Central Annals of Otolaryngology and Rhinology

Original Research *Corresponding author Dr. Rui Mamede, Department of Otorhinolaryngology and Head and Neck Surgery, Ribeirão Preto Medical Comparative Histological School, University of São Paulo, Avenida Bandeirantes 3900, 14049-900, Ribeirão Preto, SP, Brazil, Tel: 55-16-362- Analysis of Collagen and Elastic 313-50; Fax: 55-16-360-223-53;Email: Submitted: 12 June 2015 Fibers Present in the Ventricular Accepted: 01 July 2015 Published: 03 July 2015 Copyright Folds and the Vocal Folds of © 2015 Mamede et al. Cadaveric Larynges OPEN ACCESS Keywords André Silva Lucas1, Devandir Antonio de Souza Júnior2, Rui • Ventricular fold Celso M Mamede1*, Maria Célia Jamur2 • Vocal fold • Collagen 1 Department of Otorhinolaryngology and Head and Neck Surgery, University of São Paulo, • Elastic fibers Brazil • Lamina propria 2Department of Cell and Molecular Biology and Pathogenic Bioagents, University of São Paulo, Brazil

Abstract Introduction: Knowledge of the structural features of the vocal fold and the ventricular fold may help to understand the phonation process, and consequently may facilitate the use of the ventricular fold in phonation after laryngeal damage. Objective: To compare the histology and the distribution of collagen and elastic fibers between the ventricular folds and the vocal folds. Methods: Hemilaringes from 14 male cadavers were collected, processed for histology, sections stained for collagen and elastic fibers and analyzed. Results: The major part of the ventricular fold was lined with pseudostratified ciliated columnar epithelium containing goblet cells. In the ventricular folds the collagen fibers are homogeneously distributed in layers. In contrast, in the vocal foldthe collagen fibers are unorganized and have a heterogeneous distribution. The percent of total collagen and of type I and type III collagen is similar between the vocal fold and the ventricular fold. However, the percent of type I collagen is increased in the ventricular fold in comparison to type III collagen. In the ventricular fold the elastic fibers are found in all layers of lamina propria, while in the vocal fold these elastic fibers are preferentially found in the deep layer of the lamina propria. The percent of elastic fibers is similar between the vocal folds and the ventricular folds. Conclusion: The percentage of collagen fibers and elastic fibers is similar between the vocal fold and the ventricular fold. However, in the ventricular fold the percentage of type I collagen is greater than type III collagen.

