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

Abstract

Background: Donor nerve myelinated axon counts correlate with functional outcomes in reanimation procedures; however, there exists no reliable means for their intraoperative quantification. In this article, the authors 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 were compared against conventional osmium-postfixed, 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 10 minutes of tissue harvest. Although mean myelinated axon diameter appeared larger using the rapid fresh-frozen as compared to conventional nerve processing techniques (mean ± SD; rapid, 9.25 ± 0.62 μm; conventional, 6.05 ± 0.71 μm; p < 0.001), no difference in axon counts was observed on high-power fields (rapid, 429.42 ± 49.32; conventional, 460.32 ± 69.96; p = 0.277). Whole nerve myelinated axon counts using the rapid protocol herein (8435.12 ± 1329.72) were similar to prior reports using conventional osmium processing of rat sciatic nerve.

Conclusions: A rapid protocol for quantification of myelinated axon counts from peripheral nerves using widely available equipment and techniques has been described, rendering possible intraoperative assessment of donor nerve suitability for reanimation.

Figure 1.. Protocol for rapid resolution of myelinated axons from cross-sections of peripheral nerve.

Specimens may be transported from the operating room (OR) in 0.01 M phosphate buffered saline (PBS) on ice for fresh cyrosection, or cryosectioned following brief fixation in 4% paraformaldehyde (PFA) in 0.01 M PBS.

Figure 2.. Comparison of various fixation and FluoroMyelin Red™ staining protocols on imaging of rat sciatic nerve myelinated axons.

Specimens were immediately placed in fixative or 0.01M phosphate-buffered saline (PBS) following harvest, prior to axial cryosection at 1 μm. Tow row: FluoroMyelin Red™ dilution of 1:300 with 20 minute stain time. A: Specimen fixed with neutral buffered formalin (NBF) for 24 hours. B: Specimen fixed with 4% paraformaldehyde (PFA) in 0.01 M PBS for 24 hours. C: No fixation (fresh frozen sectioned). Bottom row: FluoroMyelin Red™ dilution of 1:50 with 1 minute stain time. D: Specimen fixed with NBF for 10 min. E: Specimen fixed 4% PFA in 0.01 M PBS for 10 min. F: No fixation (fresh frozen sectioned). (Widefield epifluorescent microscopy, 40x/1.3 NA oil objective, scale bar = 20um)

Figure 3.. Comparison of conventional nerve processing versus the herein reported rapid protocol on automated histomorphometry of rat sciatic nerve.

Top row: Osmium post-fixed, resin embedded, 1 μm sectioned, and toluidine blue stained specimen. Bottom row: Fresh, optimal cutting temperature compound embedded, 1 μm sectioned, and FluoroMyelin Red™ stained (1:50 for 1 min) specimen. A, D: Original images. B, D: Image pre-processing by 8-bit grayscale down-sampling, inversion, and background subtraction. C, Automated surface mapping of myelinated axons; color map represents outer diameter of mapped surfaces.

Figure 4.. Rapid intraoperative quantification of myelinated axon count in zygomatic donor branch of human facial nerve.

The patient was undergoing first-stage cross-facial nerve grafting procedure for planned free gracilis transfer for smile reanimation. A: Original wide-field epifluorescence image of fresh 1 μm cryosection stained with 1:50 FluoroMyelin Red™ for one minute. B: Automated surface mapping and quantification of myelinated axons; color map represents outer diameter of myelinated axons.