University of Veterinary Medicine Hannover
Histomorphometrical analysis of the fibrous components of the porcine vocal folds –
Stratigraphical features and their relevance for models in phoniatry
Thesis
Submitted in partial fulfilment of the requirements for the degree – Doctor of Veterinary Medicine –
Doctor medicinae veterinariae ( Dr. med. vet. )
by Anja Lang
Hamburg
Hannover 2014
Academic supervision: Univ.-Prof. Dr. med. vet. Hagen Gasse Institute of Anatomy
1. Referee: Univ.-Prof. Dr. med. vet. Hagen Gasse Institute of Anatomy
2. Referee: Univ.-Prof. Dr. med. vet. Karl-Heinz Waldmann Clinic for Swine and Small Ruminants,
Forensic Medicine and Ambulatory Services
Day of the oral examination: 21. 05. 2014
peer review system:
- LANG, A., R. KOCH and H. GASSE:
Histomorphometric analysis of collagen and elastic fibres in the cranial and caudal fold of the porcine glottis.
Anatomia, Histologia, Embryologia.
(accepted 05. 05. 2014, doi: 10.1111/ahe.12125) - LANG, A., R. KOCH and H. GASSE:
The histological components of the phoniatrical body-cover model in minipigs of different age.
(ready for submission)
Partial results of this thesis were presented at the following congresses:
- LANG, A., R. KOCH and H. GASSE (2012 a):
A comparison of the porcine and human glottis with emphasis on the elastic fibres.
In: 29. Arbeitstagung der Anatomischen Gesellschaft, Würzburg, 26.- 28. 09. 2012 (Poster 12).
Internet: URL: www.anatomische-gesellschaft.de/Tagungen- ag3/abstract- archive.html
- LANG, A., R. KOCH and H. GASSE (2012 b):
Histomorphometric analysis of fibre the contents of the cranial and caudal folds of the porcine glottis.
In: XXIXth Congress of the European Association of Veterinary Anatomists, Stara Zagora, Bulgaria, 25. - 28. 07. 2012 (Poster 37).
Bulgarian Journal of Veterinary Medicine 15, Suppl. 1, 79.
Der Bindegewebsapparat der Glottis beim Schwein unter besonderer Berücksichtigung des Alters.
In: 16. Workshop des Arbeitskreises Respiratorisches System der Deutschen Veterinärmedizinischen Gesellschaft (DVG) in Kooperation mit der Deutschen Gesellschaft für Pneumologie und Beatmungsmedizin (DPG).
Pneumologie 67, 345.
- LANG, A., R. KOCH and H. GASSE (2013 a):
Histomorphometry of non-corpuscular structures – potential and limitations.
In: Proceedings of the 7th Meeting of the Young Generation of Veterinary Anatomists, Lehmanns Media-Verlag, Berlin, 26.
- LANG, A., R. KOCH and H. GASSE (2013 b):
The elastic system of the porcine glottis: a model for human phoniatry?
A histomorphometric study of age-related changes in pigs.
In: 30. Arbeitstagung der Anatomischen Gesellschaft, Würzburg, 25. - 27. 09. 2013 (Poster 16).
Internet: URL: www.anatomische-gesellschaft.de/Tagungen- ag3/abstract-archive.html
List of abbreviations
1 Introduction ... 9
2 Survey on literature ... 12
2.1 Histomorphometry of non-corpuscular objects ... 12
2.2 The unique structure of the porcine glottis ... 15
2.3 The stratigraphical organisation of the lamina propria ... 22
2.4 The structural features of maturation and ageing of the vocal folds ... 28
3 Materials and Methods ... 32
3.1 Animals ... 32
3.2 Histological procedures ... 34
3.3 Histomorphometrical analysis of fibre amounts ... 35
3.4 Scoring of the diameters of collagen structures ... 39
3.5 Data analysis ... 41
4 Results... 45
Paper I ... 46
Paper II ... 94
5.1 The minipigs – classification of age groups ... 148
5.2 Histomorphometry of non-corpuscular objects: Potentials and limitations ... 150
5.3 Minipigs as experimental models in human phoniatry ... 152
5.4 Stratigraphical organisation of the porcine vocal folds ... 153
5.5 Maturation and ageing of the porcine vocal folds ... 156
5.6 Function of the porcine vocal folds ... 158
6 Summary ... 160
7 Zusammenfassung ... 163
8 References ... 166
9 Annex ... 183
9.1 Anatomy and terminology of relevant features of the human glottis with regard to species differences ... 183
apa.coll amounts per area of collagen structures apa.elast amounts per area of elastic fibres
approx. approximately
BMZ basement membrane zone
CauF caudal fold of the porcine glottis, caudal vocal fold CraF cranial fold of the porcine glottis, cranial vocal fold
DL deep layer
DLLP deep layer of the lamina propria e.g. exempli gratia (Latin): for example et al. et alii, et aliae (Latin): and others Fig. figure
i.e. id est (Latin): that is
IL intermediate layer
ILLP intermediate layer of the lamina propria kg kilogramme(s)
min minute(s) ml millilitre(s)
MLLP middle layer of the lamina propria µm micrometre(s)
n number of samples
SL superficial layer
SLLP superficial layer of the lamina propria Tab. table
ROI(s) Region(s) of Interest Z zone
1 Introduction
Four items shall be highlighted as the key elements of this study on the fibrous components of the porcine vocal folds.
(1) Histomorphometry of non-corpuscular objects (Paper I)
The quantitative evaluation of non-corpuscular structures (e.g. fibres) has remained a great challenge in morphological sciences. These structures lack certain pre-requisites which could make the procedure easy and efficient: The outlines of fibres are not well-demarcated, and fibres often overlap (LANG et al.
2012 b, 2013 a). Also, fibre staining may be irregular, of varying intensity, or weak.
Such properties make the Object Definition – the first, essential step of the histomorphometrical procedure – very problematic. In particular, the above qualities of non-corpuscular objects may interfere with the performance of automated procedures for the recognition of objects (LANG et al. 2013 a). In dealing with such challenges, certain steps of visual control and manual adjustment are required (LANG et al. 2013 a). The elaboration of an adequate, semi-automated procedure and its implementation into the study of the fibre apparatus of the vocal folds is described in Paper I.
(2) The unique structure of the porcine glottis (Paper I)
The pig’s glottis comprises two vocal folds – a cranial and a caudal fold, CraF and CauF – on each side of the larynx, as the porcine vocal ligament is split longi- tudinally into a cranial and a caudal part (WAIBL 2004). Each part of the vocal ligament is incorporated in a separate fold, CraF and CauF (KOCH et al. 2010).
These features distinguish the glottis of the pig from the glottis of other mammalian species, including humans (KOCH et al. 2010; LANG et al. 2012 a, b). The neglect
of this peculiarity has led to false assumptions of homology of the porcine cranial fold with the human vestibular fold (see e.g. JIANG et al. 2001; PEREIRA et al.
2009; FONSECA et al. 2010; JOHANES et al. 2011). Moreover, it seems that the cranial fold has not been considered at all in comparative studies of vocal fold histology (see e.g. KURITA et al. 1983; GARRETT et al. 2000; HAHN et al. 2005, 2006 a, b), despite the fact that ALIPOUR and JAISWAL (2008, 2009) have emphasised its role as the main oscillator of the porcine glottis. Consequently, a detailed histological study of CraF and CauF appeared desirable as a substantial basis for inter-species comparisons. Accordingly, a histological study of CraF and CauF as well as a comparison with data from literature on the human vocal fold were performed at the beginning of this project (Paper I).
