INTRODUCTION: Injury to the facial nerve can lead to functional and aesthetic sequelae in patients. Though surgical interventions are available to restore lost motor and sensory function, outcomes are often suboptimal due to inadequate or disorganized axonal regeneration. While engineering improvements to the standard of care are underway, gaps remain in our molecular understanding of peripheral nerve injury to translate these efforts clinically. Over the last few decades, advancements in intravital imaging such as the development of fluorescent reporter mice and use of multiphoton excitation techniques have allowed for markedly enhanced characterization of biological phenomena at higher resolutions, at greater depths, and for longer timescales. Challenges in reliably and serially imaging in vivo within murine models have been overcome through the development of chronic imaging windows in various settings of the body. However, there are very few techniques available presently for imaging the peripheral nerve microenvironment and no prior work detailing use in the facial nerve setting. OBJECTIVE: Longitudinal studies employing intravital imaging techniques carry potential to improve understanding of peripheral nerve regeneration and function. Using multiphoton microscopy and fluorescent reporter mice, we propose a prototype, surgical protocol of implantation, and initial safety and efficacy testing of a facial nerve window to enable chronic imaging for enhanced characterization of the peripheral nerve microenvironment. METHODS: A stainless-steel implant with an affixed glass coverslip and aluminum external fixation component was developed for implantation in a transgenic reporter mouse model to enable chronic intravital imaging of the facial nerve buccal and marginal mandibular branches. A qualitative observational study and clinical assessment scoring study was performed post-surgical implantation to monitor behavior, physical appearance, weight loss, and reactivity to animal handling over the typical time-course of nerve regeneration. Segments of facial nerve branches were harvested from control and window-implanted mice and imaged using widefield epifluorescence microscopy for axon quantification to determine any adverse effects from window compression onto axonal fibers. Two-photon microscopy (2PM) and Simulated Raman Scattering (SRS) were also performed through the window to visualize axon tracts, myelin sheaths, and surrounding collagen matrix in wild-type and transgenic mice models. RESULTS: Qualitative serial observational studies and assessment scoring indicated no obvious functional deficits over the time-course of typical nerve regeneration and normal scores for weight, behavior, physical appearance, and reactivity. Neural histomorphometric analysis indicated no significant difference in mean myelinated axon count of buccal (mean ± SD; control buccal, 947.6 ± 129.9; window-implanted buccal, 799.3 ± 128.6; p = .136) and marginal mandibular branches (control marginal mandibular, 801.3 ± 145.1; window-implanted marginal mandibular, 738.0 ± 197.2; p = .599) between control and window-implanted mice, suggesting that neuropathy was not induced from the window itself. High-resolution images of nerve morphology in healthy and injured transgenic and wild-type mice were obtained using 2PM and SRS. CONCLUSION: Herein, we describe a novel and replicable platform for longitudinal intravital imaging of murine facial nerve. Future studies will evaluate viability of this model for imaging the facial nerve microenvironment, particularly Schwann cell-axon interactions, in the setting of severe nerve injury over a period of several weeks to months. Improved understanding gained through such studies of the structural peripheral nerve microenvironment may allow for advancements in viral vector therapeutics, nerve graft scaffold design, as well as advanced injury diagnostics and tracking. / 2022-06-02T00:00:00Z