Porcine larynx as a suitable model for training in laryngeal surgery

Summary

Background: Surgical training represents fundamental pillars of successful laryngeal framework surgery. Experimental training on animal laryngeal models is a cheap and affordable method to gain knowledge, experience, and skills for laryngeal surgery. This study was focused on the porcine larynx and its possible applications for training in laryngeal surgery for both open and microlaryngeal techniques. Methods: Five porcine larynges were dissected following our own dissection manual and subsequently studied. Key steps of the dissection were documented by photos. Results: All extra -⁠ and intralaryngeal muscles that are present in humans are present in the porcine larynx as well, but the oblique arytenoid muscle is missing. Cricothyroid muscle consists of three bellies in contrast to two bellies in humans. The superior laryngeal nerve is divided into two main branches before entering the larynx in all cases. The recurrent laryngeal nerve is divided into two main branches in 4 of the 5 larynges. When simulating the endoscopic view into the laryngeal inlet, prominent vestibular folds must be taken into consideration. Conclusion: The porcine larynx is a suitable model for training in laryngeal surgery –⁠ open-techniques as well as for phonosurgery and microlaryngeal surgery. The branching pattern of the recurrent laryngeal nerve in pigs resembles the human larynx and could be used for re-innervation technique studies or laryngeal transplantation.

Keywords:

Larynx – recurrent laryngeal nerve – animal model – laryngeal microsurgery – superior laryngeal nerve – surgical training

Introduction

Laryngeal surgery represents a difficult challenge for young otorhinolaryngologists due its physiological and anatomical complexity. Understanding of laryngeal anatomy is crucial to successfully performing laryngeal surgery, for both –⁠ open and endoscopic approaches. Dissection of cadaver larynges presents one of the possibilities to gain such knowledge, but due to ethical and financial reasons, it is not always available. Previous studies used various animal models such as sheep [1–4], cats [5], dogs [6, 7], mice [8], and pigs [9]. We aimed to prove that the porcine larynx is a suitable animal model for studies on the larynx as stated in previous studies and it provides similar anatomy to the human larynx –⁠ cartilages, muscles, and distribution of nerve supply, namely the recurrent laryngeal nerve. Such a model could be used in training of ENT residents for open techniques that are performed less often, as well as for training of the endolaryngeal microsurgery itself [10]. Endolaryngeal microsurgery is mostly one-handed and there is little space to be trained by the senior ENT doctor [11]. Lastly, such models could be used for re-innervation techniques in bilateral recurrent laryngeal nerve (RLN) palsy [8, 12], laryngeal transplantation, or for studies of laryngeal electromyography (LEMG).

Materials and methods

Five porcine larynges along with the tongue, oesophagus, and trachea were obtained from a slaughterhouse and were deep frozen for 2 weeks. Dissection after defrosting followed our own dissection manual (Tab. 1). No special staining was used. The anatomy of the larynx, hypopharynx, and oesophagus was studied –⁠ the base of tongue, pyriform recesses, and cartilages of the larynx, infrahyoid muscles as well as laryngeal muscles and musculature of the oesophagus –⁠ inferior pharyngeal constrictor, were identified. RLN was identified in the tracheoesophageal groove and followed till it pierced the posterior cricoarytenoid muscle close behind the cricothyroid joint. Superior laryngeal nerve was found close to the cricothyroid membrane, accompanied by the superior laryngeal artery and vein. Oesophagus was then separated as well as the tongue, and dissection of the larynx solely followed. Extrinsic and intrinsic laryngeal muscles were identified. RLN and superior laryngeal nerve (SLN) and their branches were dissected. The cartilages were separated from the muscles and studied. The key points of the dissection were documented in photos.

