Eur Arch Otorhinolaryngol DOI 10.1007/s00405-014-3101-8
MISCELLANEOUS
The effect of temperature on basal tension and thyroarytenoid muscle contraction in an isolated rat glottis model Hsing-Won Wang • Yueng-Hsiang Chu Pin-Zhir Chao • Fei-Peng Lee
•
Received: 5 March 2014 / Accepted: 8 May 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract The pitch of voice is closely related to the vocal fold tension, which is the end result of coordinated movement of the intralaryngeal muscles, and especially the thyroarytenoid muscle. It is known that vocal quality may be affected by surrounding temperature; however, the effect of temperature on vocal fold tension is mostly unknown. Thus, the aim of this study was to evaluate the effect of temperature on isolated rat glottis and thyroarytenoid muscle contraction induced by electrical field stimulation. In vitro isometric tension of the glottis ring from 30 Sprague–Dawley rats was continuously recorded by the tissue bath method. Electrical field stimulation was applied to the glottis ring with two wire electrodes placed parallel to the glottis and connected to a direct-current stimulator. The tension changes of the rat glottis rings that H.-W. Wang P.-Z. Chao Department of Otolaryngology, Taipei Medical UniversityShuang Ho Hospital, No. 291, Jhong-Jheng Road, Jhonghe District, 23561 New Taipei City, Taiwan, ROC e-mail: [email protected] H.-W. Wang Department of Preventive and Community Medicine, Taipei Medical University-Shuang Ho Hospital, No. 291, Jhong-Jheng Road, Jhonghe District, New Taipei City, Taiwan H.-W. Wang P.-Z. Chao Graduate Institute of Clinical Medicine, School of Medicine, Taipei Medical University, New Taipei City, Taiwan H.-W. Wang Y.-H. Chu Department of Otolaryngology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan F.-P. Lee (&) Department of Otolaryngology, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan e-mail: [email protected]
were either untreated or treated with electrical field stimulation were recorded continuously at temperatures from 37 to 7 °C or from 7 to 37 °C. Warming from 7 to 37 °C increased the basal tension of the glottis rings and decreased the electrical field stimulation-induced glottis ring contraction, which was chiefly due to thyroarytenoid muscle contraction. In comparison, cooling from 37 to 7 °C decreased the basal tension and enhanced glottis ring contraction by electrical field stimulation. We concluded that warming increased the basal tension of the glottis in vitro and decreased the amplitude of electrical field stimulation-induced thyroarytenoid muscle contraction. Thus, vocal pitch and the fine tuning of vocal fold tension might be affected by temperature in vivo. Keywords Glottis Temperature Electrical field stimulation
Introduction The pitch of voice is closely related to vocal fold (VF) tension, which is an end result of complex coordinated movements of the intralaryngeal muscles and the viscoelastic properties of the VF. The contraction/relaxation of the thyroarytenoid (TA) muscle, which is innervated by the recurrent laryngeal nerve (RLN), constitutes the ‘‘body’’ of the VF and it increases the VF tension by exerting anterior traction on the vocal process. There are five intrinsic laryngeal muscles: the TA, cricothyroid (CT), interarytenoid (IA), posterior cricoarytenoid (PCA), and lateral cricoarytenoid (LCA). Of these muscles, the CT and TA are primarily involved in length and tension control. The TA, LCA, and IA are all adductors, whereas PCA is the sole abductor of the VF [1, 2]. Vocal fold tension during
123
Eur Arch Otorhinolaryngol
phonation is generated by coordinated contraction of the intrinsic laryngeal muscles. The TA muscle has been found to have increased stiffness at various levels of strain compared with the other intrinsic laryngeal muscles. The contraction of the CT muscle, which results in a decrease in the distance between the thyroid and cricoid cartilages, is considered to be the main factor in lengthening the vocal folds [1, 2]. As demonstrated by Yanagi et al. [3] and Hillel [4], the CT muscle not only lengthens the true vocal fold, but also thins the vocal fold and increases the longitudinal tension. The net effect of this action is to increase the pitch of the phonation. The contraction of the TA muscle is thought to increase the lateral tension of the vocal fold. It works to set the vocal folds into position for phonation and maintains tone of vocal folds during phonation. Work by Titze et al. [5] has even demonstrated decreased pitch with TA muscle activation in the setting of large cricothyroid activity with elongated vocal folds due to the reduced tension of the mucosal cover. It is known that vocal quality may be affected by the surrounding temperature. However, the effect of temperature on VF tension is mostly unknown. It is difficult to evaluate the effect of temperature on the basal tension of the VF in vivo since warming/cooling (temperature) has effects at multiple levels (e.g., nerve firing rate, neurotransmitter release, and ability of the muscles to contract/relax). In this study, we measured the isometric tension of isolated rat glottis rings with electrical field stimulation (EFS) as an experimental tool to simulate in vitro tissues receiving action potential, i.e. the TA muscle receiving RLN innervation, in a controlled firing rate manner. The aim of this study was to investigate the effect of temperature on VF basal tension and EFS-induced spike contraction of the TA muscle.
