Surg Radiol Anat (2006) 28: 125–128 DOI 10.1007/s00276-006-0075-2

O R I GI N A L A R T IC L E

Y.-H. Liu Æ S.-C. Xu Æ L.-L. Tu Æ K.-L. Zhang D.-H. Lu Æ M. Zhang

A rich lymphatic network exists in the inferior surface of the vocal cord

Received: 7 April 2005 / Accepted: 15 December 2005 / Published online: 15 February 2006 Ó Springer-Verlag 2006

Abstract Conservation laryngeal surgery is an increasingly available alternative for the treatment of laryngeal cancer. Understanding anatomy of laryngeal lymph drainage is essential for clinicians to diagnose, grade and surgically manage the laryngeal cancer. Although the lymphatic drainage of the larynx has been extensively studied, few studies revealed the relationship of the lymphatic drainage between various parts of the larynx. The distribution of lymphatic vessels in the inferior surface of the vocal cord also remains unclear. The aim of this study was to investigate the communication of the lymphatic networks between the vocal cord, the supraglottic and subglottic parts of the larynx, paying special attention to the lymphatic drainage of the inferior surface of the vocal cord. Eighteen larynx specimens from 18 fresh fetal cadavers were manually injected with prassion blue solution into the mucosal or submucosal layer of the larynx in order to reveal the lymphatic vessels in the inner larynx. We found that a rich lymphatic network is present in the inferior surface of the vocal cord, and the lymphatic networks in the superior and inferior surfaces of the vocal cord appear as two different patterns. These findings provide an anatomical basis for the design of a partial or conservation laryngeal surgery, particularly when considering the precise resection margin.

Y.-H. Liu Æ K.-L. Zhang Æ D.-H. Lu Department of Otolaryngology, First Affiliated Hospital of Anhui Medical University, Hefei, China S.-C. Xu Æ L.-L. Tu Department of Anatomy, Anhui Medical University, Hefei, China M. Zhang (&) Department of Anatomy and Structural Biology, University of Otago, PO Box 913, 9001 Dunedin, New Zealand E-mail: [email protected] Tel.: +64-3-4797378 Fax: +64-3-4797254

Keywords Larynx Æ Lymphatic drainage Æ Inferior surface of vocal cord Æ Laryngectomy

Introduction Conservation surgery of the larynx is an increasingly available alternative for the treatment of laryngeal cancer [7, 16]. Understanding anatomy of laryngeal lymph drainage is essential for clinicians to diagnose, grade and surgically manage the laryngeal cancer. The larynx consists of three regions: supraglottis, glottis and subglottis. The glottic region includes two vocal cords and anterior and posterior commissures. Each vocal cord has a superior surface, a free margin, an interior surface [15]. The boundary between the superior surface of the glottis and supraglottic region is the line through the lateral angle of ventricle [17]. However, the demarcation between the inferior surface of the glottis and the subglottis has been the subject of controversy [4, 12]. The National Cancer Institutes (UK; USA) have suggested that the upper limit of the subglottis begins about 10 mm below the free margin of the vocal cord [15], whereas others considered that the limit should be 5 mm below the free margin of the vocal cord [13] or be along the upper border of the cricoid cartilage [3], or begin from the free margin of the vocal cord as the inferior surface of the vocal cord should be regarded as the roof of the subglottis [5]. The lymphatic drainage of the larynx has been extensively studied [1, 6, 18–20]. It is generally accepted that (1) the supraglottis has a very rich lymphatic network, (2) there is no lymphatic vessel in the free margin of the vocal cord, (3) the superior surface of the vocal cord or the floor of the ventricle has several lymphatic vessels running parallel to the free margin of the cord, and (4) the lymphatics in the subglottis are smaller and less compact than that in the supraglottis although most studies did not clearly define the boundaries of the subglottis [1, 6, 10, 17–20]. However, few studies revealed the relationship of the lymphatic drainage

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between various parts of the larynx. The distribution of lymphatic vessels in the inferior surface of the vocal cord also remains unclear. The aim of this study was to investigate the communication of the lymphatic networks between the vocal cord, the supraglottic and subglottic parts of the larynx, paying special attention to the lymphatic drainage of the inferior surface of the vocal cord.

