Central Annals of Otolaryngology and Rhinology

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.

*Corresponding authorDr. Rui Mamede, Department of Otorhinolaryngology and Head and Neck Surgery, Ribeirão Preto Medical School, University of São Paulo, Avenida Bandeirantes 3900, 14049-900, Ribeirão Preto, SP, Brazil, Tel: 55-16-362-313-50; Fax: 55-16-360-223-53;Email:

Submitted: 12 June 2015

Accepted: 01 July 2015

Published: 03 July 2015

Copyright© 2015 Mamede et al.

OPEN ACCESS

Keywords•Ventricular fold•Vocal fold•Collagen•Elasticfibers•Lamina propria

Original Research

Comparative Histological Analysis of Collagen and Elastic Fibers Present in the Ventricular Folds and the Vocal Folds of Cadaveric LaryngesAndré Silva Lucas1, Devandir Antonio de Souza Júnior2, Rui Celso M Mamede1*, Maria Célia Jamur2

1Department of Otorhinolaryngology and Head and Neck Surgery, University of São Paulo, Brazil2Department 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 fold the 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.

INTRODUCTIONThe ventricular folds or false vocal cords are located above

the vocal folds (cords) and separated from them by the laryngeal ventricle [1]. The function of the ventricular folds in phonation is controversial. The ventricular folds are associated with closure of the laryngeal lumen during swallowing, coughing, gagging, and other [1]. The ventricular folds are also responsible for lubrication of the vocal folds by secreting mucous produced by their mucosal glands. This secretion is important in providing an antimicrobial defense.2 According to Bertelli3the ventricular fold has a passive role that helps avoid airflow turbulence when

air enters into its ventricles. In contrast, Kutta et al. [2] believe that the ventricular fold has an active role in voice resonance. The structure of the ventricular fold contributes to its phonatory and non phonatory functions [3-7]. The ventricular fold is a complex histological structure composed of muscular, glandular, adipose and connective tissues [3-9]. In an attempt to better understand the physiological functions of the vocal folds, many morphological studies have been performed. Kotby et al. [5] describe the epithelial, glandular and muscular structures of the ventricular fold in a study of the microstructure of the human laryngeal ventricle and ventricular fold. Their analyses showed

Central

Mamede et al. (2015)Email:

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

that the ventricle was composed of mucosa and muscle. The mucosa included an epithelium, and a lamina propria with a loose layer of elastic and collagenous fibers. The histological structure of the ventricular fold contributes to its mechanical properties and behavior during articulation and phonation. However, it is not clear how this structure contributes to phonation. Studies by Alipour et al. [10] suggested a biomechanical model of phonation with provision for ventricular control. But, it still is not clear how these structures contribute to adduction/abduction before, during, and after phonation. Other functions are also attributed to the ventricular fold in phonatory rehabilitation [3-9]. Behlau et al. [6] stated that, in some cases, the voice generated in the supraglottis by the ventricular folds is a good option for voice restoration in patients with vocal fold paralysis, subglottic stenosis or sequelae from oncologic larynx surgeries. It has been demonstrated that during ventricular fold vibration a low frequency component is present in the spectral data. These studies also showed that in dog larynges without a supraglottis, the sound has more pressure and is louder. However, the majority of the morphological features described for ventricular folds are inconsistent with sound production and the histological characteristics of the ventricular folds described in the literature are divergent [2, 3, 5, 6, 8].

In the present study the histological features of the ventricular fold were studied in comparison to the vocal folds. The distribution of collagen and elastic fibers in both structures were analyzed and quantified. Understanding the structural features of the vocal and ventricular folds may facilitate the use of the ventricular folds in phonation after laryngeal damage.

METHODSLarynges were removed from male cadavers ranging in age

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 laryngeal diseases and had not required orotracheal intubation for more than 3 days and the individuals were non-smokers with a negative history of laryngeal disease. The evaluation was based on interviews with the relatives and/or persons responsible for reviewing the patients’ medical report for submission to autopsy, and macroscopic analysis for the identification of injuries. The study was approved by the Research Ethics Committee of the Ribeirão Preto Medical School, USP (protocol nº 12475/2004) and all persons responsible for the patients gave written informed consent.