Materials and methods Tissue preparation This study was approved by the Animal Experiment Review Board of our hospital (LAC-100-0020). All chemical reagents were purchased from Sigma (St. Louis, MO, USA). Thirty Sprague–Dawley rats (weighing 200–300 g) were killed with intraperitoneal administration of pentobarbital (45 mg/kg) and the glottis rings were removed. The superior and inferior levels of the glottis ring were defined as the base of the epiglottis and the subglottis, respectively. The preparation totally excluded the cricoid cartilage. The glottis ring included some thyroid cartilage, TA muscle, and arytenoid cartilage (Fig. 1). The isometric tension recordings of the glottis rings were performed as follows (Fig. 2). The upper side (anterior commissure) of the glottis ring was attached to a Grass FT-03 force
123
Fig. 1 Preparation of the glottis ring. The superior and inferior levels of the glottis ring were the base of the epiglottis and subglottis, respectively. Bar 2 mm
displacement transducer (AstroMed, West Warwick, RI, USA) with curved stainless steel and a 3–0 silk ligature. The other side (posterior commissure) of the glottis ring was fixed to an adjustable micrometer (Mitutoyo, Japan) to allow for the fine adjustment of passive tension. This device was used with some modifications from previous reports [6, 7]. Briefly, the glottis ring was placed in a water-jacketed 30 mL glass chamber containing Krebs– Ringer buffer solution of the following composition: 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11.1 mM glucose. The solution was aerated continuously with 95 % O2 ? 5 % CO2 gas. The tension of the glottis ring was continuously recorded by DigiLog (for Windows V5.10.7) software (Singa Technology, Taiwan). The temperature range (37–7 °C) was set according to the surrounding room temperature (in Taipei). At the beginning of each experiment, a passive tension of 0.035 N was applied to the glottis ring. The passive tension was made by an adjustable micrometer (Mitutoyo, Japan) for the stability of the preparation. The passive tension was allowed to equilibrate for 30–45 min at 37 °C. The temperature of the experiment setting was achieved by a thermo-controlled water pump (Firstek Scientific, Taipei, Taiwan) to circulate water through the water-jacketed glass chambers. The rapid reduction or increase of temperature was achieved through insertion of an adequate amount of ice or 74 °C water into the water reservoir. Approximately 2–3 min were required to reach the desired temperature. A thermometer placed
Eur Arch Otorhinolaryngol Fig. 2 Schematic diagram and the actual photo of tension measurements in an isolated rat glottis ring
within the water reservoir confirmed the actual temperature of the preparation at any given time. In each experiment, one untreated glottis ring served as a control. Electrical field stimulation EFS (5 Hz, 5 ms pulse duration, at a voltage of 50 V, trains of stimulation for 5 s) was applied to the glottis ring with two wire electrodes placed parallel to the glottis and connected to a direct-current stimulator (Grass S44, Quincy, MA, USA). An interval of 2 min was imposed between each stimulation period to allow for recovery from the response. EFS was applied using the Grass stimulator and controlled by a timer. The stimulus intensity was always supramaximal. EFS was applied continuously to the glottis ring at 37 °C for 8–10 min. The temperature of the tissue bath was then decreased to 7 °C for 8 min. Once the basal tension of the glottis ring was stabilized at 7 °C, the temperature was again increased to 37 °C. The EFS was applied continuously during the changes in temperature. Statistical analysis The results were expressed as mean ± standard error of the mean (SEM). The Student’s paired t test was used for statistical analysis. Differences were assumed to be significant at P \ 0.01.
Results Basal tension and spike tension evoked by EFS were measured respectively at both 37 and 7 °C. Cooling (from 37 to 7 °C) produced a reduction in basal tension in the
Fig. 3 Original recording of the basal tension of the glottis ring with (upper) or without (bottom) EFS at different temperatures. Cooling reduced the basal tension and enhanced EFS-induced spike contraction, chiefly due to the TA muscle, of the glottis ring. N Newton
glottis ring. In comparison, warming (from 7 to 37 °C) caused the tension to elevate in both the absence and presence of EFS (Fig. 3). At 37 °C, the basal glottis tension was 0.035 ± 0.006 N (n = 8), and at 7 °C the basal glottis tension was 0.019 ± 0.002 N (n = 8) (Fig. 4). The basal glottis tension was significantly reduced when the temperature was reduced from 37 to 7 °C (P \ 0.01). The glottis ring contraction induced by EFS, chiefly due to the TA muscle, was increased when cooling (from 37 to 7 °C) (Fig. 3). The tension peak of the glottis ring evoked by EFS at 37 °C was 0.010 ± 0.001 N (n = 7), and at 7 °C it was 0.018 ± 0.003 N (n = 7) (Fig. 5). The tension peak of the glottis evoked by EFS at 7 °C was significantly higher than that at 37 °C (P \ 0.01).