INTRODUCTION air enters into its ventricles. In contrast, Kutta et al. [2] believe that the ventricular fold has an active role in voice resonance. The ventricular folds or false vocal cords are located above The structure of the ventricular fold contributes to its phonatory the vocal folds (cords) and separated from them by the laryngeal and non phonatory functions [3-7]. The ventricular fold is a ventricle [1]. The function of the ventricular folds in phonation is complex histological structure composed of muscular, glandular, controversial. The ventricular folds are associated with closure adipose and connective tissues [3-9]. In an attempt to better of the laryngeal lumen during swallowing, coughing, gagging, and other [1]. The ventricular folds are also responsible for understand the physiological functions of the vocal folds, many lubrication of the vocal folds by secreting mucous produced by morphological studies have been performed. Kotby et al. [5] their mucosal glands. This secretion is important in providing describe the epithelial, glandular and muscular structures of the an antimicrobial defense.2 According to Bertelli3the ventricular ventricular fold in a study of the microstructure of the human laryngeal ventricle and ventricular fold. Their analyses showed fold has a passive role that helps avoid airflow turbulence when Cite this article: Lucas AS, de Souza Júnior DA, Mamede RCM, Jamur MC (2015) Comparative Histological Analysis of Collagen and Elastic Fibers Present in the Ventricular Folds and the Vocal Folds of Cadaveric Larynges. Ann Otolaryngol Rhinol 2(6): 1045. Mamede et al. (2015) Email: Central that the ventricle was composed of mucosa and muscle. The trichloroacetic acid (TCA), dehydrated in an ascending ethanol mucosa included an epithelium, and a lamina propria with a loose in benzol. Finally, the hemilarynges were separated, cut into of the ventricular fold contributes to its mechanical properties coronalseries (50 sections to 100%), and divided immersed into inthree terpineol layers, oil,anterior, and clearedmiddle andlayer behavior of elastic during and collagenous articulation fibers. and phonation.The histological However, structure it is not clear how this structure contributes to phonation. Studies by Alipour et al. [10] suggested a biomechanical model of phonation and posterior, and embedded in paraffin. with provision for ventricular control. But, it still is not clear A totalSemi of sequential 9 sections 6 wereμm thick obtained sections from were each obtained segment with of 14an how these structures contribute to adduction/abduction before, hemilaryngesinterval of 10 for μm a betweeneachtotal of 378 sections. series of The 3 sequentialsections were sections. then during, and after phonation. Other functions are also attributed to the ventricular fold in phonatory rehabilitation [3-9]. Behlau hematoxylin and eosin (H.E.) for morphological analysis. For et al. [6] stated that, in some cases, the voice generated in the deparaffinized, hydrated and 126 sections were stained with supraglottis by the ventricular folds is a good option for voice 0.1 g Sirius Red in 100 ml of saturated picric acid, pH 2.0, for 20 restoration in patients with vocal fold paralysis, subglottic the identification of collagen, 126 sections were stained with stenosis or sequelae from oncologic larynx surgeries. It has for 60 minutes with Weigert’s elastic stain. After staining, the been demonstrated that during ventricular fold vibration a low sectionsminutes [11].were For dehydrated, elastic fiber cleared, analysis, mounted 126 sections with Permount were stained and frequency component is present in the spectral data. These observed with an Olympus BX50 microscope. The specimens studies also showed that in dog larynges without a supraglottis, stained with picrosirius red were analyzed by polarized light the sound has more pressure and is louder. However, the majority of the morphological features described for ventricular stained with picrosirius red are birefringent. Type I collagen folds are inconsistent with sound production and the histological microscopy [12,13]. Under these conditions collagen fibers characteristics of the ventricular folds described in the literature are divergent [2, 3, 5, 6, 8]. werefibers acquired show a with yellow, a Nixon orange DXM or 1200 red digital birefringence. camera using Type the III In the present study the histological features of the samecollagen level fibers of light have intensity a green for or greenishall samples. birefringence. The images Images of the ventricular fold were studied in comparison to the vocal folds. lamina propria of the vocal and ventricular folds stained with picrosirius red and with Weigert’s elastic stain were analyzed using Image-Pro Plus 5.1.2.59 (Media Cybernetics, Inc., Bethesda, featuresThe distribution of the vocal of collagen and ventricular and elastic folds fibers may in facilitate both structures the use MD). The intensity of collagen birefringence and the presence of ofwere the ventricular analyzed and folds quantified. in phonation Understanding after laryngeal the damage. structural