Materials and methods Due to the difficulty to obtain fresh adult cadavers, eighteen larynx specimens were collected from 18 fresh human fetuses aged from 7 to 10 months old (eight males and ten females). These cadavers were bequeathed for the medical education and research purposes and approved by the Medical Ethic Committee of Anhui Medical University. The specimens were divided into three groups and the larynx of each group was opened along posterior median line (six specimens), or along anterior median line (six specimens) or along right lateral line (six specimens). Thirty percent of prassion blue in chloroform solution (CHCl3) was used to demonstrate lymphatic vessels, as

Fig. 1 a A general lateral view of the right inner larynx after multiple injections of prassion blue solution into the mucosal and submucosal layers of a fetal larynx, showing three sub-regions of the larynx: the supraglottic region (sg), subglottic region (ig) and glottic region which consists of the vocal cord (vc), anterior commissure (ac) and posterior commissure (pc). White and black arrows point to the lymphatic vessels in the superior and inferior surfaces of the vocal cord, respectively. Bar=600 lm. b An inferior view of the lymphatic network (arrows and arrowheads) in the inferior surface of the vocal cord (vc) and the subglottic region (ig). The prassion blue solution was introduced at the inferior part of the posterior commissure (asterisks). Note that the lymphatic

described previously [9, 14]. Briefly, the specimen was bathed in 50°C warm water. With a fine needle (Gauge no. 5) and a syringe (1 ml), the dye was manually injected into the mucosal or submucosal layer of a subregion of the larynx. Injection pressure and quantity of injected dye varied between individuals. Satisfactory of the injection was achieved by a good and thorough illustration of lymphatic networks. A mucosal injection normally revealed the lymphatic capillaries (Fig. 1d), whereas a submucosal injection demonstrated both lymphatic capillaries and collecting vessels (Fig. 1a, b). In this study, three specimens from each group were used for the mucosal injection and the other three for the submucosal injection. Following the injection, the spreading of the injected dye in each whole-mount specimen was closely observed and photographically recorded. The injected specimen was then washed in running tap water for 5–10 min, fixed and stored in 10% neutral buffered formalin.

Results Lymphatic vessels were clearly traced following the injection of prassion blue solution (Fig. 1a). The rich

network consists of a superficial layer (focused; and appointed by arrows) and a deep layer (arrowheads, slightly out of focus) of lymphatic vessels. Double arrowheads indicate sudden stops of dye spreading towards the free margin of the vocal cord. Bar=600 lm. c A superior view of the lymphatic networks in the superior surface (arrows) of the vocal cord (vc), the supraglottic region (sg) and the anterior commissure (ac). Bar=600 lm. d A posterior view of the lymphatic network in the posterior commissure (pc), where the lymphatic vessels from both the sides of the inferior surface (arrows) of the vocal cord (vc), subglottic region (ig) and posterior subglottic wall (asterisks) communicate with each other. Bar=600 lm.

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lymphatic networks were found in the subglottic (Fig. 1b) and supraglottic areas, particularly in the laryngeal ventricle (Fig. 1c), whereas no lymphatic vessels were found in the free margin of the vocal cord (Fig. 1a, c). These findings are consistent with the previous reports [1, 20]. The most interesting finding of this study was that a rich lymphatic network was present in the inferior surface of the vocal cord (Fig. 1b), which was bounded inferiorly by the upper border of the cricoid cartilage [3]. Both intramucosal and submucosal injections revealed that this area contained not only lymphatic capillaries (Fig. 1b, d) but also numbers of large collecting lymphatic vessels (Fig. 1b). Some lymphatic capillaries extended towards the free margin of the vocal cord (Fig. 1b), particularly in the posterior region (Fig. 1d). However, the boundary of such an extension of the lymphatic vessels was difficult to be clearly defined because of the variation between the specimens (Fig. 1b vs. Fig. 1a) and sudden stops of dye spreading in various vessels of the same specimen (Fig. 1b) which may be due to blockage of lymphatic microvalves [1]. The lymphatic network in the inferior surface of the vocal cord showed a different pattern from that in the superior surface of the vocal cord. In the superior surface, only several collecting lymphatic vessels ran parallel to the free margin of the vocal cord (Fig. 1c). The injection of the dye solution in this region resulted in the dye flowing towards the posterior commissure and then to the supraglottic and subglottic lymphatic networks. In contrast, in the inferior surface of the vocal cord, those lymphatic vessels anastomosed with each other to form a highly densed network (Fig. 1b). The network communicated inferiorly with the subglottic lymphatic network (Fig. 1a, b, d) and anteriorly and posteriorly with the supraglottic network via anterior and posterior commissures (Fig. 1d). This study also found that the posterior commissure served as an important interconnection site between the various regions of the larynx. When the injection on either right or left side of the commissure, the dye was not limited on the same side but some dye were found on the opposite side. When the injection at the posterior commissure, the dye spread to both sides (Fig. 1d). The similar situations were found following an injection in the anterior commissure but less obvious than that in the posterior commissure.

