Online Poster Portal

OBJECTIVE
Recurrent laryngeal nerve (RLN) injury leads to neuromuscular junction (NMJ) degeneration, contributing to vocal fold atrophy and dysfunction. While reinnervation procedures are increasingly utilized, the timeline and extent of NMJ recovery remain poorly defined. This study characterizes NMJ remodeling following denervation and reinnervation in a rabbit model.

METHODS
Eight New Zealand rabbits underwent recurrent laryngeal nerve (RLN) transection with or without immediate ansa cervicalis–to–RLN reinnervation. Animals were humanely sacrificed at four timepoints (30, 60, 90, and 120 days); data analysis is currently complete for the 30- and 60-day timepoints, which are the focus of this report. Thyroarytenoid muscles were analyzed using whole-mount immunohistochemistry to label pre- and post-synaptic neuromuscular junction (NMJ) components. Confocal microscopy was used for image acquisition.

NMJs were quantitatively assessed using 3D reconstruction and analysis in Aivia (Leica Microsystems), enabling volumetric measurement of NMJ endplates and acetylcholine receptor (AChR) aggregates. Preliminary blinded classification of postsynaptic morphology into pretzel-like, fragmented, collapsed, and dead states is ongoing. To control for inter-animal variability in baseline NMJ size, treated-side measurements were normalized to the contralateral healthy-side mean per animal, yielding a ratio where 1.0 indicates full recovery to baseline. Between-group comparisons used the Mann-Whitney U test with Bonferroni correction (α = 0.006); effect size is reported as rank-biserial correlation (r), where 1.0 indicates complete group separation.

RESULTS
Preliminary analysis demonstrated progressive decline in NMJ endplate and AChR aggregate volume in the RLN transection group over time. NMJ morphology descriptively transitioned from organized, pretzel-like structures to increasingly fragmented forms. AChR aggregate volume declined significantly between 1 and 2 months post-surgery (median normalized ratio 0.16 vs. 0.07; p < 0.001).

Reinnervated vocal folds demonstrated early NMJ reinnervation and reconstitution. At 1 month, the anastomosis group had significantly larger NMJ endplate volumes than the RLN transection group (median normalized ratio 0.27 vs. 0.16; p = 0.002, r = 0.29). By 2 months, the anastomosis group demonstrated further significant recovery in both endplate volume (median ratio 0.90; p < 0.001, r = 0.89) and AChR aggregate volume (median ratio 0.90; p < 0.001, r = 0.90), approaching healthy contralateral baseline levels. NMJ morphology showed increased structural complexity compared to the RLN transection group, though heterogeneity persisted with coexistence of fragmented and more organized forms.

CONCLUSIONS
These preliminary findings suggest that RLN transection results in progressive NMJ degeneration characterized by endplate fragmentation and synaptic volume loss. Ansa cervicalis–to–RLN reinnervation promotes early structural recovery, with NMJ volumes approaching healthy baseline by 2 months. Future work will incorporate electromyography to correlate structural remodeling with functional outcomes.