123
Eur Arch Otorhinolaryngol
Fig. 4 The basal tension (mean ± SEM) of an isolated glottis ring at different temperatures. N Newton. *P \ 0.01
Fig. 5 The EFS-induced spike contraction (mean ± SEM) of an isolated glottis ring at different temperatures. N Newton. *P \ 0.01
Discussion Several studies have been conducted on the tension of VF muscles. In one study, canine vocalis muscles were harvested for the in vitro study of active and passive isometric tension, and the tension was recorded at various levels of elongation and stimulation rate [8]. Johns et al. [9] found that the thyroarytenoid muscle exhibited higher passive tension when generating maximal isometric tension than the digastric muscle in an in vitro feline model. Hast et al. [10] measured in situ VF muscle tension with variable electrical stimulation frequency and different passive VF length in an anesthetized mongrel dog model. They concluded that the maximum tension of the VF muscle was reached at 130 % of the muscle’s resting length with high RLN stimulation frequency of 120 pulses/s. However, in
123
these well-designed studies the effect of temperature on VF muscles was not evaluated. In our previous study, we demonstrated that cooling induced a ‘‘relaxation’’ response in isometric tension recording of human nasal mucosa strips, and that this response was a structural factor rather than a contractile tissue response [11]. We hypothesized that elastin is responsible for this cooling-induced relaxation. Similarly, cooling-induced relaxation has been reported in isolated rat aortas, and the authors concluded that the mechanism was not dependent on local nervous or known mediators [12]. It has also been reported that blood vessels with a large amount of elastic fibers show dilatation when cooling [13]. Interestingly, elastin has been shown to undergo an ‘‘inverse temperature transition’’ such that this hydrophobic molecule becomes more ordered as the temperature increases [14]. This mechanism of elastin has also been shown under conditions of isometric tension recording [15]. It is well known that elastic fibers are a major component of the lamina propria in the VF, and it has been estimated that elastin constitutes about 8.5 % of the human lamina propria of the VF [16]. The current study demonstrated that the basal tension of the VF is temperaturedependent, and that warming increased the basal tension of the VF and possibly elevated the vocal pitch in vivo. On the other hand, the elastin may be responsible for coolinginduced relaxation of the VF to some degree. The speed of contraction of the intralaryngeal muscles, the level of their activation, and their time-dependent stress–strain relationship has a major influence on all aspects of voice production. Of these muscles, the CT and TA are primarily involved in length and tension control. The TA, LCA, and IA are all adductors, whereas PCA is the sole abductor of the VF. The TA muscle has been found to have increased stiffness at various levels of strain compared with other intrinsic laryngeal muscles [1, 2]. During warming, the basal tension of the glottis was increased and the pitch may also have been increased too. This is not always true, however, as actual phonation is much more complicated. The effective overall tension of the VF depends on the coupling of the vocal cover to the adjustable vocal body which is affected by muscle contraction. During isolated contraction of the TA muscle, the vocal body is stiffened by muscle shortening while the cover becomes more lax and pliable. Because of the incongruent tension at different layers of the VF, the combination of longitudinal stretching and the contraction of the muscle mass of the VF during TA activity affects the depth of movement in the fold. The interaction between the TA and CT contractions is the main regulatory factor of fundamental frequency [17]. The TA muscle has been reported to have the smallest motor unit size among the intrinsic laryngeal muscles, suggesting that the TA muscle has a greater capacity to
Eur Arch Otorhinolaryngol
fine-tune its total force compared with the other intrinsic laryngeal muscles [18]. The results of the current study also demonstrated that the EFS-induced TA contraction was reduced by a relatively high temperature (37 °C). In other words, a smaller TA muscle contraction was elicited by each action potential at 37 °C compared to that at 7 °C. Thus, our findings suggest that warming of the VF, either by warm ambient air or vocal warm-up exercises, may be beneficial to the fine-tuning of VF tension. Our findings also demonstrated that the basal tension of the VF was increased by warming (from 7 to 37 °C), and that this was possibly due to structural factors of the VF itself rather than TA muscle contraction. There have been several investigations of isotonic shortening contractions in mammalian muscle over a similar wide temperature range. Many muscles exhibit an increased temperature sensitive at low temperatures [19, 20]. The range of temperatures studied here was realistic compared to that of air during normal free breathing. Yet, the temperature inside the TA muscle was unlikely to be reduced down to 7 °C, owing to very strong buffering by the continuous blood supply. The real temperature of the walls of the glottis during breathing cold air needs further studies. There are some limitations to this study. More precise measurements of TA muscle contraction can be achieved by tension recording of the VF strip itself, i.e. when the force–displacement transducer is attached on both ends of the VF. However, the rat VF is too small to allow for this, and the change in tension of the rat VF strip was beyond the measurement limit of our instrument. Instead, we used the glottis ring since the structure of the ring generates more force both in the basal tension and EFS-induced contraction compared to the VF strip.
