Larynx preparation

This experiment was carried out on cadavers donated to the Anatomy Laboratory of the French Alps (Laboratiore d’Anatomie des Alpes Françaises). Twelve larynges (six from men and six from women) were dissected within 48 hours post-mortem. The larynges were kept intact from the epiglottis to the fifth tracheal ring. None of the epilaryngeal structures were removed. An X-ray computed tomography (CT) scan made of one larynx dissected in this way was used to make a 3D visualisation of its cartilage, shown in Figure 5-1 (Chiche, 2012).

7 I acquired the data for the experiments in this chapter, except where otherwise noted in the

acknowledgements. I performed all of the data analysis for this chapter under the supervision of Nathalie Henrich Bernardoni. Significant portions of this chapter have been submitted for publication as ‘Laryngeal control of fundamental frequency in excised human larynges’ with co-authors N. Henrich Bernardoni, F.

Bennani, L. Morand, M. Pelloux, A. Mancini, X. Laval, T. Legou, P. Chaffanjon (Hanna et al., 2014a). A pilot study based on data from a single larynx (LF4) was presented in ‘Singing excised human larynges: relationship between subglottal pressure and fundamental frequency,’ International Workshop on Models Analysis of Vocal Emissions for Biomedical Analysis (MAVEBA), Florence, Italy (Hanna et al., 2013a), and further preliminary portions of this work were presented in ‘Singing excised human larynges: Investigating

Figure 5-1 (left) 3D visualisation of the cartilage of one of the dissected larynges from a CT scan, reproduced with permission from Chiche (2012). The epiglottis is coloured blue, the hyoid bone is red, the thyroid cartilage is purple, the cricoid cartilage is yellow, and the tracheal rings are yellow and aqua. (middle right) Illustration of a larynx attached to the experimental bench by the cricoid cartilage. (lower right) A sketch of the the upstream air supply system.

Details of the larynges are given in Table 5-1. For each experiment, the larynx was secured to the experimental bench posteriorly and anteriorly at the cricoid cartilage (shown in Figure 5-1). An intubation tube was used to supply a source of heated humidified air from a pressurised reservoir (Mecafer, LT50 Compressor) with adjustable constriction. Crico-thyroid tension was maintained by securing the epiglottis. The aryepiglottic folds were separated by the use of a Beckman-Eaton laminectomy retractor. To examine the behaviour of the supraglottal larynx, additional measurements were made without this separation.

15.5cm

41cm Humidifier

Pressurised air supply

Inflated intubation tube provides airtight connection to air supply

EGG electrodes Larynx position fixed at

cricoid cartilage Pressure probe inserted

into crico-thyroid membrane Crico-thyroid tension maintanted by tension

on epiglottis

Aryepiglottic folds separated

Table 5-1 Description of the excised larynges tested, including the recorded f0 ranges in hertz and musical notation (in square brackets). Women’s larynges are denoted LF, and men’s larynges are denoted LM. The modes correspond to two distinct regimes in the Psg, f0 plane, following the terminology of Alipour and

Dehydration of the larynx was observed by colour changes in the tissue and eventually as an increase in stiffness, which can result in tears and holes in the soft tissue. The larynges were disposed of appropriately once they were no longer able to sustain self-oscillation.

artificial vocal tract would provide a more realistic setting but would also introduce similar constraints on access to the larynx as an in vivo study and therefore remove some of the advantage of an ex vivo approach.

With no supraglottal vocal tract, the influence of the subglottal impedance may be expected to cause bifurcations in the P_sg, f₀ oscillation (Zhang et al., 2006). The impedance of the experimental setup used was estimated using the geometry of the plumbing upstream of the vocal folds, in a similar way to 4.2.1.4. The intubation tube connected to the larynx is 15.5 cm (diameter 1.6 cm), and is connected a larger diameter tube (2.1 cm) of length 41 cm that is terminated by several litres of air in the humidifier. A sketch of the plumbing is included in Figure 5-1.

5.1.2.Data acquisition

Data were acquired at a sampling rate of 6.25 kHz using a specialised aerodynamic workstation (EVA2, S.Q.Lab, Aix-en-Provence, France), then analysed using Matlab. The measurement and analysis processes for each variable are described below.

