Questo lavoro è stato supportato da una sovvenzione APART dell'Accademia austriaca delle scienze (CTH), l'agenzia di tecnologia della Repubblica Ceca nessun progetto. TA04010877 (CTH, VH e GDC), e la Fondazione di scienza ceca (GACR) nessun GA16-01246S (al GDC) del progetto. Ringraziamo W. Tecumseh Fitch per suo suggerimento di usare la crema fissante della protesi dentaria e Ing. P. Liska dal servizio di foresta dell'esercito ceco per il suo aiuto nell'acquisire la larynges di cervi asportato.

Gli esemplari di animali analizzati in questa carta sono stati trattati in conformità con i requisiti standard etici dell'Università Palacky in Olomouc, Repubblica Ceca. Essi derivano da red deer selvaggiamente vivono nelle foreste, che sono stati cacciati dal servizio forestale esercito ceco durante una stagione di caccia regolare.
1. preparazione del campione Hemi-laringe
Nota: Solo i campioni correttamente preparati devono essere utilizzati, come indicato in4 . Rapido congelamento della laringe25 immediatamente dopo l'asportazione e conservazione a-80 ° C riduce al minimo il potenziale di degradazione del tessuto e l'alterazione delle proprietà biomeccaniche e permette di eseguire gli esperimenti in qualsiasi momento conveniente.
<ol><li>
Lo sbrinamento della laringe
	<ol>
<li>Inserire la laringe congelata in due sacchetti di autoclave o eventuali altri sacchetti in plastica con guarnizione impermeabile. Sigillare i sacchetti e metterli in una vasca di acqua riscaldata a 30 ° C fino a quando la laringe è completamente scongelata. La durata necessaria varia da poche ore a più di un giorno, a seconda della dimensione del laringe e temperatura di congelamento.</li>
	</ol>
</li>
	<li>
Pulizia della laringe
	<ol>
<li>Dopo la laringe è scongelata, rimuoverlo dal sacchetto e pulirlo accuratamente con soluzione fisiologica (0,9% NaCl).</li>
		<li>Rimuovere attentamente il tessuto superfluo come applicabile (cioè esterno collo muscoli, osso ioide ecc.) senza danneggiare le strutture principali della laringe e accorciare la trachea a una lunghezza adeguata per il montaggio della laringe in un tubo di alimentazione aria (solitamente ca. 4-5 cm).</li>
		<li>Controllare il tessuto laringeo per potenziali anomalie del tessuto, quali ferite, deformazioni organiche o crepe potenzialmente che accade dal processo di congelamento, che poteva rendere la laringe inadatto per l'esperimento.</li>
	</ol>
</li>
	<li>
Esposizione delle cartilagini tiroide e cricoide
	<ol>
<li>Rimuovere le parti del tessuto muscolo laringeo esterno intorno la cartilagine tiroidea e cricoidea usando un bisturi, esponendo così le cartilagini in preparazione per il taglio sagittale creando hemi-laringe. Questa fase di preparazione è raffigurata in Figura 1A e 1B.</li>
	</ol>
</li>
	<li>
Taglio sagittale attraverso la cartilagine tiroidea
	<ol>
<li>Fare un taglio verticale iniziale attraverso la parte anteriore della cartilagine tiroidea.</li>
