The correlation between the stratigraphical organisation of the human vocal fold’s lamina propria and its oscillatory characteristics is well understood. However, the effects of the porcine vocal folds’ specific structural features on (porcine) vocal fold oscillation are still unknown. In this respect, the findings of the present study can be interpreted as follows:
The fibro-elastic subepithelial layer (SEL) present in CraF and CauF was placed like a membrane (approx. 20 µm thick) over the loose, i.e. soft, superficial layer (SL).
According to the body-cover theory of vocal fold vibration (see chapter 2.3: The stratigraphical organisation of the lamina propria), the loose superficial tissue of the vocal fold represents the ‘cover’, which moves relatively freely upon the underlying
‘body’ (HIRANO 1974; YUMOTO and KADOTA 1998). The aforementioned fibro-elastic membrane covering this loose tissue can be assumed to sustain the shape of the ‘cover’ and thus limit its range of movement. There is a similar structure, i.e. the basement membrane zone (BMZ), in humans, but there is no data reporting on how thick it is, nor has there been an attempt to incorporate it into functional considerations (see Paper II, Discussion: Functional considerations – The phoniatrical body-cover model).
In the ‘old’ minipigs, the superficial layer (SL) of CraF and CauF was no longer as loose (i.e. sparse in fibres) as before, but rather contained high amounts of fibres (Paper II). A related loss of flexibility may be further increased by the ageing of the fibres: Aged fibres become stiffer and less elastic (GRAY et al. 2000; BAILEY 2001;
SHERRATT 2009).
Consequently, the entire ‘cover’ could be assumed to be stiffer and less elastic in the
‘old’ animals. These changes appeared much more pronounced in the minipigs compared to humans, as in the latter, the cover’s structure mostly remained relatively loose (see chapter 2.4: The structural features of maturation and ageing of the vocal folds; and Paper II).
The intermediate layer (IL) of the ‘young’ minipigs resembled the human ILLP with regard to the location of the peak values of elastic fibre amounts (Paper II). In humans, this distinct structural item is an essential component of the vocal ligament.
Therefore, the structure of the IL of the ‘young’ minipigs may reflect the CraF’s role during phonation: ALIPOUR and JAISWAL (2008, 2009) have proposed the cranial vocal fold as the main oscillator of the porcine glottis.
Finally, the dense accumulations of collagen fibre bundles in the depth of the CraF deserve major attention in terms of function, as they occurred in a location equivalent to that of the muscle in the CraF and also in the human vocal fold.
Apparently, the phoniatrical ‘body’ of the CraF is completely fibrous, rather than fibro-muscular (see Paper II).
6 Summary
Anja Lang
Histomorphometrical analysis of the fibrous components of the porcine vocal folds – Stratigraphical features and their relevance for models in phoniatry
The stratigraphical organisation of the Cranial and the Caudal Vocal Fold (CraF, CauF) of the porcine glottis was examined with regard to the criteria of the phoniatrical body-cover model. The aim of this examination was the elaboration of histological features of tissue maturation and aging, in order to elucidate the potentials of the pig as an animal model in human phoniatry. With regard to the procedure of Object Definition, the potentials and limitations of histomorphometry of non-corpuscular structures (i.e. fibres) are emphasised.
For this purpose, the collagenous and elastic fibre apparatus of CraF and CauF was examined in ‘young’ (2-3 months, n = 6), ‘adult’ (11-27 months, n = 11), and ‘old’ (54-84 months, n = 6) female minipigs. Cross sections of the glottis were selectively stained by Masson’s trichrome for collagen structures, or resorcin-fuchsin for elastic fibres. The amounts of these fibres were examined by semi-automated histo-morphometry in Adobe Photoshop, in which Object Definition was based on colour selection of collagen and elastic fibres. Technical problems related to differences in staining intensities were solved by visual control and manual adjustment of the system. A different – presumably biological – problem of Object Definition occurred when collagen fibres the CraF of the ‘old’ specimens were unexpectedly stained reddish (instead of green). These two kinds of problems are discussed with special emphasis on the limitations of histomorphometrical procedures. In addition to the analysis of fibre amounts, the diameters of the collagen structures were analysed with the aid of a semiquantitative scoring system. The distinction of layers and the determination of their boundaries required extensive statistical data analysis. A key
element of this procedure was the assignment of proportional subunits and hypothetical zones.
