Effect of unilateral nasal obstruction on tongue protrusion forces in ...

J Appl Physiol 118: 1128–1135, 2015. First published March 12, 2015; doi:10.1152/japplphysiol.01152.2014.

Effect of unilateral nasal obstruction on tongue protrusion forces in growing rats Karin Harumi Uchima Koecklin, Chiho Kato, Yukiha Funaki, Maya Hiranuma, Takayoshi Ishida, Koichi Fujita, Tadachika Yabushita, Satoshi Kokai, and Takashi Ono Orthodontic Science, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan Submitted 24 December 2014; accepted in final form 10 March 2015

Uchima Koecklin KH, Kato C, Funaki Y, Hiranuma M, Ishida T, Fujita K, Yabushita T, Kokai S, Ono T. Effect of unilateral nasal obstruction on tongue protrusion forces in growing rats. J Appl Physiol 118: 1128 –1135, 2015. First published March 12, 2015; doi:10.1152/japplphysiol.01152.2014.—Mouth breathing caused by nasal obstruction affects the normal growth and development of craniofacial structures, including changes in the orofacial muscles. Tongue muscles play an important role in patency of the pharyngeal airway, and changes in the breathing pattern may influence tongue function. The purpose of this study was to evaluate the effect of unilateral nasal obstruction during growth on contractile properties of the tongue-protruding muscles. Sixty 6-day-old male Wistar albino rats were divided randomly into control (n ⫽ 30) and experimental (n ⫽ 30) groups. Rats in the experimental group underwent a unilateral nasal obstruction after cauterization of the external nostril at the age of 8 days, and muscle contractile characteristics were measured at 5, 7, and 9 wk of age. The specific parameters measured were twitch force, contraction time, half-decay time, tetanic force, and fatigue index. Repeated-measures multivariate analysis of variance was used for intergroup and intragroup statistical comparisons. Twitch contraction force and half-decay time were significantly increased in the experimental group at all ages. Tetanic forces at 60 and 80 Hz were significantly higher in the experimental group at all ages. The fatigue index was decreased significantly in the experimental group at the age of 5 wk. These results suggest that early unilateral nasal obstruction may increase the contraction force of the tongue-protruding muscles and prolong the duration of muscle contraction, which may influence the shape and development of the craniofacial complex. muscle contraction; electric stimulation; mouth breathing; hypoglossal nerve

from birth (36), and this normal respiratory activity favors the harmonious growth and development of craniofacial structures (37). Nevertheless, this respiratory pattern could be affected and changed into mouth breathing, a common cause of which is the obstruction of the upper airways due to hypertrophy of the adenoids (14, 15, 36). In humans, this adaptation process implies modification of the breathing pattern, suggesting morphophysiological changes in the craniofacial complex that affect normal growth and development and may result in aberrant facial growth patterns (14, 15, 32, 36). Mouth breathing is correlated with muscle alterations and changes in the electromyographic (EMG) activity of specific craniofacial muscles (9). Mouth-breathing patients can present orthodontic problems, such as open bite, due to alterations in

HUMAN BEINGS ARE NOSE BREATHERS

Address for reprint requests and other correspondence: K. H. Uchima Koecklin, Tokyo Medical and Dental Univ., 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan (e-mail: [email protected]). 1128

