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

Journal name:
Nature Medicine
Volume:
18,
Pages:
166–171
Year published:
DOI:
doi:10.1038/nm.2600
Received
Accepted
Published online

Abstract

Studying regeneration in the central nervous system (CNS) is hampered by current histological and imaging techniques because they provide only partial information about axonal and glial reactions. Here we developed a tetrahydrofuran-based clearing procedure that renders fixed and unsectioned adult CNS tissue transparent and fully penetrable for optical imaging. In large spinal cord segments, we imaged fluorescently labeled cells by 'ultramicroscopy' and two-photon microscopy without the need for histological sectioning. We found that more than a year after injury growth-competent axons regenerated abundantly through the injury site. A few growth-incompetent axons could also regenerate when they bypassed the lesion. Moreover, we accurately determined quantitative changes of glial cells after spinal cord injury. Thus, clearing CNS tissue enables an unambiguous evaluation of axon regeneration and glial reactions. Our clearing procedure also renders other organs transparent, which makes this approach useful for a large number of preclinical paradigms.

At a glance

Figures

  1. Clearance allows high-resolution imaging of the unsectioned spinal cord.
    Figure 1: Clearance allows high-resolution imaging of the unsectioned spinal cord.

    (a) Clearance with THF renders the adult mouse spinal cord transparent (outlined by black arrowheads) while ethanol-treated tissue remains opaque (photographic images are shown). (b) Schematic illustration of the two-photon–imaged regions and depths: only dorsal (z1) or entire dorsoventral (z3) spinal cord. (c) Comparison between uncleared (left) and cleared (right) spinal cords of GFP-M mice imaged with two-photon microscopy. Values are mean ± s.d. (dg) Horizontal projections from the cleared spinal cord at different depths marked in c: dorsal (~100 μm) (d), mid (~500 μm) (e) and ventral (~1,200 μm) (f). (g) Higher magnification of the area marked in green in e. (h) Higher magnification of the area marked in green in g. Yellow arrowheads in g and h depict some of the axonal boutons in the gray matter.

  2. Imaging large regions of the spinal cord of GFP-M mice.
    Figure 2: Imaging large regions of the spinal cord of GFP-M mice.

    (a) Entire spinal cord of a GFP-M mouse cut into 3- to 4-mm-long segments, cleared and imaged with ultramicroscopy. (b) Drawing of the ultramicroscopy setup showing tissue positioning and the light path. (c) A spinal cord segment (length 4 mm, T12 to L2 spine level) of a GFP-M mouse scanned with ultramicroscopy shown in a horizontal view. (d) Cross-view projection (50-μm thickness) of the indicated region in c. White arrows in c,d mark individual axons in the white matter; red arrows mark cell bodies in the gray matter. (e) Traced white and gray matter boundaries and axon bundles (yellow arrows).

  3. Visualization of regenerating axons in the unsectioned spinal cord.
    Figure 3: Visualization of regenerating axons in the unsectioned spinal cord.

    Regeneration of conditioned axons in GFP-M mice imaged by ultramicroscopy 10 d after injury and subsequent clearing. (a,b) 3D reconstruction in sagittal views; b shows a higher magnification of the green boxed region in a, indicating several regenerating axons (white arrows) crossing the lesion site (yellow rectangle). The distal parts of the injured axons were fragmented and mostly removed by Wallerian degeneration, providing an unambiguous view of regenerating axons beyond the lesion (asterisks). (c) 3D reconstruction with a corner-cut view of the spinal cord. The start and end of each traced axon are marked by correspondingly colored arrows and arrowheads, referring to the same axons in all images. (d) The tips of the traced axons are shown in xy and xz projections. (e) Length of conditioned regenerating axons crossing the lesion site (green dots, n = 6 mice) versus stalled unconditioned axons (red dots, n = 7 mice) at 10 d after injury. Negative values represent the degeneration distance from the lesion. Positive values represent the length of regenerating axons from the lesion in rostral direction. Each dot represents average values from one mouse. Mean and s.e.m. of each group are indicated by black lines. **P < 0.01, t test. (f,g) The trajectories of regenerating axons in the white and gray matter of segmented spinal cord in horizontal (f) and cross (g) views. The yellow and blue marked axons regenerate within the white matter, whereas the red marked axon regenerates through the gray matter.

  4. Conditioned axons grow through the lesion, whereas unconditioned axons avoid the lesion.
    Figure 4: Conditioned axons grow through the lesion, whereas unconditioned axons avoid the lesion.

