Types and Patterns of Response in the Larynx Following Inhalation

Abstract

The laryngeal mucosa responds to insult similarly to other epithelial tissues but the response depends on location within the larynx since important anatomic differences exist, even within rodent species. Although dogs and nonhuman primates are also utilized for inhalation toxicology studies, little published information is available regarding sites of injury from inhaled toxicants in these species. Accurate and consistent fixation, trimming, and microtomy of laryngeal sections allow the pathologist to compare the morphology of laryngeal mucosa from exposed and control animals and detect and interpret subtle differences resulting from inhalation exposure. There are anatomic landmarks that are keys to providing consistent sections through important areas of the laryngeal mucosa. Repeated inhalation of toxic concentrations of chemicals, drugs, or environmental contaminants induces a wide range of responses, depending on the physical properties and concentration of the toxic substance and duration of exposure. Responses include edema, acute to chronic inflammation, fibrosis, mucosal ulceration, degeneration, and necrosis. Attempts at repair include regeneration, hyperplasia, squamous metaplasia, hyperkeratosis, and neoplasia. Awareness of normal histology and zones of transition from squamous to respiratory epithelium in different areas of the larynx in different species is critical to avoid confusing normal epithelium with metaplasia or hyperplasia. Microscopic examination of laryngeal mucosa from animals exposed via inhalation and necropsied following a recovery period provides the opportunity to determine the degree of regression or progression of exposure-induced laryngeal lesions.

Introduction

The larynx, although not as complicated as the nasal cavity, is not simply a wide spot on the way to the trachea and lungs. It is a bilaterally symmetrical organ lined by several types of epithelium and complicated by various protuberances, pouches, folds, and cartilages, with considerable anatomic and histologic variation among laboratory animal species used for inhalation studies. The distribution of the types of epithelium lining the laryngeal lumen is similar to the nasal airway, in that the mucosal epithelium changes from stratified squamous cranially to pseudostratified cilated columnar (respiratory) epithelium caudally. These areas of transition from a relatively durable stratified squamous epithelium to a much more fragile respiratory epithelium are the most sensitive sites for cellular changes in rodents inhaling xenobiotics (Gopinath et al., 1987; Lewis, 1991). As in the nose, the predilection of these areas in the larynx for exposure-induced lesions in rodents is probably also related to airflow characteristics as well as regional epithelial sensitivity (Chevalier and Dontenwill, 1972; Gopinath et al., 1987; Lewis, 1981, 1991). Descriptions of the normal laryngeal histology and the sites most susceptible to injury from inhalation of toxicants in rats, mice, and hamsters are available in the literature (Lewis, 1991; Renne and Miller, 1996). Exposure-induced laryngeal lesions are frequently reported in inhalation studies in rodents, and there is considerable information available on laryngeal lesions in these species (Lewis, 1991; Miller and Renne, 1996).

In order to detect, describe, and accurately interpret microscopic laryngeal lesions induced by inhaled toxicants, it is critical to be able to examine consistent sections through areas most susceptible to injury from exposure. Consistent fixation, trimming, and microtomy of laryngeal sections allow the pathologist to compare the morphology of laryngeal mucosa from exposed and control animals. There are anatomic landmarks that are keys to obtaining consistent sections through important areas of the laryngeal mucosa of laboratory rodents. Although dogs and nonhuman primates are also utilized for inhalation toxicology studies, detailed information regarding laryngeal histology is limited and very little published information was found on type or location of laryngeal lesions from inhaled toxicants in these species.

Repeated inhalation of toxic concentrations of chemicals, drugs, or environmental contaminants induces a wide range of responses in laryngeal mucosa, depending on concentration of the toxic substance and duration of exposure. Responses include edema, acute to chronic inflammation, fibrosis, mucosal ulceration, degeneration, and necrosis. Attempts at repair include regeneration, hyperplasia, squamous metaplasia, hyperkeratosis, and neoplasia.

Materials and Methods

Similar but not identical methods were utilized to provide transverse or sagittal sections of larynx from Fischer (F344/N) or Sprague–Dawley-derived CRL: CD rats, Syrian golden hamsters, B6C3F1, C57Bl/6, or ICR mice, laboratory-bred Beagle dogs of both sexes, and male Cynomolgus monkeys. Animals utilized were from cage or sham controls for prechronic or chronic inhalation studies. Rodents ranged in age from 8 weeks to 25 months and dogs ranged in age from 6 to 11 months; Cynomolgus monkeys were young mature wild-captured males whose exact ages were unknown.

Sections were taken from throughout the larynx, but emphasis was placed on preparation and examination of sections through regions of transition from squamous to other types of epithelium, such as the base of the epiglottis, vocal processes of the arytenoid cartilages, and the ventral pouch, since these areas are known to be most susceptible to effects of inhaled materials in rodents (Lewis, 1981, 1991; Miller and Renne, 1996). The junction of the ventral pouch (rodents) or lateral ventricles (dogs and monkeys) with the lumen of the larynx and the vocal folds in the caudal larynx were also examined. Multiple transverse sections were utilized routinely; sagittal sections were utilized for orientation and comparison purposes.