(3) The stratigraphical organisation of the lamina propria (Paper I)
The characteristic histological composition of different connective tissue layers of the vocal fold has an essential functional impact on phonation (GRAY et al. 2000;
CHAN et al. 2007). This phenomenon is described by means of the so-called body-cover model: A loose and flexible ‘cover’ oscillates upon a more rigid ‘body’
during phonation (HIRANO 1974; STORY and TITZE 1995). Although the body-cover model classically reflects the functional characteristics of the human vocal folds, it has also been applied to describe porcine phonation (BLAKESLEE et al. 1995). However, this had been done only with regard to the caudal vocal fold (CauF). As emphasised above, this fold cannot be functionally homologised with the human vocal fold. For this reason, a detailed histological analysis of the stratigraphical organisation of both CraF and CauF appeared mandatory for a precise evaluation of the full potential of the pig as a model in human phoniatry (Paper I).
(4) The structural features of maturation and ageing of the vocal folds (Paper II)
The connective tissue elements of the human vocal fold are known to be subject to drastic structural changes throughout life, i.e. between infant, juvenile, adult, and senescent stages (see e.g. HAMMOND et al. 1998, 2000); the same may be true for the pig. However, the life-span of humans and animals (in this study: Minipigs) differs, and so do the chronobiological phases and ages (in years) in which states like adolescence or senescence are reached. Considering this, specimens of minipigs of three different age groups (2-3 months, presumably ‘young’; 11-27 months, presumably ‘adult’; 4-7 years, presumably ‘old’) were studied in order to elaborate structural criteria suited to characteristically distinguish states of maturation and ageing (Paper II).
2 Survey on literature
2.1 Histomorphometry of non-corpuscular objects
Histomorphometry is defined as the ‘quantitative study of the microscopical organisation and structure of a tissue (as bone) especially by computer-assisted analysis of images formed by a microscope’ (MERRIAM-WEBSTER 2014).
Properties to be measured include diameters, areas, lengths, angles of orientation etc. (FARLEX 2012). The term histomorphometry also applies to procedures involving the manual performance of certain sub-steps. As the development of computer-assisted image-recognition software has brought forth considerable advances in histomorphometry (EGAN et al. 2012), especially increases in capacity and reproducibility (LAURINAVICIUS et al. 2012) of automated or semi-automated procedures seem to be in the focus of histomorphometrical attempts.
As already mentioned in chapter 1 (Introduction), the measurement of non-cor- puscular structures is particularly problematic (LANG et al. 2012 b, 2013 a): Poorly demarcated outlines of fibres, overlapping fibre networks, and irregularities in staining intensities complicate automated Object Definition. These difficulties arise from in the algorithms at the core of the image-analysis programs, which are created on the basis of subjective human decisions (TADROUS 2010). Thus, while the capacity and the reproducibility of automated histomorphometry are very high, its accuracy – i.e. the closeness of agreement between a measured quantity value and a true quantity value of a measurand* – per se is not superior to manual procedures (JOINT COMMITTEE FOR GUIDES IN METROLOGY 2008; LAURINAVICIUS et al.
2012).
*A measurand is defined as ‘the quantity intended to be measured’ (JOINT COMMITTEE FOR GUIDES IN METROLOGY 2008)
The measurement of amounts of fibres (rather than diameters or numbers), however, is a common application of (semi-)automated histomorphometry (see e.g.
HAMMOND et al. 1998, 2000; THIBEAULT et al. 2002; HAHN et al. 2006 a, b).
To do so, digital microscopical images are taken of sections containing selectively stained fibres. Then, the size of the tissue area taken up by the stained fibres is measured within these images.
This type of analysis was previously performed with regard to the amounts elastic fibres (HAMMOND et al. 1998) and collagen fibres (HAMMOND et al. 2000) in human vocal folds. In two separate studies, elastic fibres were stained black by Verhoeff’s elastic tissue stain, while collagen fibres were stained red by picric acid.
Under a microscope, the sought-after fibres (elastic or collagen) were further accentuated by application of optical interference filters, and were then recorded in digital images. These were then transformed into black-and-white images. The actual measurement of fibre amounts was achieved by an automated recognition of the area taken up by dark objects (elastic or collagen fibres) in each image. With regard to the accuracy of this method of Object Definition, the authors stated that manual discrimination of unwanted structures was necessary, and that ‘knowledge of the histological appearance is crucial to optimal performance of this function’
(HAMMOND et al. 1998).
A similar study on the amounts of collagen and elastic fibres in vocal folds of several different species was performed by HAHN et al. (2006 a, b). Here, fibres were also stained selectively (elastic fibres: Anti-alpha elastin antibody BA-4; collagen fibres:
Picrosirius acid), and greyscale images of this stained tissue were produced.
However, in contrast to the procedure applied by HAMMOND et al. (1998, 2000), the amounts of fibres were assessed semi-quantitatively by a single observer who evaluated the relative staining intensity of the fibres in several defined regions of the vocal folds.
In preparation for the present study, the above methods were considered, and a similar setup of microscope and corresponding digital imaging software was tested (microscope: Axioskop, Carl Zeiss AG, Oberkochen; digital camera: Model DP 70, Olympus, Hamburg; digital imaging software: DP Soft 5.0, Olympus, Hamburg).
However, a comparison of this (unpublished) data with data created with the commercial graphics editing program ‘Photoshop’ (Adobe Photoshop CS 3 Extended 10.0.1, Adobe Systems, San Jose, CA, USA) revealed that Photoshop was better suited for the recognition and analysis of selectively stained fibres (LANG et al.
2012 b, 2013 a). This judgement was supported by previous studies of several other research groups (see e.g. LEHR et al. 1999; LAHM et al. 2004; EGAN et al. 2012), who had used Photoshop for histomorphometrical purposes. LEHR et al. (1999) emphasised the superior results of Photoshop-based image analysis compared to colour separation by optical filters (as used by HAMMOND et al. [1998, 2000]).
However, regardless of Photoshop’s suitability for the measurements of fibre amounts, it was not able to analyse the numbers or diameters of fibres – an important criterion to describe the biomechanical properties of a tissue. For reasons explained above, this task was much more difficult to solve ‘automatically’. Complex mathematical processes such as Fourier Analysis or Distance Mapping are suited to describe fibre orientation or spacing, but these procedures cannot count and measure numbers or diameters of fibres (VAN ZUIJLEN et al. 2002; WONG and BUENFELD 2006; VERHAEGEN et al. 2012). VERHAEGEN et al. (2012) stated that there were no valid and reliable techniques for an automated measurement of fibre bundle thickness at that time.
2.2 The unique structure of the porcine glottis
The most prominent feature distinguishing the porcine glottis from that of other mammalian species, including humans, is the fact that its vocal ligament is split longitudinally into two parts, i.e. a cranial and a caudal part.
Both parts of the split vocal ligament originate from the cricothyroid ligament at the ventral part of the thyroid cartilage and insert on the vocal process of the arytenoid cartilage (WAIBL 2004).
With respect to this bi-parted organisation of the porcine vocal ligament, special attention is to be paid to the laryngeal mucosa: The mucosa forms a lateral recess, i.e. ventriculus laryngis (INTERNATIONAL COMMITTEE ON VETERINARY GROSS ANATOMICAL NOMENCLATURE 2005), also called ventriculus laryngis lateralis (KÖNIG and LIEBICH 2009). However, in pigs, the opening of this pouch lies between the cranial and the caudal part of the vocal ligament (WAIBL 2004), and not between the vocal ligament and the vestibular ligament (as it does, e.g., in dogs, horses, or humans; see WAIBL [2004], STANDRING [2005]).
These unique features – bi-partition of the vocal ligament with a lateral outgrowth of laryngeal mucosa between them – have contributed to some inconsistent definitions of what the so-called vocal fold really is in pigs.
In the past, BUROW (1902) and PRODINGER (1940) suggested that there was only one single vocal fold (one on each side – right and left – of the larynx), which contained the two parts of the split vocal ligament. In contrast, the vocal fold described by WAIBL (2004) contains only the caudal part of the split vocal ligament.