the position of the lips, tongue, and mandible (32, 36). Upper airway patency depends on a balance between the contraction force of upper dilating muscles, such as pharyngeal muscles and the genioglossus (GG) muscle, and the collapsing force of negative pressure generated by the diaphragm (27, 44). Thus the tongue is a key contributor to respiratory actions, and tongue-protruding muscle activity is increased with mouth breathing (10, 27, 42). When breathing through the mouth, the hyoid bone moves more anteriorly, making the entire mass of the tongue advance more anteriorly. This leads to an increase in tongue pressure measured at the lower incisors, as reported by Takahashi et al. (41). Furthermore, after mouth breathing, the GG EMG activity is increased, which advances the base of the tongue and dilates the upper airway (16, 41). Evidence suggests that GG EMG activity and tongue protrusion force are related (28). To maintain this forward position of the tongue, the tongueprotruding force may increase by increasing its contraction force. Miller et al. (26) reported that, after nasal obstruction in monkeys, to adapt the oral passage for chronic respiratory work, there was a rhythmical discharge of the GG muscle, which resulted in active muscle recruitment (26). There is also an increase in the inspiratory activation of GG after nasal obstruction (16). To help maintain the airway patency in mouth breathers, a more constant and longer contraction duration of the tongue-protruding muscles would be expected. Based on this, we hypothesized that the contraction force and contraction duration of tongue-protruding muscles would be enhanced after nasal obstruction. The activity of the tongue-protruding muscles after bilateral nasal obstruction has been well studied. Nevertheless, there is no in-depth understanding of the influence of partial nasal obstruction on the actual properties of the tongue-protruding muscles. The aim of this study was to evaluate the effect of unilateral nasal obstruction during growth on the contractile properties of the tongue-protruding muscles. The hypothesis was that the contraction force of tongue-protruding muscles was increased, and the duration of muscle contraction was prolonged, after unilateral nasal obstruction. MATERIALS AND METHODS

The experimental procedures described herein were approved by the Institutional Animal Care and Use Committee (nos. 0130064A, 0140228A, and 0150161) and performed in accordance with the Animal Care Standards of Tokyo Medical and Dental University. Animal preparation. Sixty 6-day-old male Wistar albino rats were randomly divided into control (n ⫽ 30) and experimental (n ⫽ 30)

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Unilateral Nasal Obstruction and Craniofacial Development

groups. At the age of 8 days, rat pups were anesthetized by hypothermia by placing them inside a chamber with an internal temperature of ⫺18°C for 10 min. Unilateral nasal obstruction was performed in the experimental group by cauterization of the left external nostril, a simple procedure for causing nasal obstruction (13). Briefly, cauterization was performed by burning the surrounding tissues of the nostril by placement of the self-constructed cauterization instrument (1 mm in diameter, stainless-steel wire, Tomy, Tokyo, Japan) on the nostril, which occluded the orifice of the nostril without mechanical or chemical damage to the olfactory mucosa. To prevent infection, the tissue was coated with 3% chlortetracycline (Aureomycin Ointment; Pola Pharma, Tokyo, Japan) after cauterization. The pups were kept warm (37°C) for 30 min and then returned to their mothers. The control group underwent a sham operation involving placement of the cauterizing instrument 1 to 2 mm above the left nostril. Body weight measurements were performed throughout the experimental period and measured every week. Rats were checked every week to verify that there was no reopening of the cauterized nostril. Surgical methods. Samples were collected at 5, 7, and 9 wk after birth (n ⫽ 10 for each week). After anesthesia (ketamine 70 mg/kg; xylazine 7 mg/kg; intraperitoneal injection), the hypoglossal nerves were exposed bilaterally by a ventral approach, as described in previous studies (29). A middle incision at the anterior neck was performed, and bifurcation of the hypoglossal nerve was made deep to the intersection of the lateral edge of the anterior belly of the digastric muscle and the edge of the mylohyoid muscle. The hypoglossal nerve has medial and lateral branches. The medial branch innervates the GG and intrinsic muscles of the tongue, and the lateral branch innervates the retraction muscles of the tongue. Following isolation, a 2- to 5-mm section of the lateral branch of the hypoglossal nerve was removed bilaterally to allow stimulation of only the tongue-protruding muscles. Nerve stimulation and recording of contraction properties. Tongue contraction forces and temporal properties of muscle contraction were investigated. Parameters of muscle contraction that were measured included twitch force, contraction time (CT), half-decay time (HDT), tetanic force at 60 Hz, tetanic force at 80 Hz, and fatigue index (FI). Peak twitch force is the maximum tension generated from a single supramaximal stimulation (1.5 times maximal stimulation) of the specific motor nerve. Tetanic force is generated by repeated stimulations applied before complete relaxation occurs (23) and is a fused force signal. The FI represents the reduction of tetanic force over time, as a muscle is continuously driven to contract. FI is calculated as the muscle force following 2 min of stimulation, divided by the initial force, following the Burke fatigue test (5). Temporal variables were measured from the twitch force signal. CT is the duration between the onset of the stimulation and the point of 50% peak force. HDT is the duration between the onset of stimulation and the point of 50% decay from peak force. To measure these parameters, we attached a 20-cm-long 3-0 black silk suture (Matsuda Sutures, Tokyo, Japan) to the tip of the tongue and connected the end of the suture to an isometric force transducer (MLT0420; ADInstruments, Dunedin, New Zealand). The animal was placed onto a stage with the body and head fixed and the tongue extended at a preloaded force of ⬃3 g, as described previously (29). Parameters were measured in response to bilateral electrical stimulation of the medial branches of the hypoglossal nerves by 0.1-ms rectangular pulses at a supramaximal current (generally around 500 ␮A), as described in previous studies (7, 29). The electrical stimulation was supplied by an electric stimulator (SEN-7203, Nihon Kohden, Tokyo, Japan) with two tungsten electrodes (0.010 in., 250-␮m-diameter, epoxylite insulation, 9 M⍀ of impedance measured at 1,000 Hz; FHC, Bowdoin, ME). Both electrodes were placed simultaneously, and the tip of the electrode was inserted into the medial branch proximal to the bifurcation of the hypoglossal nerve, with one electrode on each side.

