Surgical landmarks for identification and preservation of the internal branch of the superior laryngeal nerve

To determine the topographical anatomic features of the internal branch of the superior laryngeal nerve (ibSLN) at the thyrohyoid membrane entrance area in relation to certain consistent anatomical structures. Twenty-two fresh adult head cadavers (9 male, 13 female; age range 52–95 years) with no signs of abnormality in the neck were dissected to determine the anatomic relationship of ibSLN and superior border of thyroid cartilage, thyroid notch, carotid bifurcation, hyoid corpus, and hyoid greater cornu. The topographical relationship between ibSLN and superior border of thyroid cartilage, thyroid notch, carotid bifurcation, hyoid corpus, and hyoid greater cornu was identified bilaterally in all cadavers. According to the measures, danger zone and safe zone areas for surgical could be predicted and for surgical manipulations as well. We provided the surgical anatomy and important landmarks for determining the internal branch of superior laryngeal nerve in the thyrohyoid membrane entrance region to avoid surgical damage during surgeries of this region.


Introduction
Internal branch of superior laryngeal nerve (ibSLN) provides sensory innervation for the major part of the laryngeal mucosa above the vocal cords [16,19]. During swallowing, vomiting, and coughing, the glottic closure reflex mechanism protects us from aspiration. Damage or dysfunction of ibSLN may cause insufficient mucosal sensation and lack of glottic closure reflex that may lead to aspiration of secretions/foreign bodies to the lungs [2,7,10,21].
The risk of damaging of ibSLN is higher in laryngeal surgeries, neck dissections and thyroid surgeries, and cervical spine surgeries [1,8,9,11,20]. For providing the healthy glottic closure reflex during coughing, vomiting, and swallowing that protects us from aspiration of secretions, preservation of ibSLN is very important. Especially during supra-glottic and supracricoid partial laryngectomies, neck dissections and mass excisions from this region, identification and preservation of this nerve should be done to maintain the protective function of larynx.
The topographical anatomic features should be determined so as to identify and prevent damage of the nerve. For the otolaryngologists, preservation of the nerve at least on one side is very important for the protective functions of the larynx especially in partial laryngectomy surgeries, neck dissections, and thyroid surgeries. In this study, we aimed to investigate the relationship between the ibSLN at the point of penetrating the thyrohyoid membrane and certain consistent anatomical landmarks that are superior border of thyroid cartilage, thyroid notch, carotid bifurcation, hyoid corpus, and hyoid greater cornu in order not to identify and preserve the nerve.

Materials and methods
The research protocol was approved by the local ethics committee (protocol no: 523; 25.05.2021) and was conducted in accordance with the Declaration of Helsinki. Between June 2021 and December 2021, 22 fresh human head cadavers were used to determine the topography of 1 3 ibSLN. The cadavers were placed in supine position, and bilateral dissection (44 sides in total) was performed under a Zeiss Extaro 300 stereoscopic dissection microscope.
The laryngeal region was carefully dissected, and thyrohyoid membrane was preserved to fully demonstrate the entrance of the ibSLN and its relationship between other consistent structures. The sternocleidomastoid muscle was laterally deviated, and carotid sheet and then the vagus nerve were identified. Following the tracing of the vagus nerve, ibSLN and its penetrating point in the thyrohyoid membrane were identified. Five different distances were measured via using ruler and recorded from where the nerve pierced the membrane. The distances between penetrating point of the ibSLN to the membrane and corpus of the hyoid bone, the greater cornu of the hyoid bone, carotid bifurcation, vertical distance to superior border of thyroid cartilage, and horizontal distance to thyroid notch were all measured by means of ruler and noted (Figs. 1, 2, 3, 4). The ruler was preferred for the measurements due to its easy utility.

Statistical analyses
Data were analyzed by means of SPSS v. 25 software. All data are presented as mean ± standard deviation. Continuous variables were visually assessed for normality using histograms and Q-Q plots. Independent sample Student's t test was used to compare the mean values of parameters between men and women. Paired sample test was applied to compare right-and left-side measurements of cadavers. A p value of < 0.05 was considered statistically significant.

Results
Of the 22 fresh human head cadavers, 9 were male and 13 were female with an age range 52-95 years. Demographical features of the cadavers were noted in Table 1. The ibSLN was dissected bilaterally in each cadaver, and the most inferior ramus of each ibSLN at its entry point to thyrohyoid membrane was selected for measurements. The measured  parameters of all cadavers are listed in Table 2, and the parameters according to the gender are listed in Table 3.
Vertical distance between the penetration point of ibSLN to thyrohyoid membrane and the superior border of thyroid cartilage (TS) was 0.60 ± 0.14 SD cm for all measurements. There was no statistically difference between male and female cadavers (Table 3).
We draw a vertical line from the penetrating point of the ibSLN to the superior border of the thyroid cartilage. The horizontal distance between the midpoint of the thyroid notch and the intersecting point of the line we draw at the superior border of the thyroid cartilage (TN) was measured. The distance was 1.97 ± 0.29 SD cm for all cadavers. It was statistically longer in male cadavers. The mean distance was 2.10 ± 0.19 SD cm in male cadavers, and it was 1.88 ± 0.24 SD cm in female cadavers (p = 0.034) ( Table 3).
The distance from the midpoint of hyoid corpus (HC) to the penetration point of the ibSLN was 2.04 ± 0.30 SD cm for all cadavers. There was no statistically difference between genders (Table 3).
We found the distance between greater horn of the hyoid (HH) and the penetration point of the ibSLN as 1.65 ± 0.38 SD cm. There was no statistically difference between genders ( Table 3).
The distance between the carotid bifurcation (CB) and the penetration point of ibSLN was 1.67 ± 0.32 SD cm. It was also found to be longer in male cadavers that was statistically   (Table 3).
In all cadavers, we observed the penetrance points of the SLA and ibSLN were common at the thyrohyoid membrane area.

