Scanning Electron Microscopy of the Tongue,

Pharynx, and Larynx of Rats Exposed

to Cigarette Smoke

Regina Helena Garcia Martins, Tatiana Maria Goncalves, Sergio Luis Madeira, Norimar Hernandes Dias,

and Graziela de Oliveira Semenzati, Botucatu, S~ao Paulo, Brazil

Summary: Objective. To examine, by using scanning electron microscopy, the surface of the tongue, pharynx, and

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larynx of animals exposed to tobacco.Study Design. Experimental study.Methods. Twenty rats were allocated to two groups: group I, control group: 10 rats not exposed to any inhalingpollutant and group II, tobacco group: 10 rats exposed to smoke from 10 cigarettes twice a day for 260 days. Animalsof both groups had no restriction of food or water. After those 260 days, their aerodigestive segment was removed, andfragments of their tongue, hypopharynx, and right vocal fold were immersed in 2.5% glutaraldehyde and prepared forscanning electron microscopy.Results. The filiform tongue papillae of the tobacco group were irregularly displayed, flattened, and adhered to eachother. The hypopharynx mucosa was highly irregular, thickened, rough and had increased superficial peeling. The mu-cosa of the vocal folds had deep furrows surrounding the cells. These alterations were not identified for the controlgroup.Conclusion. Several changes were recorded for the tongue, pharynx, and larynx of tobacco group animals, confirmingthe harmful effects of smoking to the respiratory and digestive epithelium.Key Words: Tobacco–Smoking–Scanning electron microscopy–Rats–Tongue–Pharynx–Larynx.

INTRODUCTION

The oral cavity, the larynx, and the pharynx are responsible for30–40%, 25%, and 15%, respectively, of head and neck carci-nomas, and smoking has been reported by 95% of these pa-tients.1–4 Cigarette smoke contains a large number of harmfulchemicals, especially carbon monoxide, nicotine, and tar.Cellular absorption of tobacco’s noxious agents may lead toDNA mutations, triggering the carcinogenesis process.5

Experimental studies have used different methods to repro-duce lesions in the airways of animals by exposing the latterto cigarette smoke. Mucosal responses to these harmfulagents include hyperplasia, hyperkeratosis, dysplasias, andmetaplasia of the epithelium.6–12 Some of these lesionswere already shown by one of our studies exposing rats tocigarette smoke for 60 and 260 days12,13 and confirmed bydifferent animal studies.8–11 Those authors, however, did notinclude electron microscopic analysis, which may addimportant information.

The aim of this study was to examine, by using scanningelectron microscopy, the surface of the tongue, pharynx, andlarynx of animals exposed to cigarette smoke.

ted for publication September 24, 2013.udy was financially supported by Foundation of support research of the State ofo (FAPESP).he Departamento de Oftalmologia, Otorrinolaringologia e Cirurgia de Cabeca eBotucatu Medical School, S~ao Paulo State University, Botucatu, S~ao Paulo,ESP.

ss correspondence and reprint request to Regina Helena Garcia Martins, Departa-Oftalmologia, Otorrinolaringologia e Cirurgia de Cabeca e Pescoco, Faculdadeina de Botucatu - UNESP, Distrito de Rubi~ao J�unior, 18618-970, Botucatu, S~aorazil. E-mail: rmartins@fmb.unesp.brl of Voice, Vol. 28, No. 3, pp. 287-290997/$36.004 The Voice Foundationx.doi.org/10.1016/j.jvoice.2013.09.006

MATERIAL AND METHODS

This project was approved by the Animal ExperimentationEthics Committee of Botucatu Medical School, UNESP-UnivEstadual Paulista (698/2008). Twenty adult Wistar ratsweighed approximately 180–200 g were kept in individual ca-ges under acclimatized environment (temperature of23�C ± 2�C and humidity of 60% ± 5%) and allocated totwo study groups:

Group I, control: 10 rats received water and animal food adlibitum during 260 days;Group II, tobacco: 10 rats were exposed to smoke from 10cigarettes twice a day during 260 days, without foodrestriction.

