Morphological analyses are key outcome assessments for nerve regeneration studies but are historically limited to tissue sections. Novel optical tissue clearing techniques enabling three-dimensional imaging of entire organs at a subcellular resolution have revolutionized morphological studies of the brain. To extend their applicability to experimental nerve repair studies we adapted these techniques to nerves and their motor and sensory targets in rats. The solvent-based protocols rendered harvested peripheral nerves and their target organs transparent within 24 h while preserving tissue architecture and fluorescence. Optical clearing is compatible with conventional laboratory techniques, including retrograde labelling studies, and computational image segmentation, providing fast and precise cell quantitation. Further, optically cleared organs enable three dimensional morphometry at an unprecedented scale including dermatome wide innervation studies, tracing of intramuscular nerve branches or mapping of neurovascular networks. Given their wide-ranging applicability, rapid processing times and low costs, tissue clearing techniques are likely to be a key technology for next-generation nerve repair studies.
bioRxiv Subject Collection: Neuroscience