METHODS elastic fibers were quantified. For collagen quantitation, using Larynges were removed from male cadavers ranging in age vocala 10x and objective, ventricular areas fold. of 22,475The total pixels collagen were in identifiedthese areas in was the superficial, middle and deep layers of the connective tissue of from 43 to 67 years (mean age: 54.71 years) with a maximum post mortem time of 24 h. The causa mortis was not related to birefringence. Areas similar to those used for collagen analysis laryngeal diseases and had not required orotracheal intubation quantified using Image-Pro Plus by measuring the intensity of the for more than 3 days and the individuals were non-smokers with stained dark brown with Weigert’s elastic stain, were quantitated a negative history of laryngeal disease. The evaluation was based bywere selecting selected the for appropriate elastic fiber coloranalysis. range The using elastic Image-Pro fibers, which Plus. on interviews with the relatives and/or persons responsible for The statistical comparison between paired samples was done reviewing the patients’ medical report for submission to autopsy, using Student’s t-test. Data were expressed as the mean±SD of a minimum of three separate samples. p values <0.05 were study was approved by the Research Ethics Committee of the and macroscopic analysis for the identification of injuries. The RESULTS and all persons responsible for the patients gave written considered significant. informedRibeirão Pretoconsent. Medical School, USP (protocol nº 12475/2004) Histological characteristics The ventricular folds presented invaginations of the laryngeal tracheal ring to avoid structural alteration. Immediately after mucosa in the direction of the midline. The folds had one surface Larynges were removed with the hyoid bone and the first facing the lumen of the larynx and another surface forming the superior epiglottis cartilage, hyoid bone and excess surrounding medial wall of the laryngeal ventricle. The vocal fold is located tissueremoval, were they carefully were fixed removed. in 10% formaldehyde. An incision was Then, made half inof the inferior to the ventricular fold (Figure 1A). The vocal fold and superglottis at the superior border of the thyroid cartilage in the free margin of the ventricular fold are lined with non- a horizontal plan to remove the superior half of the epiglottis cartilage, hyoid bone and surrounding soft tissue. A cut was made in the superior border of the anterior ring of the cricoid cartilage, epitheliumkeratinized withstratified goblet squamous cells (Figure epithelium, 1A and B). in The contrast subepithelial to the in a horizontal plane, 1 cm above the free border of the vocal fold. larynx which is lined with ciliated pseudostratified columnar This piece was cut longitudinally in the posterior medium line and divided in two hemi larynges by cutting the thyroid cartilage onportion the free of the margin ventricular of the fold,fold consistsconsists ofof uniform,a thick, uniform nonstratified layer permeatedfibroelastic by tissue. blood The and mucosa, lymph located vessels, below adipose the tissueepithelium and at the anterior medium line. The pieces were decalcified in 10% Ann Otolaryngol Rhinol 2(6): 1045 (2015) 2/5 Mamede et al. (2015) Email: Central countless seromucous glands in the deeper region (Figure 2A). deepensThe vocal laterally fold is in characterized the direction byof the the vocal presence muscle of (Figure a stratified 2B). lamina propria with a gradual increase in fibro elastic tissue as it Collagen distribution in the ventricular and vocal folds

The distribution of the collagen fibers in the mucosa of the (Figureventricular 3A). fold This appears distribution to be is homogeneous heterogeneous and in fibers the vocal are present from the superficial to the deep layer of lamina propria lamina propria (Figure 3B). Type I and Type III collagen can be fold and collagen fibers formed a stratum in the deep layer of distinguished in the ventricular fold (Figure 3C) and the vocal Figure 2 Histological characteristics of the ventricular and vocal fold (Figure 3D) by picrosirus red staining and polarized light folds. A. Ventricular fold showing the mucosal epithelium (arrow), microscopy. Quantitative analysis shows no significant differences fibroelastic tissue (FIB), seromucous glands (GLD) and adipose tissue (ADI). B. Vocal fold showing stratification of the lamina propria in the percentage of total collagen in the vocal fold (5.66%±2 DLP – Deep lamina propria) and the vocal muscle (VM). Also there is no difference in the percentage of collagen type I (SLP – Superficial lamina propria; ILP Intermediate lamina propria; .44%) and in the ventricular fold (7.93%±1 .21%) (Figure 4). between the vocal (4.30%±1 .81%) and the ventricular folds (5.63%±1 .03%). Similar results were found for collagen type III in the vocal (1.40%±0 .60%) and the ventricular folds (2.64%±0 .36%). However, in the ventricular fold the percentage of type I collagen (5,63%±1,03%) was significantly higher compared to thatDistribution found in the of vocal elastic fold (2.86%±1 fibers in .9) the (Figure ventricular 5). and vocal folds

