A novel workflow for 3D imaging and spatial analysis of nerves in bone

Peripheral sensory and sympathetic nerves innervating bony tissues are of interest for their role in modulating bone function and responding to physiologic stimuli. However, due to their filamentous structure and small diameter, peripheral nerves are difficult to visualize and characterize in dense tissues such as bone. Recent advances in imaging have resulted in novel techniques to reconstruct the 3D bone microenvironment following fluorescent staining, using methods such as whole-mount immunostaining, tissue clearing, and lightsheet microscopy (LSM). While these methods have significantly improved visualization of peripheral nerves within the bony skeleton, current practices in skeletal nerve segmentation rely on manual tracing methods that are both subjective and time consuming. Further, due to excess background signal and moderate off-target staining of skeletal nerves, traditional methods for nerve segmentation remain inadequate for effective quantification. Here, we describe a methods-focused workflow for 3D imaging and spatial analysis of nerves in bone that consists of combining LSM with nerve segmentation using the Ilastik® open-source software. First, LSM was used to acquire high-resolution 3D images of nerves in uninjured and injured murine calvaria. Next, we employed Ilastik® to generate accurate segmentations of peripheral nerves in bone. This approach reduced the need for manual segmentation and overcame the limitations of conventional segmentation methods. We then demonstrated the utility of this workflow as a broadly applicable tool, by performing Ilastik®-based segmentation for three distinct skeletal bone contexts (i.e. use cases) with varied bone samples, tissue clearing, processing protocols, inflammatory states, and imaging modalities. Collectively, this workflow enabled the characterization of spatial nerve patterning in bone and has the potential to provide novel insights into spatially regulated phenomena in bone homeostasis, injury, and disease.