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

Plast Reconstr Surg. 2019 Mar;143(3):771-778. doi: 10.1097/PRS.0000000000005338.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Axons / pathology
  • Axons / transplantation
  • Clinical Decision-Making / methods
  • Cost-Benefit Analysis
  • Facial Expression*
  • Facial Nerve / cytology
  • Facial Nerve / pathology
  • Facial Nerve / transplantation*
  • Facial Paralysis / surgery*
  • Femoral Nerve / cytology
  • Femoral Nerve / pathology
  • Fluorescent Dyes
  • Frozen Sections
  • Humans
  • Models, Animal
  • Myelin Sheath / pathology
  • Nerve Transfer / economics
  • Nerve Transfer / instrumentation
  • Nerve Transfer / methods*
  • Rabbits
  • Rats
  • Rats, Wistar
  • Sciatic Nerve / cytology
  • Sciatic Nerve / pathology
  • Staining and Labeling / economics
  • Staining and Labeling / instrumentation
  • Staining and Labeling / methods*
  • Time Factors
  • Treatment Outcome


  • Fluorescent Dyes