A Rapid Protocol for Intraoperative Assessment of Peripheral Nerve Myelinated Axon Count and its Application to Cross-Facial Nerve Grafting

Background: Donor nerve myelinated axon counts correlate with functional outcomes in reanimation procedures, however there exists no reliable means for their intraoperative quantification. Herein, we report a novel protocol for rapid quantification of myelinated axons from frozen sections, and demonstrate its applicability to surgical practice. Methods: The impact of various fixation and FluoroMyelin Red™ staining strategies on resolved myelin sheath morphology from cryosections of rat and rabbit femoral and sciatic nerves was assessed. A protocol comprising fresh cryosection and rapid staining was developed, and histomorphometric results compared against conventional osmium post-fixed, resin-embedded, toluidine blue-stained sections of rat sciatic nerve. The rapid protocol was applied for intraoperative quantification of donor nerve myelinated axon count in a cross-facial nerve grafting procedure. Results: Resolution of myelinated axon morphology suitable for counting was realized within ten minutes of tissue harvest. Though mean myelinated axon diameter appeared larger using the rapid fresh-frozen as compared to conventional nerve processing techniques (mean ± standard deviation; rapid, 9.25 ± 0.62; conventional, 6.05 ± 0.71; p

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