The study revealed the presence of a four-layered stratigraphy in both CraF and CauF, comprising subepithelial, superficial, intermediate, and deep layers (SEL, SL, IL, DL).
The subepithelial layer (SEL) was thin, but characterised by dense accumulations of fibres located directly underneath the epithelium in both folds of all age groups. This fibro-elastic membrane resembled the so-called basement membrane zone of humans described in the literature. It was assumed to limit the range of movement of the underlying tissue during vocal fold oscillation in pigs.
Underneath the SEL, a superficial layer (SL) was present in CraF and CauF. It was histologically similar to the loose and flexible superficial layer of humans (Reinke’s space), which is supposed to act as a phoniatrically relevant part of the ‘cover’. In the
‘old’ minipigs, the loose composition of the SL was lost due to a strong increase in fibre amounts. In the CauF, this increase was encountered in the entire lamina propria, as has been reported to occasionally occur in geriatric humans.
In the CauF, the tissue underneath the SL (i.e. IL and DL) displayed no clearly demarcated accumulations of thick fibre bundles which could be interpreted as a
‘vocal ligament’. Elastic fibres – known to be superimposed on collagen fibres in the human vocal ligament – were rather homogeneously mixed with the collagen fibres.
Consequently, the application of the term ‘vocal ligament’ to the intermediate and deep layers (IL, DL) of the CauF did not appear appropriate.
Only in the CraF of the ‘young’ minipigs, peak values of elastic fibre amounts were detected in the intermediate layer (IL), i.e. spread over the collagen fibres of the deep layer (DL). This similarity between porcine CraF and human vocal fold was presumed to reflect the CraF’s role as the main oscillator. With increasing age, however, this
similarity between pigs and humans vanished due to a strong increase in elastic fibre amounts throughout all layers, but with a certain concentration in the superficial regions of the porcine vocal folds.
Large amounts of thick collagen fibre bundles were present in the depth of the CraF, i.e. in a location equivalent to that of the vocal muscle in the CauF and in the human vocal fold. This fibre arrangement in the CraF was discussed to adopt a functional role (similar to that of the vocal muscle) as part of the phoniatrical ‘body’ during vocal fold oscillation. This assumption was supported by the fact that these bundles developed as a result of histological maturation rather than of ageing.
In conclusion, the histological data support the assumption (from the literature) that the CraF is suited to act as the main oscillator of the porcine glottis. Considering Reinke’s space, however, it is the CauF which deserves major attention, because its superficial layer was twice as thick as that of the CraF.
7 Zusammenfassung
Anja Lang
Histomorphologische Untersuchungen am Faserapparat der Stimmfalten des Schweins – Stratigraphische Merkmale und deren Relevanz für phoniatrische Modelle
Die stratigraphische Organisation der Kranialen und der Kaudalen Stimmfalte (CraF, CauF) des Schweines wurde mit Blick auf die Kriterien des phoniatrischen Body-Cover-Modells untersucht. Hierbei sollte die Darstellung der Eigenschaften der Reifung und Alterung des Bindegewebes eine Einschätzung des Potentials eines Modells „Schwein“ für die Human-Phoniatrie ermöglichen. Im Mittelpunkt dieser Arbeit standen insbesondere die Möglichkeiten und die Grenzen der Histomorpho-metrie nicht-korpuskulärer Strukturen.
Der kollagene und elastische Faserapparat von „jungen“ (2-3 Monate, n = 6),
„adulten“ (11-27 Monate, n = 11) und „alten“ (54-84 Monate, n = 6) weiblichen Minipigs wurde untersucht, indem Querschnitte ihrer Glottis entweder mittels Masson-Trichrom für Kollagenfasern oder Resorcin-Fuchsin für elastische Fasern gefärbt wurden. Die Fasergehalte in diesen Querschnitten wurden mit Hilfe eines semiautomatischen histomorphometrischen Verfahrens im Bildbearbeitungspro-gramm Adobe Photoshop bestimmt. Die Object Definition beruhte dabei auf der Erfassung der kollagenen und elastischen Fasern anhand ihrer selektiven Farbewerte. Technische Probleme (unterschiedliche Intensität der Färbungen) konnten mit Hilfe von visuellen Kontrollen und manuellen Eingriffen in die Routine gelöst werden. Ein anderes – vermutlich biologisches – Problem trat in der CraF der
„alten“ Tiere auf. Hier wurden Kollagenfasern unerwarteterweise rötlich (anstatt grün) gefärbt. Die Auswirkungen dieser Art von Problemen wurden besonders mit Blick auf die Grenzen der Histomorphometrie diskutiert. Zusätzlich zu der Analyse der
Fasergehalte wurden die Durchmesser der Kollagenfasern und -faserbündel mit Hilfe eines semiquantitiativen Scoring-Systems ausgewertet. Die Bestimmung von Schichten und deren Grenzen bedurften einer aufwändigen statistischen Datenanalyse; ein wesentliches Element dieser Prozedur war eine Unterteilung in
„Proportionale Subunits“ und hypothetische „Zonen“.