Exposure to cigarette smoke was carried out by transferringgroup II animals (n¼ 10) to a chamber connected to a ‘‘smok-ing device.’’ Puffs of smoke were vacuum aspirated from ciga-rettes and introduced into the chamber during 30 minutes twicea day for 260 days; then, the chamber was opened to allow thesmoke out.8 The used cigarettes had the following composition:1.1 mg nicotine, 14 mg tar, and 15 mg carbon monoxide.

The animals were killed at 260 days after the beginning of theexperiment by means of intraperitoneal pentobarbital sodium(50 mg/kg). The aerodigestive segment containing the tongue,the pharynx, the larynx, and the first tracheal rings wereremoved and dissected (Figure 1). Each specimen was macro-scopically examined throughout its extension and, in theabsence of lesions, standardized biopsies were performed forthe following sites: base of tongue, right lateral hypopharynx,and right vocal fold. The obtained 0.6–0.8 cm fragments wereimmediately immersed in 2.5% glutaraldehyde solution andsubjected to processing.

FIGURE 1. Removed airway segment and biopsied sites. FIGURE 3. Tongue surface of a group II animal (tobacco). Dysmor-

phic and asymmetrical aspect of the filiform papillae. Scanning elec-

tron microscope, 3443.

Journal of Voice, Vol. 28, No. 3, 2014288

Preparation for scanning electron microscopy

After fixation using 2.5% glutaraldehyde for 48 hours, frag-ments were washed in phosphate buffer, 0.1 M, pH 7.3, fixedwith 1% osmium tetroxide solution, washed in phosphatebuffer, dehydrated in alcohol solutions at increasing concentra-tions, 75–100%, and dried in a critical point device (BalzersCPD-020; Union, Liechtenstein) using liquid carbon dioxide.The specimens were mounted on a metal base using silverglue and covered with gold in a Balzers MED-010 device(Liechtenstein). Then, each quadrant was blindly examinedby an experienced pathologist under a scanning electron micro-scope at increasing magnifications (model Quanta 200 FEG;FEI Company, EUA), under 15 KV tension.

RESULTS

Tongue

Control animals had their tongue surface covered with regularlydisplayed filiform tongue papillae (Figure 2). However, allsmoke-exposed animals had irregularly displayed papillaethat were frequently flattened and adhered to each other(Figures 3 and 4). Accumulation of food debris and mucus onthe papillae were also found.

FIGURE 2. Tongue surface of a group I animal (control). Uniform

arrangement of filiform papillae. Scanning electron microscope,

3400.

Hypopharynx

For control animals, the hypopharynx fragments examinedunder a scanning electron microscope showed highly foldedsurface, which allows this organ to distend during feeding,and some cells were detaching from the surface (Figure 5).For smoke-exposed animals, the hypopharynx surface wasgreatly irregular, thickened, rough and had increased superficialpeeling (Figures 6 and 7). The covering mucosa had ‘‘brilliant,impermeable, and smooth’’ aspect.

Larynx

For control animals, the surface of vocal folds was less foldedand had some detaching cells (Figure 8). On the other hand,smoke-exposed animals had deep furrows surrounding the cellson the surface of vocal folds, similarly to microclefts (Figure 9).

DISCUSSION

Smoking is responsible for the development of several lesions inthe airways including inflammation, acanthosis, hyperkeratosis,dysplasias, leukoplakias, erythroplakias, and carcinoma. These

FIGURE 4. Tongue surface of a group II animal (tobacco). Flattened

filiform papillae adhered to each other. Food debris and mucus depos-

ited on the papillae. Scanning electron microscope, 31499.

FIGURE 5. Hypopharynx of a group I animal (control). Light and

uniform mucosal folding and some detaching cells. Scanning electron

microscope, 31600.

FIGURE 7. Hypopharynx of a group II animal (tobacco). Irregular

aspect of the mucosae and intense desquamation. Scanning electron

microscope, 3500.