Conclusion Warming increased the basal tension of the glottis in vitro and decreased the amplitude of EFS-induced TA muscle contraction. Thus, the vocal pitch and the fine tuning of VF tension may also be affected in vivo. Acknowledgments This work was supported in part by Taipei Medical University, Shuang-Ho Hospital (102TMU-SHH-17).
2. Hirano M, Ohala J, Vennard W (1969) The function of laryngeal muscles in regulating fundamental frequency and intensity of phonation. J Speech Hear Res 12:616–628 3. Yanagi E, Slavit DH, McCaffrey TV (1991) Study of phonation in the excised canine larynx. Otolaryngol Head Neck Surg 105:586–595 4. Hillel AD (2001) The study of laryngeal muscle activity in normal human subjects and in patients with laryngeal dystonia using multiple fine-wire electromyography. Laryngoscope 111(Suppl 97):1–47 5. Titze IR, Luschei ES, Hirano M (1989) Role of the thyroarytenoid muscle in regulation of fundamental frequency. J Voice 3:213–224 6. Jackson RT (1980) An in vitro technique for testing nasal vasodilating agents. Otolaryngol Head Neck Surg 88:434–438 7. Wang H-W, Wu C-C (2008) Effects of oxymetazoline on isolated rat’s tracheal smooth muscle. Eur Arch Otorhinolaryngol 265:695–698 8. Alipour-Haghighi F, Titze IR, Perlman AL (1989) Tetanic contraction in vocal fold muscle. J Speech Hear Res 32:226–231 9. Johns MM, Urbanchek M, Chepeha DB, Kuzon WM Jr, Hogikyan ND (2004) Length-tension relationship of the feline thyroarytenoid muscle. J Voice 18:285–291 10. Hast MH (1966) Physiological mechanisms of phonation: tension of the vocal fold muscle. Acta Otolaryngol 62:309–318 11. Chu YH, Wu CC, Kao CH, Wang HW (2006) Low temperature decreased tension in isolated hypertrophic human nasal mucosa. Am J Rhinol 20:84–86 12. Mustafa S, Thulesius O (2001) Cooling is a potent vasodilator of deep vessels in the rat. Can J Physiol Pharmacol 79:899–904 13. Herrera B, Desco MM, Eisenberg G, Garcı´a-Barreno P, Del Can˜izo JF (2002) Role of elastic fibers in cooling-induced relaxation. Cryobiology 44:54–61 14. Li B, Alonso DO, Daggett V (2001) The molecular basis for the inverse temperature transition of elastin. J Mol Biol 305:581–592 15. Urry DW, Parker TM (2002) Mechanics of elastin: molecular mechanism of biological elasticity and its relationship to contraction. J Muscle Res Cell Motil 23:543–559 16. Hahn MS, Kobler JB, Starcher BC, Zeitels SM, Langer R (2006) Quantitative and comparative studies of the vocal fold extracellular matrix. I: elastic fibers and hyaluronic acid. Ann Otol Rhinol Laryngol 115:156–164 17. Jiang J, Lin E, Hanson DG (2000) Vocal fold physiology. Otolaryngol Clin North Am 33:699–718 18. Santo Neto H, Marques MJ (2008) Estimation of the number and size of motor units in intrinsic laryngeal muscles using morphometric methods. Clin Anat 21:301–306 19. Ranatunga KW (1982) Temperature-dependence of shortening velocity and rate of isometric tension development in rat skeletal muscle. J Physiol 329:465–483 20. Asmussen G, Beckers-Bleukx G, Mare´chal G (1994) The forcevelocity relation of the rabbit inferior oblique muscle; influence of temperature. Pflu¨gers Arch 426:542–547
References 1. Alipour F, Ingo R, Titze IR, Hunter E, Tayama N (2005) Active and passive properties of canine abduction/adduction laryngeal muscles. J Voice 19:350–359
123
MISCELLANEOUS
The effect of temperature on basal tension and thyroarytenoid muscle contraction in an isolated rat glottis model Hsing-Won Wang • Yueng-Hsiang Chu Pin-Zhir Chao • Fei-Peng Lee
•
Received: 5 March 2014 / Accepted: 8 May 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract The pitch of voice is closely related to the vocal fold tension, which is the end result of coordinated movement of the intralaryngeal muscles, and especially the thyroarytenoid muscle. It is known that vocal quality may be affected by surrounding temperature; however, the effect of temperature on vocal fold tension is mostly unknown. Thus, the aim of this study was to evaluate the effect of temperature on isolated rat glottis and thyroarytenoid muscle contraction induced by electrical field stimulation. In vitro isometric tension of the glottis ring from 30 Sprague–Dawley rats was continuously recorded by the tissue bath method. Electrical field stimulation was applied to the glottis ring with two wire electrodes placed parallel to the glottis and connected to a direct-current stimulator. The tension changes of the rat glottis rings that H.