Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury (original) (raw)

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Acknowledgements

We thank K. Dornmair, J. Enes, C. Hojer, A. Kania, D. Neukirchen, K. Olsen, M. Stiess and S. Tahirovic for critically reading the manuscript, V. Duc Ha for technical assistance and R. Brand for his help with Amira. We are indebted to A. Borst and M. Sheng for their support and to J. Sanes (Harvard University) for the GFP-M mice. A.E. was supported by the Marie Curie Association (European Union; RTN MRTN–CT–2,003–504,636). C.P.M. was supported by the Hertie Stiftung. N.J. was supported by the Theodor Körner Fonds. F.B. is a recipient of a Career Development Award from the Human Frontier Science Program. This work was supported by the Max Planck Society, the International Foundation for Research in Paraplegia and additional grants from the Deutsche Forschungsgemeinschaft and the Hertie Stiftung.

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Authors and Affiliations

  1. Max Planck Institute of Neurobiology, Axonal Growth and Regeneration, Martinsried, Germany
    Ali Ertürk, Farida Hellal & Frank Bradke
  2. Department of Neuroscience, Genentech, South San Francisco, California, USA
    Ali Ertürk
  3. Max Planck Institute of Psychiatry, Munich, Germany
    Christoph P Mauch
  4. Deutsches Zentrum für Neurodegenerative Erkrankungen, Axonal Growth and Regeneration, Bonn, Germany
    Farida Hellal & Frank Bradke
  5. Max Planck Institute of Neurobiology, Systems and Computational Neurobiology, Martinsried, Germany
    Friedrich Förstner
  6. Max Planck Institute of Neurobiology, Cellular and Systems Neurobiology, Martinsried, Germany
    Tara Keck & Mark Hübener
  7. Medical Research Council Centre for Developmental Neurobiology, King's College London, Guy's Hospital Campus, London, UK.,
    Tara Keck
  8. Department of Bioelectronics, Technical University Vienna, Institute of Solid State Electronics, Vienna, Austria
    Klaus Becker, Nina Jährling & Hans Ulrich Dodt
  9. Center for Brain Research, Medical University of Vienna, Section of Bioelectronics, Vienna, Austria
    Klaus Becker, Nina Jährling & Hans Ulrich Dodt
  10. Department of Neurobiology, University of Oldenburg, Oldenburg, Germany
    Nina Jährling
  11. Department of Neurogenetics, Glial Physiology and Imaging, Max Planck Institute of Experimental Medicine, Göttingen, Germany
    Heinz Steffens & Frank Kirchhoff
  12. Development and Maintenance of the Nervous System, Centre for Molecular Neurobiology, Hamburg, Germany
    Melanie Richter & Edgar Kramer
  13. Molecular Physiology, Institute of Physiology, University of Saarland, Homburg/Saar, Germany
    Frank Kirchhoff

Authors

  1. Ali Ertürk
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  2. Christoph P Mauch
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  3. Farida Hellal
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  4. Friedrich Förstner
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  5. Tara Keck
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  6. Klaus Becker
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  7. Nina Jährling
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  8. Heinz Steffens
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  9. Melanie Richter
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  10. Mark Hübener
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  11. Edgar Kramer
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  12. Frank Kirchhoff
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  13. Hans Ulrich Dodt
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  14. Frank Bradke
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Contributions

A.E. initiated the project, designed the experiments, developed and performed the clearing protocol, performed the surgeries, performed the in vivo confocal and two-photon imaging, analyzed the data, made the figures and videos, performed the statistical tests, and wrote the paper. C.P.M. developed and performed the clearing protocol and performed ultramicroscopy imaging. F.H. performed surgeries and analysis for two-dimensional glia quantification and performed the rat tracing. F.F. developed the automated segmentation, tracking software and analyzed the 3D Sholl data. T.K. and M.H. performed initial two-photon experiments. M.R. and E.K. performed the deep-tissue antibody staining. H.S. and F.K. performed two-photon imaging on double- and triple-transgenic mice. K.B. and N.J. performed the histochemical screen and developed the clearing protocol. H.U.D. constructed ultramicroscopy and supervised C.P.M. F.B. initiated the project, designed experiments, coordinated and supervised the project and wrote the paper. All authors edited the paper.

Corresponding author

Correspondence toFrank Bradke.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14 and Supplementary Methods (PDF 2110 kb)

Supplementary Video 1

Uncleared (left) and cleared (right) spinal cords of GFP-M mice were imaged with two-photon microscopy. (MOV 6487 kb)

Supplementary Video 2

A cleared spinal cord tissue section of a GFP-M mouse imaged with confocal microscopy. (MOV 17618 kb)

Supplementary Video 3

3D rotation of the sample shown in Figure 2c. (MOV 13075 kb)

Supplementary Video 4

3D imaging of the unsectioned spinal cord and caudal section of the medulla from a GFP-M mouse in rostro-caudal direction. (MOV 10132 kb)

Supplementary Video 5

The CST of a rat after tracing with biotin dextran amine conjugated to rhodamine, cleared and imaged with two-photon microscopy. (MOV 3905 kb)

Supplementary Video 6

Visualization of a single injured spinal cord of a GFP-M mouse in three different orientations: horizontal, sagittal and cross. (MOV 8073 kb)

Supplementary Video 7

Two-photon stack of the injured spinal cord from a GFP-M mouse in its entire depth in dorsoventral orientation. (MOV 3250 kb)

Supplementary Video 8

3D reconstruction and animation of the spinal cord from a GFP-M mouse shown in Figure 3. (MOV 14446 kb)

Supplementary Video 9

3D reconstruction and animation of the spinal cord from a GFP-M mouse shown in Figure 4, 15 months after injury. (MOV 32719 kb)

Supplementary Video 10

3D reconstruction and animation of the unlesioned spinal cord from a (TgH(CX3CR1-EGFP)) mouse shown in Figure 5a. (MOV 35105 kb)

Supplementary Video 11

Two-photon scan of unlesioned spinal cord from a astrocyte-GFP mouse (TgN(hGFAP-EGFP)) in dorsoventral orientation. (MOV 10149 kb)

Supplementary Video 12

The astrocytes in the spinal cord of TgN(hGFAP-EGFP) mouse scanned by two-photon microscopy in high resolution. (MOV 3910 kb)

Supplementary Video 13

3D imaging of the cleared spinal cord from a double transgenic animal. (MOV 10763 kb)

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Ertürk, A., Mauch, C., Hellal, F. et al. Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury.Nat Med 18, 166–171 (2012). https://doi.org/10.1038/nm.2600

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