A Surgical Mouse Model for Advancing Laryngeal Nerve Regeneration Strategies

Abstract

Iatrogenic recurrent laryngeal nerve (RLN) injury is a morbid complication of anterior neck surgical procedures. Existing treatments are predominantly symptomatic, ranging from behavioral therapy to a variety of surgical approaches. Though laryngeal reinnervation strategies often provide muscle tone to the paralyzed vocal fold (VF), which may improve outcomes, there is no clinical intervention that reliably restores true physiologic VF movement. Moreover, existing interventions neglect the full cascade of molecular events that affect the entire neuromuscular pathway after RLN injury, including the intrinsic laryngeal muscles, synaptic connections within the central nervous system, and laryngeal nerve anastomoses. Systematic investigations of this pathway are essential to develop better RLN regenerative strategies. Our aim was to develop a translational mouse model for this purpose, which will permit longitudinal investigations of the pathophysiology of iatrogenic RLN injury and potential therapeutic interventions. C57BL/6J mice were divided into four surgical transection groups (unilateral RLN, n = 10; bilateral RLN, n = 2; unilateral SLN, n = 10; bilateral SLN, n = 10) and a sham surgical group (n = 10). Miniaturized transoral laryngoscopy was used to assess VF mobility over time, and swallowing was assessed using serial videofluoroscopy. Histological assays were conducted 3 months post-surgery for anatomical investigation of the larynx and laryngeal nerves. Eight additional mice underwent unilateral RLN crush injury, half of which received intraoperative vagal nerve stimulation (iVNS). These 8 mice underwent weekly transoral laryngoscopy to investigate VF recovery patterns. Unilateral RLN injury resulted in chronic VF immobility but only acute dysphagia. Bilateral RLN injury caused intraoperative asphyxiation and death. VF mobility was unaffected by SLN transection (unilateral or bilateral), and dysphagia (transient) was evident only after bilateral SLN transection. The sham surgery group retained normal VF mobility and swallow function. Mice that underwent RLN crush injury and iVNS treatment demonstrated accelerated and improved VF recovery. We successfully developed a mouse model of iatrogenic RLN injury with impaired VF mobility and swallowing function that can serve as a clinically relevant platform to develop translational neuroregenerative strategies for RLN injury.

Keywords: Animal model; Deglutition; Deglutition disorders; Dysphagia; Electrical stimulation; Laryngeal nerve; Regeneration.

Fig. 1

Size comparison between a human and mouse larynx. A photograph of side-by-side specimens of a human and mouse larynx next to a US penny (approximately 1.9 cm diameter), overlaid on a proportionately scaled schematic of a human head and neck. The cartilaginous/bony framework is drawn on both specimens for anatomical clarity. A 2-cm scale bar is shown for added size perspective

Fig. 2

Microsurgical approach. a Our microsurgical approach in mice to access the laryngeal nerves via a midline ventral neck incision is typically a bloodless procedure. Micro-retractors (white asterisks) are used to maintain the large salivary glands out of the surgical field. The strap muscles obscure the RLN (b) and SLN (c), which are shown at higher magnification in images b and c, respectively. b To access the RLN, the strap muscles are carefully divided along the midline fascia and gently retracted with micro-forceps to expose the tracheal rings (numbered) and RLN (yellow arrows). The inset image shows a close-up of the target tracheal ring region, with the RLN (yellow arrows) isolated from the inferior thyroid artery (black asterisks) with a micro-hook in preparation for surgical transection. c To access the SLN, the strap muscles covering the lateral aspect of the larynx are gently elevated with micro-forceps to expose the SLN (yellow arrows) traveling alongside the superior laryngeal artery (black arrows) near the bifurcation of the common carotid artery (black asterisk). The inset image shows a close-up of the SLN (yellow arrow) isolated from the fascia with micro-forceps

Fig. 3

Laryngoscopy assay. a Lateral view of our custom endoscopy suite for mice, with labeled components. b An anesthetized mouse in dorsal recumbency undergoing laryngoscopy, with the head gently secured in ear bars. The micromanipulator in image a is used to precisely guide oral insertion, gentle advancement, and precise positioning of the sialendoscope (fitted with a custom laryngoscope) to visualize the larynx. c Representative endoscopic image of the murine larynx at maximum VF abduction during spontaneous breathing, taken from a 30 fps video at baseline (i.e., before surgery). Visible laryngeal structures of interest include the bilateral VFs (black asterisks), epiglottis, aryepiglottic folds (AEF), and pyriform sinuses (PS). In contrast to the human larynx, the VFs of mice retain midline proximity at the dorsal commissure (yellow asterisk), and the ventral commissure is consistently obscured by the epiglottis