		<li>Posizionare con attenzione il taglio leggermente più sul lato che sta per essere rimosso, per non danneggiare il popolare vocale che deve rimanere conservato. Se possibile, utilizzare un bisturi per il taglio. Se la cartilagine è ossificata, utilizzare una piccola sega.</li>
	</ol>
</li>
	<li>
Taglio della cartilagine cricoide
	<ol>
<li>Portare il taglio in verticale (inferiormente) da tra le cartilagini arytenoid e poi attraverso la cartilagine cricoide ad un livello circa orizzontale della tacca inferiore della tiroide.</li>
	</ol>
</li>
	<li>
Rimozione di una piega vocale, creando un'incisione a forma di L nella laringe
	<ol>
<li>Rendere un orizzontale tagliare a partire dall'estremità inferiore del taglio verticale precedentemente fatto nella cartilagine cricoide e portare il nuovo taglio verso la tacca inferiore della tiroide. Anteriormente e piegare il lato della laringe che sta per essere rimosso.</li>
		<li>Fare una verticale tagliare attraverso i tessuti molli sul lato interno della cartilagine tiroidea - attenzione mentre conduceva il taglio tra l'attacco anteriore dei popolare vocali alla cartilagine di tiroide, evitando così danni al popolare vocale.</li>
	</ol>
</li>
	<li>
Raffinatezza del taglio attraverso la cartilagine tiroidea
	<ol>
<li>Uso un bisturi, una sega o un file, al fine di applicare un taglio proprio dritto in cartilagine tiroidea e ottenere come vicino possibile alla parte anteriore del popolare vocale precedentemente ispezionato.</li>
		<li>Rimuovere anche una piccola parte della cartilagine tiroidea posteriore, al fine di creare spazio per inserire il Polo per avvicinare arytenoid cartilagine e così il vocal fold (Vedi sotto). Questa fase di preparazione è raffigurata in Figura 1 e D 1. Nota: A seconda della domanda di ricerca, esposizione completa del popolare complesso vocale può essere necessaria per consentire la visibilità dall'alto. In tal caso, le strutture sopra la piega vocale (vera) (cioè, la ventricolare o vestibolare piega, come applicabile dato l'anatomia dell'esemplare) dovrebbero essere rimosso. In alcuni esemplari il tessuto laringeo molle interno sopra le corde vocali potrebbe perdere la connessione con la cartilagine tiroidea e interferisce con il popolare vocale durante la vibrazione, causando potenzialmente spurie modelli oscillatorie (per lo più irregolari). In tal caso attenta rimozione di quel tessuto è inevitabile.</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55303/55303fig1.jpg"> Figura 1 : Montaggio e preparazione di Hemi-laringe. (A) e (B) ripuliti laringe esemplare, Vista mediale e posteriore, prima della rimozione del popolare vocale di sinistra; (C) e (D) preparati hemi-laringe con incisione a forma di L (sinistra popolare vocale rimosso), mediale e dalla vista posteriore. <a href="//cloudflare.jove.com/files/ftp_upload/55303/55303fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>

