Abstract
INTRODUCTION/AIMS: Peripheral nerve surgery entails repairing or transferring nerves to restore sensory or motor functions. Functional outcomes correlate with axon counts within operated nerves, but there exists no reliable means for intraoperative assessment of injured nerves. Conventional histologic assessment of regenerating peripheral nerve is labor-intensive, requiring multiday processing for tissue immunohistochemistry. Here, we describe a high-throughput protocol for histomorphometry of regenerating peripheral nerves. METHODS: Healthy and regenerating murine and human peripheral nerve sections were stained with commercial fluorescent membrane-specific dyes, one of which (MemBrite) was discovered to internalize within axons. Samples were imaged using confocal microscopy, and regenerating axons were quantified using commercial segmentation software. Quantification results using the novel staining protocol were compared against conventional neurofilament immunohistochemistry. The protocol was employed to guide intraoperative decision-making in a case of free functional muscle transfer for treating a patient with facial paralysis. RESULTS: Axon quantification within healthy and regenerating nerve sections in human and murine samples via rapid staining yielded similar results when compared to antineurofilament labeling. The relative difference in axon counts for the healthy murine facial nerve for antineurofilament and MemBrite 594 staining was < 1%. For the regenerating human nerve, the relative difference was 8.7%. DISCUSSION: A rapid quantification protocol for regenerating axons has been described, which may inform intraoperative decision-making and advance knowledge in peripheral nerve surgery. Minimizing tissue processing time during surgery would allow surgeons to receive immediate feedback regarding axon counts in the donor nerve for cross-facial nerve surgeries, ultimately leading to improved clinical outcomes for their patients.