Elastic fibers were distributed in the mucosa of the vocal and the ventricular folds. In the ventricular fold the elastic fibers were localizedfound from in the the superficial deep layer to ofthe the deep lamina layer of propria the lamina (Figure propria 6B). However,(Figure 6A). no In differences contrast, in are the found vocal in fold the the percentage elastic fibers of elastic were fibers between the vocal (2. 23%±1 .01%) and the ventricular DISCUSSION folds (1.23%±0 .18%) (Figure 7). Figure 3 Collagen distribution in the ventricular and vocal folds. In

to the deep layer of lamina propria (arrows). In the vocal fold (B) the ventricular fold (A) the collagen is localized from the superficial Histological analysis of the vocal fold confirms previous findings which clearly show the stratification of the lamina of lamina propria (arrows). Type I collagen, yellow, red or orange birefringence,the collagen appearsand Type stratified III collagen, and green is localized birefringence, in the can deep be layerseen in the ventricular fold (C) and in the vocal fold (D). Polarized light microscopy. Staining: Picrosirius Red.

propria and are compatable with the body-cover theory of Hirano [14]. The histological features of the ventricular folds have been described in many previous studies [3, 5] but the descriptions differ from study to study [2, 4, 9]. In the present study a ciliated

pseudostratified columnar epithelium with goblet cells was Figure 1 Microstructure of the ventricular and vocal folds. A. keratinized squamous epithelium, similar to that found in the found lining most of the ventricular fold. The stratified non- vocal fold, is observed only at the free margin of the ventricular squamous epithelium (NSSE) in the margin close to the laryngeal ventricleVentricular (LV), fold free (VenF), margin vocal of the fold vocal (VF); and nonkeratinized ventricular folds, stratified and epithelium, but in the ventricular fold the connective tissue is fold [14]. Collagen and elastic fibers are found just below the ventricle. B. Detail of the ventricular fold showing the transition of the not organized into distinct layers [13, 14] as was reported for the ciliated pseudostratified columnar epithelium (CPCE) inside the vocal fold [15,16]. stratified to cylindrical epithelia (arrow). Ann Otolaryngol Rhinol 2(6): 1045 (2015) 3/5 Mamede et al. (2015) Email: Central

Figure 7 Figure 4 Quantitative analysis of collagen in the ventricular and the and vocal folds. The sections were stained with stained with Weigert’s vocal folds. Sections were stained with picrosirius red and analyzed by Quantitative analysis of the elastic fibers in the ventricular polarized light microscopy. The intensity of the collagen birefringence elastic stain and the elastic fibers were analyzed using Image-Pro Plus. No significant differences were found between samples. Mean±SD. was quantified using Image-Pro Plus. No significant differences were p≤0.3895. found between samples. Mean±SD. p ≤0.4529. picrosirius red staining in conjunction with polarized microscopy Collagen fibers were detected by their birefringence using

[11,17]. Type I collagen fibers are formed by the aggregation of whichcollagen interferes fibrils. They with are bundle thick and formation show yellow, [11]. These orange collagen or red birefringence. In type III collagen, the fibrils are glycosylated, method it was possible to distinguish between collagen type I and fibers are thin and the birefringence has a green color. Using this

type III fibers. Although there are some detractors [15] to the use of picrosirius red to identify collagen fibers, it remains as a widely acceptedChan method et al. [16] to distinguish examined between the elasticity types ofof thecollagen ventricular fibers. folds from six male larynges and six female larynges. Based on tests for the determination of expansibility to uniaxial tension

Figure 5 Quantitative analysis of type I and type III collagen in the ventricular and vocal folds. Sections were stained with picrosirius theand samepost-stretching study, the deformation,authors obtained they sections observed from a 20% the greatermiddle red and analyzed by polarized light microscopy. The intensity and thirdflexibility of these in male three ventricular larynges, foldstwo from compared females to andfemale one folds. from In a the color of the collagen birefringence were quantified using Image- the ventricular fold stained with Masson Trichromic and Van Pro Plus. Means ±SD. p ≤ 0,0448. * = Significant. male, and quantified the composition of the lamina propria of