Discussion An accurate assessment and treatment for primary laryngeal carcinoma must consider the lymphatic drainage patterns of the larynx, particularly the inner larynx [4,15]. During last decades, anatomy of lymphatic drainage of the larynx has been extensively studied [1, 6, 18–20]. Several conclusions come out of those studies. Firstly, the supraglottic and subglottic portions of the larynx have very rich lymphatic networks; secondly,

there is no lymphatic vessel in the free margin of the vocal cord, and thirdly, the superior surface of the vocal cord has several lymphatic vessels running parallel to the free margin of the cord [1, 6, 18–20]. Using the method of injection of prassion blue solution into the mucosal or submucosal layer of various sub-regions of the larynx, this study reveals that a rich lymphatic network is present in the inferior surface of the vocal cord (Fig. 1b), and the lymphatic networks in the superior (Fig. 1c) and inferior (Fig. 1b) surfaces of the vocal cord appear as two different patterns. The lymphatic network in the inferior surface of the vocal cord has not been specifically described before, although several previous studies have demonstrated that a rich lymphatic network exists in the subglottic region [1, 18–20]. One of difficulties to interpret and compare those findings is that the anatomical demarcation between the sub-regions of the inner larynx is somewhat ill defined. The most controversial subject is the boundary between the vocal cord and subglottic region. At the histology level, the insertion of the fibers of the elastic lamina into the vocal cord has been used to demarcate the inferior margin of the vocal cord and the upper limit of the subglottis [2]. At the gross anatomy level, it has been suggested the above boundary can be roughly estimated by the depth of the protrusion of the vocal cord into the laryngeal cavity [2]. According to Bryce’s summary, the depth of the vocal cord is about 5 mm posteriorly, tapering to 1 mm at the anterior commissure [2]. Therefore, some studies adopted an imaginary circle 5 mm below the free margin of the vocal cord as the upper limit of the subglottic region [4, 12, 13]. However, as suggested by the National Cancer Institutes (UK; USA), the glottis extends for 10 mm below the free margin of the vocal cord [15], which in adults is almost equivalent to the upper border of the cricoid cartilage [3]. Nevertheless, it is generally accepted that there is an anatomical area sandwiched in between the free margin of vocal cord and the upper border of the subglottis. So far, little report has specifically described the lymphatic drainage of this anatomical area, which is usually defined as the inferior surface of the vocal cord [12]. In this study, we demonstrated that the lymphatic vessels, particularly lymphatic capillaries exist in the inferior surface of the vocal cord (Fig. 1b, d). However, several cautions should be taken when interpreting our results. (1) As our study was based on the fetus larynx, either a 5 mm or 10 mm imaginary zone that was mainly derived from adult observation may not be applicable. We have conducted an extensive literature review and found that no anatomical data were available for the depth of the vocal cord at various age groups. As in adults an imaginary circle 10 mm below the free margin of the vocal cord [15] is almost equivalent to the upper border of the cricoid cartilage [3], in this study we used the upper border of cricoid cartilage as the demarcation between the inferior surface of the vocal cord and the subglottic region. (2) The demarcation between the free

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margin and the inferior surface of the vocal cord has also not been clearly defined in any literature. Previous reports often mixed up the terminology of the free margin [12], upper free margin [4] and inferior surface [8] of the vocal cord. (3) This study may underestimate the density of the lymphatic network because of the limitation of the method used in this study. Single injections of prassion blue solution into the mucosal or submucosal layer of various sub-regions of the larynx may not be able to reveal the whole lymphatic vessels, as there are numerous valves in the lymphatic system [1]. As shown in Fig. 1b, some sudden stops of the spreading dye were seen along the free margin of the vocal cord, implying that some lymphatic vessels may extend more into the free margin of the vocal cord than what we have observed.