In recent years, authors have proposed to distinguish two vocal folds (i.e. two on each side of the larynx): ALIPOUR and JAISWAL (2008, 2009) referred to them as the superior and inferior vocal fold, while – respecting the quadruped organisation of the porcine body – KOCH et al. (2010) spoke of the cranial and caudal fold of the
porcine glottis. Both of these folds are part of the glottis, which is the vocal apparatus of the larynx (SCHALLER 2007), and therefore should possess phonatory properties;
this has been demonstrated by ALIPOUR and JAISWAL (2008, 2009). Consequently, the folds’ assignment as a cranial vocal fold and a caudal vocal fold – as in a previous study (LANG et al. 2013 b) – appears equally appropriate.
In all parts of the following text, the simplified terms ‘cranial fold’ (CraF) and ‘caudal fold’ (CauF) are applied.
The porcine vestibular ligament, also called ligamentum vestibulare (INTER- NATIONAL COMMITTEE ON VETERINARY GROSS ANATOMICAL NOMEN- CLATURE 2005) or ligamentum ventriculare (BUROW 1902; PRODINGER 1940) lies far cranial of the vocal ligament. It extends from the base of the epiglottis to the lateral surface of the arytenoid cartilage up to its corniculate process (WAIBL 2004).
Its structure is rather membrane-like than string-like, and, as such, differs significantly from its human equivalent. The vestibular ligament is not involved in the formation of a vestibular fold in pigs (WAIBL 2004); this means that there is no vestibular fold in the porcine larynx (BUROW 1902; PRODINGER 1940; WAIBL 2004).
According to WAIBL (2004), the porcine larynx contains a fibroelastic membrane: It originates at the lateral parts of the cricothyroid ligament and ends on the caudal part of the vocal ligament. WAIBL (2004) equates it with the human conus elasticus.
However, this assumption does not appear appropriate, as the fibroelastic membrane in humans has two components (STANDRING 2005): Firstly, the conus elasticus (in inferior position); and secondly, the membrana quadrangularis (in superior position).
WAIBL (2004) does not make reference to such a membrana quadrangularis as part of the fibroelastic membrane in the porcine larynx. Nevertheless, he does describe a fibrous sheet located between the vestibular ligament and the cranial part of the split vocal ligament (i.e. supraglottically), which extends from the thyroid lamina to the ventral border of the arytenoid cartilage. In principle, this fibrous sheet appears to resemble the human membrana quadrangularis, except that it is smaller in dimension (it does not line the entire supraglottic wall of the larynx).
Lateral to this sheet/membrane lies the lumen of the laryngeal ventricle. Next to its opening, it is shaped like an oval duct, but then continues into a pouch-like recess reaching up to the level of the vestibular ligament (PRODINGER 1940).
The thyroarytenoid muscle is the main muscle in the porcine glottis (WAIBL 2004). Its broad and flat body extends from the ventro-caudal part of the thyroid cartilage to the arytenoid cartilage (WAIBL 2004). The muscle is located inside the cartilaginous skeleton of the larynx, but lies lateral to the split vocal ligament, and also lateral to the laryngeal ventricle (PRODINGER 1940). As the lumen of the laryngeal ventricle extends cranially (past the cranial part of the split vocal ligament), it ‘pushes in between’ the cranial part of the split vocal ligament and the thyroarytenoid muscle (PRODINGER 1940).
Due to this anatomical situation, there is no muscular component in the cranial fold (CraF) of the porcine glottis. The thyroarytenoid muscle lies directly lateral to the caudal part of the split vocal ligament and thus constitutes a large part of the caudal fold (CauF).
Unlike in other species (e.g. in horses or dogs), the thyroarytenoid muscle of pigs shows no sign of a subdivision into a vocal muscle and a ventricular muscle (PRODINGER 1940; WAIBL 2004).
In summary: What seems to resemble the ventricular fold in humans is in fact the cranial fold of the porcine glottis. This fold is supposed to be the main oscillator in the pig’s glottis (ALIPOUR and JAISWAL 2008, 2009). Understanding this striking anatomical and functional difference is essential for avoiding misinterpretations and confusion when comparing the results of histological and phoniatrical studies on pigs and other mammalian species, including humans.
Table 1 gives a survey on synonyms and definitions of terms related to the human and porcine glottis. They were applied in the literature of which reference is made in the text of this thesis.
A further, more detailed description of the human glottis is to be found in Annex 9.1:
Anatomy and terminology of relevant features of the human glottis with regard to species differences.
Tab. 1. Synonyms and definitions of terms related to the human and porcine glottis;
data in brackets [ ] are explained at the end of the table.
Official term
Synonyms Description
Vocal fold [TA]
Human Plica vocalis [TA], vocal cord [1], true vocal cord [1], Stimmfalte* [2],
Stimmlippe* [3]
Vocal ligament and vocal muscle covered by mucosa
Porcine Cranial and caudal (vocal) fold [4], CraF and CauF [4], superior and inferior vocal fold [5]
Cranial and caudal part of the vocal ligament and thyro- arytenoid muscle; covered by mucosa and divided by the opening into the laryngeal ventricle
Vocal ligament [TA]
Human Ligamentum vocale [TA], Stimmband* [3]
Ligament between thyroid cartilage and arytenoid
cartilage; upper free edge of the conus elasticus
Porcine Ligamentum vocale [NAV], Stimmband* [6]
Ligament between cricothyroid ligament and arytenoid
cartilage; split into a cranial and a caudal part
Vestibular fold [TA]
Human Plica vestibularis [TA], false vocal fold [1], ventricular fold, Taschenfalte* [2], Vorhoffalte* [7]
Vestibular ligament covered by mucosa
Porcine Term does not apply (see explanation on the right)
Absent
* Term commonly used in German language
Official term
Synonyms Description
Vestibular ligament [TA]
Human Ligamentum vestibulare [TA], Taschenband* [3], falsches Stimmband* [3]
Thickened lower body of the quadrangular membrane
Porcine Ligamentum vestibulare [NAV], ligamentum
ventriculare [8, 9]
Ligament between the base of epiglottis and the lateral sur- face of the arytenoid cartilage Laryngeal
ventricle [TA]
Human Ventriculus laryngis [TA], Morgagni’s ventricle [10], ventricle of Galen [10], sinus of Morgagni [10]
A slit between the vestibular fold and the vocal fold opens into a fusiform recess; it extends upwards into the laryngeal wall lateral to the vestibular fold Porcine Ventriculus laryngis [NAV],
ventriculus laryngis lateralis [11]
Opening between the two divisions of the vocal ligament;
extends cranially to the level of the vestibular ligament
Thyro- arytenoid muscle [12]
Human Musculus thyroarytenoideus [TA], thyro-arytenoid [TA]
Main muscle lateral to the vocal fold, the cricovocal membrane, and the laryngeal ventricle Porcine Musculus thyroarytenoideus
[NAV], musculus
thyreoarytaenoideus [NAV]
Main muscle lateral to the vocal folds and the laryngeal ventricle
Vocal muscle [10]
Human Vocalis [TA], musculus vocalis [TA]
Medial portion of the thyro- arytenoid muscle; lateral to the vocal ligament
Porcine Term does not apply (see explanation on the right)
The thyroarytenoid muscle is not subdivided, therefore there is no separate vocal muscle
* Term commonly used in German language
[TA] Terms listed in Terminologia Anatomica (FEDERATIVE COMMITTEE ON ANATOMICAL TERMINOLOGY 2011), i.e. anatomy of humans
[NAV] Terms listed in Nomina Anatomica Veterinaria (INTERNATIONAL COMMITTEE ON VETERINARY GROSS ANATOMICAL NOMEN-
CLATURE 2005)
[1] STANDRING (2005)
[2] SOBOTTA and WELSCH (2009) [3] DRUNCKER and KUMMER (2008) [4] LANG et al. (2012 a)
[5] ALIPOUR and JAISWAL (2009) [6] WAIBL (2004)
[7] LIPPERT (1990)
[8] BUROW (1902)
[9] PRODINGER (1940) [10] REUTER and REUTER (1996) [11] KÖNIG and LIEBICH (2009) [12] CLEMENTE (1997)
2.3 The stratigraphical organisation of the lamina propria
A profound understanding of the stratigraphical composition of the vocal fold’s lamina propria is of great importance in human phoniatry, as disturbances in this com- position can lead to severe voice disorders (NAWKA and HOSEMANN 2005).
