Structure of the normal rat larynx

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1 Laboratory Animals (1977) 11, Structure of the normal rat larynx G. SMITH Grollp Research & Development Centre British-American Tobacco Co. Ltd, Regent's Park Road, SOllthampton, S09 IPE Summary Recent studies have shown the rat larynx to be an important organ in the evaluation of irritancy of inhaled materials. Serial sections and scanning electron microscope observations have clarified its basic structure. In particular the cartilage pattern l1,ndthe distribution of the epithelia lining the lumen have been determined. There are 2 specialised epitheliastratified squamous (found in areas subject to wear) and ciliated pseudostratified columnar (which produces and transports mucus). Always intervening between these is an unspecialised epithelium, the non-ciliated columnar. The marked variation in type of epithelium over a short distance within the larynx emphasises the need to take sections in the same plane when comparing different specimens. Inhalation toxicity studies with smoke carried out in these laboratories have produced consistent pathological changes in the larynxes of animals (Walker, Wilton & Binns, 1977). These changes (mainly squamous metaplasia and hyperplasia, often with keratinisation) have been studied by conventional histological techniques and more recently by scanning electron microscopy (Smith, (977). Treatment-induced changes have been observed after relatively short-term exposures to smoke, suggesting that studies of the larynx may allow the development of a rapid screening method for comparing irritancy of inhaled materials. Tn order to maximise any observations, an appreciation of the detailed structure of the larynx is essential. Of particular interest are the types of epithelium and their distribution in the lumen. Also of importance is a knowledge of the cartilage distribution, since the exact orientation of material can be determined from the pattern of cartilages exposed during sectioning. Although there have been some studies on the rat larynx (Broyles, ] 959; Ewen, Bussolati & Pearse, 1972; Andrews, ] 974), there is no description of its basic structure or of epithelial distribution in the organ. The purpose of this study was to examine the normal rat larynx and to describe the distribution of those major features which have been shown to be useful in the evaluation of inhalation toxicity studies. It also forms part of a preliminary study to see if the scanning electron microscope (SEM) could be used routinely to examine features previously evaluated by conventional histological techniques only. Ventral Dorsal Lateral Cricoid Pig. 1. Ventral, loteral and dorsal aspects of the rat larynx.

2 224 Smith TG - Thvroid P - Pharynx Tracheal Cricoid L - Lilrynll. Fig. 2. Internal structure of the larynx, illustrating cartilage distribution as seen by transverse and sagittal sections. TG thyroid gland. P pharynx. L larynx. Areas of seromucous glands indicated by broken lines. Materials and methods Adult specified-pat hogen-free (SPF) white rats were killed by an intraperitoneal injection of sodium pentabarbitone and the larynx dissected out into fixative as quickly as possible. Any blood entering the laryngeal lumen was washed out prior to fixation. For light microscopy, tissue was fixed in 4 % buffered formalin and 5 Ilm paraffin wax sections were stained with Ehrlich's haematoxylin and eosin. The sections were serial in the transverse, sagittal or coronal plane. These preparations were used to determine the distribution of the cartilages and the shape of the laryngeal lumen. For scanning electron microscopy the larynxes were fixed for 24 h in 5 % cacodylate buffered glutaraldehyde ph 7,3 at 4 C, washed for 72 hours at 4 C in buffer, brought t,o room temperature and dehydrated in a series of water-acetone solutions. The larynx was dissected under acetone along a near mid-sagittal plane. This exposed the main laryngeal structures and was used for all SEM observations. The dissected larynx was critical-point dried and coated with a conductive layer of gold-palladium alloy. Results The larynx is a part of the conducting system of the respiratory tract and connects the pharynx and the trachea. Its fundamental function is to prevent particulate matter from entering the lower respiratory tract and it also plays an important part in the production of sound. Laryngeal cartilages (Figs 1 and 2) The cartilages maintain the shape of the larynx and prevent its collapse during inhalation. There are 6 groups of cartilages, I, A plate-like cartilage supporting the flat triangular epiglottis. 2. The thyroid cartilage. The largest in the larynx, supporting the majority of the ventral and lateral aspects. The lateral surfaces of this cartilage are Trachea Ventral ridge Aryepiglottic Ventra, depression Fig. 3. Slructure and epithelial distribution 01 the rat lar~'nx as shown by sagiltal section. ~ Squamous epithelium l'lli8 Nonciliated pseudostralified columnar epithelium D Ciliated pseudostratified columnaor epithelium fold