P_sg was measured with a probe inserted via a catheter into the crico-thyroid membrane. AC (audio) component of the signal was removed by low pass filtering (2^nd order Butterworth filter at 5 Hz) to provide the DC (aerodynamic) pressure.

The vocal fold vibratory behaviour was monitored by an electroglottograph (EGG) (EG2-PCX2, Glottal Enterprises, Syracuse, New York, USA). f₀ was estimated from the EGG signal using the YIN algorithm (de Cheveigné and Kawahara, 2002), with a frequency detection range of 65-1000 Hz and the absolute threshold parameter set to 0.5. Periods of aphonia and erratic quasi-periodic behaviour were removed from further analysis.

The sound pressure level was measured by the EVA2 workstation microphone at a distance of 30 cm from the larynx. The background noise level was ~45 dB, so the lower range of phonation sound pressure level was not recorded with sufficient signal to noise ratio. Consequently the reported dynamic ranges of each larynx are conservative.

Each experimental sequence was recorded with a Sony HVR-V1 HD video camera at 60 interlaced frames per second with a vertical resolution of 1920x1080 pixels. A Nikon EOSENS Cube 7 high speed camera was used to record a few sequences at 2000 frames per second for two of the larynges, LF1 and LM1, in order to assess vocal fold closure.

5.1.3.Experimental protocol

The study was conducted with two sweeping (increasing then decreasing) control conditions: air supply and mechanical. Saline solution was applied before each sweep.

For the air supply control condition, air from the pressurised reservoir was released by varying the output constriction, allowing air to flow through the humidifier and on to the larynx. The pressure P_sg, measured just below the glottis depends on the load at the glottis, and so is not constant on the time scale of the vocal fold oscillations. P_sg, was increased then decreased by hand, while the laryngeal geometry was kept fixed by applying one of three different applied tensions to produce a low, medium or high level of vocal fold adduction.

For the mechanical control condition, the air supply constriction was fixed to a value that allowed phonation with a minimum tension applied to the vocal folds. Two methods of simulating in vivo biomechanical laryngeal control were tested. For the majority of larynges, sutures were secured in the muscular processes of the arytenoid cartilages and pulled as illustrated in Figure 5-2, similar to the approach of Berry et al. (1996). For larynges LF4, LM5 and LM6, adjustment of vocal folds geometry was performed manually with metal probes inserted into the arytenoid cartilage, also shown in Figure 5-2.

Figure 5-2 (left) A schematic view from above showing the sutures used to simulate vocal fold tension adjustments. (right) A photograph showing the adjustment of vocal fold tension with metal probes inserted in the arytenoid cartilage.

The aim of the mechanical control condition is to mimic a combined action of lateral crico-arytenoid and thyro-crico-arytenoid muscular tension. The direction of applied tension was adjusted to achieve the most complete vocal fold adduction during oscillation, an example is shown in Figure 5-3. Tension was applied symmetrically by pulling the sutures from the same support via a dynamometer, then releasing. The applied tension was increased to a maximum of 10 N depending on the larynx. This approach adjusts the vocal fold geometry but does not directly activate the muscles, and since it is not possible to measure their internal stress, the effect may not be representative of the muscular laryngeal control performed in vivo. Furthermore, increasing the vocal fold adduction in this way increases P_sg and may cause other effects, e.g. adducting the vocal folds medially and therefore changing their effective mass.

Figure 5-3 Example still images extracted from the 2000 fps video, showing the closed and open phases of vocal fold oscillation in larynx LM1 with the laryngeal configuration controlled by the sutures shown in Figure 5-2.

5.2. Results and Discussion

Firstly, the range of phonation achieved by each larynx is presented, and the limits of repeatability determined. Then the dependence of f₀ on air supply and vocal fold geometry is investigated, looking for qualitatively different regimes and quantifying the variation in each regime. An empirical power law for the P_sg, f₀ relationship is compared with a linear fit.

Finally, the passive behaviour of aryepiglottic larynx is examined.