Consequently, extensive research has been done in this field of human medical science. In contrast, the interest in the vocal folds of animals is (almost exclusively) limited to their suitability as models in human phoniatry (see e.g. GARRETT et al.
2000; JIANG et al. 2001; HAHN et al. 2005). This correlation gives reason to briefly explain the stratigraphical organisation of the human vocal fold and its functional significance, before summarising the relatively sparse information on pigs from the literature.
In terms of macroscopical anatomy, the human vocal fold is divided into three components (Tab. 2), i.e. mucosa, vocal ligament, and vocal muscle (see e.g.
CLEMENTE 1997; STANDRING 2005). However, in terms of histology, it is divided into three different histological layers (Tab. 2): Epithelium, lamina propria, and vocal muscle (see e.g. HIRANO 1981; BÜHLER et al. 2011). Thus, the vocal ligament is – histologically – a part of the vocal fold’s lamina propria. This lamina propria contains varying amounts of collage and elastic fibres, which are used to distinguish stratigraphical subdivisions (Tab. 2) as follows: The superficial layer of the lamina propria (SLLP) is sparse in fibres, the intermediate layer (ILLP) is particularly rich in elastic fibres, and the deep layer of the lamina propria (DLLP) contains large amounts of collagen fibres (see e.g. HIRANO 1977, 1981; GARRETT et al. 2000;
SATO et al. 2002; BÜHLER et al. 2011). Some authors additionally propose a fourth layer of the lamina propria (Tab. 2): A basement membrane zone (BMZ), located adjacent to the subepithelial basement membrane (HAHN et al. 2005, 2006 a;
TATEYA et al. 2006); it is described as a thin layer or band made up of densely
arranged collagen fibres (HAMMOND et al. 2000; MADRUGA DE MELO et al. 2003;
HAHN et al. 2006 b; TATEYA et al. 2006) and elastic fibres (HAHN et al. 2006 a).*
The sparseness of fibres in the superficial layer of the lamina propria (SLLP), and the resulting abundance in Ground Substance (HIRANO 1977, 1981; HIRANO and KAKITA 1985; REMACLE et al. 1996; HAMMOND et al. 1998, 2000; HAHN et al.
2006 a, b) account for the SLLP’s loose texture susceptible to the pathological condition of Reinke’s edema (see e.g. REINKE 1895; REMENAR et al. 1984;
MARCOTULLIO et al. 2002; VEČERINA-VOLIĆ and IBRAHIMPAŠIĆ 2004).
Correspondingly, the healthy SLLP is also referred to as Reinke’s space (HIRANO 1977; FINCK 2005; SATO et al. 2010).
The elastic fibres of the intermediate layer of the lamina propria (ILLP) (HIRANO 1981; HAMMOND et al. 1998; HAHN et al. 2006 a) form an important component of what is called the ‘vocal ligament’ in macroscopical anatomy. The ILLP is placed on top of the collagenous deep layer of the lamina propria (DLLP), which represents the vocal ligament’s deep component (HIRANO 1977; HIRANO et al. 1983) (Tab. 2). The collagen fibres of the DLLP contribute tensile strength to the vocal ligament (GRAY et al. 2000; CHAN et al. 2007).
* The term ‘basement membrane zone’ is applied inconsistently in the literature:
Occasionally, it is used as a synonym for the basement membrane (GRAY et al.
1994; GRAY 2000) – a structure of only approx. 0.05 µm thickness separating the epithelial cells from the lamina propria, and visible only in an electron microscope (LEONHARDT 1990; STEVENS and LOWE 1997).
Tab. 2. Structure of the human vocal fold according to different criteria.
[1] CLEMENTE (1997) [2] STANDRING (2005) [3] HIRANO (1981)
[4] BÜHLER et al. (2011) [5] GARRETT et al. (2000) [6] SATO et al. (2002)
[7] HAHN et al. (2005) [8] HAHN et al. (2006 b) [9] TATEYA et al. (2006)
While oscillating, the vocal fold’s surface seems to ripple in a wavelike motion referred to as the Mucosal Wave over a more rigid base (YUMOTO and KADOTA 1998; McCOY and HALSTEAD 2004; KRAUSERT et al. 2011). The so-called body-cover model of vocal fold vibration attempts to describe these processes of oscillation: In its initial version (Tab. 3), the ‘cover’ was made up of the mucous membrane of the vocal fold, while the conus elasticus and the vocal muscle formed the ‘body’ (HIRANO 1974). This model was further elaborated by the author when he first described the layered composition (SLLP, ILLP, and DLLP) of the lamina propria (HIRANO 1977). In the course of this refinement, a ‘transition’ was added to the body-cover model (Tab. 3). Accordingly, a new pattern of functional stratigraphy was created: The ‘cover’ now consisted of epithelium and SLLP, the ‘transition’ was made up of the ILLP and DLLP, and the ‘body’ was made up of the vocal muscle (Tab. 3).
This three-component version of the cover-body model is most commonly Anatomical structures Histological layers Histological subdivisions
of layers
Mucosa [1, 2]
Epithelium [3, 4] Epithelium [3-9]
Lamina propria [3, 4]
BMZ [7-9]
SLLP [3-9]
Vocal ligament [1, 2] ILLP [3-9]
DLLP [3-9]
Vocal muscle [1, 2] Vocal muscle [3, 4] Vocal muscle [3-9]
encountered in the literature (see e.g. BLAKESLEE et al. 1995; GARRETT et al.
2000; NAWKA and HOSEMANN 2005). Nonetheless, several variations of this model (summarised in Tab. 3) have been proposed by other authors. All versions of the body-cover model have in common that the tissue comprising the ‘cover’ is of much looser composition than the tissue of the ‘body’, thereby allowing the cover’s relatively free movement upon the ‘body’ (HIRANO 1974, 1977; TITZE 1994;
HAMMOND et al. 1998; GRAY et al. 2000).
Tab. 3. Different stratigraphical features of the body-cover model reported in the literature on the human vocal fold (terminology as applied by the authors).*
HIRANO (1974)
HIRANO (1977)
TITZE (1994)
HAMMOND et al. (1998)
GRAY et al. (2000)
COVER
Mucous Epithelium Epithelium Epithelium Epithelium
membrane SLLP SLLP SLLP SLLP
ILLP most of the
ILLP
TRANSITION -- ILLP
-- -- MLLP***
DLLP
BODY
Conus elasticus
remainder of the ILLP
DLLP DLLP DLLP Vocalis
muscle
Vocalis muscle
Thyro- arytenoid muscle**
Vocal muscle
Vocalis muscle
* Data refers to adult individuals
** The thyroarytenoid muscle lies lateral to the vocal fold’s lamina propria. The vocalis muscle, or vocal muscle, is its medial portion (see also Annex 9.1:
Anatomy and terminology of relevant features of the human glottis with regard to species differences)
*** MLLP: Middle layer of the lamina propria, used by the authors as a synonym for the ILLP
A different version of the body-cover model has been developed with respect to the distinct characteristics of the vocal fold’s composition in newborn children. According to HIRANO et al. (1983), the vocal fold of newborns has not yet developed a vocal ligament; the lamina propria is still uniformly loose, resembling the superficial layer of the adult vocal fold. Consequently, HIRANO and KAKITA (1985) defined the ‘cover’
as consisting of the entire mucosa of newborns, while the ‘body’ was made up of the vocal muscle alone. A ‘transition’ was not included in this model (HIRANO and KAKITA 1985).