2. esperimento Hemi-laringe
<ol><li>
Installazione di Hemi-laringeo
	<ol>
<li>Utilizzare un tubo di alimentazione dell'aria che trasporta l'aria riscaldata e umidificata nella laringe.</li>
		<li>Costrutto due disposti perpendicolarmente lastre trasparenti come una sostituzione per pezzi rimossi laringei.</li>
		<li>Punte di uso 4 per aumentare la stabilità della laringe e creazione di una configurazione corretta pre-phonatory laringe di avvicinare la piega vocale rimanente per la lastra di vetro verticale (Vedi Figura 2A). Nota: Teoricamente, le corde vocali anche potrebbe essere addotta da suture e pesi su una puleggia-leva sistema 26 . Tuttavia, tale approccio è, per la migliore conoscenza di questi autori, non ancora stato tentato per una preparazione hemilarynx.</li>
	</ol>
</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55303/55303fig2.jpg">
Figura 2 : Installazione di Hemi-laringe. (A) strutture di sostegno: aria tubo di alimentazione, arrangiamento di piastra di vetro a forma di L, rebbi di adduzione. (B) montata preparazione hemi-laringe con rebbi di adduzione. (C) e (D) primi piani di hemi-laringe-preparazione, visto dal lato e dalla parte superiore, rispettivamente. <a href="//cloudflare.jove.com/files/ftp_upload/55303/55303fig2large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<ol start="2"><li>
Montaggio hemi-laringe
	<ol>
<li>Coprire il tubo di alimentazione dell'aria con fissativo protesi crema e montare la laringe utilizzando la parte restante della sua trachea. La crema fissante funziona come un adesivo e chiude gli spazi potenziali, creando così una tenuta ermetica.</li>
		<li>Fissare la trachea con una cinghia o una fascetta di serraggio di plastica.</li>
		<li>Anche coprire i bordi del taglio attraverso la cartilagine tiroidea con il fissativo crema, evitando di spalmare la crema fissante il popolare vocale o l'interno dei tessuti molle del laringeo.</li>
		<li>Fissare le piastre trasparente.</li>
	</ol>
</li>
	<li>
Stabilizzazione della cartilagine tiroidea, adduzione del popolare vocale usando i rebbi
	<ol>
<li>Utilizzare i rebbi del complesso popolare vocale alla piastra e stabilizzare la cartilagine tiroidea.</li>
		<li>Dopo la crema fissante è impostato, è possibile applicare il flusso d'aria al fine di stabilire l'oscillazione del popolare vocale e controllare per eventuali perdite tra hemi-laringe e le lastre di vetro.</li>
		<li>Sigillare le lacune eventualmente presenti in natura aggiungendo più crema fissante.</li>
	</ol>
</li>
</ol>