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Supramaximal stimulation was considered as 1.5 times the current level required to obtain maximal twitch force at the supramaximal magnitude. Stimuli were delivered at 1 Hz for twitch force. Stimuli for tetanic force were at 60 and 80 Hz for 200-ms trains. FI was recorded with a 2-min stimulation of a 100-Hz train for 500 ms. The schematic representation of the experiment is shown in Fig. 1. After the supramaximal stimulation was determined for the whole experiment, five stimuli for each test (twitch force, tetanic force at 60 Hz, and tetanic force at 80 Hz) were performed. The final result for each test was the median of the five measurements. For the FI, it was a continuous 2-min stimulation, and the median of the first five stimuli and the last five stimuli were used to obtain the FI. The total time for the experiment was around 20-30 min per rat. Statistical analysis. All data of the contractile properties of the tongue-protruding muscles are expressed as means ⫾ SD. The data for weight are expressed as means ⫾ SD in Table 1. The unpaired Student’s t-test was used for statistical comparison of the weight between the two groups. Repeated-measures multivariate analysis of variance was used for intergroup and intragroup statistical comparisons of the muscle contractile properties. Simple main-effects analysis with the Sidak adjustment was used for multiple comparisons. Statistical analysis was performed with SPSS Statistics for Windows, version 13.0J (SPSS, Chicago, IL), and statistical significance was established at P ⬍ 0.05. RESULTS