Results

The gross anatomy of the oropharynx (base of the tongue, valecullae), hypopharynx (pyriform sinus, retrocricoid region, and lateral wall), and oesophagus is very similar to human anatomy. The laryngeal inlet (aditus) is marked with a prominent and differently shaped arytenoid cartilage and a small epiglottis. Under endoscopic view, the arytenoids are larger, and the vestibular folds are more prominent than the vocal cords compared to the human larynx (Fig. 1, 2). Besides the different shape of the arytenoid cartilage and epiglottis, the thyroid and cricoid cartilage are very similar in size and shape.

Musculature

Inferior pharyngeal constrictor attaches to the dorsal part of the larynx and continues into the oesophagus. It takes additional nerve supply from the external branch of the SLN. Main nerve supply comes through the pharyngeal plexus of the IX and X nerves. After the cross--section of the inferior pharyngeal constrictor (IPC), the posterior cricoarytenoid (PCA) comes fully into view, with further dissection and identification of the thyroarytenoid (TA), interarytenoid (IA), and lateral cricoarytenoid (LCA) muscles. Cricothyroid muscle (CT, extralaryngeal muscle) stretches anteriorly between the thyroid and cricoid cartilages, having three bellies in contrast to two bellies in humans, but the oblique arytenoid muscle is missing in the porcine larynx (1). The origins, insertions, nerve supply, and function are depicted in Tab. 2.

Nerve supply

Although some authors [6, 8] used modified Sihler’s staining technique to visualize the nerves (the nerves then appear dark purple), we were able to follow the nerves till their fine branches in the muscles with no staining method or special preparation of the specimens –⁠ e. g., macroscopically only.

Superior laryngeal nerve

The SLN branches off the vagal nerve and descends accompanied by the laryngeal artery and vein to the thyrohyoid membrane (Fig. 3). Before entering the larynx, it is divided into two main branches –⁠ the external laryngeal nerve (extSLN) descended to the cricothyroid muscle, and in its cranial portion it often gave an additional nerve supply to the IPC. The internal laryngeal nerve (intSLN) pierced the thyrohyoid membrane and provided many fine branches to the laryngeal mucosa; it anastomosed with the ending branches of the RLN. In three of the larynges, the anastomosis between intSLN and the posterior branch of RLN, called Gallen’s anastomosis, was found.

Recurrent laryngeal nerve

The RLN arises from the vagal nerve, loops around the aortic arch on the left side, around the subclavian artery on the right side and ascends to the larynx in the tracheoesophageal groove. During its course, it gave off branches to the trachea and oesophagus. It entered the larynx through the PCA close behind the cricothyroid joint. In 4 of the 5 larynges, it divided into two branches before entering the larynx –⁠ a smaller posterior branch, entered the IPC and supplied the hypopharyngeal and subglottic mucosa. The anterior branch pierced the PCA, where it mostly divided into 2–3 branches (Fig. 4). One branch dissolved in the PCA, the other (upper branch) continued to the other muscles –⁠ IA, LCA, forming a dense plexus, and lastly TA, where it formed the densest plexus (Fig. 5) –⁠ due to the rapidity of the glottic closure reflex and delicate movements during phonation [13]. The IA accepted branches of the RLN from both sides. The ending branches of the RLN (upper branch of the anterior RLN) anastomosed with branches of the intSLN.

Discussion

Practical training plays an important role in the education of young surgeons [14]. Some of the previously used techniques of open laryngeal surgery were replaced by microlaryngeal endoscopic surgery and became less frequent [1]. Endoscopic surgery is often “one-handed” with little possibility to learn under direct supervision of more experienced colleagues. A study by Ghirelli et al. [11] showed that time of surgery of ENT residents with no previous experience with microlaryngeal surgery was shorter after already 10 training sessions on an animal model without a higher rate of complications. These facts bring the need of finding more suitable models for training that would provide similar anatomy of the human larynx to understand the principles, define the key points and moments of surgical procedures, and thus achieve a lower complication rate [10, 11, 15]. Furthermore, practical training brings a higher confidence to the young surgeon.

