Complex 3D cytoarchitecture of the anterior horn of the human sacral spinal cord revealed by light sheet fluorescence microscopy

This study provides a unique three-dimensional characterization of the cytoarchitecture of the anterior horn of the human sacral spinal cord, with a specific focus on the S2 segment. This region is of particular interest because it constitutes the transition between the motoneurons that innervate the muscles of the lower limbs and those that control the pelvic floor. The S2 segment also contains a distinct motor nucleus, Onuf’s nucleus (ON), involved in sphincter control. Using a tissue clearing protocol combined with light sheet fluorescence microscopy, we analyzed samples of sacral spinal cords from ten human cadavers to obtain high-resolution 3D images. Segmentation revealed seven distinct motoneuron groups organized into medial and lateral divisions surrounding ON. ON itself displayed a complex, multilobed architecture, with a denser central region and more fragmented extremities. A robust cytoarchitectural pattern of specific neuron columns was identified along the rostrocaudal axis of ON and characterized by their progressive disappearance, fusion, or emergence. Notably, lateral columns predominated at the level of ON rostral half, whereas medial columns appeared more caudally. Additionally, a significant sexual dimorphism in ON length was observed, being longer in males. Overall, our findings demonstrate that the classical columnar model of spinal motoneuron organization underestimates the structural complexity of the anterior horn. This study highlights the interest of light sheet fluorescence microscopy for revealing fine anatomical organization and provides a framework for future investigations of SC cytoarchitecture.