The mean body weight of all rats increased normally throughout the experimental period, and there was no significant difference between the two groups at the same age (Table 1). Analysis of the main effect between the overall age groups showed significant differences in the maximal twitch force. Analysis of the main effect overall between the control and experimental groups showed significant differences in maximal twitch force, HDT, maximal tetanic force at 60 Hz, maximal tetanic force at 80 Hz, and FI. Analysis of the interaction across age and groups (control and experimental) showed no significant difference in any of the parameters. Twitch force. Twitch force signals from control and experimental groups are shown in Fig. 2A. For the 5-wk-old animals, the twitch force was 2.52 ⫾ 0.75 gram-force (gf) for the control group and 3.80 ⫾ 0.98 gf for the experimental group. At 7 wk, the twitch force was 2.57 ⫾ 0.53 gf for the control group and 5.07 ⫾ 0.72 gf for the experimental group. At 9 wk, the twitch force was 3.54 ⫾ 1.37 gf for the control group and 5.58 ⫾ 1.50 gf for the experimental group. There were significant differences between the control and experimental groups at all ages (Fig. 2B). Intragroup comparisons among 5-, 7-, and 9-wk-old rats revealed significant differences between the 5and 7-wk-old rats, and between the 5- and 9-wk-old rats of the experimental group. CT and HDT. CT for the 5-wk-old animals was 24.45 ⫾ 2.25 ms for the control group and 26.26 ⫾ 2.58 ms for the experimental group. At 7 wk, CT was 24.92 ⫾ 1.24 ms for the control group and 25.46 ⫾ 1.78 ms for the experimental group. In 9-wk-old animals, CT was 25.40 ⫾ 2.12 ms for the control group and 26.22 ⫾ 3.06 ms for the experimental group. CT was not significantly different between the control and experimental groups at any age. Intragroup comparisons among 5-, 7-, and 9-wk-old animals revealed no significant difference in either the control or experimental group (Fig. 2C). For the 5-wk-old animals, HDT was 56.27 ⫾ 2.49 ms in the control group and 64.12 ⫾ 6.4 ms in the experimental group. At 7 wk, HDT was 56.02 ⫾ 3.01 ms in the control group and

J Appl Physiol • doi:10.1152/japplphysiol.01152.2014 • www.jappl.org

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A Mandible

Masseter muscle

Electrode

Fig. 1. A: hypoglossal nerves were exposed bilaterally after a midsagittal incision at the anterior neck. Bifurcation of the hypoglossal nerve into the medial and lateral branches was found deep in the intersection of the lateral edge of the anterior belly of digastric muscle and the edge of the myohyoid muscle. The lateral branch of the hypoglossal nerve was transected, and only the medial branch of the hypoglossal nerve was stimulated. Simultaneous stimulation was performed. B and C: scheme of the methods utilized for stimulating the hypoglossal nerve. The rat was held in a supine position, and the tip of the tongue was connected to the force transducer by placement of a suture. The rat before stimulation (B) and after stimulation (C) of the medial branch of the hypoglossal nerve is shown.

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62.28 ⫾ 7.28 ms in the experimental group. At 9 wk, HDT was 55.08 ⫾ 4.05 ms in the control group and 62.18 ⫾ 7.00 ms in the experimental group. HDT was significantly different at 5, 7, and 9 wk of age between the control and experimental groups (Fig. 2D). Intragroup comparisons among 5-, 7-, and 9-wk-old animals revealed no significant difference in either the control or experimental group. Tetanic force. Representative tetanic force signals are shown in Fig. 3A. The tetanic force at 60 Hz for the 5-wk-old animals was 6.77 ⫾ 1.00 gf in the control group and 8.00 ⫾ 1.01 gf in the experimental group. At 7 wk, the tetanic force at 60 Hz was 6.99 ⫾ 0.63 gf in the control group and 8.44 ⫾ 0.36 gf in the experimental group. At 9 wk, the tetanic force at 60 Hz was 7.31 ⫾ 0.78 gf in the control group and 8.52 ⫾ 0.39 gf in the experimental group. The tetanic force at 60 Hz was significantly different between groups at 5, 7, and 9 wk after birth (Fig. 3B). Intragroup comparisons of tetanic forces at 60 Hz among 5-, 7-, and 9-wk-old animals revealed no significant difference in either the control or experimental group. The tetanic force at 80 Hz for the 5-wk-old animals was 7.08 ⫾ 1.09 gf in the control group and 8.22 ⫾ 0.80 gf in the

