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Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation

Nature Neuroscience volume 16, pages 580–586 (2013)Cite this article

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Abstract

Astrocytes are thought to have important roles after brain injury, but their behavior has largely been inferred from postmortem analysis. To examine the mechanisms that recruit astrocytes to sites of injury, we used in vivo two-photon laser-scanning microscopy to follow the response of GFP-labeled astrocytes in the adult mouse cerebral cortex over several weeks after acute injury. Live imaging revealed a marked heterogeneity in the reaction of individual astrocytes, with one subset retaining their initial morphology, another directing their processes toward the lesion, and a distinct subset located at juxtavascular sites proliferating. Although no astrocytes actively migrated toward the injury site, selective proliferation of juxtavascular astrocytes was observed after the introduction of a lesion and was still the case, even though the extent was reduced, after astrocyte-specific deletion of the RhoGTPase Cdc42. Thus, astrocyte recruitment after injury relies solely on proliferation in a specific niche.

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Figure 1: Live imaging of astrocyte responses to a punctate lesion.
Figure 2: Live imaging of astrocyte responses to a stab wound.
Figure 3: Astrocyte proliferation adjacent to blood vessels.
Figure 4: Juxtavascular locations of proliferating astrocytes.
Figure 5: Live imaging of Cdc42−/− astrocytes following stab wounding.
Figure 6: Proliferation defect in Cdc42−/− astrocytes following injury.

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Acknowledgements

We are indebted to C. Brakebusch for the Cdc42loxP/loxP mice, S. Robel for initial help in setting up the two-photon live-imaging procedure, and C. Straube and S. Falkner for sharing their imaging expertise. We would also like to thank R. Waberer, C. Meyer, D. Franzen, I. Mühlhahn and G. Jäger for technical assistance, and M. Hübener, D.E. Bergles, J. McCarter and P. Hardy for critical comments on the manuscript. We thank the DFG (German Research foundation) whose support (particularly via the Leibniz Prize and SFB 870) allowed us to invest into this new experimental area and for funding M.G. and F.J.T. by SPP1356 and I.B. by DFG-FOR 1336. In addition, this work was supported by the BMBF (Ministry of Science and Education) to M.G. and F.J.T., the Helmholtz Association (Helmholtz Alliance ICEMED to M.G., I.B. and F.J.T.; Helmholtz Alliance on Systems Biology to M.G. and F.J.T.), the Emmy Noether Program of the DFG (ME 3542/1-1 to M.M.-L.), the European Research Council (starting grant 'LatentCauses' to F.J.T.) and Munich Cluster for Systems Neurology. The Initial Training Network Edu-GLIA (PITN-GA-2009-237956) funded by the European Commission under the Seventh Framework Program (FP7) provided a wonderful discussion platform for glial research.

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Authors and Affiliations

  1. Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, Munich, Germany

    Sophia Bardehle, Leda Dimou & Magdalena Götz

  2. Institute for Stem Cell Research, Helmholtz Zentrum Munich, Neuherberg, Germany

    Sophia Bardehle, Julia Schwausch, Jovica Ninkovic, Leda Dimou & Magdalena Götz

  3. Institute of Anatomy, University of Leipzig, Leipzig, Germany

    Martin Krüger & Ingo Bechmann

  4. Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum Munich, Neuherberg, Germany

    Felix Buggenthin & Fabian J Theis

  5. Department of Mathematics, Technical University Munich, Germany

    Felix Buggenthin & Fabian J Theis

  6. Hubrecht Institute, Koninklijke Nederlandse Akademie van Wetenschappen and University Medical Center Utrecht, Connecticut Utrecht, The Netherlands

    Hans Clevers & Hugo J Snippert

  7. Department of Biochemistry, Adolf Butenandt Institute, Ludwig-Maximilians University Munich, Munich, Germany

    Melanie Meyer-Luehmann

  8. Department of Neurology, Neurocenter, University of Freiburg, Freiburg, Germany

    Melanie Meyer-Luehmann

  9. Munich Cluster for Systems Neurology (SyNergy), Munich, Germany

    Magdalena Götz

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Contributions

S.B. performed all of the experiments and data analyses (except for electron microscopy) and wrote the manuscript. M.K. and I.B. carried out electron microscopy. F.B. and F.J.T. assisted in data processing and volume analysis (Supplementary Figs. 2 and 3). J.S., J.N., H.C. and H.J.S. supplied the GLAST/confetti mouse strain (Supplementary Fig. 9). M.M.-L. provided access to the 2pLSM and expert advice on imaging. L.D. initially established the in vivo two-photon microscopy technique and taught it to S.B. M.G., together with L.D., designed the project and experiments, discussed the results and wrote the manuscript. M.G. coordinated and directed the project.

Corresponding author

Correspondence to Magdalena Götz.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 11813 kb)

Supplementary Video 1

Live imaging of GFP+ astrocytes and TexasReddextran-labeled blood vessels in the cerebral cortex grey matter of a GLAST/eGFP mouse. The optical sections are 5 μm thick and total stack depth is 250 μm. Magnification: 20x zoom 2 (MOV 1245 kb)

Supplementary Video 2

Live imaging of a juxtavascular astrocyte contacting an injured blood vessel that was undergoing division upon injury on 0dpo (see Fig. 3a, b and Movie 3). The optical sections are 5 μm thick and total stack depth is 175 μm. Magnification: 20x zoom2 (MOV 473 kb)

Supplementary Video 3

A juxtavascular astrocyte in contact to an injured blood vessel and forming a duplet, imaged 7 days after lesion (see Fig. 3c). The optical sections are 5 μm thick and total stack depth is 75 μm. Magnification: 20x zoom 2 (MOV 243 kb)

Supplementary Video 4

Live imaging of astrocyte polarization 7 days after stab wound. The optical sections are 5 μm thick and total stack depth is 100 μm. Magnification: 20x zoom 5 (MOV 417 kb)

Supplementary Video 5

Superimposition of 3D images after image registration reveals astrocytes that remain stationary after acute lesion. Overlay of GFP+ astrocytes (green: 0dpo; white: 7dpo) and blood vessels (red: 0dpo; blue: 7dpo) after image registration (see Suppl. Fig. 2 and Methods). Theoptical sections are 5 μm thick and total stack depth is 150 μm. Magnification: 20x zoom 2 (MOV 3353 kb)

Supplementary Video 6

Live imaging of GFP+ astrocytes and TexasReddextran-labeled blood vessels in the cerebral cortex grey matter of an Aldh1L1-eGFP mouse after stab wound (0dpo). The optical sections are 5 μm thick and total stack depth is 450 μm. Magnification: 20x (MOV 3896 kb)

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Bardehle, S., Krüger, M., Buggenthin, F. et al. Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation. Nat Neurosci 16, 580–586 (2013). https://doi.org/10.1038/nn.3371

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