Regina Helena Garcia Martins, et al Tongue, Pharynx, and Larynx Injuries Caused by Smoking 289

lesions have been shown by several histologic studies8–13;however, investigation using scanning electron microscopy isscarce.

In the present study, smoke-exposed animals had importantchanges in their airway mucosae, such as marked folding andextreme epithelial peeling in the hypopharynx, in addition todeep furrows and microclefts in the larynx. Because thesechanges were not seen for control animals, we assume thatthey were due to the exposure to cigarette smoke. Similarchanges in the epithelial stratification were also reported byIsik et al7 for the vocal folds of rats exposed to cigarette smokeduring 2 hours a day for 60 days. Using scanning electron mi-croscopy, those authors identified desmosome connection lossand enlargement of intercellular junctions. In another morpho-logic study, Isik et al8 examined the trachea of rats, identifyingepithelial hyperplasia and areas of cilium loss.

Another important change observed for our smoke-exposedanimals was the ‘‘impermeable, brilliant, and smooth’’ aspectof the pharynx mucosa. This same change was already identi-fied and described in one of our previous scanning electron

FIGURE 6. Hypopharynx of a group II animal (tobacco). Intense

mucosal folding. Scanning electron microscope, 3260.

microscopic study of the vocal folds of Reinke edema patients,whose lesion is also caused by smoking.14 Correlation with his-tologic analysis indicated that these changes corresponded toepithelial thickening with hyperkeratosis, in which epithelialcells proliferate and the keratin layer increases, giving the mu-cosa the aspect of a ‘‘smooth’’ surface. B�an�oczy et al15 exam-ined under a scanning electron microscope oral fragments ofhumans with leukoplakia areas and noted this same keratinizedaspect for their mucosa. According to Reichart and Althoff,16

the hyperkeratotic mucosa, the folding irregularities, and theincreased detaching of surface cells are frequent findings in leu-koplakias and dysplasias of the squamous epithelium. Mucosalirregular folding and pleomorphism were also detected byWor-awongvasu17 in a scanning electron microscopic analysis of theoral mucosa of humans with dysplastic areas close to the squa-mous carcinoma.

Bolton et al11 experimentally showed the aggressions causedto the respiratory epithelium after exposure of rats to cigarettesmoke for 6 hours a day, three times a week during 14 weeks,and noted important areas of squamous metaplasia. Similar

FIGURE 8. Surface of the vocal fold of a group I animal (control).

Less folded mucosae and some detaching cells. Scanning electron mi-

croscope, 3500.

FIGURE 9. Surface of the vocal fold of a group II animal (tobacco)

showing deep sulcus around the cells. Scanning electron microscope,

32000.

Journal of Voice, Vol. 28, No. 3, 2014290

findings were reported by Duarte et al,10 who carried out anexperimental study exposing rats to the smoke of 10 cigarettes,three times a day for 25, 50, and 70 days. Those authors foundhyperplasia and squamous metaplasia of the free edge of vocalfolds in all three study periods.

The filiform papillae of smoke-exposed animals in the pre-sent study were highly altered, had irregular distribution andshapes, and were adhered to each other. Greater damage tothe tongue papillae, including destruction and disorganization,were reported by Harada et al6 in an experimental study repro-ducing oral lesions in the palate, tongue, and pharynx ofhamsters exposed to cigarette smoke. Caldeira et al9 used dailyintraperitoneal injections of nicotine to rats (0.250 mg nicotine/100 g body weight) and examined their oral mucosa by meansof transmission electron microscopy, after 90 days, notingepithelial atrophy, disorganization of cell membranes, and tis-sue damage. Those authors emphasized that these changesmay predispose the epithelium of the oral mucosa to the actionof other carcinogenic agents contained in the cigarette.

CONCLUSIONS

This study showed the results of scanning electron microscopicanalyses of the surface of tongue, pharynx, and larynx of ani-mals exposed to cigarette smoke. Several changes were re-corded for those sites, especially disorder of the filiformpapillae, irregular pharynx mucosal folds, and deep furrowsin the larynx epithelium, confirming the harmful effects of cig-arettes to the respiratory and digestive mucosae.

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