experimental group. At 7 wk, the tetanic force at 80 Hz was 7.16 ⫾ 0.99 gf in the control group and 8.59 ⫾ 0.58 gf in the experimental group. At 9 wk, the tetanic force at 80 Hz was 7.40 ⫾ 0.93 gf in the control group and 8.62 ⫾ 0.68 gf in the experimental group. The tetanic force at 80 Hz was significantly different between the groups at 5, 7, and 9 wk after birth (Fig. 3C). Intragroup comparisons of tetanic forces at 80 Hz among 5-, 7-, and 9-wk-old animals revealed no significant difference in either the control or experimental group. Fatigue index. Representative signals used to obtain the FI are shown in Fig. 4. For the 5-wk-old animals, FI was 76.50 ⫾ 8.48% in the control group and 61.68 ⫾ 10.57% in the experimental group. At 7 wk, FI was 70.35 ⫾ 8.16% in the control group and 65.14 ⫾ 6.07% in the experimental group. At 9 wk, FI was 73.20 ⫾ 9.59% in the control group and 67.23 ⫾ 12.30% in the experimental group. There was a significant difference at 5 wk of age between the groups (Fig. 4C). Intragroup comparisons among 5-, 7-, and 9-wk-old animals revealed no significant difference in either the control or experimental group.

Table 1. Mean body weight 5 wk

Weight, g

7 wk

9 wk

Control

Experimental

Control

Experimental

Control

Experimental

143.85 ⫾ 7.42

136.60 ⫾ 10.95

236.10 ⫾ 16.82

230.90 ⫾ 15.51

304.80 ⫾ 8.31

299.50 ⫾ 5.99

Values are means ⫾ SD. J Appl Physiol • doi:10.1152/japplphysiol.01152.2014 • www.jappl.org


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Fig. 2. A: representative twitch contraction curves from control and experimental rats at 9 wk of age. Solid line, results from the control group; shaded line, results of the experimental group. Arrow head ⫽ stimulation point. B: comparison of mean maximal twitch force [gram-force (gf)] between groups at 5, 7, and 9 wk after birth. *Significant difference between groups at all ages, P ⬍ 0.05. #Significant difference in the intragroup results between 5 and 7 wk and between 5 and 9 wk in the experimental group, P ⬍ 0.05. C: comparison of mean contraction time (ms) between groups at 5, 7, and 9 wk after birth. D: comparison of mean half-decay time (ms) between groups at 5, 7, and 9 wk after birth. *Significant differences between groups at all ages, P ⬍ 0.05. Solid bars, control group; shaded bars, experimental group. Error bars 95% ⫽ standard deviations. CI, confidence interval.

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DISCUSSION

Unilateral nasal obstruction model. We conducted a unilateral nasal obstruction to change the breathing pattern of the rats in a manner that could be well tolerated. In humans, chronic nasal obstruction is a nonspecific condition observed from childhood that can lead to mouth breathing (14, 42). Impaired nasal breathing can be divided into two components: the chronic absence of active nasal respiration with olfactory deprivation, and chronic mouth-opening (14). These types of patients are usually associated with a low tongue position, a narrow upper dental arch, increased lower facial height, excessively erupted molars, malocclusion such as anterior open bite, and a posterior crossbite (32, 36). Chronic mouth breathing is a contributing factor to deviant facial growth patterns and is associated with alterations in specific EMG activities of orofacial muscles (9, 14). Nevertheless, most cases present a combination of oral and nasal breathing, and only 4.3% breath from the mouth alone (43). When mouth breathing was induced in infant monkeys by bilateral obstruction of the nasal passages, some animals increased the oral airway rhythmically, while others maintained the mandible in a lower position with or without tongue protrusion (17, 25). All experimental animals gradually acquired a different facial appearance and dental occlusion. Rats rarely survive bilateral nasal obstruction and seem unable to learn to breathe through the mouth (15). Unilateral

