Using tissue clearing and light sheet fluorescence microscopy for the three-dimensional analysis of sensory and sympathetic nerve endings that innervate bone and dental tissue of mice.

Bone and dental tissues are richly innervated by sensory and sympathetic neurons. However, the characterization of the morphology, molecular phenotype, and distribution of nerves that innervate hard tissue has so far mostly been limited to thin histological sections. This approach does not adequately capture dispersed neuronal projections due to the loss of important structural information during three-dimensional (3D) reconstruction. In this study, we modified the immunolabeling-enabled imaging of solvent-cleared organs (iDISCO/iDISCO+) clearing protocol to image high-resolution neuronal structures in whole femurs and mandibles collected from perfused C57Bl/6 mice. Axons and their nerve terminal endings were immunolabeled with antibodies directed against protein gene product 9.5 (pan-neuronal marker), calcitonin gene-related peptide (peptidergic nociceptor marker), or tyrosine hydroxylase (sympathetic neuron marker). Volume imaging was performed using light sheet fluorescence microscopy. We report high-quality immunolabeling of the axons and nerve terminal endings for both sensory and sympathetic neurons that innervate the mouse femur and mandible. Importantly, we are able to follow their projections through full 3D volumes, highlight how extensive their distribution is, and show regional differences in innervation patterns for different parts of each bone (and surrounding tissues). Mapping the distribution of sensory and sympathetic axons, and their nerve terminal endings, in different bony compartments may be important in further elucidating their roles in health and disease.