Recurrent Laryngeal and Ansa Cervicalis Nerves Morphometry in a Rabbit Model

Introduction: Ansa cervicalis–to–recurrent laryngeal nerve (RLN) anastomosis is commonly employed for laryngeal reinnervation. The rabbit model has been used to study laryngeal reinnervation; however, detailed morphometric characterization of these recipient and donor nerves is limited. Accurate quantification of nerve architecture is necessary to inform reinnervation strategies and optimize experimental models. This study used automated image analysis to perform a morphometric characterization of these nerves in a rabbit model.

Methods: The RLN and ansa cervicalis nerves were harvested from 10 adult rabbits. Nerve cross-sections were stained with FluoroMyelin and imaged using confocal microscopy. Axon count was quantified in all nerves. Axon diameter, myelination thickness, and g-ratio were measured in a subset of RLNs and ansa cervicalis nerves using automated image analysis. G-ratio distributions were analyzed descriptively within and between nerves.

Results: Axon counts varied among RLNs, with a mean of 725 ± 304, and a median of 826 (IQR 550-925). The ansa cervicalis nerve demonstrated substantially fewer axons, with a median count of 148. RLN axon diameters ranged from 0.2 – 13.3 µm with a mean of 4.0 ± 0.4 µm and a median of 4.1 µm (IQR 3.8-4.2). Ansa cervicalis axon diameters ranged from 0.1 to 16.8 µm with a mean of 4.7 ± 2.5 µm and a median of 4.4 µm (IQR 3.2-5.8). The G-ratio distribution for the RLNs was unimodal, with a mean of 0.5 ± 0.1 and a median of 0.6 (IQR 0.5-0.6). The ansa cervicalis axons demonstrated a broader distribution with greater dispersion (mean g-ratio 0.5 ± 0.1; median of 0.53 (IQR 0.4–0.6).

Conclusions: In a rabbit model, the RLN exhibited higher axon counts than the ansa cervicalis nerve, whereas axon diameters were greater in the ansa cervicalis. The g-ratio values—reflecting relative myelination thickness—were comparable between nerves. Automated image analysis enabled reproducible morphometric quantification. These complementary morphologic features may inform donor–recipient matching in laryngeal reinnervation, although larger studies are needed support definitive comparisons.