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<li>Baer, T. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBaer%2C+T+%22Vocal+Fold+Physiology%22">Vocal Fold Physiology</a>. Stevens, K. N., Hirano, M. , University of Tokyo Press. Tokyo. 119-133 (1981).</li>
<li>Pelorson, X., Hirschberg, A., van Hassel, R. R., Wijnands, A. P. J., Auregan, Y. <a target="_blank" href="http://asa.scitation.org/doi/abs/10.1121/1.411449">Theoretical and experimental study of quasisteady-flow separation within the glottis during phonation. Application to a modified two-mass model.</a> J Acoust Soc Am. 96 (6), 3416-3431 (1994).</li>
<li>Alipour, F., Scherer, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+separation+in+a+computational+oscillating+vocal+fold+model%5BTitle%5D)+AND+%22J+Acoust+Soc+Am%22%5BJournal%5D)">Flow separation in a computational oscillating vocal fold model.</a> J Acoust Soc Am. 116 (3), 1710-1719 (2004).</li>
<li>Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+flow+separation+location+on+phonation+onset%5BTitle%5D)+AND+%22J+Acoust+Soc+Am%22%5BJournal%5D)">Influence of flow separation location on phonation onset.</a> J Acoust Soc Am. 124 (3), 1689-1694 (2008).</li>
<li>Kaburagi, T., Tanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-dimensional+models+of+the+glottal+flow+incorporating+viscous-inviscid+interaction%5BTitle%5D)+AND+%22J+Acoust+Soc+Am%22%5BJournal%5D)">Low-dimensional models of the glottal flow incorporating viscous-inviscid interaction.</a> J Acoust Soc Am. 125 (1), 391-404 (2009).</li>
<li>Sidlof, P., Doaré, O., Cadot, O., Chaigne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+flow+separation+in+a+human+vocal+folds+model%5BTitle%5D)+AND+%22Exp+Fluids%22%5BJournal%5D)">Measurement of flow separation in a human vocal folds model.</a> Exp Fluids. 51 (1), 123-136 (2011).</li>
<li>Smith, S. L., Thomson, S. L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356320/">Effect of inferior surface angle on the self-oscillation of a computational vocal fold model.</a> J Acoust Soc Am. 131 (5), 4062-4075 (2012).</li>
<li>Khosla, S., Oren, L., Ying, J., Gutmark, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+simultaneous+measurement+of+intraglottal+geometry+and+velocity+fields+in+excised+larynges%5BTitle%5D)+AND+%22Laryngoscope%22%5BJournal%5D)">Direct simultaneous measurement of intraglottal geometry and velocity fields in excised larynges.</a> Laryngoscope. 124, S1-S13 (2014).</li>
<li>Brücker, C., Kirmse, C., Triep, M. <a target="_blank" href="http://www.ingentaconnect.com/contentone/dav/aaua/2016/00000102/00000002/art00007">Feedback of the glottal jet flow with supraglottal wall oscillations.</a> Acta Acustica United With Acustica. 102 (2), 240-243 (2016).</li>
<li>Herbst, C. T., Fitch, W. T., Lohscheller, J., Svec, J. G. <a target="_blank" href="NULL">AQL 2013, Proceedings of the 10th International Conference on Advances in Quantitative Laryngology, Voice and Speech Research</a>. Deliyski, D. D. , AQL Press. Cincinnati, Ohio, USA. 75-76 (2013).</li>
<li>Berke, G. S., Gerratt, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laryngeal+biomechanics%3A+an+overview+of+mucosal+wave+mechanics%5BTitle%5D)+AND+%22J+Voice%22%5BJournal%5D)">Laryngeal biomechanics: an overview of mucosal wave mechanics.</a> J Voice. 7 (2), 123-128 (1993).</li>
<li>Boessenecker, A., Berry, D. A., Lohscheller, J., Eysholdt, U., Doellinger, M. <a target="_blank" href="http://www.ingentaconnect.com/contentone/dav/aaua/2007/00000093/00000005/art00015">Mucosal wave properties of a human vocal fold.</a> Acta Acustica United With Acustica. 93 (5), 815-823 (2007).</li>
<li>Hirano, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHirano%2C+M+%22Clinical+examination+of+voice%22">Clinical examination of voice</a>. 5, Springer-Verlag. Wien, Austria. (1981).</li>
<li>Jing, B., Tang, S., Wu, L., Wang, S., Wan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+the+Vibration+of+Laryngeal+Tissue+during+Phonation+Using+Ultrafast+Plane+Wave+Ultrasonography%5BTitle%5D)+AND+%22Ultrasound+in+Med+BIol%22%5BJournal%5D)">Visualizing the Vibration of Laryngeal Tissue during Phonation Using Ultrafast Plane Wave Ultrasonography.</a> Ultrasound in Med BIol. 42 (12), 2812-2825 (2016).</li>
<li>Herbst, C. T. Ch. 6. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHerbst%2C+CT+%22Vertebrate+Sound+Production+and+Acoustic+Communication%22">Vertebrate Sound Production and Acoustic Communication</a>. Suthers, R. A., Fitch, W. T., Fay, R. R., Popper, A. N. , Springer International Publishing. Switzerland. 159-189 (2016).</li>
</ol>