Gieson’s stains. According to these authors, the female ventricular fold had 2.5 times more seromucous glands, 36% more collagen fold,fibers since and theless elasticity than one ofthird glandular of the elastictissue isfibers reduced. of the Despite male fold. the differencesThis finding in is methodology compatible with used the in greaterour study elasticity and the of absence the male of

in both the ventricular and the vocal folds. However, we did female larynges, we also found large amounts of collagen fibers the ventricular fold and the vocal fold. In the present study we not find differences in the percent of elastic fibers present in layers of the connective tissue of ventricular folds and vocal Figure 6 demonstrated that most of the collagen fibers are in the deeper deeper layers of the connective tissue. In the ventricular fold the Elastic fiber distribution. In the ventricular fold (A) the percentagefolds. The amountof type I ofcollagen elastic is fibers greater also than increased type III collagen. towards The the areelastic concentrated fibers (arrows) in the are deep present layer from of the the lamina superficial propria. to theStaining: deep layer of the lamina propria. In the vocal fold (B) the elastic fibers increase in type I collagen could make the ventricular fold more Weigert’s elastic stain. resistant and help in vibration.

Ann Otolaryngol Rhinol 2(6): 1045 (2015) 4/5 Mamede et al. (2015) Email: Central

Over the last few years, studies have shown the active 2. participation of the ventricular folds in various styles of vocal folds -- an analysis of antimicrobial defense mechanisms. Anat EmbryolKutta H, (Berl).Steven 2002;P, Kohla 205: G, 315-323. Tillmann B, Paulsen F. The human false certain Tibetan mantras [7]. These studies demonstrated that 3. Bertelli AP. Câncer da laringe. Editora Manole.1980; 356. itlaryngeal is possible songs to (biphonic train the ventricular songs) such fold as Gregorian and thus to chants modify or the voice generated. Ricz et al. [18] also demonstrated that the 4. Reidenbach MM. The muscular tissue of the vestibular folds of the larynx. Eur Arch Otorhinolaryngol. 1998; 255: 365-367. musculature of the ventricular fold used in glottis reconstruction after cordectomy shows recruitment of motor units during sound 5. Kotby MN, Kirchner JA, Kahane JC, Basiouny SE, El-Samaa M. emission, with action potentials morphologically similar to those Histo-anatomical structure of the human laryngeal ventricle. Acta of the remaining vocal fold. The same author also observed that in Otolaryngol. 1991; 111: 396-402. cases in which the fold had been used for phonation for a longer 6. period of time there was a reduction of fundamental frequency, suggesting the possible presence of local muscle hypertrophy –Behlau Voice M, Conservation, Gonçalves MI, Treatment Pontes and P. Physiology Restoration of after sound Laryngeal source Carcinoma.following partial Digest. vertical 1994; 32-33.laryngectomy VII Pacific Voice Conference specialization of the muscle. Hoh [19] has reported that chronic 7. and changes in muscle fiber type in order to increase the Sygyt singing: A hypothesis. International Conference on Voice PhysiologyTsai CG, Shau and YW, Biomechanics. Hsiao TW. 2004;False vocal18-20. fold surface waves during stimulation of fast twitch muscle fibers with low frequency 8. The use of the ventricular fold for phonation may also impulses can transform them into slow twitch fibers. the Human Vestibular Fold. Int. J. Morphol. 