-W. Wang P.-Z. Chao Department of Otolaryngology, Taipei Medical UniversityShuang Ho Hospital, No. 291, Jhong-Jheng Road, Jhonghe District, 23561 New Taipei City, Taiwan, ROC e-mail: [email protected] H.-W. Wang Department of Preventive and Community Medicine, Taipei Medical University-Shuang Ho Hospital, No. 291, Jhong-Jheng Road, Jhonghe District, New Taipei City, Taiwan H.-W. Wang P.-Z. Chao Graduate Institute of Clinical Medicine, School of Medicine, Taipei Medical University, New Taipei City, Taiwan H.-W. Wang Y.-H. Chu Department of Otolaryngology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan F.-P. Lee (&) Department of Otolaryngology, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan e-mail: [email protected]
were either untreated or treated with electrical field stimulation were recorded continuously at temperatures from 37 to 7 °C or from 7 to 37 °C. Warming from 7 to 37 °C increased the basal tension of the glottis rings and decreased the electrical field stimulation-induced glottis ring contraction, which was chiefly due to thyroarytenoid muscle contraction. In comparison, cooling from 37 to 7 °C decreased the basal tension and enhanced glottis ring contraction by electrical field stimulation. We concluded that warming increased the basal tension of the glottis in vitro and decreased the amplitude of electrical field stimulation-induced thyroarytenoid muscle contraction. Thus, vocal pitch and the fine tuning of vocal fold tension might be affected by temperature in vivo. Keywords Glottis Temperature Electrical field stimulation
Introduction The pitch of voice is closely related to vocal fold (VF) tension, which is an end result of complex coordinated movements of the intralaryngeal muscles and the viscoelastic properties of the VF. The contraction/relaxation of the thyroarytenoid (TA) muscle, which is innervated by the recurrent laryngeal nerve (RLN), constitutes the ‘‘body’’ of the VF and it increases the VF tension by exerting anterior traction on the vocal process. There are five intrinsic laryngeal muscles: the TA, cricothyroid (CT), interarytenoid (IA), posterior cricoarytenoid (PCA), and lateral cricoarytenoid (LCA). Of these muscles, the CT and TA are primarily involved in length and tension control. The TA, LCA, and IA are all adductors, whereas PCA is the sole abductor of the VF [1, 2]. Vocal fold tension during
123
Eur Arch Otorhinolaryngol
phonation is generated by coordinated contraction of the intrinsic laryngeal muscles. The TA muscle has been found to have increased stiffness at various levels of strain compared with the other intrinsic laryngeal muscles. The contraction of the CT muscle, which results in a decrease in the distance between the thyroid and cricoid cartilages, is considered to be the main factor in lengthening the vocal folds [1, 2]. As demonstrated by Yanagi et al. [3] and Hillel [4], the CT muscle not only lengthens the true vocal fold, but also thins the vocal fold and increases the longitudinal tension. The net effect of this action is to increase the pitch of the phonation. The contraction of the TA muscle is thought to increase the lateral tension of the vocal fold. It works to set the vocal folds into position for phonation and maintains tone of vocal folds during phonation. Work by Titze et al. [5] has even demonstrated decreased pitch with TA muscle activation in the setting of large cricothyroid activity with elongated vocal folds due to the reduced tension of the mucosal cover. It is known that vocal quality may be affected by the surrounding temperature. However, the effect of temperature on VF tension is mostly unknown. It is difficult to evaluate the effect of temperature on the basal tension of the VF in vivo since warming/cooling (temperature) has effects at multiple levels (e.g., nerve firing rate, neurotransmitter release, and ability of the muscles to contract/relax). In this study, we measured the isometric tension of isolated rat glottis rings with electrical field stimulation (EFS) as an experimental tool to simulate in vitro tissues receiving action potential, i.e. the TA muscle receiving RLN innervation, in a controlled firing rate manner. The aim of this study was to investigate the effect of temperature on VF basal tension and EFS-induced spike contraction of the TA muscle.