3 Rat larynx 225 extended into a posterior horn joining on the cricoid cartilage, and an anterior horn supporting the hypopharynx. 3. The cricoid cartilage is a complete ring which is a narrow band ventrally and laterally but a broad plate on the dorsal surface. It is often called the 'signet-ring' cartilage. 4. A pair of arytenoid cartilages which are approximately V-shaped and support the vocal cords. 5. A pair of small cartilages, the cuneiform, found in the dorsal aryepiglottic folds anterior to the vocal cords. 6. The ventral depression of the larynx has around it a small V-shaped cartilage. The laryngeal lumen is divided into 3 areas: the vestibule, which opens into the pharynx by the laryngeal opening; the glottis, which is slit-like dorso-ventrally and contains the vocal cords and Fig. 4 (a) Squamous epithelium on leading edge of vocal cord. (b) Microvilli on surface squamous cells. (c) Ventral depression (VD) surface (V) and ridge (R) invested in nonciliated columnar epithelium. (d) Detail of noneiliated columnar epithelium.

4 226 Smith ventral depression; and the infraglottis, which is caudal to the glottis. A mid-sagittal section of the larynx displays its main structures (Fig. 3). The rostral part of the larynx is the plate-like epiglottis. Other outstanding features are the vocal cords below which are the ridge and depression in the ventral surface of the larynx. Caudal to the vocal cords, the larynx has a more circular lumen which leads into the trachea (Fig. 2). The shape of the lumen here is maintained by the cricoid cartilage. Epithelia 3 types of epithelia line the laryngeal lumen, stratified squamous, ciliated pseudostratified columnar, and non-ciliated pseudostratified columnar. Their general distribution is shown in Fig. 3. Stratified squamous epithelium is found on the epiglottis, the Fig. 5. (a) Medial aspect of the vocal cord showing relativc distribution of squamous (S) and nonciliated (NC) epithclium. (b) Sccrctory cclls of mid-trachca, with surfaccs extcndcd into blcbs (8) by production of sccretory granulcs. (c) Cilated epithelium with ciliated (CC) and microvillous cells (MC): one cell type may be undergoing ciliogenesis (CG). (d) Underlying cells obscured by complete covering of cilia.

5 Rat larynx 227 leading edge of the vocal cords (Fig. 4a) and occasionally on the ventral ridge. Of the remaining types, ciliated pseudostratified columnar is the most widespread. It occurs caudal to the vocal cords, lining the lower part of the larynx and extending into the trachea (Fig. 5, b and c). Small areas of this type of epithelium are sometimes observed near the base of the epiglottis. Intervening between the squamous and ciliated epithelium is the nonciliated columnar epithelium (Fig. 4d). It is seen on the lateral and ventral aspects of the larynx including the ventral depression (Fig. 4c). The vocal cords also have this type of epithelium, again found between the other types (Fig. 5a). Although observations of the laryngeal epithelium were concerned mainly with determining the types and distribution of the epithelia present, some observations have been made on the surface appearance of the epithelial cell types. Stratified squamous epithelium appears as an area of flat or slightly convex cells whose intercellular boundary is often emphasised by a thickening of the cell wall. The cell surface is formed into numerous short nub-like microvilli (Fig. 4b). At low magnifi cations cells are often seen being sloughed from the surface (Fig. 4a). Non-ciliated pseudostratified columnar epithelium is often formed into ridges and consists of a single cell type (Fig. 4d). The cells are markedly convex and, although they often possess microvilli these are not so prominent as in the other cell types of the larynx. Ciliated pseudostratijied columnar epithelium has 4 basic cell types that can be recognised, all of which have prominent microvilli. (i) Ciliated cells. The most easily recognisable cell type with the cell surface of microvilli obscured by numerous cilia. Ciliated cells often form a complete covering to large areas of the mucous membrane (Fig. 5d). (ii) Microvillous cells. The most common cell type observed. The majority of cells have closely packed microvilli which decrease towards the periphery of the cell. On occasions this cell type can be seen undergoing what may be ciliogenesis (Fig. 5c). (iii) Secretory cells. Distinguished from microvillous cells during active secretion as they have a markedly convex free surface formed into numerous blebs. They appear to be more numerous in the midtrachea (Fig. 5b) but this is uncertain. (iv) Brush cells. Form less than 1% of the cell population and could be confused with a cell undergoing ciliogenesis. The cell surface is produced into many elongate microvilli superficially resembling cilia. Discussion The combined use of both conventional histological techniques and scanning electron microscopy has provided a means of determining the basic structure of the rat larynx. For general microscopy the plane of section can be standardised using the cartilage distribution to fix the orientation of specimens with a high degree of consistency. The structure of the larynx is readily observed using the SEM and the relative positions of the epiglottis, aryepiglottic folds, vocal cords, ventral ridge and depression can be visualised in 3 dimensions in a single field. The epithelial distribution (although varying slightly from specimen to specimen) follows a basic pattern: it is formed of 2 main areas of squamous and ciliated epithelium with non-ciliated epithelium always intervening between them. As the majority of observations in this study were at low magnifications only a limited amount of information was available on the cells making up the 3 types of epithelium. The squamous cells are flat and microvillous and similar to those described for the rat epiglottis (Andrews, 1974) and human larynx (Biondi & Biondi-Zappala, ] 974). The nonciliated columnar cells, which are markedly convex and only slightly microvillous, have not previously been described in detail. They may be equivalent to the microvillous cells found at the base of the epiglottis (Andrews, 1974), but no mention was made of them forming a uniform area as observed in this study. A type of stratified or pseudostratified columnar epithelium found at the boundary of the squamous and ciliated epithelium in the ventricle of the monkey larynx corresponds in relative position to the nonciliated epithelium noted in the rat, but light-microscope observations show a marked difference in the cell types making up the membrane (Rhodin, 1974). The basic structure of the ciliated pseudostratified columnar epithelium follows previous observations in the rat larynx (Andrews, 1974), trachea (Alexander, Ritchie, Maloney & Hunter, 1975), and bronchioles (Ebert & Terracio, ] 975). The microvillous cell is the most common type in the ciliated epithelium, supporting observations of Jeffery & Reid (1975). In the present study the secretory cells could not be further differentiated but may represent a mixed population of goblet and serous cells (Lynne Reid, personal communication). In fact, preliminary transmission electron microscope observations show the majority to be serous. The secretory and brush cells correspond with previous observations with the SEM by Andrews (1974) and Alexander et al. (1975). The appearance of the secretory cells varied with the