As noted above, one key to accurately determining the effects of inhalation exposure on laryngeal tissues is the availability of consistent sections through areas most susceptible to injury. This requires consistent preparation of tissue sections to allow the pathologist to compare the morphology of identical areas of laryngeal mucosa from exposed and control animals. Visualization of cartilages, glands, and adjacent structures in unstained sections at microtomy by adjusting the light diffraction in the microscope (Figure 1), allows the microtomist to accurately determine the location of the sections and provide the pathologist with consistent sections through key areas of the larynx.

Sections were routinely stained with hematoxylin and eosin. Alcian blue/periodic acid stains were used in some studies to assess goblet cell populations. Morphometry using Image Pro Plus software was performed on sections through the base of the epiglottis in some rodent studies to quantify total area of mucosal epithelium lining the ventral mucosal surface at this site.

Results

The laryngeal epithelium responds in the same basic sequence of events as the epithelium lining the nose, trachea, and airways. The response depends on the physical, chemical, and toxic properties of the inhaled material, concentration of the material in the inhaled air, and duration/frequency of exposure. As in the case in the nose and other mucosal surfaces, the most rostral areas of the larynx lined by stratified squamous epithelium are those most exposed to trauma by direct contact with inhaled substances. Although the thickness of this epithelium and the inherent resistance to damage of its squamous surface layers provide more protection than other epithelial types, the stratified squamous epithelium lining the rostral larynx of rodents is still more susceptible than that in other areas of the oropharyngeal cavity because under normal conditions it lacks keratin or is poorly keratinized compared to oral or nasal mucosa. Thus, exposure to irritants can induce edema, inflammation, and if prolonged and severe enough, blistering, necrosis, and epithelial sloughing. Death due to occlusion of the lumen from edema and inflammation may occur (Miller and Renne, 1996). Prolonged irritation may induce hyperplasia of stratified squamous epithelium, often accompanied by formation of a thickly keratinized surface layer (hyperkeratosis).

The epithelium lining the base of the epiglottis in the area of transition from stratified squamous epithelium to respiratory epithelium of rodents is the area of the laryngeal epithelium most susceptible to damage from inhaled materials. The normal mucosal epithelium at the base of the rodent epiglottis consists of a mixture of ciliated and nonciliated columnar to round cells 2–3 cells thick, with no definite basal cell layer. A small area in the ventral midline at the rostral and caudal borders of the submucosal glands may be covered by squamous epithelium (Renne et al., 1992), but these areas do not have the prominent basal cell layer typical of stratified squamous epithelium.

Loss of cilia from surface epithelium and slight flattening of the epithelium lining the base of the epiglottis are subtle morphologic changes in response to inhaled materials. Detection and diagnosis of this degenerative change requires careful fixation and comparison of exposed with control tissues at precisely the same level of the larynx. Acute inflammation with edema and a suppurative inflammatory infiltrate in the lumenal epithelium and submucosa may lead to necrosis and ulceration, most frequently at the base of the epiglottis in rodents. If no further insult occurs, the epithelium will regenerate by epithelial cells migrating in from either side of the ulcer, then proliferating, eventually restoring normal mucosal morphology.

Repeated inhalation of materials sufficiently irritating to induce an inflammatory response and/or loss of cilia will eventually stimulate squamous metaplasia of the mixed ciliated and nonciliated (transitional) epithelium. Depending on severity and duration of exposure, this metaplastic epithelium also becomes hyperplastic and hyperkeratotic, with up to 10 or more layers of nucleated cells forming rete pegs, covered by a thick stratum corneum. Morphometry to determine the total area of epithelium lining the ventral floor of the base of the epiglottis has proven to be useful in quantitating hyperplasia and hyperkeratosis in rodents.

Although usually seen first and most severe at the base of the epiglottis in rodents, squamous metaplasia and hyperplasia may occur throughout the laryngeal mucosa. Areas that are normally lined by stratified squamous epithelium such as the medial surface of the vocal processes of the arytenoids, may undergo hyperplasia and hyperkeratosis. Areas normally covered with a thin layer of squamous epithelium such as the mucosa adjacent to the ventral pouch, may also become hyperplastic and hyperkeratotic. Metaplastia and hyperplasia may be accompanied by granulocytic or mononuclear inflammatory cell infiltrates in the adjacent submucosa, again depending on severity and duration of the stimulus. If squamous metaplasia of surface epithelium extends downward into the ducts of submucosal glands, the ducts may plug with hyperkeratotic debris, causing dilatation and inflammation of the affected glands. The granulomatous inflammatory response may lead to formation of polypoid lesions extending into the lumen and potentially decreasing airflow (Bucher et al., 1990).

