Three-Dimensional Mapping of Facial Motor Neurons with Tissue Clearing for Facial Nerve Nucleus of Guinea Pig via Long Distance Retrograde Tracing

Facial nerve palsy profoundly affects facial expression and quality of life, with recovery frequently hindered by aberrant nerve regeneration leading to synkinesis. A comprehensive understanding of three-dimensional (3D) cytoarchitectural organization and adaptive changes within the facial motor nucleus remains constrained by conventional two-dimensional analyses. In this study, retrograde neural tracers (DiI, DiO, Dextran Alexa Fluor^TM 488/546) were integrated with tissue clearing techniques (CUBIC and LUCID) to reconstruct and analyze the 3D distribution of neurons innervating the orbicularis oris and orbicularis oculi muscles in guinea pigs. Cleared brainstem tissues were imaged using two-photon microscopy, revealing distinct region-specific neuronal clusters within the facial motor nucleus. In a facial nerve injury model, disrupted regional specificity and disorganized neuronal distribution were observed, suggesting misdirected central reinnervation. This novel 3D imaging method enables high-resolution spatial neuronal analysis and reorganization following nerve injury. This approach provides a valuable tool for elucidating facial nerve regeneration and synkinesis, and may facilitate the development of improved therapeutic strategies.