6 228 Smith loss of microvilli as secretory granules accumulated prior to their release.. The SEM observations of the 3 types of epithelium show that the laryngeal mucosa is specialised for specific functions. Yet it remains plastic enough to adapt to a changing environment, and the epithelium varies in relation to the function carried out by the different regions of the larynx. In areas subject to wear and tear, such as the epiglottis and free edge of vocal cords, there is stratified squamous epi. thelium. More posterior there is again a specialised epithelium, the ciliated columnar. It is this type which produces (secretory cells) and transports (ciliated cells) mucus. Intervening between the 2 specialised epithelia is the ciliated columnar epithelium. unspecialised, non- It forms a band in the region of the vocal cords, on the cords themselves, and on the lateral and ventral aspects of the larynx. As an unspecialised epithelium may more readily respond to an external stimulus, the relatively narrow band of nonciliated columnar epithelium may be of particular importance in inhalation toxicity studies. Thus the area in the region of the vocal cords and ventral depression could be of special interest. The distinct change in epithelial types over a short distance in this region emphasises the need to know their distribution pattern, since a slight variation in level of section will show a different epithelial pattern from slide to slide. Lack of standardisation in section c\,;tting may make the evaluation of any treatment-induced changes extremely difficult, par ticularly in short-term studies in which epithelial changes may be relatively minor and restricted. Acknowledgements I would like to thank Dr R. Binns for advice in preparing this script, and Mr F. Hunt and Miss S. Murthwaite for their excellent technical assistance. References Alexander, I., Ritchie, B. C., Maloney, J. E. & Hunter, C. R. (1975). Epithelial surfaces of the trachea and principal bronchi in the rat. Thorax 30, Andrews, P. M. (1974). A scanning electron microscopy study of the extrapulmonary respiratory tract. American Journal of Anatomy 139, Biondi, S. & Biondi-Zappala, M. (1974). Surface of laryngeal mucosa seen through the scanning electron microscope. Folia phoniatrica 26, Broyles, E. N. (1959). Anatomical observations concerning the laryngeal appendix. Annals of Otology, Rhinology and Laryngology 68, Ebert, R. B. & Terracio, M. J. (1975). Observation 0 the secretion on the surface of the bronchioles with the scanning electron microscope. American Review of Respiratory Di~eases 112, Ewen, S. W. B., Bussolati, G. & Pearse, A. G. E. (1972). Uptake of L-DOPA and L-5-hydroxytryptophan by endocrine like cells in the rat larynx. Hi~tochemical Journal 4, Jeffery, P. K. & Reid, L (1975). New observations of rat airway epithelium: a quantitative and electron microscope study. Journal of Anatomy 120, Rhodin, J. A. G. (1974). Hi~tology: a text and atlas. Oxford: Oxford University Press.. Smith, G. (1977). Scanning electron microscope obser vations of the rat larynx. Proceedings of the European Society of Toxicology, 18, Walker, D., Wilton, LV. & Binns, R. (1977).The acute pathological effects of modified cigarette smoke inhaled by rats. Proceedings of the European Society of Toxicology 18,