Relation of ILN & superior laryngeal artery (SLA): In the majority of cases, ILN was found posterior to the SLA, followed by cranial and caudal relations, as depicted in Table 2 ( Fig. 1a,b, and c). Branches of ILN: in the majority of the specimens, ILN was single and did not give any branch before piercing the TM, as shown in Table 3 ( Fig. 2a, b, c & d).

On the right side of one cadaver, the ILN pierced the lamina of the thyroid cartilage, 4 mm below its upper border ( Fig. 3).

Branches of ELN: In all specimens, it supplied the cricothyroid muscle (CTM). However, in one left-sided specimen, the ELN communicated with a branch of the superior cervical sympathetic ganglion. ( Fig. 4)

Relation between ELN & ICM: •

In 90.6% of specimens, ELN was superficial to ICM,

Distance between ELN & apex of thyroid gland: Fig. 5a & b.

In the majority of specimens (90.6%), the distance between the ELN and AT was less than 1 cm (average 5.22 mm). These distances are tabulated in Table 4.

In 68.75% of specimens, the RLN was in the tracheoesophageal groove, whereas in 31.25%, it was lateral to the trachea.

Relation between RLN & inferior thyroid artery (ITA):

In the majority of specimens, the RLN was posterior to the loop of the ITA, as shown in Table 5 ( Fig. 6a, b, c and d).

Branches of RLN: In the majority of specimens, RLN provided three branches, as tabulated in Table 6 ( Fig. 7a, b and c).

The knowledge of anatomical localization of ILN is a prerequisite for surgeries involving sensory denervation of the larynx for odynophagia or neuralgia of superior larynx, supraglottic laryngectomy and laryngeal transplantation (Cernea et al., 1992; Yanagisawa et al., 1970; Schmidt et al., 1981; Strome et al., 2001) ^{7 , 15 – 17} The average length of ILN in previous studies was 44.9 mm (Furlan et al., 2003) ¹⁸ and 57.2 mm (Kiray et al., 2006). ¹⁹ The mean length of ILN was 33.8 mm in our study ( Table 1).

ILN and its relation with Thyrohyoid membrane

The identification of the area of the TM pierced by the ILN is crucial in the dissection of the neck for hypopharyngeal squamous cell carcinoma because any injury to this nerve may prove to be fatal due to loss of cough reflex (Sun et al., 2022). ²⁰ We observed a distance of 2 mm to 12 mm (0.2-1.2 cm), between the ILN from the point of penetrating the TM to the upper border of the thyroid lamina, with an average of 6.33 mm (0.63 cm). Paraskevas et al. (2012) noticed that this distance ranged between 0-1.8 cm, but in 87.5% of cases, it ranged from 0.1 to 1.2 cm. ²¹ They described the 0-1.8 cm range as the ‘wide zone’ and 0.1 to 1.2 cm as the ‘danger zone’ between TM penetration of ILN and thyroid cartilage. Familiarity with the ‘danger zone’ and any deviation from the usual pattern can be helpful in preserving ILN during surgical procedures in this region. Guven et al., 2021 have observed this distance ranging from 6-16 mm (0.6-1.6 cm) with the mean of 12 ±2.61 mm (1.2±0.26 cm) in patients undergoing laryngectomy. ²²

Relation between SLA and ILN

Although a varied relationship between ILN and SLA was observed in our study, in majority of the specimens, the ILN was posterior to the SLA before piercing the TM (51.56%) (( Table 2; Fig. 1a). It was above (cranial) the SLA in 29 specimens (45.31%) ( Fig. 1b) and below (caudal) the SLA in two specimens (3.1%) ( Fig. 1c). In contrast, Kiray et al. (2006) observed SLA crossing the ILN and piercing the TM above the ILN in 75% and below it in 25% of cases. ¹⁹ Furlan et al. (2003) reported an ILN medial to the SLA in 89% and distal to the SLA in 11%. ¹⁸ Paraskevas et al. (2012 have observed SLA parallel caudal and anterior to the ILN in 88.89% and superior and anterior to the ILN in 11.11% of cases. ²¹

Branching pattern of ILN ( Table 3)