Regarding pigs, the concept of a body-cover model has only been described for the caudal vocal fold (CauF), but again in a modified version (BLAKESLEE et al. 1995):
The lamina propria of the CauF was assumed to consist of only two layers (KURITA et al. 1983; BLAKESLEE et al. 1995), rather than three as in humans. Accordingly, the ‘cover’ in pigs comprised the epithelium and the superficial layer, while the ‘body’
was made up of the deeper layer of the lamina propria and the muscle (BLAKESLEE et al. 1995). Although this concept of a body-cover model of the porcine CauF is still the only existing version, more recent studies on the stratigraphical composition of the CauF have proposed the existence of four layers, rather than two: In their comparative studies, HAHN et al. (2005, 2006 a, b) applied the human terminology of layers and distinguished BMZ, SLLP, ILLP, and DLLP. With regard to collagen fibres, HAHN et al. (2006 b) found their distribution to be similar in the human vocal fold and the porcine CauF (highest amounts of collagen fibres in BMZ and DLLP, few fibres in the SLLP). In contrast, the distribution of elastic fibres differed in the two species:
Elastic fibres were concentrated in the ILLP of humans, but were relatively evenly distributed throughout the entire lamina propria of the porcine CauF (HAHN et al.
2006 a). In a preliminary study of limited sample size, KOCH et al. (2010) confirmed this even distribution of elastic fibres in the CauF, but could only distinguish three, rather than four layers (KOCH et al. 2010). As mentioned above, no attempts were made by HAHN et al. (2005, 2006 a, b) or KOCH et al. (2010) to incorporate their concepts of layers into an updated version of the porcine body-cover model which could be compared with the human model.
In summary, the abundance of information on the stratigraphical composition of the human vocal fold and on its functional significance contrast with the comparatively little information on the pig – particularly regarding the pig’s cranial vocal fold (CraF).
2.4 The structural features of maturation and ageing of the vocal folds
The structural composition of the human vocal fold changes with age (see e.g.
HIRANO et al. 1983; HAMMOND et al. 1998, 2000; ISHII et al. 2000). Medical interest in these processes is focused on their negative functional effects, as e.g. the
‘ageing of the voice’ (BIEVER and BLESS 1989; HIRANO et al. 1989). As already mentioned in the previous chapter 2.3 (The stratigraphical organisation of the lamina propria), the phoniatrical interest in animals mostly focuses on their use as models for humans (see e.g. GARRETT et al. 2000; JIANG et al. 2001; HAHN et al. 2005). In this context, a lack of knowledge on age-related changes in the vocal folds of pigs (KOCH et al. 2010) may limit the potential benefit from their use as an animal model.
Several studies on the human vocal fold deal with maturation (see e.g. HIRANO et al.
1983; HAMMOND et al. 1998, 2000; ISHII et al. 2000; SATO et al. 2001 a). The findings of these studies are rather inconsistent, which is probably due to the heterogeneity of specimens: Most examinations were either limited to very young (foetal to infantile) specimens (see e.g. SATO et al. 2001 a; NITA et al. 2009), or compared specimens of newborns or infants with those of adult or old humans (see e.g. HAMMOND et al. 1998, 2000; HIRANO et al. 1999; FAYOUX et al. 2004); other studies lacked specimens of a broad age span (e.g. no samples of 6-11 year-old children were examined by ISHII et al. [2000]). Nevertheless, all of these data taken together served well to describe the basic features of maturation as follows:
In newborn children, the entire lamina propria is made up of abundant Ground Substance and only sparse homogeneously distributed fibres (HIRANO et al. 1983;
ISHII et al. 2000; SATO et al. 2001 a). In this regard, the entire lamina propria resembles the SLLP of the adult vocal fold (HIRANO et al. 1983). Accordingly, the vocal fold of newborns does not contain a structure which can be designated as a vocal ligament (HIRANO et al. 1983; ISHII et al. 2000; SATO et al. 2001 a).
Between infancy and adulthood, the amounts of collagen and elastic fibres increase significantly (HAMMOND et al. 1998, 2000). These fibres are mainly produced by Vocal Fold Stellate Cells (SATO et al. 2001 b). This special type of fibroblast is accumulated in the maculae flavae, which are located in the anterior and posterior ends of the membranous part of the vocal fold (HIRANO et al. 1983; SATO et al.
2001 b). The fibres produced in the maculae flavae extend towards the middle of the vocal fold (HIRANO et al. 1983; SATO et al. 2001 a). According to SATO et al.
(2001 a), collagen and reticular fibres are produced early in infancy (age not further specified), and serve as stabilising scaffolds for the extension of elastic fibres. They thus appear in the midportion of the vocal fold prior to the elastic fibres (SATO et al.
2001 a).
The stages of development of the adult fibre stratigraphy were examined in detail by HIRANO et al. (1983) and ISHII et al. (2000). An immature ligamentous structure – characterised by a homogeneous distribution of collagen and elastic fibres (rather than an arrangement in clearly differentiated layers) – was occasionally found in children of 1-4 years of age, and was always present in children older than 4 years (HIRANO et al. 1983). According to ISHII et al. (2000), the development of the distinct adult stratigraphical organisation begins later in life: In specimens of 5-year- old children, longitudinally arranged collagen and elastic fibres were still evenly distributed throughout all layers of the lamina propria (ISHII et al. 2000). Later on, first signs of a differentiation of layers were found in individuals aged 12 years (ISHII et al. 2000). However, no samples were examined of children aged 6-11. The completion of the mature vocal fold structure was fairly consistently described as occurring at 16 years (HIRANO et al. 1983), or 17 years (ISHII et al. 2000), respectively.*
* These data refer to male specimens in both studies (HIRANO et al. 1983; ISHII et al. 2000). No reference was made to female individuals.
The process of ageing of the vocal fold appears difficult to assess, as there is no unanimous agreement on the age at which ’ageing’ of the vocal fold actually begins (see e.g. HIRANO et al. 1983, 1989; KAHANE 1983; ISHII et al. 1996; HAMMOND et al. 1998, 2000; BUTLER et al. 2001; SATO et al. 2002; ROBERTS et al. 2011):
Some authors attribute structural changes encountered at the age of 40 years to processes of ageing (HIRANO et al. 1983), while others have classified the status of 63-year-old specimens as ‘adult’ instead of ‘old’ (ISHII et al. 1996).
The main feature of ageing is a decrease in number and activity of the Vocal Fold Stellate Cells in the maculae flavae (HIRANO et al. 2000; SATO et al. 2010). This decrease causes a decrease of the turnover rate of the fibrous components in the lamina propria: Production of fibres is slowed down, as is their breakdown (SATO and HIRANO 1995; GRAY, 2000). In the aged vocal fold, fibres thus become older before being degraded and replaced by new fibres (GRAY, 2000). As a consequence, ageing is characterised by changes in the structure and in the amounts of fibres (KAHANE 1983; HIRANO et al. 1983, 1989; SATO et al. 2002;
SATO and HIRANO 1995, 1997; HAMMOND et al. 1998, 2000; ROBERTS et al.
2011).
Structural alterations in collagen and elastic fibres result in an increase in fibre diameter (SATO et al. 2002; HAMMOND et al. 1998), and also in a greater variation of fibre diameters (SATO and HIRANO 1997; SATO et al. 2002). With age, the arrangement of fibres becomes less orderly (HIRANO et al. 1983; ISHII et al. 1996;
SATO and HIRANO 1997); an increased number of cross links develops between fibres (SATO and HIRANO 1997; BAILEY 2001), and collagen fibres display irregular outlines (SATO et al. 2002).
An increase in the amounts of collagen fibres in the aged vocal fold of humans is most pronounced in the DLLP (HIRANO et al. 1983, 1989; HAMMOND et al. 2000;
SATO et al. 2002; ROBERTS et al. 2011). Yet in some individuals, collagen fibre amounts have been reported to increase evenly throughout the entire lamina propria.
In these cases, a layered organisation was no longer visible in the aged vocal fold (SATO et al. 2002).
The effect of ageing on the amounts of elastic fibres is described inconsistently (see HIRANO et al. 1983; KAHANE, 1983; HAMMOND et al. 1998; ROBERTS et al.