Laryngeal anatomy and histology are quite different in non-rodent laboratory animal species. In dogs and monkeys the stratified squamous epithelium lining the epiglottal cartilage is much thicker than in rodents and extends further caudally, resulting in a more formidable barrier to inhaled materials impacting the base of the epiglottis. Diverticula (lateral ventricles and saccules) extend laterally rather than ventrally and are lined by stratified squamous epithelium in dogs and respiratory epithelium in monkeys. In some non-human primate species, large air sacs extend laterally into the adjacent neck and thoracic musculature. Arytenoid cartilages are more prominent in dogs and monkeys than in rodents. In these species the transition from stratified squamous to respiratory epithelium occurs in the epithelium lining the vocal processes of the arytenoids cartilages, just caudal to the junction of the lateral ventricles with the laryngeal lumen (Figures 2 and 3). In dogs and nonhuman primates the epithelium lining the caudal larynx is similar to rodents; slightly flattened ciliated columnar epithelium dorsally, sparsely ciliated tall columnar epithelium ventrally.

Based on these histologic differences and investigations of the responses of rodent larynges to inhaled toxicants, one would expect to find lesions further caudally in the larynges of dogs and monkeys compared to rodents. However, our search of published literature found very few descriptions of inhalation exposure-induced laryngeal lesions in either of these species. Squamous metaplasia of laryngeal epithelium was reported in rhesus monkeys inhaling formaldehyde for 6 weeks (Monticello et al., 1989). Our experience includes observation of a few lesions in the larynges of dogs and monkeys. These were foci of inflammation and/or ulceration of the squamous epithelium in or near the junction of the lateral ventricles with the laryngeal lumen (Figure 4).

Discussion

Squamous metaplasia and hyperplasia of laryngeal epithelium have been described in many chronic inhalation studies in rodents but progression to laryngeal neoplasia has been reported only a few times. Benign and malignant squamous neoplasms have been induced in rodent larynges by repeated intratracheal instillation of polycyclic aromatic hydrocarbons (PAH) such as benzo(a)pyrene (Saffiotti et al., 1972; Kaufman and Madison, 1974) or dimethylbenzanthracene (Della Porta et al., 1958), and by lifetime inhalation exposure to high concentrations of tobacco smoke (Dontenwill, 1972; Dontenwill et al., 1973; Bernfeld et al., 1974; Homburger et al., 1974; Homburger, 1975) or acetaldehyde (Feron et al., 1982). Laryngeal lesions described in these studies included squamous metaplasia, hyperplasia, and hyperkeratosis of the transitional epithelium. Conversely, there are numerous published reports of chronic inhalation exposures of rodents to irritant compounds such as tobacco smoke (Dalbey et al., 1980), ozone (Boorman et al., 1994), hexachlorocyclopentadiene (NTP, 1994), and molybdenum trioxide (NTP, 1997) in which laryngeal squamous metaplasia was induced but progression to neoplasia was not present, even after lifetime exposure. Squamous metaplasia of the laryngeal epithelium is described as occurring in at least half of the healthy human population, yet carcinoma of the larynx is very uncommon (Stell et al., 1982). It appears that, although severe squamous metaplasia and hyperplasia of laryngeal epithelium often precede neoplastic lesions, the metaplastic change by itself is simply a response to repeated irritation in which a resistant type of epithelium replaces a susceptible one (Gopinath et al., 1987; Burger et al., 1989). Holding rodents for recovery periods of 6 to 13 weeks following inhalation exposure to cigarette smoke consistently results in partial or complete regression of squamous metaplasia and hyperplasia of laryngeal epithelium to normal laryngeal histology (Coggins et al., 1989; Wehner et al., 1990).

Although inhalation studies using non-rodent species are often required by regulatory agencies as part of development and testing of candidate pharmaceuticals and chemicals, relatively little published information is available on the types of lesions to expect or the more frequent locations of upper respiratory tract injury from inhaled materials in dogs or primates. This is especially true for the larynx; a recent literature search located very little information on laryngeal lesions induced in nonhuman primates or dogs by inhalation. Although much information is available on inhalation-induced laryngeal injury in rodents, the differences in laryngeal anatomy and histology between rodents and other laboratory species are significant and toxicology data on effects in rodents may not apply to other species. Additional mapping and descriptive histology of canine and other nonrodent laboratory animal larynges and publication of detailed descriptions of laryngeal lesions induced in non-rodent species would be useful to fill in these gaps.

There are wide interspecies variations in mammalian laryngeal anatomy related to differences in normal environment, diet, and means of respiration. These interspecies variations are described in detail in excellent treatises on comparative laryngeal anatomy and physiology of various animal species (Negus, 1949) and of mammals (Harrison, 1995).