Larynges were removed with the hyoid bone and the first tracheal ring to avoid structural alteration. Immediately after removal, they were fixed in 10% formaldehyde. Then, half of the superior epiglottis cartilage, hyoid bone and excess surrounding tissue were carefully removed. An incision was made in the superglottis at the superior border of the thyroid cartilage in 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, in a horizontal plane, 1 cm above the free border of the vocal fold. This piece was cut longitudinally in the posterior medium line and divided in two hemi larynges by cutting the thyroid cartilage at the anterior medium line. The pieces were decalcified in 10%

trichloroacetic acid (TCA), dehydrated in an ascending ethanol series (50 to 100%), immersed in terpineol oil, and cleared in benzol. Finally, the hemilarynges were separated, cut into coronal sections and divided into three layers, anterior, middle and posterior, and embedded in paraffin.

Semi sequential 6 μm thick sections were obtained with an interval of 10 μm betweeneach series of 3 sequential sections. A total of 9 sections were obtained from each segment of 14 hemilarynges for a total of 378 sections. The sections were then deparaffinized, hydrated and 126 sections were stained with hematoxylin and eosin (H.E.) for morphological analysis. For the identification of collagen, 126 sections were stained with 0.1 g Sirius Red in 100 ml of saturated picric acid, pH 2.0, for 20 minutes [11]. For elastic fiber analysis, 126 sections were stained for 60 minutes with Weigert’s elastic stain. After staining, the sections were dehydrated, cleared, mounted with Permount and observed with an Olympus BX50 microscope. The specimens stained with picrosirius red were analyzed by polarized light microscopy [12,13]. Under these conditions collagen fibers stained with picrosirius red are birefringent. Type I collagen fibers show a yellow, orange or red birefringence. Type III collagen fibers have a green or greenish birefringence. Images were acquired with a Nixon DXM 1200 digital camera using the same level of light intensity for all samples. The images of the 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, MD). The intensity of collagen birefringence and the presence of elastic fibers were quantified. For collagen quantitation, using a 10x objective, areas of 22,475 pixels were identified in the superficial, middle and deep layers of the connective tissue of vocal and ventricular fold. The total collagen in these areas was quantified using Image-Pro Plus by measuring the intensity of the birefringence. Areas similar to those used for collagen analysis were selected for elastic fiber analysis. The elastic fibers, which stained dark brown with Weigert’s elastic stain, were quantitated by selecting the appropriate color range using Image-Pro Plus. The statistical comparison between paired samples was done using Student’s t-test. Data were expressed as the mean±SD of a minimum of three separate samples. p values <0.05 were considered significant.

RESULTS

Histological characteristics

The ventricular folds presented invaginations of the laryngeal mucosa in the direction of the midline. The folds had one surface facing the lumen of the larynx and another surface forming the medial wall of the laryngeal ventricle. The vocal fold is located inferior to the ventricular fold (Figure 1A). The vocal fold and the free margin of the ventricular fold are lined with non-keratinized stratified squamous epithelium, in contrast to the larynx which is lined with ciliated pseudostratified columnar epithelium with goblet cells (Figure 1A and B). The subepithelial portion of the ventricular fold consists of uniform, nonstratified fibroelastic tissue. The mucosa, located below the epithelium on the free margin of the fold, consists of a thick, uniform layer permeated by blood and lymph vessels, adipose tissue and

Central

Mamede et al. (2015)Email:

Ann Otolaryngol Rhinol 2(6): 1045 (2015) 3/5

countless seromucous glands in the deeper region (Figure 2A). The vocal fold is characterized by the presence of a stratified lamina propria with a gradual increase in fibro elastic tissue as it deepens laterally in the direction of the vocal muscle (Figure 2B).