Materials and methods Tissue preparation This study was approved by the Animal Experiment Review Board of our hospital (LAC-100-0020). All chemical reagents were purchased from Sigma (St. Louis, MO, USA). Thirty Sprague–Dawley rats (weighing 200–300 g) were killed with intraperitoneal administration of pentobarbital (45 mg/kg) and the glottis rings were removed. The superior and inferior levels of the glottis ring were defined as the base of the epiglottis and the subglottis, respectively. The preparation totally excluded the cricoid cartilage. The glottis ring included some thyroid cartilage, TA muscle, and arytenoid cartilage (Fig. 1). The isometric tension recordings of the glottis rings were performed as follows (Fig. 2). The upper side (anterior commissure) of the glottis ring was attached to a Grass FT-03 force
123
Fig. 1 Preparation of the glottis ring. The superior and inferior levels of the glottis ring were the base of the epiglottis and subglottis, respectively. Bar 2 mm
displacement transducer (AstroMed, West Warwick, RI, USA) with curved stainless steel and a 3–0 silk ligature. The other side (posterior commissure) of the glottis ring was fixed to an adjustable micrometer (Mitutoyo, Japan) to allow for the fine adjustment of passive tension. This device was used with some modifications from previous reports [6, 7]. Briefly, the glottis ring was placed in a water-jacketed 30 mL glass chamber containing Krebs– Ringer buffer solution of the following composition: 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11.1 mM glucose. The solution was aerated continuously with 95 % O2 ? 5 % CO2 gas. The tension of the glottis ring was continuously recorded by DigiLog (for Windows V5.10.7) software (Singa Technology, Taiwan). The temperature range (37–7 °C) was set according to the surrounding room temperature (in Taipei). At the beginning of each experiment, a passive tension of 0.035 N was applied to the glottis ring. The passive tension was made by an adjustable micrometer (Mitutoyo, Japan) for the stability of the preparation. The passive tension was allowed to equilibrate for 30–45 min at 37 °C. The temperature of the experiment setting was achieved by a thermo-controlled water pump (Firstek Scientific, Taipei, Taiwan) to circulate water through the water-jacketed glass chambers. The rapid reduction or increase of temperature was achieved through insertion of an adequate amount of ice or 74 °C water into the water reservoir. Approximately 2–3 min were required to reach the desired temperature. A thermometer placed
Eur Arch Otorhinolaryngol Fig. 2 Schematic diagram and the actual photo of tension measurements in an isolated rat glottis ring
within the water reservoir confirmed the actual temperature of the preparation at any given time. In each experiment, one untreated glottis ring served as a control. Electrical field stimulation EFS (5 Hz, 5 ms pulse duration, at a voltage of 50 V, trains of stimulation for 5 s) was applied to the glottis ring with two wire electrodes placed parallel to the glottis and connected to a direct-current stimulator (Grass S44, Quincy, MA, USA). An interval of 2 min was imposed between each stimulation period to allow for recovery from the response. EFS was applied using the Grass stimulator and controlled by a timer. The stimulus intensity was always supramaximal. EFS was applied continuously to the glottis ring at 37 °C for 8–10 min. The temperature of the tissue bath was then decreased to 7 °C for 8 min. Once the basal tension of the glottis ring was stabilized at 7 °C, the temperature was again increased to 37 °C. The EFS was applied continuously during the changes in temperature. Statistical analysis The results were expressed as mean ± standard error of the mean (SEM). The Student’s paired t test was used for statistical analysis. Differences were assumed to be significant at P \ 0.01.
Results Basal tension and spike tension evoked by EFS were measured respectively at both 37 and 7 °C. Cooling (from 37 to 7 °C) produced a reduction in basal tension in the
Fig. 3 Original recording of the basal tension of the glottis ring with (upper) or without (bottom) EFS at different temperatures. Cooling reduced the basal tension and enhanced EFS-induced spike contraction, chiefly due to the TA muscle, of the glottis ring. N Newton
glottis ring. In comparison, warming (from 7 to 37 °C) caused the tension to elevate in both the absence and presence of EFS (Fig. 3). At 37 °C, the basal glottis tension was 0.035 ± 0.006 N (n = 8), and at 7 °C the basal glottis tension was 0.019 ± 0.002 N (n = 8) (Fig. 4). The basal glottis tension was significantly reduced when the temperature was reduced from 37 to 7 °C (P \ 0.01). The glottis ring contraction induced by EFS, chiefly due to the TA muscle, was increased when cooling (from 37 to 7 °C) (Fig. 3). The tension peak of the glottis ring evoked by EFS at 37 °C was 0.010 ± 0.001 N (n = 7), and at 7 °C it was 0.018 ± 0.003 N (n = 7) (Fig. 5). The tension peak of the glottis evoked by EFS at 7 °C was significantly higher than that at 37 °C (P \ 0.01).