Fig. 4

Videofluoroscopic swallow study (VFSS) assay. a A custom VFSS test chamber (black asterisk) is positioned in lateral view between the X-ray source (white asterisk) and image intensifier (yellow arrow) of our custom, miniaturized c-arm fluoroscope. The X-ray beam is turned on only when mice are actively drinking, identified via a webcam positioned above the test chamber. A remote-controlled positioning lift is used to readily maintain the mouse’s aerodigestive tract within the fluoroscopy field of view. b Close-up of the VFSS test chamber in image a, designed to promote voluntary drinking of liquid contrast agent by mice with minimal behavioral distractions. Note the bowl within the test chamber is filled using a syringe delivery system that is manually controlled a few feet away from the fluoroscope. c, d Representative X-ray images from a 30 fps video of a mouse drinking in lateral view. Image c shows the oropharyngeal stage of swallowing, immediately prior to triggering of the swallow reflex. Note the liquid contrast agent accumulating in the vallecula within the pharynx, which is the stereotypical swallow trigger point in mice. Image d shows the esophageal stage of swallowing. Note the swallowed bolus traversing the distal esophagus into the stomach, while liquid contrast continues to accumulate in the vallecula prior to triggering a subsequent swallow

Fig. 5

VF immobility after RLN transection. Endoscopic images showing the bilateral VFs of an anesthetized mouse during spontaneous breathing of room air after transection of the right RLN. Top Maximal VF abduction during inspiration. Bottom Maximal VF adduction during expiration. Note the paralyzed (immobile) right VF during the inspiratory and expiratory phases of the respiratory cycle

Fig. 6

Effect of laryngeal nerve transection injury on swallow function. Of the four surgical groups investigated, only unilateral RLN transection and bilateral SLN transection had a statistically significant effect on swallow function. At the acute (1-week post-surgery) time point, lick rate (i.e., tongue motility) was significantly slower after unilateral RLN transection (red line, Oral Stage—left panel), and pharyngeal transit time was significantly longer after bilateral SLN transection (purple line, Pharyngeal Stage—middle panel). In addition, esophageal transit time was longer for the unilateral RLN and bilateral SLN transection groups (red and purple lines, respectively, Esophageal Stage—right panel); however, results did not reach statistical significance. At the chronic (3-month post-surgery) time point, swallow function was not significantly different from baseline function. Asterisk denotes statistical significance (p < 0.05) based on change scores; error bars = ± 1 SEM

Fig. 7

Effect of iVNS on VF mobility after RLN crush injury in aged mice. VF mobility improved in a stair step pattern after RLN crush injury in iVNS-treated mice > 1 year of age. Untreated age-matched mice fluctuated in VF mobility, with minimal improvement after a 12-week post-surgical recovery period. VF mobility was scored using a 3-point Likert scale. Error bars = ± 1 SEM; n = 3 mice per group. Time points with collapsed error bars indicate no within group variation of VF mobility scores. Asterisk = p = 0.047, based on a single t test at the final time point

Fig. 8

Sihler staining for laryngeal nerve mapping. A representative Sihler stained sample from a mouse in the bilateral SLN transection group demonstrates that the murine laryngeal framework and laryngeal nerve branching pattern are remarkably similar to humans. Red X indicates the location of SLN transection. Black arrows show the origin of nerve branches from the RLN trunk bilaterally. CN X Cranial nerve 10 (i.e., vagus nerve), RLN recurrent laryngeal nerve, SLN superior laryngeal nerve

Fig. 9

Post-mortem dissection demonstrating RLN branching. The left side shows that with minimal retraction of the soft tissues, as is used in our surgical approach, RLN branching is not visible. Instead, only the RLN trunk (gray arrow) can be seen running between the inferior thyroid artery (gray asterisk) and trachea. As shown on the right side, RLN branching is visible only during extreme lateral retraction of the midline strap muscles and fascia. In this specimen, the right RLN trunk (black arrow) has been pulled away from the inferior thyroid artery (black asterisk) to expose a single RLN branch (between the green arrows) near the 6th tracheal ring

Fig. 10

Murine laryngeal framework. a Hematoxylin and eosin (H&E) stained transverse Sect. (10 μm) of a mouse larynx at the level of the VFs, with labeled structures. b Endoscopic image of the murine VFs, with corresponding labeled structures from image a. In contrast to the human larynx, mice have proportionately larger arytenoid cartilages (black asterisks) and a proportionately smaller mucosal region extending beyond the vocal processes (VP) to the ventral commissure (red asterisk). In the mouse, the ventral commissure (which is obscured during laryngoscopy) is framed by a U-shaped alar cartilage (AC) that does not exist in humans. During spontaneous breathing in the mouse, the most dorsal portion of the arytenoids (yellow asterisk; dorsal commissure) remains relatively fixed near midline, serving as a pivot point for VF abduction and adduction. As a result, VF movement in the mouse is more readily apparent at the ventral (yellow bidirectional arrow in image b rather than the dorsal (posterior) region as in humans. TC thyroid cartilage, AC alar cartilage, VP vocal process, TA thyroarytenoid muscle (M medial belly, L lateral belly, O oblique belly), S strap muscles; blue asterisk: glottis; black asterisk: arytenoid cartilage; yellow asterisk: dorsal commissure; red asterisk: ventral commissure. Scale bar 500 μm