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
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- Supplementary Text and Figures (2M)
Supplementary Figures 1–14 and Supplementary Methods
- Supplementary Video 1 (7M)
Uncleared (left) and cleared (right) spinal cords of GFP-M mice were imaged with two-photon microscopy.
- Supplementary Video 2 (18M)
A cleared spinal cord tissue section of a GFP-M mouse imaged with confocal microscopy.
- Supplementary Video 3 (13M)
3D rotation of the sample shown in Figure 2c.
- 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.
- 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.
- Supplementary Video 6 (8M)
Visualization of a single injured spinal cord of a GFP-M mouse in three different orientations: horizontal, sagittal and cross.
- Supplementary Video 7 (3M)
Two-photon stack of the injured spinal cord from a GFP-M mouse in its entire depth in dorsoventral orientation.
- Supplementary Video 8 (15M)
3D reconstruction and animation of the spinal cord from a GFP-M mouse shown in Figure 3.
- 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.
- Supplementary Video 10 (36M)
3D reconstruction and animation of the unlesioned spinal cord from a (TgH(CX3CR1-EGFP)) mouse shown in Figure 5a.
- Supplementary Video 11 (10M)
Two-photon scan of unlesioned spinal cord from a astrocyte-GFP mouse (TgN(hGFAP-EGFP)) in dorsoventral orientation.
- Supplementary Video 12 (4M)
The astrocytes in the spinal cord of TgN(hGFAP-EGFP) mouse scanned by two-photon microscopy in high resolution.
- Supplementary Video 13 (11M)
3D imaging of the cleared spinal cord from a double transgenic animal.