In beiden Falten (CraF und CauF) der Glottis des Schweins wurde eine Vierschichtigkeit festgestellt. Diese zeichnete sich aus durch Subepithelial, Superficial, Intermediate und Deep Layer (SEL, SL, IL, DL).
Der Subepithelial Layer (SEL) bestand aus einem schmalen Saum dicht angehäufter Fasern direkt unterhalb des Epithels, und zwar in beiden Falten und in allen Altersgruppen. Diese fibro-elastische Membran ähnelte der aus dem Schrifttum bekannten sogenannten Basement Membrane Zone des Menschen. Faserart und Faserdichte des SEL legten nahe, dass er den Bewegungsspielraum des darunter liegenden lockeren Gewebes (d.h. des phoniatrischen „Covers“) während der Oszillation der Stimmfalten beeinflusst.
Unterhalb des SEL befand sich in CraF und CauF der Superficial Layer (SL).
Histologisch ähnelte er dem lockeren Superficial Layer in der Stimmfalte des Menschen (Reinke-Raum); von ihm wird angenommen, dass er den wesentlichen Anteil des „Covers“ ausmacht. Dieser lockere Bau des SL verlor sich jedoch in Folge eines starken Anstiegs der Fasermengen in beiden Falten der „alten“ Minipigs. In der CauF fand eine solche Zunahme der Fasermengen über die ganze Tiefe der Lamina propria statt; ähnliches wird im Schrifttum gelegentlich von geriatrischen Menschen berichtet.
Die CauF zeigte unterhalb des SL (also in IL und DL) keine klar abgegrenzte Ansammlung dicker Faserbündel welche man als Stimmband bezeichnen könnte.
Elastische Fasern, die beim menschlichen Stimmband den Kollagenfasern aufge-lagert sind, waren in der CauF des Schweins homogen mit den Kollagenfasern
vermischt. Demnach erschien es nicht angebracht, IL und DL als Stimmband (Ligamentum vocale) anzusprechen.
Nur in der CraF der „jungen“ Schweine wurden im Intermediate Layer (IL) Spitzenwerte an Mengen elastischer Fasern festgestellt; sie waren den Kollagenfasern des Deep Layers (DL) aufgelagert. Hierin besteht eine Gemein-samkeit von CraF und menschlicher Stimmfalte, die die Annahme einer Rolle der CraF als Hauptoszillator stützt. Mit zunehmendem Alter verschwand jedoch diese Gemeinsamkeit zwischen Schweinen und Menschen, und zwar aufgrund eines starken Anstiegs der Gehalte elastischer Fasern vor allem im oberflächlichen Bereich der Falte.
In der Tiefe der CraF – dort wo sich in der CauF und in der menschlichen Stimmfalte der Stimmmuskel befindet – wurden sehr viele dicke Kollagenfaserbündel angetroffen. Diese Bündel übernehmen vermutlich die Funktion des phoniatrischen
„Bodys“, ähnlich dem Stimmmuskel. Die Entwicklung der Bündel im Zuge der Reifung (statt der Alterung) unterstützt diese Annahme.
Aus den Befunden wird abgeleitet: Die histologische Zusammensetzung und die stratigraphische Organisation der kranialen Falte (CraF) unterstützen die im Schrifttum formulierte These, wonach die kraniale Falte (CraF) in der Glottis des Schweins als Hauptoszillator fungieren kann. Hinsichtlich des Reinke-Raum sollte jedoch die kaudale Falte im Zentrum der Aufmerksamkeit stehen, da der Superficial Layer hier doppelt so dick ist wie in der CraF.
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