The branching pattern of the ILN before it pierces the TM has been studied by several authors. Stephen et al. (1999) reported that ILN branched before piercing TM in 16% and after penetration in 84% of cases, ²³ whereas according to Kiray et al., it was 37.5% and 62.5%, respectively. ¹⁹ In a study by Guven et al. (2021), ILN was divided into three branches in 58.62% of cases and two branches in 41.37% of cases. ²² Paraskevas et al. (2012) reported three branches of ILN before it pierces the TM in 72.22% of cases and two branches in 27.78% of cases. ²¹ In our study, ILN did not give any branch before piercing the TM in 84.37% ( Fig. 2a), in 10.93% it was divided into two branches ( Fig. 2b), in 3.1% of the three branches ( Fig. 2c), and in only one right-side specimen, it was divided into multiple branches ( Fig. 2d). Awareness of the branching pattern of the ILN is important during surgical procedures of the larynx, such as laryngectomy. Even if a single branch of the ILN is removed during these procedures, it may cause significant postoperative complications; therefore, re-innervation of these branches is crucial in restoring the reflex function of the larynx. Additionally, we observed a rare variation in ILN piercing the lamina of the thyroid cartilage in one right-sided specimen ( Fig. 3). This variant course may result in some unexplained complaints due to nerve compression, leading to misinterpretation by the clinician if he/she is unaware of such a variation.

The principles of thyroidectomy are based on the identification and preservation of the ELN, as injury to the ELN is detrimental to the patient by producing a weak and hoarse voice. The close relationship between the ELN and the upper pole of the thyroid gland makes nerves vulnerable to injury during thyroid surgery. The lower the deviation of the nerve from the STA, the higher is the risk of injury during dissection (Potenza et al., 2017). ²⁴

In our study, the mean length of the ELN was 58 mm ( Table 1), whereas Furlan et al. (2003) reported a mean length of 62.6 mm, ¹⁸ Thomassin et al., (1982) ²⁵ as 65 mm, and Kambic et al., (1984) as 80 mm. ²⁶ The course of the ELN has been studied by several authors (Cernea et al., 1995; Kierner et al., 1998; Cha et al., 2017; Friedman et al., 2002). ^{27 – 30} Cernea et al. (1995) were the first to describe a classification of ELN with respect to its relationship with the STA. ²⁷ Accordingly, they classified 3 types of ELN wherein, type 1 crosses STA more than 1 cm above the apex of the thyroid gland (AT), type 2a crosses STA less than 1 cm above the AT and Type 2b crosses STA under AT cover. They mentioned that type 2b has a higher risk of damage than type 1 because, in this type, ELN is very close to STA. Kierner et al., (1998) revised nomenclature of Cernea et al., (1995) and described four types of ELN, where type 1 (42%), type 2 (30%) and type 3 (14%) are the same as type 1, type 2a and type 2b, respectively of Cernea et al., classification. ²⁸ In type 4, the ELN descends posterior to the STA and crosses its branches just above the AT. In our study commonest finding of ELN was type 2 (< 1 cm) of Kierner et al, in 82.81% ( Fig 5a) followed by type 1 (> 1 cm) in 9.37% and type 3 in 7.81% ( Table 4). We did not encounter Kierner et al. type 4 in our study. Cha et al. (2017) found ELN <1 cm distance from AT in 51.7%, >1 cm distance from AT in 27.6%, and under AT in 20.7%. ²⁹

ELN and inferior constrictor (ICM) & cricothyroid muscle (CTM)

Friedman et al. (2002) described three types of ELN in relation to ICM, CTM and STA. ³⁰ In type 1, ELN runs superficial to ICM throughout, descending with STA until its termination in CTM. In type 2, the ELN penetrates the ICM in its lower part, whereas in type 3, the ELN penetrates the ICM in its upper part. Type 2 and type 3 ELN of Friedman et al. (2002) are difficult to identify during thyroid surgery as they are deep to the ICM but are safer than type 1, as type 1 is more prone to injury. We observed type 1 of Friedman et al., in 90.6% of the specimen, type 2 in 6.26%, and type 3 in 3.1% of the specimens in our study. Few authors have reported these as unidentified ELN (Jonas et al., 2000; Lore et al., 1998). ^{31 , 32} In such situations, the ELN can be identified by nerve stimulation at the junction of the ICM and CTM (Thomassin JM., 1982). ²⁵ Additionally, we observed a rare variation wherein the ELN communicates with the superior cervical ganglion ( Fig. 4). Such variation might lead to disturbances in sympathetic function if the ELN is injured during thyroid or other neck surgeries.