2011), even though all of the reviewed studies applied similar methods: Selectively stained elastic fibres in paraffin sections were examined by light microscopy.
However, HIRANO et al. (1983) and KAHANE (1983) performed a descriptive analysis, while HAMMOND et al. (1998) and ROBERTS et al. (2011) chose a histomorphometrical procedure. The studies by HIRANO et al. (1983) and KAHANE (1983) revealed a decrease in elastic fibre amounts throughout the entire lamina propria due to a degradation of fibres with age. In contrast, HAMMOND et al. (1998) described a significant age-dependent increase in elastic fibre amounts throughout the lamina propria, but predominantly in the ILLP. Finally, an almost complete absence of elastic fibres in the SLLP combined with high amounts of elastic fibres in the DLLP was reported by ROBERTS et al. (2011).
The human vocal folds are subject to extensive structural stages during maturation and ageing. Considering this, similar extents of age-related changes can be suspected in the porcine vocal folds. (An investigation of different stages of structural development of the porcine vocal folds may thus enable the determination of the adequate age of the pig to be used as a model.)
3 Materials and Methods
3.1 Animals
Twenty-three female minipigs (Tab. 4) were included in this study; they were assigned to three age groups by considering data from the literature on skeletal development (SWINDLE 2007; GUNDLACH 2012), sexual maturation (SWINDLE and SMITH 2008), and weight development (SWINDLE et al. 2006) as follows:
‘Young’ = 2-3 months (n = 6), ‘adult’ = 11-27 months (n = 11), and ‘old’ = >4 years (n = 6).
The minipigs were euthanised either by injection of 1.5 ml/10 kg Euthadorm® (Pento- barbital Sodium) or by captive bolt stunning and consecutive bleeding.
The Guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (86/609/EEC) were observed.
As the primary designated use of the minipigs was dissection in the anatomical classes of the Veterinary University of Hannover, approval by the Lower Saxony State Office for Consumer Protection and Food Safety was not necessary.
Tab. 4. Data on the female minipigs included in the present study.
Item Age Age group Breed
1 2 months
‘Young’
Göttinger x Minnesota
2 3 months Göttinger x Minnesota
3 3 months Göttinger x Minnesota
4 3 months Göttinger x Minnesota
5 3 months Göttinger x Minnesota
6 3 months Göttinger x Minnesota
7 11 months
‘Adult’
Göttinger
8 11 months Göttinger
9 12 months Mini Lewe
10 13 months Göttinger
11 13 months Mini Lewe
12 14 months Mini Lewe
13 14 months Mini Lewe
14 14 months Mini Lewe
15 27 months Mini Lewe
16 27 months Mini Lewe
17 27 months Mini Lewe
18 54 months (4 years)
‘Old’
Göttinger
19 58 months (4 years) Göttinger
20 65 months (5 years) Mini Lewe
21 70 months (5 years) Göttinger
22 72 months (6 years) Göttinger
23 84 months (7 years) Göttinger
3.2 Histological procedures Fixation:
Immediately after euthanasia of the animals, their larynges were excised and immersion-fixed in Bouin’s solution (saturated aqueous picric acid, filtered acid-free formaldehyde 37 %; acetic acid 98 %; 15:5:1); fixation time was 48 hours at room temperature.
Embedding:
The left side of the glottis was cut transversely at its midportion; both resulting specimens were embedded in paraffin (Paraplast®, Leica Biosystems), according to the standard procedure (BÖCK 1989).
Staining:
Serial cross sections (5-8 µm) were alternately stained either with Masson’s trichrome (for collagen structures*) according to BÖCK (1989) or with resorcin- fuchsin (for elastic fibres) according to WEIGERT (1898). Occasionally, some variations in the protocol of Masson’s trichrome stain were necessary due to irregularities in the colour of the collagen fibres. For instance, when fibres were stained reddish instead of green, the incubation time of Ponceau + Acid fuchsin was shortened (3 minutes instead of 5 minutes), while the incubation time of Phosphotungstic acid + Orange G, and the incubation time of Light green were prolonged (7 minutes instead of 5 minutes).
* ‘Collagen structures’ is a term used to address single collagen fibres as well as collagen fibres accumulated in bundles.
3.3 Histomorphometrical analysis of fibre amounts Creation of microscopical images:
Images of the stained cross sections (1 section stained with Masson’s trichrome, 1 section stained with resorcin-fuchsin of each minipig) were recorded utilising the following setup: Light microscope (Axioskop, Carl Zeiss AG, Oberkochen), digital camera (Model DP 70, Olympus, Hamburg), and digital imaging software (DP Soft 5.0, Olympus, Hamburg).
Selection of Regions of Interest (ROIs):
In the digital images, a midline was placed through the connective tissue (lamina propria) of each fold, i.e. CraF and CauF (Fig. 1). The midline through the CraF was twice as long as the midline through the corresponding CauF, because the connective tissue of the CraF was thicker (due to the lack of the muscle). Along each midline, pairs of circular regions of interest (ROIs) with a diameter of 35 µm (Fig. 1) were placed at a distance of approx. 5 µm. The first pair of ROIs was always placed directly underneath the epithelium (Fig. 1). Afterwards, these ROIs were extracted from the digital images (Fig. 2) with ‘Photoshop’ (Adobe Photoshop CS 3 Extended 10.0.1, Adobe Systems, San Jose, CA, USA). Within the ROIs, the procedures of Object Definition and measurements were performed as described below.
Fig. 1. Graphical illustration of the porcine glottis with its cranial and caudal fold, CraF and CauF (inset upper left corner). The laryngeal ventricle is located between the folds. Pairs of ROIs were placed along the midlines of the CraF and CauF. The midline through the CraF was twice as long as the midline through the neighbouring CauF.
M: Thyroarytenoid muscle.
Fig. 2. Example of a circular region of interest (ROI) with a diameter of 35 µm. Pairs of such ROIs were placed along the midlines though the connective tissue/lamina propria of CraF and CauF (see Fig 1).
Object Definition:
The distinct colour of the selectively stained collagen structures (green in Masson’s trichrome) and elastic fibres (dark purple in resorcin-fuchsin) was used for Object Definition. In every section of CauF and CraF, a so-called ‘Middle Green’, or ‘Middle Purple’ colour was defined in Photoshop as follows: The RGB-values* of a typically stained fibre were recorded by placing the Photoshop tool ‘eyedropper’ on it. Then, all pixels within a set range (tolerance: ‘80’) of the previously defined colour were selected with the tool ‘color range’. In the next step, the average colour of these selected pixels was determined by using the tool ‘average’.
* Photoshop RGB Color mode uses the RGB model, assigning an intensity value to each pixel. In 8‑bits-per-channel images, the intensity values range from 0 (black) to 255 (white) for each of the RGB (red, green, blue) components in a color image. For example, a bright red color has an R value of 246, a G value of 20, and a B value of 50. When the values of all three components are equal, the result is a shade of neutral gray. When the values of all components are 255, the result is pure white;
when the values are 0, pure black. (http://help.adobe.com)
This procedure was performed four times, and the resulting average colour (RGB- values) was named ‘Middle Green’ (Masson’s stain) or ‘Middle Purple’ (resorcin- fuchsin stain), respectively. This ‘Middle Green’, or ‘Middle Purple’, was then applied in the subsequent measurement procedure.
Measurement procedure:
The first step of the actual measurement of fibre amounts per area was the selection of all pixels exhibiting the colour ‘Middle Green’ or ‘Middle Purple’ in each ROI. For this purpose, the tool ‘color range’ was applied at a fixed tolerance ‘100’ in each ROI.
This procedure accurately selected (green) collagen structures or (purple) elastic fibres, respectively.