123
Eur Arch Otorhinolaryngol
Fig. 4 The basal tension (mean ± SEM) of an isolated glottis ring at different temperatures. N Newton. *P \ 0.01
Fig. 5 The EFS-induced spike contraction (mean ± SEM) of an isolated glottis ring at different temperatures. N Newton. *P \ 0.01
Discussion Several studies have been conducted on the tension of VF muscles. In one study, canine vocalis muscles were harvested for the in vitro study of active and passive isometric tension, and the tension was recorded at various levels of elongation and stimulation rate [8]. Johns et al. [9] found that the thyroarytenoid muscle exhibited higher passive tension when generating maximal isometric tension than the digastric muscle in an in vitro feline model. Hast et al. [10] measured in situ VF muscle tension with variable electrical stimulation frequency and different passive VF length in an anesthetized mongrel dog model. They concluded that the maximum tension of the VF muscle was reached at 130 % of the muscle’s resting length with high RLN stimulation frequency of 120 pulses/s. However, in
123
these well-designed studies the effect of temperature on VF muscles was not evaluated. In our previous study, we demonstrated that cooling induced a ‘‘relaxation’’ response in isometric tension recording of human nasal mucosa strips, and that this response was a structural factor rather than a contractile tissue response [11]. We hypothesized that elastin is responsible for this cooling-induced relaxation. Similarly, cooling-induced relaxation has been reported in isolated rat aortas, and the authors concluded that the mechanism was not dependent on local nervous or known mediators [12]. It has also been reported that blood vessels with a large amount of elastic fibers show dilatation when cooling [13]. Interestingly, elastin has been shown to undergo an ‘‘inverse temperature transition’’ such that this hydrophobic molecule becomes more ordered as the temperature increases [14]. This mechanism of elastin has also been shown under conditions of isometric tension recording [15]. It is well known that elastic fibers are a major component of the lamina propria in the VF, and it has been estimated that elastin constitutes about 8.5 % of the human lamina propria of the VF [16]. The current study demonstrated that the basal tension of the VF is temperaturedependent, and that warming increased the basal tension of the VF and possibly elevated the vocal pitch in vivo. On the other hand, the elastin may be responsible for coolinginduced relaxation of the VF to some degree. The speed of contraction of the intralaryngeal muscles, the level of their activation, and their time-dependent stress–strain relationship has a major influence on all aspects of voice production. Of these muscles, the CT and TA are primarily involved in length and tension control. The TA, LCA, and IA are all adductors, whereas PCA is the sole abductor of the VF. The TA muscle has been found to have increased stiffness at various levels of strain compared with other intrinsic laryngeal muscles [1, 2]. During warming, the basal tension of the glottis was increased and the pitch may also have been increased too. This is not always true, however, as actual phonation is much more complicated. The effective overall tension of the VF depends on the coupling of the vocal cover to the adjustable vocal body which is affected by muscle contraction. During isolated contraction of the TA muscle, the vocal body is stiffened by muscle shortening while the cover becomes more lax and pliable. Because of the incongruent tension at different layers of the VF, the combination of longitudinal stretching and the contraction of the muscle mass of the VF during TA activity affects the depth of movement in the fold. The interaction between the TA and CT contractions is the main regulatory factor of fundamental frequency [17]. The TA muscle has been reported to have the smallest motor unit size among the intrinsic laryngeal muscles, suggesting that the TA muscle has a greater capacity to
Eur Arch Otorhinolaryngol
fine-tune its total force compared with the other intrinsic laryngeal muscles [18]. The results of the current study also demonstrated that the EFS-induced TA contraction was reduced by a relatively high temperature (37 °C). In other words, a smaller TA muscle contraction was elicited by each action potential at 37 °C compared to that at 7 °C. Thus, our findings suggest that warming of the VF, either by warm ambient air or vocal warm-up exercises, may be beneficial to the fine-tuning of VF tension. Our findings also demonstrated that the basal tension of the VF was increased by warming (from 7 to 37 °C), and that this was possibly due to structural factors of the VF itself rather than TA muscle contraction. There have been several investigations of isotonic shortening contractions in mammalian muscle over a similar wide temperature range. Many muscles exhibit an increased temperature sensitive at low temperatures [19, 20]. The range of temperatures studied here was realistic compared to that of air during normal free breathing. Yet, the temperature inside the TA muscle was unlikely to be reduced down to 7 °C, owing to very strong buffering by the continuous blood supply. The real temperature of the walls of the glottis during breathing cold air needs further studies. There are some limitations to this study. More precise measurements of TA muscle contraction can be achieved by tension recording of the VF strip itself, i.e. when the force–displacement transducer is attached on both ends of the VF. However, the rat VF is too small to allow for this, and the change in tension of the rat VF strip was beyond the measurement limit of our instrument. Instead, we used the glottis ring since the structure of the ring generates more force both in the basal tension and EFS-induced contraction compared to the VF strip.