Collagen distribution in the ventricular and vocal folds

The distribution of the collagen fibers in the mucosa of the ventricular fold appears to be homogeneous and fibers are present from the superficial to the deep layer of lamina propria (Figure 3A). This distribution is heterogeneous in the vocal fold and collagen fibers formed a stratum in the deep layer of lamina propria (Figure 3B). Type I and Type III collagen can be distinguished in the ventricular fold (Figure 3C) and the vocal fold (Figure 3D) by picrosirus red staining and polarized light microscopy. Quantitative analysis shows no significant differences in the percentage of total collagen in the vocal fold (5.66%±2 .44%) and in the ventricular fold (7.93%±1 .21%) (Figure 4). Also there is no difference in the percentage of collagen type I 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 that found in the vocal fold (2.86%±1 .9) (Figure 5).

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

DISCUSSIONHistological analysis of the vocal fold confirms previous

findings which clearly show the stratification of the lamina

Figure 1 Microstructure of the ventricular and vocal folds. A. Ventricular fold (VenF), vocal fold (VF); nonkeratinized stratified squamous epithelium (NSSE) in the margin close to the laryngeal ventricle (LV), free margin of the vocal and ventricular folds, and ciliated pseudostratified columnar epithelium (CPCE) inside the ventricle. B. Detail of the ventricular fold showing the transition of the stratified to cylindrical epithelia (arrow).

Figure 2 Histological characteristics of the ventricular and vocal folds. A. Ventricular fold showing the mucosal epithelium (arrow), fibroelastic tissue (FIB), seromucous glands (GLD) and adipose tissue (ADI). B. Vocal fold showing stratification of the lamina propria (SLP – Superficial lamina propria; ILP Intermediate lamina propria; DLP – Deep lamina propria) and the vocal muscle (VM).

Figure 3 Collagen distribution in the ventricular and vocal folds. In the ventricular fold (A) the collagen is localized from the superficial to the deep layer of lamina propria (arrows). In the vocal fold (B) the collagen appears stratified and is localized in the deep layer of lamina propria (arrows). Type I collagen, yellow, red or orange birefringence, and Type III collagen, green birefringence, can be seen 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 found lining most of the ventricular fold. The stratified non-keratinized squamous epithelium, similar to that found in the vocal fold, is observed only at the free margin of the ventricular fold [14]. Collagen and elastic fibers are found just below the epithelium, but in the ventricular fold the connective tissue is not organized into distinct layers [13, 14] as was reported for the vocal fold [15,16].

Central

Mamede et al. (2015)Email:

Ann Otolaryngol Rhinol 2(6): 1045 (2015) 4/5

Figure 4 Quantitative analysis of collagen in the ventricular and the vocal folds. Sections were stained with picrosirius red and analyzed by polarized light microscopy. The intensity of the collagen birefringence was quantified using Image-Pro Plus. No significant differences were found between samples. Mean±SD. p ≤0.4529.

Figure 5 Quantitative analysis of type I and type III collagen in the ventricular and vocal folds. Sections were stained with picrosirius red and analyzed by polarized light microscopy. The intensity and the color of the collagen birefringence were quantified using Image-Pro Plus. Means ±SD. p ≤ 0,0448. * = Significant.

Figure 7 Quantitative analysis of the elastic fibers in the ventricular and vocal folds. The sections were stained with stained with Weigert’s elastic stain and the elastic fibers were analyzed using Image-Pro Plus. No significant differences were found between samples. Mean±SD. p≤0.3895.

Figure 6 Elastic fiber distribution. In the ventricular fold (A) the elastic fibers (arrows) are present from the superficial to the deep layer of the lamina propria. In the vocal fold (B) the elastic fibers are concentrated in the deep layer of the lamina propria. Staining: Weigert’s elastic stain.