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La preparazione di hemi-laringe condivide i vantaggi dell'installazione "convenzionali" laringe asportata (completo): tale approccio sperimentale, possono essere fisiche e fisiologiche condizioni al contorno e parametri (quali la pressione subglottidale o allungamento del popolare vocale) abbastanza ben controllata. Il comportamento della hemilarynx è omologo a quella di una laringe completa con una perfetta laterale simmetria, con l'eccezione che magnitudini di alcuni parametri (ad es., portata d'aria, pressio...

Voce viene in genere creato dalla vibrazione del tessuto laringeo (principalmente le corde vocali), che converte un flusso d'aria costante, fornito dai polmoni, in una sequenza di impulsi di flusso d'aria. La forma d'onda di pressione acustica (vale a dire, il suono primario) emergono da questa sequenza di impulsi di flusso acusticamente eccita il tratto vocale che li filtra, e il suono risultante è irradiato dalla bocca e (in una certa misura) dal naso1 . La composizione spettrale del suono generato è in gran parte influenzata dalla qualità della vibrazione del popolare vocale, sotto l'egida della laringe biomeccanica e interazioni con il flusso d'aria tracheale2. Sia in una clinica e un contesto di ricerca, documentazione e valutazione delle vibrazioni del popolare vocale è quindi di interesse soprattutto in quando si studia la produzione vocale.
In esseri umani, indagine endoscopica diretta della laringe durante la produzione del suono in vivo è impegnativo, ed è praticamente impossibile in mammiferi non umani, dati mezzi tecnologici attuali. Pertanto e in ordine alla garanzia attentamente controllato fisico e/o fisiologico sperimentale condizioni al contorno, l'uso di asportato larynges3,4 è in molti casi un'adeguata sostituzione per indagine su in vivo meccanismi di produzione di voce.
Vibrazione del popolare vocale è un fenomeno complesso tridimensionale5. Mentre i metodi di indagine convenzionali come endoscopia laringea (in vivo) o preparazioni laringe asportata in genere forniscono solo una superior vista dei popolare vocali vibrante6, non consentono per analisi tridimensionale completa di movimento del popolare vocale. In particolare, nella vista superiore i margini inferiori (caudali) dei popolare vocali sono invisibili durante una parte importante del ciclo vibratorio. Ciò è dovuto il ritardo di fase tra il inferior (caudale) e il bordo superiore (craniale) dei popolare vocali, un fenomeno che si verifica in genere durante popolare vocale oscillazione5. Come diretta evidenza empirica per il backup risultati da modelli matematici e fisici è scarso, conoscenza della geometria e movimento del canto inferiore piegare bordo7e quindi la geometria del canale subglottidale8,9 , 10 è cruciale per comprendere meglio l'interazione tra il flusso d'aria laringeo, il tessuto popolare vocale e la risultante delle forze e pressioni11,12. Un altro aspetto della vibrazione del popolare vocale che è nascosto dal consueta superior vista è la profondità verticale (caudo-craniale) del contatto tra le due corde vocali. La profondità di contatto verticale è relativo allo spessore verticale dei popolare vocali, che è un indicatore potenziale del registro vocale usato in canto ("petto" vs "falsetto" registro)13,14.
Al fine di superare le carenze delle preparazioni convenzionali laringe asportata (completo), può essere utilizzato un programma di installazione di cosiddetto hemi-laringe, dove metà della laringe è rimosso, facilitando così la valutazione delle caratteristiche vibratorie delle rimanenti del popolare vocale in tre dimensioni. Sorprendentemente, dopo l'introduzione di questa configurazione nel 1960s15 una convalida iniziale del concetto nel 199316, non molti laboratori hanno eseguito esperimenti con questo promettente approccio sperimentale17,la18 ,19,20,21,22,23. Una spiegazione per questo potrebbe essere trovata nelle difficoltà di creazione di una preparazione valida hemi-laringe. Mentre la preparazione convenzionale asportato laringe (completo) è ben documentato4, nessun tali istruzioni approfondite sono ancora disponibili per la creazione di un setup di hemi-laringe. È dunque lo scopo di questa carta per fornire un tutorial per la creazione di una configurazione riproducibili in modo affidabile hemi-laringe, completata da risultati sperimentali da esemplari di cervo.
Un programma di installazione di hemi-laringe condivide molte caratteristiche con un setup di laringe asportata "convenzionali", come apparecchiature di misurazione, ad alta velocità o altra tecnologia di imaging per documentare adeguatamente le vibrazioni delle strutture laringee durante la generazione del suono, o corretto fornitura di aria riscaldata e umidificata. Queste considerazioni generali di setup sono descritte in dettaglio in un libro capitolo4 e una relazione tecnica dal centro nazionale di voce e discorso24. Reiterazione di queste istruzioni potrebbe essere oltre la portata di questo manoscritto. Qui, solo le direttive specializzate per la generazione di un programma di installazione di hemi-laringe sono presentate.

