

    Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    CAS  Article  Google Scholar 

  35. 35

    Hirasawa, T. et al. Visualization of microglia in living tissues using Iba1-EGFP transgenic mice. J. Neurosci. Res. 81, 357–362 (2005).

    CAS  Article  Google Scholar 

  36. 36

    Klingstedt, T. et al. Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates. Org. Biomol. Chem. 9, 8356–8370 (2011).

    CAS  Article  Google Scholar 

  37. 37

    Wegenast-Braun, B.M. et al. Spectral discrimination of cerebral amyloid lesions after peripheral application of luminescent conjugated oligothiophenes. Am. J. Pathol. 181, 1953–1960 (2012).

    CAS  Article  Google Scholar 

  38. 38

    Mahler, J. Ex vivo and in vivo analysis of protein aggregates in transgenic mouse models of cerebral amyloidosis using luminescent conjugated oligothiophenes. MSc thesis, University of Tübingen (2012).

  39. 39

    Collet, D. Modelling Survival Data in Medical Research 2nd edn. (Chapman & Hall, New York, 2003).

Download references

Acknowledgements

We thank L. Walker, G. Kempermann and M. Staufenbiel, as well as all other laboratory members, for help and comments on the manuscript, and P. Nilsson (Linköping University) for providing the LCO amyloid dye. This work was supported by internal institutional funds from the Ministry of Science, Research and the Arts, Baden-Wuerttemberg (M.J.), by a Ph.D. fellowship of the Studienstiftung des Deutschen Volkes (A.-C.W.), by National Institute of Health grants AG021494 and AG027854 (S.S.S., K.V.) and by the Cure Alzheimer's Fund (S.S.S.).

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P.F., J.K.H., J.J.N., A.S., M.J. designed the study. P.F., C.S., A.-C.W., U.O., B.M.W.-B. and A.S. performed the experimental imaging work. K.V., S.K., M.T., R.F. and S.S.S. generated the mouse models. A.S. and P.M. did the statistical analysis. The manuscript was prepared by P.F., J.J.N., A.S. and M.J. with the help of all authors.

Corresponding authors

Correspondence to Angelos Skodras or Mathias Jucker.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Genetic labeling of microglia

Male and female 2-3 month-old CreERT2;R26-tdTomato mice or CreERT2;R26-tdTomato;Iba1-eGFP transgenic mice were treated with 1x, 2x (48 h apart), or 5x (each 24 h apart) Tamoxifen (TAM; 100 μg per gram bodyweight) and analyzed 4 weeks later. One hemisphere was used for flow cytometry (only the neocortex was used, see methods for details), the second hemisphere was used for histology. (a) Exemplary gating strategy of an animal injected with 2xTAM. Cells were gated based on forward (FSC) and backward scatter (BSC). FSC-width and height was used to determine single cells. 1.55% of single cells were tdTomato positive, of which the vast majority (approximately 97%) were microglia, identified as the CD11bhigh and CD45low cell population by flow cytometry. tdTomato-positive microglia were not distinguishable from tdTomato-negative microglia in size and granularity, as indicated by their similar position in the FSC/BSC blot (tdTomato-positive cells colour-coded in green) and were distributed across the microglial population based on CD11b and CD45 intensity. (b) Flow cytometric analysis of the neocortex revealed a dose-dependent increase in the induction of tdTomato-positive cells, of which the vast majority were microglia (n=3, 4, 3 mice per group, for 1x, 2x, and 5x TAM, respectively). (c) Histological analysis of tdTomato-positive microglia (arrows) revealed an apparent random distribution throughout the neocortex (shown in the frontal cortex). Morphologically, tdTomato-labeled microglial cells revealed all aspects of mature microglia (insert). Scale bars 100 μm and 10 μm for the insert. Some perivascular macrophages also showed recombination, but were clearly distinguishable from microglia due to their elongated shape and lack of processes. (d) The total number of tdTomato-positive cells per neocortex was estimated and related to the total number of Iba-1-positive microglia in the neocortex (the latter was assessed in Supplementary Figure 4 and the mean number of 880,000 was used for the calculation). In accordance with the flow cytometry analysis, a dose-dependent and comparable increase was found (n=3, 4, 3 mice per group, for 1x, 2x, and 5x TAM, respectively).

