Title: Identification of valid anatomical landmarks to locate and protect recurrent laryngeal nerve during thyroid surgery: A cadaveric study Abstract: Purpose: Recurrent laryngeal nerve (RLN) is the most crucial structure in terms of intricacy, anatomic variations of the nerve may further make thyroid surgery cumbersome. The present study was undertaken to provide comprehensive knowledge about the soundness of Berry’s ligament (BL), tracheoesophageal groove (TEG) and inferior thyroid artery (ITA) and the posterior border of the thyroid gland as anatomical landmarks to identify the nerve intraoperatively. Methods: Thirty adult cadavers were dissected to identify the RLN in the neck and to locate it in relation to the aforementioned anatomical landmarks. Results: The RLN/BL relationship: RLN was most often located superficial to the BL (88.3%), followed by deep to the BL in 8.4%, and piercing the BL in 3.3 % of cases respectively. The RLN/TEG relationship: the RLN was located inside the TEG in most of the cases (71.7%), followed by RLN lying outside the TEG in 28.3%. When located outside the groove, it was most commonly found lateral to the TEG (64.7%). RLN/ITA relationship: the nerve was passing deep to the artery in most of the cases (65%), followed by superficial (30%) and rarely (5%) in-between the branches. RLN/posterior border of thyroid relationship: In 57 (95%) cases, RLN was coursing in the area posterior to the posterior border of the gland with an average distance of 6.31 mm ranging between 3.12 mm to 10.30 mm. Conclusions: Both the BL and TEG are potentially crucial for safeguarding of RLN. Although in results, BL turns out to be more consistent than TEG, we propose the utilization of both these anatomical landmarks together for complication-free neck surgeries. Whereas, Posterior border of thyroid turns out to be the single most consistent landmark for identification of RLN. Keywords: Groove; Landmark; Ligament; Nerve injury; Thyroidectomy Introduction: The recurrent laryngeal nerve (RLN) is a branch of the vagus. RLN may have variations during the course from thorax to larynx and it is highly vulnerable during thyroid surgery. It has been observed that RLN enters larynx after dividing into two or more branches. The prevalence of RLN injury is 0.1-8% during thyroid surgery. During surgery, RLN damage is associated with its improper identification and difficult isolation due to adhesion. The course of injury to the recurrent laryngeal nerve is a critical but avoidable complication of thyroidectomy and other neck surgeries. This complication may be avoided by prior identification of the nerve with the assistance of important anatomical landmarks found along its course [10, 14]. A reliable landmark and method for identifying the RLN are imperative to avert postoperative complications such as hoarseness of voice and vocal cord paralysis. Intraoperatively, RLN can be identified by several techniques, such as palpation, intraoperative nerve monitoring (IONM), and direct inspection felicitated by crucial anatomical landmarks such as Berry’s ligament (BL), the Tracheoesophageal Groove (TEG), Inferior thyroid artery (ITA) and Zukerkandl’s tubercle. A volatile relationship exists among these parameters [12]. The lucid identification of these landmarks improves the sensitivity of IONM. BL is a fibrous structure that anchors the thyroid gland to the first three tracheal rings and the most common site of injury to the RLN, where the nerve penetrates into the larynx [1, 2]. Few authors have proposed BL as a reliable anatomical landmark to locate the RLN intraoperatively [5, 21], however, this has yet to be widely accepted and implemented as standard practice [2]. Previous reports revealed capacious variations in the location of the RLN with respect to the BL, the nerve coursing superficial to the BL ranges from zero to 100 % [4, 6, 21] while the penetrating pattern varies between zero [4, 6, 9, 13, 16, 21] to 31.6% [20]. A further thorough cognizance of the frequency with which nerves penetrate the BL will bring down the complications, as traction-related injuries are commonly linked with RLNs taking a piercing course [1, 13]. The TEG is a deep sulcus formed by the concourse of the trachea anteriorly and oesophagus posteriorly. It provides a fairly safe haven for the RLN as it ascends towards the larynx [20] and can also be used as a landmark for identifying the nerve [25]. During surgery, the TEG needs to be inspected routinely for the presence of the nerve, otherwise, it might be overlooked unwittingly [23]. Previous studies also revealed that the location and relationship of the RLN to the TEG have varied widely. The reported presence of an RLN coursing within the TEG has ranged from 24.9% to 100% [8, 17]. ITA is another crucial landmark for recognizing the RLN intraoperatively. The anatomical relationship between the RLN and the ITA is volatile (Fig. 1). RLN is related to the ITA differently on both sides. On the right side, RLN crosses ITA at the anterior aspect but, the same crosses ITA at the posterior aspect on the left side. The clear visualization of the relationship between artery and nerve is the necessity of precise surgical technique to avoid the damage of neurovascular structures. The fourth landmark is the posterior border of the thyroid lobe, which points toward the tracheoesophageal groove. Usually, RLN travels in the area posterior to this landmark, but in a few instances, it can course anteriorly making itself vulnerable to injury during partial thyroidectomy. RLN may have a very