Conclusion Warming increased the basal tension of the glottis in vitro and decreased the amplitude of EFS-induced TA muscle contraction. Thus, the vocal pitch and the fine tuning of VF tension may also be affected in vivo. Acknowledgments This work was supported in part by Taipei Medical University, Shuang-Ho Hospital (102TMU-SHH-17).
2. Hirano M, Ohala J, Vennard W (1969) The function of laryngeal muscles in regulating fundamental frequency and intensity of phonation. J Speech Hear Res 12:616–628 3. Yanagi E, Slavit DH, McCaffrey TV (1991) Study of phonation in the excised canine larynx. Otolaryngol Head Neck Surg 105:586–595 4. Hillel AD (2001) The study of laryngeal muscle activity in normal human subjects and in patients with laryngeal dystonia using multiple fine-wire electromyography. Laryngoscope 111(Suppl 97):1–47 5. Titze IR, Luschei ES, Hirano M (1989) Role of the thyroarytenoid muscle in regulation of fundamental frequency. J Voice 3:213–224 6. Jackson RT (1980) An in vitro technique for testing nasal vasodilating agents. Otolaryngol Head Neck Surg 88:434–438 7. Wang H-W, Wu C-C (2008) Effects of oxymetazoline on isolated rat’s tracheal smooth muscle. Eur Arch Otorhinolaryngol 265:695–698 8. Alipour-Haghighi F, Titze IR, Perlman AL (1989) Tetanic contraction in vocal fold muscle. J Speech Hear Res 32:226–231 9. Johns MM, Urbanchek M, Chepeha DB, Kuzon WM Jr, Hogikyan ND (2004) Length-tension relationship of the feline thyroarytenoid muscle. J Voice 18:285–291 10. Hast MH (1966) Physiological mechanisms of phonation: tension of the vocal fold muscle. Acta Otolaryngol 62:309–318 11. Chu YH, Wu CC, Kao CH, Wang HW (2006) Low temperature decreased tension in isolated hypertrophic human nasal mucosa. Am J Rhinol 20:84–86 12. Mustafa S, Thulesius O (2001) Cooling is a potent vasodilator of deep vessels in the rat. Can J Physiol Pharmacol 79:899–904 13. Herrera B, Desco MM, Eisenberg G, Garcı´a-Barreno P, Del Can˜izo JF (2002) Role of elastic fibers in cooling-induced relaxation. Cryobiology 44:54–61 14. Li B, Alonso DO, Daggett V (2001) The molecular basis for the inverse temperature transition of elastin. J Mol Biol 305:581–592 15. Urry DW, Parker TM (2002) Mechanics of elastin: molecular mechanism of biological elasticity and its relationship to contraction. J Muscle Res Cell Motil 23:543–559 16. Hahn MS, Kobler JB, Starcher BC, Zeitels SM, Langer R (2006) Quantitative and comparative studies of the vocal fold extracellular matrix. I: elastic fibers and hyaluronic acid. Ann Otol Rhinol Laryngol 115:156–164 17. Jiang J, Lin E, Hanson DG (2000) Vocal fold physiology. Otolaryngol Clin North Am 33:699–718 18. Santo Neto H, Marques MJ (2008) Estimation of the number and size of motor units in intrinsic laryngeal muscles using morphometric methods. Clin Anat 21:301–306 19. Ranatunga KW (1982) Temperature-dependence of shortening velocity and rate of isometric tension development in rat skeletal muscle. J Physiol 329:465–483 20. Asmussen G, Beckers-Bleukx G, Mare´chal G (1994) The forcevelocity relation of the rabbit inferior oblique muscle; influence of temperature. Pflu¨gers Arch 426:542–547
References 1. Alipour F, Ingo R, Titze IR, Hunter E, Tayama N (2005) Active and passive properties of canine abduction/adduction laryngeal muscles. J Voice 19:350–359
123