Collagen fibers were detected by their birefringence using picrosirius red staining in conjunction with polarized microscopy [11,17]. Type I collagen fibers are formed by the aggregation of collagen fibrils. They are thick and show yellow, orange or red birefringence. In type III collagen, the fibrils are glycosylated, which interferes with bundle formation [11]. These collagen fibers are thin and the birefringence has a green color. Using this method it was possible to distinguish between collagen type I and type III fibers. Although there are some detractors [15] to the use of picrosirius red to identify collagen fibers, it remains as a widely accepted method to distinguish between types of collagen fibers.

Chan et al. [16] examined the elasticity of the ventricular folds from six male larynges and six female larynges. Based on tests for the determination of expansibility to uniaxial tension and post-stretching deformation, they observed a 20% greater flexibility in male ventricular folds compared to female folds. In the same study, the authors obtained sections from the middle third of these three larynges, two from females and one from a male, and quantified the composition of the lamina propria of the ventricular fold stained with Masson Trichromic and Van Gieson’s stains. According to these authors, the female ventricular fold had 2.5 times more seromucous glands, 36% more collagen fibers and less than one third of the elastic fibers of the male fold. This finding is compatible with the greater elasticity of the male fold, since the elasticity of glandular tissue is reduced. Despite the differences in methodology used in our study and the absence of female larynges, we also found large amounts of collagen fibers in both the ventricular and the vocal folds. However, we did not find differences in the percent of elastic fibers present in the ventricular fold and the vocal fold. In the present study we demonstrated that most of the collagen fibers are in the deeper layers of the connective tissue of ventricular folds and vocal folds. The amount of elastic fibers also increased towards the deeper layers of the connective tissue. In the ventricular fold the percentage of type I collagen is greater than type III collagen. The increase in type I collagen could make the ventricular fold more resistant and help in vibration.

Central

Mamede et al. (2015)Email:

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

Over the last few years, studies have shown the active participation of the ventricular folds in various styles of laryngeal songs (biphonic songs) such as Gregorian chants or certain Tibetan mantras [7]. These studies demonstrated that it is possible to train the ventricular fold and thus to modify the voice generated. Ricz et al. [18] also demonstrated that the musculature of the ventricular fold used in glottis reconstruction after cordectomy shows recruitment of motor units during sound emission, with action potentials morphologically similar to those of the remaining vocal fold. The same author also observed that in cases in which the fold had been used for phonation for a longer period of time there was a reduction of fundamental frequency, suggesting the possible presence of local muscle hypertrophy and changes in muscle fiber type in order to increase the specialization of the muscle. Hoh [19] has reported that chronic stimulation of fast twitch muscle fibers with low frequency impulses can transform them into slow twitch fibers.

The use of the ventricular fold for phonation may also stimulate changes in the pattern of synthesis of connective tissue fibers that could help in adduction and vibration of the ventricular folds. Hartnick et al. [13] and Sato et al. [20] studied the patterns of development and maturation of the lamina propria of the vocal folds up to the adult phase and concluded that the mechanical influence of the transglottic airflow and phonation itself induce the differentiation into layers of the lamina propria. Hartnick et al. [13] also stated that, by seven years of age, it is already possible to observe a cellular stratification into three layers with different densities. Although only after 13 years of age is it possible to define layers with fibroelastic composition of different densities. More recently, Roberts and Morton [21] observed that changes in structure of the vocal folds continue into old age.

The capacity of ventricular folds to produce movement is clear but the specific structures involved in this mechanism remain to be clarified. Further analysis of the components of connective tissue from ventricular folds from patients who use them for phonation will help to elucidate this hypothesis.

CONCLUSIONAlthough the percentage of type I collagen is greater than

type III collagen in the ventricular fold, the overall percentage of collagen fibers and elastic fibers is similar between the vocal fold and the ventricular fold.

ACKNOWLEDGEMENTSWe would like to thank Vani Maria Alves Correa for technical

assistance and Dr. Constance Oliver for critical reading of the manuscript, both from the Department of Cell and Molecular Biology and Pathogenic Bioagents, FMRP-USP, Ribeirão Preto, Brazil. This work was supported by Grant No. 470814/2004-7 from CNPq (Conselho Nacional de Pesquisa) to Dr. Rui Mamede.