intimate relationship with the thyroid capsule along the posterior border. This relationship is a possible indicator in calculating the safe zone in deciding the line of incision in subtotal thyroidectomy or near-total thyroidectomy (Fig 2). Although, the distance between the RLN and posterior border of the thyroid is crucial, very little is mentioned in the literature. The high variability of the data published to date, forbidden us from making solid opinions regarding the robustness of these soft tissue structures as valid and reliable anatomical landmarks for the RLN identification. The lofty incidence of RLN injury, and the need to safeguard the structure during surgery, has persuaded substantial research during recent years. However, a reliable and uniform technique for identifying and safeguarding the nerve has yet to be conceived [12]. With such a background, the aim of the current work was to evaluate the reliability of the BL, TEG, ITA and posterior border of the thyroid gland as anatomical landmarks for identifying the RLN. Reliable landmarks would help to alleviate the frequency of iatrogenic injury and long-term postoperative complications. Methods: A total of 30 formalin-fixed adult cadavers (25 male and 5 female) aged between 45 to 75 years were dissected bilaterally at the dissection hall of Anatomy department, to investigate the anatomical relationship of the RLN to the BL, TEG, ITA and the posterior border of the thyroid gland. The dissection of the neck was done as per Cunningham’s practical manual for dissection. A wooden block with slight concavity in the middle was placed underneath the nape of the neck, so as to keep the neck in extended and fixed position without undue movement during dissection. A midline vertical incision was given thereafter, starting from the symphysis menti and continuing downwards up to the sternal notch. Then the incision was extended laterally from the sternal notch up to the tip of the acromion along the anterior border of subcutaneous clavicle. Subplatysmal flaps of the skin along with the superficial facia and subcutaneous fatty tissue were then raised and reflected laterally for better exposure of the anterior triangle of neck. The strap muscles (sternohyoid, sternothyroid and omohyoid) lying in front of the thyroid gland were identified, transacted transversely through middle of the muscle belly and reflected upward and downward. The dissection was carried out until the thyroid gland was clearly visible. Structures surrounding the gland (muscles, fatty and connective tissues) were kept away from the area of dissection by applying mechanical traction with the help of metallic hook or forceps. The RLN was visualized by applying slight medial traction on the trachea. Once, the area was clearly visible, the relationship of the nerve to the BL (superficial, piercing, or deep) was noted first (Figure 3). The nerve was then traced downwards to the TEG, and the position of the RLN with respect to this groove was recorded (inside or outside, when outside: anterior, anterolateral, lateral, and posterior) (Figure 4). After noting the position of the nerve in relation to the BL, TEG and ITA, its location and distance from the posterior border of the thyroid gland was also measured with the help of a digital vernier callipers (Mitutoyo, Japan) (Figure 3). The distance between RLN and the midpoint of the posterior border was measured. Percentage prevalence of RLN variation was calculated. The mean and standard deviation were computed for the distance between the posterior border of thyroid and RLN with the help of Microsoft excel 2019. Results: The present study was conducted on 30 formalin-fixed adult cadavers aged between 45 and 75 years. Amongst them, 25 (83.3%) were male and 5 (16.7 %) were female. The RLN was recognized reciprocally in all 30 cadavers (n=60). RLN/BL relationship: In 53 (88.3%) cases, the RLN was located superficial to the BL, in 5 cases, the nerve was piercing the ligament (Figure 3) and in 2 cases, coursing deep to it. The relationship was bilaterally symmetrical in 23 (76.7%) cadavers. RLN/TEG relationship: In 43 (71.7%) cases, the RLN was coursing within the groove (Figure 4) while in 17 (28.3%), the nerve was lying outside the groove (Figure 3). When located outside the groove, it was most commonly found lateral to the TEG (11 nerves, 64.7%) (Table 1). The relationship was bilaterally symmetrical in 19 (63.3%) cadavers. RLN/ITA relationship: The most common type of RLN was deep to the ITA (65%), while the second most common was superficial to the ITA (30%). The least common type was between the branches of the ITA, with an incidence of 5%. In addition, there were differences between the left and right sides, with the posterior type being the most common, occurring in 35.73% of the nerves on the left side and 20.78% of those on the right one (Table 3). The relationship was bilaterally symmetrical in 13 (43.3%) cadavers. RLN/ Posterior border of thyroid: In 57 (95%) cases, RLN was coursing in the area posterior border of the gland with an average distance of 4.95±2.23 mm ranging between 2.21mm to 12.1mm. Almost all the nerves were found within the range of 2 to 10 mm except in two cases, where the distance was more than 10.00 mm. In three (5%) cases, nerve was passing anteriorly to lie closely along the lateral surface of the gland. The relationship was bilaterally symmetrical in 23 (76.7%) cadavers. No significant differences were observed between subtypes based on TEG, ITA and BL. 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