Discussion
It has been reported that the inferior branch of ibSLN serves as the principal sensory nerve in the region above the level of the vocal cords and sends fibers to posterior glottis, hypopharynx, and subglottic mucosa [15,17,21]. The preservation of ibSLN is mandatory for providing sensory innervation from most of the laryngeal mucosa.
It is possible to damage the ibSLN during surgical procedures like neck dissections, partial laryngectomies, cervical spine surgery, or carotid endarterectomies [1,11]. To prevent the nerve during the surgeries of this region, knowledge about the topographic anatomy of ibSLN becomes important. More accurate documentation of the topographic anatomy of the nerve and its relation to certain anatomical landmarks should be identified, thus promoting safer handling during surgery.
Superior laryngeal nerve (SLN) arises from vagus nerve and deep into the carotid artery, and it splits into internal and external branches at the level of lesser cornu of hyoid [5,18,21]. The ibSLN courses medially and accompanied by the superior thyroid artery [5,6]. Entrance point of the ibSLN to the larynx is through thyrohyoid membrane [2]. While some authors reported that superior laryngeal artery (SPA) and ibSLN enter the thyrohyoid membrane through a common opening, others reported that SLA pierces the membrane below ibSLN from a different foramen [2][3][4]21]. In the present study, we observed the penetrance points of the SLA and ibSLN were common at the thyrohyoid membrane area.
After the penetration of thyrohyoid membrane, ibSLN was shown to be divided into two branches or three branches [9,15,17,21]. Mucosa of the epiglottis and some part of vallecula are innervated by the superior branch, and the aryepiglottic folds are innervated by the middle branch. Inter-arytenoid muscles receive branches from the inferior branches [12,13,15,17].
Knowledge about the relationship between the topographic anatomy of ibSLN and certain anatomical landmarks is very limited in the literature. The diameter of the ibSLN is thin in the thyrohyoid membrane region and thus vulnerable for damage around this area [9,12,17]. In the present study, we demonstrated its topographic anatomy at the entrance point through the thyrohyoid membrane in relation to the superior border of thyroid cartilage, thyroid notch, carotid bifurcation, hyoid corpus, and hyoid greater cornu. During the surgical procedures of this region (either dissection or the placement of retractor blades), these anatomical landmarks are frequently encountered structures.
In the present study, we found the distance between the carotid bifurcation (CB) and the penetration point of ibSLN as 1.67 ± 0.32 SD cm. It was also longer in male cadavers that was statistically different (p = 0.012). During exposure, the relation between the nerve and carotid bifurcation and the difference between genders should be taken into consideration.
Prior to piercing the thyrohyoid membrane, the ibSLN crosses the greater cornu of the hyoid bone. We found the distance between greater horn of the hyoid (HH) and We found that vertical distance between the penetration point of ibSLN to thyrohyoid membrane and the superior border of thyroid cartilage (TS) was 0.60 ± 0.14 SD cm. In another study, the authors found the same distance ranging from 0 to 1.8 cm. They noted that in 87.5% of the cases, the distance ranged from 0.1 to 1.2 cm [14]. During the incisions to thyrohyoid membrane for partial laryngectomies or laryngocele surgeries, the distance between superior border of the thyroid bone and the penetration point of the nerve should be kept in consideration.
In our study, the horizontal distance between the midpoint of the thyroid notch and the intersecting point of the line we draw at the superior border of the thyroid cartilage (TN) was 1.97 ± 0.29 SD cm. It was statistically longer in male cadavers (p = 0.034). As a result, we can conclude that the data should be remembered for male and female candidates.
For the thyroid notch and carotid bifurcation landmarks, the distances between them and the penetrating point of the nerve were significantly different between genders. One of the possible reasons for this can be the differences of thyroid cartilage anatomy between genders. This should be considered during surgery of this region.
In clinical practice, by means of knowledge of the topographical anatomy, danger zone for damaging the ibSLN can be predicted, and damage risk can be minimized. The area that should be preserved can be identified. In our study, ibSLN penetrated the thyrohyoid membrane 1.97 ± 0.29 cm lateral to the midpoint of thyroid notch and 0.60 ± 0.14 cm superior from the superior border of the thyroid cartilage. The penetrating point is also 2.04 ± 0.30 cm distance from the midpoint of hyoid corpus and 1.65 ± 0.38 cm from the greater horn. The area of the intersection of these measurements should be defined as danger zone, and the surgeons should be more delicate around the danger zone. Besides, the safe zone can also be predicted. To protect the function of the larynx, at least one ibSLN must be preserved during the manipulation of this area. According to our results, from the midpoint of thyroid notch, 1 cm lateral area seems to be the safe zone for ibSLN while there is risk of damaging the nerve lateral to 1 cm distance from thyroid notch and 0.5 cm above the superior border of thyroid cartilage.
There are some limitations in our study. First, the ethnicities of the cadavers were unknown; therefore, the measurements may vary in people from different geographic origins. Second, the number of the cadavers may not be sufficient to identify anatomical variants.

Conclusion
We provided the surgical anatomy and important landmarks for determining the internal branch of superior laryngeal nerve in the thyrohyoid membrane entrance region. With the use of this knowledge, the nerve can be identified and/ or preserved during surgical interventions, such as partial laryngectomies, neck dissections, and cervical spine surgeries.