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.
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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.
Uncleared (left) and cleared (right) spinal cords of GFP-M mice were imaged with two-photon microscopy.
A cleared spinal cord tissue section of a GFP-M mouse imaged with confocal microscopy.
3D rotation of the sample shown in Figure 2c.
3D imaging of the unsectioned spinal cord and caudal section of the medulla from a GFP-M mouse in rostro-caudal direction.
The CST of a rat after tracing with biotin dextran amine conjugated to rhodamine, cleared and imaged with two-photon microscopy.
Visualization of a single injured spinal cord of a GFP-M mouse in three different orientations: horizontal, sagittal and cross.
Two-photon stack of the injured spinal cord from a GFP-M mouse in its entire depth in dorsoventral orientation.
3D reconstruction and animation of the spinal cord from a GFP-M mouse shown in Figure 3.
3D reconstruction and animation of the spinal cord from a GFP-M mouse shown in Figure 4, 15 months after injury.
3D reconstruction and animation of the unlesioned spinal cord from a (TgH(CX3CR1-EGFP)) mouse shown in Figure 5a.
Two-photon scan of unlesioned spinal cord from a astrocyte-GFP mouse (TgN(hGFAP-EGFP)) in dorsoventral orientation.
The astrocytes in the spinal cord of TgN(hGFAP-EGFP) mouse scanned by two-photon microscopy in high resolution.
3D imaging of the cleared spinal cord from a double transgenic animal.