After the selection of the ‘Middle Green’ or ‘Middle Purple’ pixels, their numbers appeared in the window ‘histogram’. These numbers were transferred into a spreadsheet in Excel 2003 (Microsoft Corporation, Redmond, WA, USA). The entire procedure was performed manually, under visual inspection of selected pixels in each ROI. In case of an incorrect selection of structures (investigator’s visual control), the pre-selected ‘Middle Green’ or ‘Middle Purple’ values were adapted manually. In these cases, the selection of pixels was repeated in all ROIs of a fold with the new, subjective ‘Middle Green’ or ‘Middle Purple’. This divergence from the standard procedure was occasionally necessary for the recognition of fibres in very lightly stained specimens.
Finally, the numbers of selected pixels were put in proportion to the total number of pixels in a ROI (in Excel). This quotient equalled the area covered by collagen structures or elastic fibres per total area of each ROI or, in other words, the amount of fibres in each ROI. As the ROIs were arranged in pairs along the fold’s midline, the values of the two ROIs composing one pair were averaged.
The amounts per area (apa) of collagen structures were referred to as ‘apa.coll’, and the amounts per area of elastic fibres as ‘apa.elast’.
3.4 Scoring of the diameters of collagen structures
A semiquantitative scoring system was applied for the evaluation of the diameters of collagen fibres and fibre bundles. For this purpose, circular ROIs of a diameter of 70 µm (Fig. 3) were examined*. The applied scoring system distinguished three groups according to the diameter of the objects (Tab. 5): Fibres and small fibre bundles (diameter of 2-4 µm), intermediate fibre bundles (diameter of >4-10 µm), and large fibre bundles (diameter of >10 µm). Fibres and small fibre bundles were included in the same group (2-4 µm), as data from literature on their respective sizes were not unanimously uniform (compare for instance: SCHRÖDER 2000; LIEBICH 2004;
JOHANES et al. 2011).
Scoring was performed twice by the same observer (‘rater’) with histological expertise** in random order, and with a time lag of at least one week. The two respective data sets were tested for their level of agreement (‘intra-rater agreement’) by determining the weighted kappa coefficient (kappa statistics according to ALTMAN [1999]). The resulting weighted kappa coefficient was 0.9192, which is regarded as a very good intra-rater agreement (ALTMAN 1999).
* The above mentioned ROIs of 35 µm were too small, as their field of view did not reliably represent the collagen structures; e.g. when the gap between fibre bundles was very large, the selected ROI did not ‘hit’ the bundle (false negative hits).
** Special thanks go to Gudrun Wirth, veterinary medical-technical assistant, for meticulously performing this task.
Fig. 3. Example of a circular region of interest (ROI) of a diameter of 70 µm, as used for the evaluation of fibre and fibre bundle diameters.
Tab. 5. Scoring system of the numbers of collagen structures of different diameters (fibres and fibre bundles).
Diameter Score Number of objects
Fibres/
small fibre bundles 2-4 µm
0 0 1 1-4 2 5-10 3 >10 Intermediate fibre
bundles >4-10 µm
0 0 1 1-3 2 4-8 3 >8 Large fibre bundles >10 µm
0 0 1 1-2 2 3-4 3 >4
3.5 Data analysis
Standardisation of fold thickness:
The individual thickness of the folds varied significantly in the minipigs. Accordingly, a varying number of pairs of ROIs could be placed (Fig. 4). In order to enable a comparison of the data from folds of different thicknesses, their thicknesses were
‘standardised’ by subdividing them into proportional subunits of 10 % each. These subunits added up to a number of 10, which equalled 100 % (see Fig. 4).
Consequently, a single proportional subunit (of 10 % depth) in a thin fold might have contained two pairs of ROIs (Fig. 4a), while a proportional subunit in a thick fold (10 % depth) might have contained three pairs of ROIs (Fig. 4b). This was the case in the CauF. However, as the CraF’s midline was twice as long as the CauF’s midline, 20 subunits (instead of 10) were assigned in the CraF.
The subdivision into ‘proportional subunits’ (each 10 % of the total thickness) facilitated a comparison of folds even when their thickness was variable: All data of subunit 1 of all animals were pooled in one group, all data of subunit 2 were pooled in another group, and so on. Then, the group representing all subunits 1 (one subunit per animal) was compared with the group representing all subunits 2, and so on.
Fig. 4. Graphical illustration of the standardisation procedure of vocal fold thickness.
Blue digits indicate the consecutive numbers of ROI-pairs, green lines mark the borders of the subunits, and red numbers indicate the proportional sizes of the related subunits. Note that – due to different thicknesses of the CauF – the thicknesses of the subunits also differed; for instance, one proportional subunit might have contained two pairs of ROIs (Fig. 4a) or three pairs of ROIs (Fig. 4b).
M: Thyroarytenoid muscle.
Definition of hypothetical zones:
The aim of the subsequent statistical analysis was to test the hypothesis that the connective tissue/lamina propria was composed of four layers, as proposed by HAHN et al. (2005, 2006 a, b). As the thickness of each of these layers was unknown, four ‘hypothetical zones’ were defined.
These four hypothetical zones were assigned as follows: Zone 1 (Z1) represented a relatively thin band (i.e. the basement membrane zone, BMZ, of HAHN et al. [2005, 2006 a, b]), which was attributed to subunit 1. The tissue underneath zone 1 was divided into three thirds: Zone 2 (Z2) represented subunits 2-4, zone 3 (Z3) represented subunits 5-7, and subunits 8-10 made up the last third of the lamina propria, i.e. zone 4 (Z4), as illustrated in Fig. 5. This procedure was applied for the CauF.
A similar assignment of zones was performed in the CraF (Fig. 5): Zones 1 to 4 (Z1- Z4) were identical to those in the CauF. However, four additional zones were assigned, because the investigated area was twice as thick as in the CauF: Subunits 11-13 formed zone 5 (Z5), subunits 14-16 made up zone 6 (Z6), subunits 17-19 made up zone 7 (Z7), and subunit 20 made up zone 8 (Z8) (Fig. 5).
Fig. 5. Graphical illustration of the zones (Z1-Z4, Z1-Z8) supposed to represent hypothetical layers of the connective tissue of the caudal and cranial vocal fold (CauF, CraF).
Statistical analyses:
Statistical analyses of differences in apa.coll and apa.elast were performed between different locations (subunits, zones) within each age group, and between different age groups.*
Within each age group, fibre amounts were statistically analysed by pairwise comparison of single subunits with their neighbouring subunits, and by comparison of groups of subunits.
Model residuals were tested for normal distribution by means of the Kolmogorov- Smirnov test and visual assessment of q-q-plots. As the majority of tested data sets were not normally distributed, distribution-free non-parametric methods were used.
Therefore, differences between neighbouring subunits were calculated using the Wilcoxon signed-rank test for paired observations, considering the comparison-wise error rate.
Differences between the age groups were calculated by pairwise comparison of zones.
For instance: Apa.coll in zone 1 (Z1) of the ‘adult’ group was compared with apa.coll in zone 1 (Z1) of the ‘young’ group and of the ‘old’ group. These comparisons between different age groups (independent samples) were calculated using the Kruskal-Wallis test, again considering the comparison-wise error rate.
Resulting p-values of p <= 0.05 were regarded as statistically significant. All analyses were performed with the statistics program SAS (Version 9.3, SAS Institute, Cary, NC, USA).
* Special thanks go to Dr. K. Rohn, Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, for his critical comments and technical assistance with the SAS program.
4 Results
A brief listing of the essentials of the studies shall be presented prior to each of the two manuscripts.
Histomorphometric analysis of collagen and elastic fibres in the cranial and caudal fold of the porcine glottis (Paper I) – brief survey:
This paper focuses on the histomorphometrical application of the Photoshop software.
The different thicknesses of the lamina propria of the folds required the elaboration and application of a very complex procedure of data analysis. For instance, the distinction between stratigraphical subunits – each representing a certain propor- tional depth – was a necessary tool for statistical analysis.