The RLN is closely related to the ITA near the base of the thyroid gland, and inadvertent injury to the RLN may lead to hoarseness of voice and cough if unilateral. Bilateral injury may cause dyspnea or life-threatening laryngeal obstruction (Jiang et al., 2014). ³³

RLN and trachea-oesophageal groove

The course of RLN makes it vulnerable to injuries during certain surgical interventions in the neck (Culp et al., 2023). ³⁴ This can include surgeries involving the thyroid or parathyroid gland (Joliat et al., 2017), ³⁵ laryngeal malignancies (Benninger et al., 1998) ³⁶ or endotracheal intubation (Norris et al., 2011). ³⁷ We found RLN in the tracheoesophageal groove in 68.75% and lateral to the trachea in 31.25% of the patients. Sailaja (2016) reported RLN lateral to the trachea on 27 (15 right, 12 left) sides and in the tracheoesophageal groove on 23 sides (10 right, 13 left). ³⁸ Shao et al., (2016) have also reported that the right RLN is more anterolateral in position than the left RLN. ³⁹ Asgharpour et al., (2012) have seen the RLN anterior to the tracheoesophageal groove in 41.6%, in the groove in 33%, and posterior to the groove in 24.5%. ⁴⁰ They also found that the right RLN is more anterior than the left side. They opine that the right RLN is more exposed than the left. This may be due to differences in the embryological origin.

RLN and relation with inferior thyroid artery (ITA):

Surgical injuries are the most common cause of RLN palsy, accounting for 11 to 32% (Titche, 1976). ⁴¹ Surgeries which can cause injury to the RLN are thyroidectomy, parathyroidectomy, esophagectomy, tracheoplasty, mediastinoscopy, correction of patent ductus arteriosus, etc. (Titche, 1976; Friedrich et al., 1998). ^{41 , 42} Thyroidectomy is the most common cause of RLN injury, accounting for approximately 5 to 14%, according to various studies (Hayward et al., 2013; Friedrich et al., 1998, Zakaria et al., 2011). ^{8 , 42 , 43} This can occur when a branch of the ITA is accidentally injured during the procedure. In most specimens (50%), the RLN was posterior to the ITA compared to other positions ( Table 5; Fig. 6a, b, c, d) in our study. This is in accordance with the results of Tang et al. (2012) and Sailaja (2016) (86.25% and 56%, respectively). ^{12 , 38} On the contrary, Campos et al. (2000) observed RLN behind the ITA in a comparatively lesser number of specimens. ⁴⁴ However, they observed RLN passing between the branches of ITA in the majority of the cases they studied (≈50%). When the RLN passes between the branches of the ITA, as shown in Fig. 6c, it makes it more vulnerable to injury during dissection for surgical procedures. Few researchers have opinioned that RLN injury is more common when the nerve is anterior or between the branches of the ITA than posterior to it (Skandalakis et al., 1976; Steurer et al., 2002). ^{45 , 46} Surgeons should be aware of these types of variable relations between the RLN and ITA to avoid nerve damage.

Branching pattern of RLN

The number of branches from the RLN before it pierces the muscles of the larynx is clinically significant because the branches may entangle the ITA, place the nerve at risk, and paralyze the laryngeal muscles (Shao et al., 2016). ³⁹ In our study, a varied number of branches of the RLN before piercing the larynx was found, with a maximum of 64% ( Table 6; Fig. 7a, b & c). Sailaja (2016) revealed that the number of branches of the RLN was 2 (75%), 3 (9%), more than 3 in 8%, and 0 in 8%. ³⁸ Common pattern of extralaryngeal branching of the RLN is bifurcation with an incidence of 51%, and trifurcation or multiple branches have been reported by a few authors (Shao et al., 2016; Cakir et al., 2006; Henry et al., 2016). ^{39 , 47 , 48} This type of branching of RLN before its entry into the larynx is the common cause for surgical morbidity in thyroid surgery (Shao et al., 2016). ³⁹ Other than ITA, other reliable landmarks used for the identification of the RLN are distance between the entrance of RLN to the larynx and inferior cornu of the thyroid cartilage or inferior tubercle of the thyroid cartilage or anterior part of cricoid cartilage (Cakir et al., 2006). ⁴⁷ We have found the mean distance between the cricothyroid joint and the entrance of RLN into the larynx as 19.5 mm with the range of 13 mm and 30 mm. The present study has few limitations like effect of formalin on the dimensions of the nerves and gender based comparison, which was not performed.

This study was conducted on 64 dissected neck of formalin fixed cadavers, which were utilized for undergraduate teaching purpose, in the department of Anatomy of our institution. The study protocol is approved by Institutional Ethics Committee of Kasturba Medical College, Mangalore (ECR/541/Inst/KA/2014/RR-17) on 21 ^st August 2019 (IEC KMC MLR 08-19/355).

Consent form was waived by the Institutional Ethics committee, as it is a cadaveric study. However the body donors had given written consent before donating the body to our department.