REFERENCES1. Pinho SM, Pontes PA, Gadelha ME, Biasi N. Vestibular vocal fold

behavior during phonation in unilateral vocal fold paralysis. Voice. 1999; 13: 36-42.

2. Kutta H, Steven P, Kohla G, Tillmann B, Paulsen F. The human false vocal folds -- an analysis of antimicrobial defense mechanisms. Anat Embryol (Berl). 2002; 205: 315-323.

3. Bertelli AP. Câncer da laringe. Editora Manole.1980; 356.

4. Reidenbach MM. The muscular tissue of the vestibular folds of the larynx. Eur Arch Otorhinolaryngol. 1998; 255: 365-367.

5. Kotby MN, Kirchner JA, Kahane JC, Basiouny SE, El-Samaa M. Histo-anatomical structure of the human laryngeal ventricle. Acta Otolaryngol. 1991; 111: 396-402.

6. Behlau M, Gonçalves MI, Pontes P. Physiology of sound source following partial vertical laryngectomy VII Pacific Voice Conference – Voice Conservation, Treatment and Restoration after Laryngeal Carcinoma. Digest. 1994; 32-33.

7. Tsai CG, Shau YW, Hsiao TW. False vocal fold surface waves during Sygyt singing: A hypothesis. International Conference on Voice Physiology and Biomechanics. 2004; 18-20.

8. Guida HL, Zorzetto NL. Morphological and Histochemical Analysis of the Human Vestibular Fold. Int. J. Morphol. 2007; 25: 8.

9. Kucinschi BR, Scherer RC, DeWitt KJ, Ng TT. Flow visualization and acoustic consequences of the air moving through a static model of the human larynx. Biomech Eng. 2006; 128: 380-390.

10. Alipour F, Jaiswal S, Finnegan E. Aerodynamic and acoustic effects of false vocal folds and epiglottis in excised larynx models. Ann Otol Rhinol Laryngol. 2007; 116: 135-144.

11. Junqueira LC, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J. 1979; 11: 447-455.

12. Zhang C, Zhao W, Frankel SH, Mongeau L. Computational aeroacoustics of phonation, part II: Effects of flow parameters and ventricular folds. Acoust Soc Am. 2002; 112: 2147-2154.

13. Hartnick CJ, Rehbar R, Prasad V. Development and maturation of the pediatric human vocal fold lamina propria. Laryngoscope. 2005; 115: 4-15.

14. Hirano M. Morphological structure of the vocal cord as a vibrator and its variations. Folia Phoniatr (Basel). 1974; 26: 89-94.

15. Rich L, PW. Collagen and picrosirius red staining: a polarized light assessment of fibrillar hue and spatial distribution. Braz. J. morphol 2002; 22: 8.

16. Chan RW, Fu M, Tirunagari N. Elasticity of the human false vocal fold. Ann Otol Rhinol Laryngol. 2006; 115: 370-381.

17. Junqueira LC, Cossermelli W, Brentani R. Differential staining of collagens type I, II and III by Sirius Red and polarization microscopy. Arch Histol Jpn. 1978; 41: 267-274.

18. Ricz H, Mamede RCM, LN AR. Análise funcional da laringe pós cordectomia, reconstruída com retalho de vestibular fold. Rev Bras Otorrinolaringol. 2004. 70: 6.

19. Hoh JF. Laryngeal muscle fibre types. Acta Physiol Scand. 2005; 183: 133-149.

20. Sato K, Umeno H, Nakashima T, Nonaka S, Harabuchi Y. Expression and distribution of hyaluronic acid and CD44 in unphonated human vocal fold mucosa. Ann Otol Rhinol Laryngol. 2009; 118: 773-780.

21. Roberts T, Morton R, Al-Ali S. Microstructure of the vocal fold in elderly humans. Clin Anat. 2011; 24: 544-551.

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.

Cite this article