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

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 tetrahydrofuranbased 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.