<table><tbody><tr><td>Surgical blades</td><td>Surgeon</td><td>Jai Surgical Ltd., New Delhi, India</td><td></td></tr><tr><td>Saw</td><td>Hand saw (Lux, 150 mm length)</td><td>Lux, Wermelskirchen, Germany</td><td></td></tr><tr><td>Thermometer</td><td>Testo 922</td><td>Testo Ltd., Hampshire, UK</td><td>K-type Probe, Operating temperature -20 to +50 &amp;deg;C</td></tr><tr><td>Autoclave bags</td><td>Autoclave bags</td><td>vwr.com, VWR International s.r.o., Stribrna Skalice, Czech republic</td><td></td></tr><tr><td>Conductive glass plates</td><td>Custom made</td><td>UPOL - Joint laboratory of Optics&lt;br/&gt; Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.</td><td></td></tr><tr><td>Fixative cream</td><td>Denture fixative cream</td><td>Blend-a-dent Natural</td><td></td></tr><tr><td>Prongs and fastening system</td><td>Customized Kanya Al eloxed profiles</td><td>Distributor: VISIMPEX a.s.. Seifertova 33, 750 02 Prerov, the Czech Rep.;</td><td>&amp;nbsp;Combination of Kanya RVS and PVS fastening systems (http://www.kanya.cz/) + custom made prongs</td></tr><tr><td>Mounting tube</td><td>Custom made</td><td>UPOL - Joint laboratory of Optics,&lt;br/&gt; Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.</td><td></td></tr><tr><td>LED Light</td><td>Verbatim 52204 LED Lamp</td><td>Mitsubishi Chemical Holdings Corporation, Tokyo, Japan</td><td></td></tr><tr><td>Camera</td><td>Canon EOS1100D</td><td>Canon Inc.</td><td>18-55 mm lens</td></tr><tr><td>Airpump</td><td>Resun LP100</td><td>Resun</td><td></td></tr><tr><td>Strobe light</td><td>ELMED Helio-Strob micro2</td><td>ELMED Dr. Ing. Mense GmbH, Heiligenhaus, Germany</td><td></td></tr><tr><td>Humidifier</td><td>Custom made</td><td>Voice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republic</td><td></td></tr><tr><td>Subglottic tract</td><td>Custom made adjustable subglottic tract</td><td>Voice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republic</td><td>Hampala, V., Svec, Jan, Schovanek, P., and Mandat, D. Uzitny vzor c. 25585: Model subglotickeho traktu. [Utility model no. 25585: Model of subglottal tract] (In Czech) Soukup, P. 2013-27834(CZ 25505 U1), 1-7. 24-6-2013. Praha, Urad prumysloveho vlastnictvi</td></tr></tbody></table>

hemi-laryngeal setup for studying vocal fold vibration in three dimensions

La voce degli esseri umani e la maggior parte dei mammiferi non umani viene generata nella laringe attraverso self-sustaining oscillazione dei popolare vocali. Documentazione visiva diretta della vibrazione del popolare vocale è impegnativo, specialmente in mammiferi non umani. In alternativa, laringe asportata esperimenti forniscono la possibilità di indagare la vibrazione del popolare vocale in condizioni fisiologiche e fisiche controllate. Tuttavia, l'uso di una laringe completa fornisce semplicemente una vista dall'alto dei popolare vocali, escluse parti cruciali delle strutture oscillante da osservazione durante la loro interazione con le forze aerodinamiche. Questa limitazione può essere superata utilizzando un programma di installazione di hemi-laringe dove metà della laringe è metà-sagittally rimosso, fornendo sia una superior e una vista laterale del popolare vocale rimanente durante l'oscillazione autosostenuto.
Qui, una guida passo passo per la preparazione anatomica delle strutture hemi-laringeo e il loro montaggio su banco di laboratorio è dato. Fonazione esemplare della preparazione hemi-laringe è documentato con alta velocità dati video catturati da due telecamere sincronizzate (viste superiore e laterale), visualizzando tridimensionale del popolare vocale movimento e area di contatto corrispondente variabili nel tempo. La documentazione dell'installazione hemi-laringe in questa pubblicazione faciliterà l'applicazione e affidabile ripetibilità nella ricerca sperimentale, fornendo gli scienziati vocale con il potenziale per comprendere meglio la biomeccanica della produzione vocale.

Illustrazioni della preparazione hemi-laringe e suo montaggio sul tubo di alimentazione dell'aria, come indicato nella sezione precedente, sono forniti in Figura 1 e Figura 2, rispettivamente.
Documentazione di vibrazione del popolare vocale da due angolazioni
Oscillazione autosufficiente indo...

Watch this Scientific Journal Video about Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions at JoVE.com

Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

Questo documento introduce un protocollo per la preparazione di campioni di hemi-laringe facilitando una vista multi-dimensionale di vibrazione del popolare vocale, al fine di indagare i vari aspetti biofisici della produzione vocale negli esseri umani e non umani mammiferi.