    Regeneration of conditioned and unconditioned axons in the cleared GFP-M mouse spinal cord imaged by ultramicroscopy 15 months after injury. (a) 3D reconstruction of the trajectories of conditioned versus unconditioned axons (green) in corner-cut view through the lesion (red). (b,c) Projections from the indicated regions in a. The high magnifications of the axon tips marked with numbered arrowheads in b are shown in Supplementary Figure 11. Conditioned axons regenerate over long distances in both dorsal (b) and deeper (c) layers of the spinal cord (arrowheads). Some unconditioned axons regenerate over short distances and grow on the surface of the dorsal column (b) but fewer in deeper regions (c) (arrowheads). The yellow dashed rectangles in b,c indicate the lesion area. (d) Virtual segmentation of the spinal cord into nested cylindrical regions for a 3D Sholl analysis (radius increases in 75-μm increments). White dots mark positions of growing axons at the lesion plane. Conditioned axons were observed in every region of the lesion, and unconditioned regenerating axons were at the edge of the lesion. (e) Horizontal transparent view of the spinal cord, depicting the trajectories of stalled or regenerated axons (green). (f) The percentage of the axonal volume in the conditioned versus unconditioned (n = 4 mice for each) along the indicated cylinder regions (mean ± s.e.m.). (g) The average crossing number of individual axons through cylindrical regions (mean ± s.d.). *P < 0.05, **P < 0.01, t test.

  5. Microglia reaction in the injured spinal cord and simultaneous 3D visualization of neurons, astrocytes and microglia.
    Figure 5: Microglia reaction in the injured spinal cord and simultaneous 3D visualization of neurons, astrocytes and microglia.

    (a,b) 3D visualization of cleared naive (a) and injured (b) spinal cords from TgH(CX3CR1-EGFP) mice in horizontal views. (c,d) Spatial distributions of microglia density in the naive (c) and injured (d) spinal cords in cross-sections using color code (blue: low cell density; red: high cell density). The yellow rectangle in b indicates the lesion site. (e) Optical projection of the naive section indicated in c, showing microglia in the gray and white matter (white arrowheads) and adjacent to blood vessels (yellow arrowheads), which appear as black tubes. (f,g) Optical projections from the injured white matter (WM, f) and gray matter (GM, g) regions indicated in d. (h) Quantification of microglia number in the regions depicted in c and d. The values are mean ± s.e.m.; n = 8 TgH(CX3CR1-EGFP) mice per group. **P < 0.01, t test between groups. Contra, contralateral. (i,j) 3D imaging of neurons, astrocytes and microglia within the same sample using triple-transgenic mice: TgN(hGFAP-ECFP) × TgH(CX3CR1-EGFP) × TgN(Thy1-EYFP). (i) 3D visualization of an L1 spinal cord segment from a triple-transgenic mouse showing astrocytes (blue), microglia (red) and neurons (green) in cross-view. (j) Projection (about 50-μm thickness) of the indicated region in i in horizontal view; higher magnification of the boxed region.

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Author information

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. Technical University Vienna, Institute of Solid State Electronics, Department of Bioelectronics, 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. University of Oldenburg, Department of Neurobiology, Oldenburg, Germany.

    • Nina Jährling
  11. Glial Physiology and Imaging, Department of Neurogenetics, 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

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Supplementary information

PDF files

  1. Supplementary Text and Figures (2M)

    Supplementary Figures 1–14 and Supplementary Methods

Movies

  1. Supplementary Video 1 (7M)

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

  2. Supplementary Video 2 (18M)

    A cleared spinal cord tissue section of a GFP-M mouse imaged with confocal microscopy.

  3. Supplementary Video 3 (13M)

    3D rotation of the sample shown in Figure 2c.

  4. Supplementary Video 4 (10M)

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

  5. Supplementary Video 5 (4M)

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

  6. Supplementary Video 6 (8M)

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

  7. Supplementary Video 7 (3M)

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

  8. Supplementary Video 8 (15M)

    3D reconstruction and animation of the spinal cord from a GFP-M mouse shown in Figure 3.

  9. Supplementary Video 9 (34M)

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

  10. Supplementary Video 10 (36M)

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

  11. Supplementary Video 11 (10M)

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

  12. Supplementary Video 12 (4M)

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

  13. Supplementary Video 13 (11M)

    3D imaging of the cleared spinal cord from a double transgenic animal.

Additional data