nasal obstruction was performed because it is well tolerated and affects the craniofacial complex, such as the jaw-opening reflex, in rats (13). Unilateral obstruction also induces atrophy of the ipsilateral olfactory bulb, which is associated with olfactory deprivation in rats (24) and also occurs in mouthbreathing patients (42). No significant difference was observed in the weight of the rats between groups, suggesting that differences in growth and development did not influence the differences observed in muscle contractility. Tongue function in relation to respiration. Tongue musculature is composed of intrinsic and extrinsic muscles that function independently (1, 3, 8, 19, 40) and that permit rapid and accurate changes in position, shape, and stiffness of the tongue necessary for orofacial behaviors (1, 19, 35, 39), such as respiration, swallowing, mastication, and speech (1, 8, 22, 34). Study of the contractile properties of the tongue musculature may enable a deeper understanding of the pathophysiology of these behaviors, since movements of the tongue are also governed by muscle contractile properties (34). Increased airway resistance may stimulate mechanoreceptors in the upper airway and increase activities in the alae nasi muscle, orbicularis oris, GG, and mylohyoid muscles (36). Tongue muscle activities are dominated by the inhibitory effects of lung inflation at low and moderate levels of hypercapnia, leading to responses by protrusion and retraction mus-


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Fig. 3. A: representative tetanic force contraction curves at 80 Hz from both groups at 9 wk of age. Fusion of tetanic contraction was established at 60 or 80 Hz. Solid line, results from the control group; shaded line, results from the experimental group. Arrow head ⫽ stimulation point. B: comparison of mean tetanic force production at 60 Hz between groups. The experimental group had significantly increased tetanic force levels at all ages, *P ⬍ 0.05. C: comparison in mean tetanic force production at 80 Hz between groups. The experimental group had significantly increased tetanic force levels at all ages, *P ⬍ 0.05. Solid bars, control group; shaded bars, experimental group. Error bars 95% ⫽ standard deviations.

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cles. While the hyoglossus (a tongue retraction muscle) reduces inspiratory flow, the GG (a tongue-protruding muscle) is associated with an increase in flow (2, 10, 35). The GG, the main tongue-protruding muscle, pulls the tongue forward and functions as a muscle of inspiration by maintaining pharyngeal airway patency (4, 6, 8, 22). We observed that twitch and tetanic forces for protrusive tongue contractions were increased in rats with unilateral nasal obstruction. The greater force of the tongue-protruding muscles may be related to the increased GG activity that occurs in response to negative pressure of the upper airway in animals and humans (6). There is also evidence that the GG EMG activity is related to tongue-protruding strength in both normal subjects and patients with sleep apnea/hypopnea syndrome (27), which is consistent with the present findings. This increase in the contraction force of tongue-protruding muscles after unilateral nasal obstruction may decrease airway resistance and contribute to the maintenance of pharyngeal airway patency (6, 10). The anatomy, nervous innervation, and mechanical actions of the extrinsic tongue musculature are similar in rats, cats, dogs, and humans (3, 8); thus a similar pattern can also be expected in humans.

5

(weeks old)

Age

Contractile properties of tongue-protruding muscles. In this study, nerve stimulation and measurements of the tongue protrusion forces and contractile properties were performed according to procedures described in previous studies (7, 29, 30). Hyperactivity and increased EMG activity are typically related to muscle hypertrophy (33). However, this increased activity does not make reference to the muscle contractile properties or tongue position. All measurements of the contractile properties considered the force (twitch and tetanic forces), time (CT and HDT), and fatigue measurements (FI), since they are physiologically significant and reflect the muscles’ strength capabilities and resistance to fatigue (12, 29). An increase in the tongue protrusion force may also indicate a more forward tongue position (11), and the increased HDT indicates a longer maintenance of the steady force. Thus nasal obstruction may increase not only the tongue protrusion force, but also a longer maintenance of the force contraction at early ages, both of which are consistent with the increase in GG activity due to nasal obstruction (25). Twitch force is the smallest contractile response of the muscle fiber, and its increase is related to greater elongation of the muscle fiber (23). A significant increase in the rate of



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Fig. 4. A and B: representative tetanic contraction curves used in the calculation of fatigue index at 9 wk after birth. The larger force wave is the initial force signal (solid line), which degraded over 2 min of contraction and is represented by the smallest force signal (shaded line) in each panel. C: mean fatigue index was significantly different only at 5 wk between control and experimental group, *P ⬍ 0.05. Solid bars, control group; shaded bars, experimental group. Error bars 95% ⫽ standard deviations.