Supplementary Figure 2 Long-term repetitive in vivo imaging does not cause microglial activation

(a) There was no noticeable activation of microglial cells in any of the in vivo imaged areas. Shown are in vivo images (z-projections of imaged volumes) of a triple-transgenic CD11b-CreERT2;R26-tdTomato;Iba1-eGFP mouse after an imaging period of 24 weeks (6 months). Scale bar is 100 μm. (b, c) Consistently, postmortem immunohistochemical analysis of the triple-transgenic CD11b-CreERT2;R26-tdTomato;Iba1-eGFP animals after the 24 week-imaging period using an Iba1-antibody did not reveal any signs of microglial activation, regardless of whether the cortical tissue was cut horizontally (b) or coronally (c), when compared to a non-imaged control animal of the same age. Blue area indicates the location of the cranial window. Three imaged and three control mice were analysed. Scale bars are 100 μm and 20 μm for the insert.

Supplementary Figure 3 Daily imaging of neocortical microglial cells rules out rapid cell turnover

To study potential rapid replacement of microglial cells that would have been unrecognized in our long-term biweekly imaging protocol (see Fig. 1 and 2), three CD11b-CreERT2;R26-tdTomato;Iba1-eGFP mice (13 months of age; one male, two females) and two CD11b-CreERT2;R26-tdTomato mice (13 months of age; two males) were imaged daily for 10 consecutive days. (a, b) Shown are z-projections of imaged volumes of CD11b-CreERT2;R26-tdTomato;Iba1-eGFP mice where recombined microglia appeared yellow while surrounding microglia were green. An example of a recombined yellow microglial cell with surrounding green cells that are all stable over the imaging period of ten days is shown in (a). An example with a lost green microglial cell on day 5 is shown in (b). Scale bar is 40 μm. Note that in contrast to the long-term biweekly imaging (Fig. 2), the daily imaging also allowed us to track the Iba1-eGFP positive cells (green) since movement from day-to-day was minimal. (c) Average number of tdTomato-positive and Iba1-eGFP-positive cells tracked per mouse, the number of newly appearing cells (New) and the number of lost cells (Lost) during the imaging period. Given is the mean ± SEM of n=5 mice. The analysis and tracking was done as described for the long-term analysis in Figure 3. Occasionally, additional structures, which were connected to microglial processes, were observed. In two cases, these resembled a cell body in shape and size, but were identified as being formed by distinct microglial processes reminiscent of recently described extruded exophers albeit in C. elegans (Melentijevic, I. et al. C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature 542, 367–371, 2017), whereas in three other instances, their composition was ambiguous. Though rapid splitting and consecutive death of the daughter cell in these cases cannot be ruled out, these observations were excluded from the analysis. Moreover, they do not change the lifetime estimate of the microglia.

Supplementary Figure 4 No difference was observed in total microglial numbers or the number of BrdU-labeled microglia in the different mouse lines used in this study

To exclude differences in microglial number or microglial proliferation based on BrdU incorporation, 7-month-old C57BL/6J mice, CD11b-CreERT2;R26-tdTomato;Iba1-eGFP (Cre/tdTom/GFP) mice and CD11b-CreERT2;R26-tdTomato (Cre/tdTom) mice of mixed gender were selected (n=5-6 per group). The selected age corresponds to the mid-time of the biweekly imaging period (4 months to 10 months) that was used for quantification in Figure 3. Mice were intraperitoneally injected on five consecutive days with BrdU and then analysed three days later. (a-c) Immunofluorescence double staining for Iba1 (green) and BrdU (red). Scale bar is 50 μm. (d) Quantification of double-labeled (yellow) cells (Iba1/BrdU) in the neocortex revealed no differences in BrdU-labeled microglia among the three groups (Kruskal-Wallis, χ2(2) = 2.19, p>0.05); dataset was checked for outliers using Graphpad Prism’s ROUT method and an outlier value was excluded from the Cre/tdTom dataset. (e-g) Immunohistochemical staining of the neocortex using an Iba1 antibody for the three genotypes. Scale bar is 100 μm. (h) Stereological analysis of total neocortical microglia revealed no difference in microglial number between C57BL/6J control mice and the double- and triple-transgenic animals used in this study (Kruskal-Wallis, χ2(2) = 0.10, p>0.05).

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Füger, P., Hefendehl, J., Veeraraghavalu, K. et al. Microglia turnover with aging and in an Alzheimer's model via long-term in vivo single-cell imaging. Nat Neurosci 20, 1371–1376 (2017). https://doi.org/10.1038/nn.4631

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