Intraoperative Real-Time Fluorescence Labeling of Degenerated Facial Nerves with Bevonescein

Background: Bevonescein (ALM-488), a nerve-targeted peptide-dye conjugate, enables intraoperative fluorescence of degenerated peripheral nerves because of its extracellular matrix–binding mechanism. In contrast, myelin-based nerve-targeting agents would not be expected to label degenerated nerves because of demyelination. The authors compare the intraoperative fluorescence of chronically degenerated murine facial nerves produced by bevonescein and myelin-binding dye oxazine-4 and discuss its utility in peripheral nerve reconstruction. Methods: Sixteen wild-type mice underwent transection of the marginal mandibular branch of the facial nerve. At 5 months, 10 mice were co-injected with bevonescein and oxazine-4 and underwent intraoperative facial nerve exploration with fluorescence imaging. The signal-to-background ratio (SBR) was calculated by comparing the mean gray value along each nerve segment to adjacent nonnerve tissue. Results: All degenerated nerve segments were visible with bevonescein (100%; n = 20 nerves, 10 mice). In contrast, degenerated segments were invisible with oxazine-4 in 6 of 10 mice (60%; n = 12 nerves, 10 mice) and faintly perceptible in 4 of 10 mice (40%; n = 8 nerves, 10 mice). The mean SBR for oxazine-4 was lower than for bevonescein (1.27 ± 0.54 versus 3.31 ± 1.11; P < 0.001). Autonomic nerves demonstrated strong fluorescence with bevonescein (SBR, 1.77 ± 0.65; n = 7 nerves, 7 mice) but were invisible or faintly visible with oxazine-4 (SBR, 1.11 ± 0.14; n = 7 nerves, 7 mice; P = 0.02). Conclusions: Bevonescein produces successful intraoperative labeling of chronically degenerated and autonomic nerves in a rodent nerve transection model. In contrast, myelin-binding oxazine-4 does not meaningfully produce fluorescence labeling in these contexts. These findings may influence choice of nerve-labeling agents in the setting of delayed peripheral nerve construction. Clinical Relevance Statement: This study demonstrates the utility of bevonescein, a fluorescein-conjugated nerve-binding peptide, in the identification of degenerated facial nerves during fluorescence-guided surgery. This technology could benefit reconstructive surgeons who perform delayed peripheral nerve reconstruction.