Conclusion This study specifically describes that a rich lymphatic network is present in the inferior surface of the vocal cord, and the distribution patterns of the lymphatic networks in the superior and inferior surfaces of the vocal cord appear differently. These findings provide new anatomical references for the design of a partial or conservation laryngeal surgery, particularly when considering the precise resection margin. However, further studies to precisely define the anatomical subdivisions of the larynx are badly needed, particularly for various age groups. Acknowledgements The generosity of the Department of Anatomy, Anhui Medical University, for providing access to the fetal cadavers and technical assistance is acknowledged. The laryneal specimens used in this study were collected from the fetal cadavers that were bequeathed for the medical education and research purposes and approved by the Medical Ethic Committee of Anhui Medical University, Hefei, China.

References 1. Beck C, Mann W (1980) The inner laryngeal lymphatics. Acta Otolaryngol 89:265–270

2. Bryce DP (1975) The laryngeal subglottis. J Laryng Oto 89:667–685 3. Buckley JG, MacLennan K (2000) Cancer spread in the larynx; a pathologic basis for conservation surgery. Head Neck 22:265– 274 4. Dahm JD, Sessions DG, Paniell RC et al (1998) Primary subglottic cancer. Laryngoscope 108:741–746 5. Freeland AP, Nostrand AW (1975) The applied anatomy of the anterior commissure and subglottis. Can J Otolaryngol 4:644– 659 6. Johner CH (1970) The lymphatics of the larynx. Otolaryngol Clin North Am 3:439–450 7. Laramore GE, Coltrera MD (2003) Organ preservation strategires in the treatment of laryngeal cancer. Curr Treat Options Oncol 4:15–25 8. Lederman M (1970) Radiotherapy of cancer of the larynx. J Laryngol 84:867–896 9. Lin ZH, Cai YE, Wang HE (1990) Lymphatic distribution and ultrastructures of human larynx. Zhonghau Er Bi Yan Hou Ke Za Zhi 1990:275–276 10. Pressman JJ (1956) Submucosal compartmentation of the larynx. Ann Otol Rhinol Laryngol 65:766–771 11. Pressman JJ, Dowdy A, Libby R et al (1956) Further studies upon the submucosal compartments and lymphatics of the larynx by the injection of dyes and radioisotopes. Ann Otol Rhinol Laryngol 65:963–980 12. Saleh EM, Mancuso AA, Alhussaini AA (1992) Computed tomography of primary subglottic cancer: clinical importance of typical spread pattern. Head Neck 14:125–132 13. Sessions DG, Ogura JH, Fried MP (1975) Carcinoma of the subglottic area. Laryngoscope 85:1417–1423 14. Skarzynski H, Skarzynska B, Deszezynska H (1990) Now outlook on the problem of an anatomic barrier in the lymphatics of the laryngeal mucous membrane. Otolaryngol Pol 44:57–61 15. Thekdi AA, Ferris RL (2002) Diagnostic assessment of laryngeal cancer. Otolaryngol Clin N Am 35:953–969 16. Tufano RP (2002) Organ preservation surgery for laryngeal cancer. Otolaryngol Clin North Am 35:1067–1080 17. Welsh JJ, Welsh LW, Rizzo TA (1983) Laryngeal spaces and lymphatics: current anatomic concepts. Ann Otol Rhinol Laryngol 92(Suppl):19–31 18. Welsh L (1964) The normal human laryngeal lymphatics. Ann Otol 73:569–582 19. Werner JA, Schunke M, Rudert H et al (1990) Description and clinical importance of the lymphatics of the vocal fold. Otolaryngol Head Neck Surg 102:13–19 20. Werner JA, Schunke M, Lippert BM et al (1995) The laryngeal lymph vessel system of the human. A morphologic and lymphography study with clinical viewpoints. HNO 43:525–531