In our study, we proved that the porcine larynx is a suitable model for laryngeal surgery as well as for microlaryngeal endoscopic techniques. Porcine models have already been used for training of tracheostomy or paediatric laryngotracheoplasty with positive responses from experienced surgeons [2, 16]. This model provides sufficient anatomic similarity and is available at low costs (slaughterhouses). Furthermore, the branching pattern of the SLN and RLN resembles that in humans and is promising for use in studies on laryngeal re-innervation techniques and transplantation. As in humans, the SLN in pigs divides into two main branches –⁠ internal and external laryngeal nerve. RLN divides into the anterior and posterior RLN, where the ending branches of both anastomose with each other. According to Stavroulaki et al. [5], the intSLN divides into superior, middle, and inferior branches [5]. Superior branches supply the mucosa of the vallecula and epiglottis and anastomose with the thyroarytenoid branch of the RLN. Middle branches supply the mucosa of the posterior commissure and subglottis and anastomose with interarytenoid branches of the RLN. Inferior branch of the intSLN forms Galen’s anastomosis mentioned above, supplying the postcricoid area and cervical oesophagus. In most of our larynges, we could find the anastomosis of the superior and inferior branches of the intSLN and antRLN.

In the past, there have been already promising studies also on laryngeal transplantation. Besides studies of the immunological profile of the larynx [17], emphasis is placed on how to achieve successful re-innervation with low synkinesis [5]. PCA being the only abductor of vocal cords and the other intralaryngeal muscles being adductors, there is a need to re-anastomose the branches of the anterior RLN to these two functional groups separately. Re-innervation can be done with e. g. ansa cervicalis or phrenic nerve [5]. That would need a separate dissection of the trunks of the antRLN in the PCA. This would be possible in patients with benign diseases, but not in patients with laryngeal carcinoma when considering the need of surgical radicality regarding tumour removal. Re-innervating the whole antRLN might end up in synkinesis and unwanted adduction (through LCA and IA), Stavroulaki et al. [5] proposed to re-innervate the most inferior branch of the antRLN (innervates the PCA only) separately from the rest of the branches of the antRLN.

The endoscopic view differs due to the large arytenoid cartilages and prominent vestibular folds that tend to cover the actual vocal cords, since there is no interarytenoid space, as this is filled with mucosa [11]. One should keep that in mind when using the porcine model for training in microlaryngeal surgery (Fig. 1). The laryngeal ventricle –⁠ sinus Morgagni can be demonstrated on a cross-section of the larynx (Fig. 2).

Animal models are being implicated as well in phoniatric research among animals, since pigs share the most phonatory characteristics with humans, they are a suitable model for ex vivo as well as in vivo experiments [18–21]. Rohlfing et al. [18] used an ex vivo porcine model to study the effect of anterior glottoplasty for voice feminization surgery; pigs can also be used for training of needle insertion during laryngeal electromyography [4, 18].

Conclusions and significance

The porcine larynx seems to be a suitable model for training as well as in microlaryngeal endoscopic surgery of ENT residents and doctors. The gross anatomy, cartilages, musculature, and nerve supply are similar enough to the human larynx anatomy. Branching patterns of the recurrent laryngeal nerve are similar in humans, which could be used for further studies on re-innervation techniques. More studies on practical use of the porcine model in training for laryngeal surgery should be carried out.

List of abbreviations:

RLN         recurrent laryngeal nerve

SLN         superior laryngeal nerve

IPC          inferior pharyngeal constrictor muscle

PCA         posterior cricoarytenoid muscle

IA            interarytenoid muscle

TA           thyroarytenoid muscle

LCA         lateral cricoarytenoid muscle

CT           cricothyroid muscle

extSLN   
external branch of the superior laryngeal nerve

intSLN    
internal branch of the superior laryngeal nerve

antRLN   
anterior branch of the recurrent laryngeal nerve

postRLN 
posterior branch of the recurrent laryngeal nerve