A four-layered structure was the basic organisation pattern of the lamina propria in both CraF and CauF:
- A subepithelial layer (SEL) was markedly dense (rich in collagen and elastic fibres); in principle, it compared with the so-called basement membrane zone (BMZ) in humans.
- In contrast, the subsequent superficial layer (SL) displayed a loose texture and, as such, was clearly demarcated from the SEL; it shared structural characteristics with the clinically relevant Reinke’s space of humans.
- The intermediate (IL) and deep layers (DL) of both folds were never clearly demarcated from each other due to relatively high amounts of fibres.
However, they were partially different in CraF and CauF:
o CauF: No clearly outlined ‘vocal ligament’ consisting of distinct strands of thick, densely packed fibre bundles could be identified. This was due to a continuous rather than an abrupt increase in fibre amounts.
o CraF: Many thick fibre bundles were present in the depth of the CraF, i.e.
at a level which was stratigraphically identical with the location of the muscle in the CauF.
Paper I
Histomorphometric analysis of collagen and elastic fibres in the cranial and caudal fold of the porcine glottis
A. Lang1, R. Koch1,K. Rohn2 and H. Gasse1
1 Institute of Anatomy, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany;
2 Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Bünteweg 2, 30559 Hannover, Germany.
Anatomia, Histologia, Embryologia; doi: 10.1111/ahe.12125
Summary
The porcine glottis differs from the human glottis in its cranial and caudal vocal folds (CraF, CauF). The fibre apparatus of these folds was studied histomorphometrically in adult minipigs. For object definition and quantification, the colour-selection tools of the Adobe-Photoshop program were used. Another key feature was the subdivision of the cross-sections of the folds into proportional subunits. This allowed a statistical analysis irrespective of differences in thickness of the folds.
Both folds had a distinct, dense subepithelial layer, equivalent to the basement membrane zone in humans. The subsequent, loose layer was interpreted – in principle – as being equivalent to Reinke’s space of the human vocal fold. The next two layers were not clearly separated. Due to this, the concept of a true vocal ligament did not appear applicable to neither CauF nor CraF. Instead, the body-cover model was emphasized by our findings. The missing vocal muscle in the CraF is substituted by large collagen fibre bundles in a proportional depth corresponding to the position of the muscle of the CauF. The distribution of elastic fibres made the CraF rather than the CauF more similar to the human vocal fold. We suggest that these data are useful for those wishing to use the porcine glottis as a model for studying oscillatory properties during phonation.
Introduction
The pig has been used as a presumably suitable model in human phoniatry (Blakeslee et al., 1995; Garrett et al., 2000; Jiang et al., 2001; Hahn et al., 2005;
Alipour and Jaiswal, 2008; Woodson, 2012). However, little attention has been paid to basic anatomical differences between the glottis in pigs and humans (Koch et al., 2010) and to the impact they have on phonation.
The most distinct special feature of the porcine glottis is the split – or doubled – structure of its vocal ligament, which has a significant importance for the formation of the glottical folds. Both parts of this longitudinally split vocal ligament originate at the ventral part of the thyroid cartilage and end on the vocal process of the arytenoid cartilage (Waibl, 2004). The cranial part of it may be misinterpreted and regarded as equivalent to the human vestibular ligament due to its location. However, the pigs’
true vestibular ligament lies far more cranial in the larynx between the epiglottis and the arytenoid cartilage (Burow, 1902).
These facts must be kept in mind when making reference to the folds of the glottis:
The cranial and the caudal part of the porcine vocal ligament are included separately in two different folds, i.e. a cranial fold, CraF, and a caudal fold, CauF (Koch et al., 2010), with the opening of the laryngeal ventricle lying in-between. What seems to resemble – in a topographical sense – the vestibular fold in the human glottis is in fact the cranial vocal fold in the porcine one. Phoniatrically, this anatomical difference may become important if one decides to use the pig as an animal model for the human; this cranial vocal fold is not only an additional oscillator, but rather the main oscillator in porcine phonation (Alipour and Jaiswal, 2008).
The phoniatrical properties of the glottis basically depend on the histological composition and stratigraphical organization of the glottical folds (Hirano, 1981;
Yumoto and Kadota, 1998; Chan et al., 2007). The lamina propria of the true human vocal fold traditionally comprises three layers, i.e. superficial, SLLP, intermediate,
ILLP, and deep layer, DLLP, of the lamina propria (Hirano, 1977). Some authors include an additional basement membrane zone, BMZ (Hahn et al., 2005; Tateya et al., 2006). Each of these layers are characterized by distinct amounts and distributions of collagen fibres and elastic fibres. No such data have been available – so far – on the cranial vocal fold, CraF, of pigs. The aim of this study was, therefore, to compare both CraF and CauF by using identical settings of methods which put major emphasis on the morphometry of the connective tissue fibres.
Digital image analysis was the key element of this procedure. The automated detection and analysis of well-demarcated structures (cells, cell nuclei) has become a fairly common and suitable application. However, the accurate selection of structures with poorly demarcated borders like fibres and fibre networks has remained a special challenge. Previously, the graphics editing program Adobe Photoshop has been proven to be well suited for histomorphometry, as it offers highly developed and powerful tools and commands for recognising and selecting objects and colours (Lehr et al., 1999; Lahm et al., 2004; Egan et al., 2012). A similar application was used in the present study.
Materials and Methods
Female minipigs (n = 11, aged 11-27 months) of the breeds Göttinger Minipig and Mini Lewe Minipig were included in this study. All animals were obtained and euthanized for the anatomical dissection classes of the University of Veterinary Medicine Hannover. All related procedures were performed in accordance with the Guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (86/609/EEC). Respecting this, an approval by the Lower Saxony State Office for Consumer Protection and Food Safety was not necessary. The animals were euthanized either by injection (performed by a veterinarian) of 1.5 ml/10 kg Euthadorm® (Pentobarbital Sodium; CP-Pharma Handelsgesellschaft mbH, Burgdorf, Germany) or by captive bolt stunning and consecutive bleeding; they showed no signs of larynx-associated diseases upon examination.
(1) Histological procedures
The larynges of the minipigs were excised immediately after euthanasia and immersion-fixed in Bouin’s solution (saturated aqueous picric acid, filtered acid-free formaldehyde 37 %, acetic acid 98 %; 15:5:1) at room temperature for 48 hours. The left side of the glottis was then excised and cut transversely at its midportion. These specimens were then treated by the standard procedure of alcoholic dehydration and paraffin embedding. Serial cross sections (5-8 µm) were alternately stained with either Masson’s trichrome for collagen structures or resorcin-fuchsin for elastic fibres.
Both kinds of sections were submitted to quantitative analysis as described in detail below. However, specimens stained with Masson’s trichrome stain often showed irregularities in collagen-fibre staining (i.e. either reddish colour of relatively small fibres, or red cores in thick collagen fibre bundles). Multiple variations of the staining protocol served well to solve these problems in most sections, but not in all. In the latter (rare) cases, the respective sections were discriminated from the
measurements, and the adjacent section was used. The most successful modification of the staining protocol is illustrated in table 1. The resorcin-fuchsin staining was successfully conducted following the standard protocol (Weigert, 1898).
After the visual evaluation of 8-10 serial sections, the subsequent measurements were performed on two adjacent sections, i.e. one stained with Masson’s trichrome, one stained with resorcin-fuchsin, both representing the midportion of the CauF.
Tab. 1. Masson trichrome staining protocol (Böck, 1989; modified, modifications in bold letters).
standard modified
Hansen's trioxy hematoxylin 8 min 8 min wash in acetic acid solution 1 % 1 min 1 min rinse with running tap water 10 min 10 min Goldner I: Ponceau + Acid fuchsin 5 min 3 min wash in acetic acid solution 1 % 10 min 10 min Goldner II: Phosphotungstic acid + Orange G 5 min 7 min
wash in acetic acid solution 1 % 5 min 5 min Goldner III: Light green SF yellowish 5 min 7 min
wash in acetic acid solution 1 % 5 min 5 min