2007; 25: 8. stimulate changes in the pattern of synthesis of connective tissue Guida HL, Zorzetto NL. Morphological and Histochemical Analysis of 9. Kucinschi BR, Scherer RC, DeWitt KJ, Ng TT. Flow visualization and folds. Hartnick et al. [13] and Sato et al. [20] studied the patterns acoustic consequences of the air moving through a static model of the human larynx. Biomech Eng. 2006; 128: 380-390. offibers development that could and help maturation in adduction of andthe vibrationlamina propria of the ofventricular the vocal folds up to the adult phase and concluded that the mechanical 10. Alipour F, Jaiswal S, Finnegan E. Aerodynamic and acoustic effects of false vocal folds and epiglottis in excised larynx models. Ann Otol the differentiation into layers of the lamina propria. Hartnick et Rhinol Laryngol. 2007; 116: 135-144. al.influence [13] also of stated the transglottic that, by seven airflow years and of age, phonation it is already itself possible induce 11. densities. Although only after 13 years of age is it possible to tissueJunqueira sections. LC, BignolasHistochem G, J. Brentani1979; 11: RR.447-455. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in to observe a cellular stratification into three layers with different 12. More recently, Roberts and Morton [21] observed that changes in structuredefine layers of the with vocal fibroelastic folds continue composition into old of age. different densities. AcoustZhang C, Soc Zhao Am. W, 2002; Frankel 112: SH, 2147-2154. Mongeau L. Computational aeroacoustics of phonation, part II: Effects of flow parameters and ventricular folds. The capacity of ventricular folds to produce movement is 13. Hartnick CJ, Rehbar R, Prasad V. Development and maturation of the pediatric human vocal fold lamina propria. Laryngoscope. 2005; 115: 4-15. connectiveclear but thetissue specific from structuresventricular involvedfolds from in patients this mechanism who use 14. Hirano M. Morphological structure of the vocal cord as a vibrator and themremain for to phonation be clarified. will help Further to elucidate analysis this of thehypothesis. components of its variations. Folia Phoniatr (Basel). 1974; 26: 89-94. CONCLUSION 15. Rich L, PW. Collagen and picrosirius red staining: a polarized light Although the percentage of type I collagen is greater than 2002; 22: 8. assessment of fibrillar hue and spatial distribution. Braz. J. morphol type III collagen in the ventricular fold, the overall percentage of 16. Chan RW, Fu M, Tirunagari N. Elasticity of the human false vocal fold. Ann Otol Rhinol Laryngol. 2006; 115: 370-381. and the ventricular fold. 17. Junqueira LC, Cossermelli W, Brentani R. Differential staining of collagen fibers and elastic fibers is similar between the vocal fold ACKNOWLEDGEMENTS collagens type I, II and III by Sirius Red and polarization microscopy. Arch Histol Jpn. 1978; 41: 267-274. We would like to thank Vani Maria Alves Correa for technical 18. Ricz H, Mamede RCM, LN AR. Análise funcional da laringe pós assistance and Dr. Constance Oliver for critical reading of the cordectomia, reconstruída com retalho de vestibular fold. Rev Bras manuscript, both from the Department of Cell and Molecular Otorrinolaringol. 2004. 70: 6. 19. 133-149. 7 Biology from CNPq and (Conselho Pathogenic Nacional Bioagents, de Pesquisa) FMRP-USP, to Dr.Ribeirão Rui Mamede Preto,. Hoh JF. Laryngeal muscle fibre types. Acta Physiol Scand. 2005; 183: Brazil. This work was supported by Grant No. 470814/2004- 20. REFERENCES and distribution of hyaluronic acid and CD44 in unphonated human vocalSato K, fold Umeno mucosa. H, Nakashima Ann Otol Rhinol T, Nonaka Laryngol. S, Harabuchi 2009; 118: Y. 773-780. Expression 1. behavior during phonation in unilateral vocal fold paralysis. Voice. 21. Roberts T, Morton R, Al-Ali S. Microstructure of the vocal fold in 1999;Pinho 13: SM, 36-42. Pontes PA, Gadelha ME, Biasi N. Vestibular vocal fold elderly humans. Clin Anat. 2011; 24: 544-551.

Cite this article Lucas AS, de Souza Júnior DA, Mamede RCM, Jamur MC (2015) Comparative Histological Analysis of Collagen and Elastic Fibers Present in the Ventricular Folds and the Vocal Folds of Cadaveric Larynges. Ann Otolaryngol Rhinol 2(6): 1045.

Ann Otolaryngol Rhinol 2(6): 1045 (2015) 5/5