Hemi-laringeo Setup per lo studio delle vibrazioni del popolare vocale in tre dimensioni

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual ...

hemi-laryngeal-setup-for-studying-vocal-fold-vibration-three

Research

JoVE Journal

Biology

10.9K Views.  Palacky University Olomouc. Questo documento introduce un protocollo per la preparazione di campioni di hemi-laringe facilitando una vista multi-dimensionale di vibrazione del popolare vocale, al fine di indagare i vari aspetti biofisici della produzione vocale negli esseri umani e non umani mammiferi.

Video: Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties 1. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone 2 and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.
Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).
Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry 3,4, clinical instrument development 5, laryngeal aerodynamics 6-9, vocal fold contact pressure 10, and subglottal acoustics 11 (a more comprehensive list can be found in Kniesburges et al. 12)
Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds 1 that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages 3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).
In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers 1. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.

The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.

Sintetico, Multi-Layer, Auto-oscillante Fabrication Vocal modello Fold

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

Ricerca

Bioingegneria

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

Basato su chip tridimensionale coltura cellulare in perfuso Micro-bioreattori

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Biologia

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Investigare la separazione tridimensionale del flusso indotta da un modello di polipo piega vocale

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Costruzione e caratterizzazione di un Fold bioreattore Novel Vocal

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Tecniche di microiniezione per Studiare Mitosi in Drosophila melanogaster Embryo sinciziale

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Stampa 3D di modelli di corde vocali in silicone multistrato

Manufacturing Process for Non-Adhesive Super-Soft Vocal Fold Models

This study aims to develop super-soft, non-sticky vocal fold models for voice research. The conventional manufacturing process of silicone-based vocal fold models results in models with undesirable properties, such as stickiness and reproducibility issues. Those vocal fold models are prone to rapid aging, leading to poor comparability across different measurements. In this study, we propose a modification to the manufacturing process by changing the order of layering the silicone material, which leads to the production of non-sticky and highly consistent vocal fold models. We also compare a model produced using this method with a conventionally manufactured vocal fold model that is adversely affected by its sticky surface. We detail the manufacturing process and characterize the properties of the models for potential applications. The outcomes of the study demonstrate the efficacy of the modified fabrication method, highlighting the superior qualities of our non-sticky vocal fold models.&#160;The findings contribute to the development of realistic and reliable vocal fold models for research and clinical applications.

Autore in primo piano: Progressi nella fabbricazione di modelli sintetici di corde vocali per applicazioni fonetiche e robotiche

This study demonstrates the manufacturing of non-sticky and super-soft vocal fold models by introducing a specific way to create the vocal fold layers, providing a detailed description of the manufacturing procedure, and characterizing the properties of the models.

Processo di produzione per modelli di corde vocali super-morbide non adesive

This study aims to develop super-soft, non-sticky vocal fold models for voice research. The conventional manufacturing process of silicone-based vocal ...

Ingegneria

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

Preparazione della piega vocale del ratto per le analisi neuromuscolari

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

Hemi-laringeo Setup per lo studio delle vibrazioni del popolare vocale in tre dimensioni

Riepilogo

Esplora Altri Video

Sintetico, Multi-Layer, Auto-oscillante Fabrication Vocal modello Fold

Basato su chip tridimensionale coltura cellulare in perfuso Micro-bioreattori

Investigare la separazione tridimensionale del flusso indotta da un modello di polipo piega vocale

Costruzione e caratterizzazione di un Fold bioreattore Novel Vocal

Tecniche di microiniezione per Studiare Mitosi in Drosophila melanogaster Embryo sinciziale

Processo di produzione per modelli di corde vocali super-morbide non adesive

Processo di produzione per modelli di corde vocali super-morbide non adesive

Processo di produzione per modelli di corde vocali super-morbide non adesive

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

Preparazione della piega vocale del ratto per le analisi neuromuscolari