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peripheral fatigue development reflects decreases in the twitch force (18). Conversely, tetanic forces are due to subsequent stimulations, referring to the highest force that can be obtained (23). In the present study, all maximal tetanic forces were significantly increased at all ages after unilateral nasal obstruction. The 5-wk-old rats showed a significant difference in FI after unilateral nasal obstruction compared with that of control rats, but this difference did not symbolize general muscle fatigue (FI ⱖ 50%) (40). Moreover, in 5-wk-old rats, although the twitch force was significantly increased in the experimental group, twitch force maximal expression was affected by the fatigability level, leading to the difference observed between the 5-wk-old rats and older rats in the experimental group. At this age, the musculature may be in an adaptive process. Thus increasing the force could lead to more fatigue compared with more advanced ages (7- and 9-wk-old rats) when this adaptive process is complete, and the muscle is able to produce more force with no changes in fatigue. In addition, fatigue of the tongue protrusion and retraction muscles in vivo is significantly greater during systemic hypoxia compared with that during normoxia (12). The present findings may also indicate that, after unilateral nasal obstruction at younger ages (5-wkold rats), oxygenation level may be reduced over that required by this stage of life, thus leading to increased fatigability. These age-related changes may also be due to a possible potentiation process (23), which occurs when a previously fatigued muscle is stimulated, and eventually results in in-

creased force with continued activation. At more advanced ages, 7 and 9 wk, the previously fatigued muscles are still in use, causing a potentiation of the muscles, and increasing the force generated. Fatigue of a single muscle leads to an increase in muscle activity in synergistic muscles during voluntary contractions, because of a compensating effect (38). Fatigue expression and changes in twitch force expression are related to changes in the sensitivity to Ca2⫹, affecting the threshold response to nerve stimuli (23, 29). Our findings suggest that unilateral nasal obstruction alters the sensitivity of the tongue-protruding muscles to Ca2⫹. Muscle fibers have the capacity to adapt to new functional demands (21). Our results also suggest possible changes in muscle fiber composition, since there is a correlation between muscle contractile properties and myosin heavy-chain expression (14). Muscle fatigue and force expression depend on fiber type, which also depends on fiber metabolism (21, 40). Changes in the area of the muscle fibers and the motor unit size may lead to changes mainly in the fast-muscle fibers of the GG (21, 40). The possible maintenance of a forward tongue position with an increased force is critical for the effectiveness of orthodontic and myofunctional therapy in mouth-breathing children (20). Our findings of increased force and activity of the tongue-protruding muscles and a sustained contraction force, when accompanied by the lip incompetence in mouth breathing (9, 20), suggest that, in mouth-breathing patients, changes in


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the tongue musculature may contribute to several malocclusions, thus altering the shape of the craniofacial complex. In cases of nasal obstruction, there could be not only increased activity of the tongue-protruding muscles, but also a possible increase in their force. In conclusion, unilateral nasal obstruction during growth altered the contractile properties of the tongue-protruding muscles in rats. Our results suggest that early unilateral nasal obstruction may increase the force of the tongue-protruding muscles and prolong the duration of contraction of these muscles, which may have profound effects on craniofacial morphology and tongue function. ACKNOWLEDGMENTS We thank Associate Professor Yoshiyuki Sasaki for statistical expertise. We thank Professor Kumiko Sugimoto and Professor Keiji Moriyama for helpful comments. GRANTS This study was financially supported in part by Grants-in-Aid for Scientific Research (24593081) from the Japanese Ministry of Education, Culture, Sports, Science and Technology. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: K.H.U.K., C.K., Y.F., M.H., T.I., K.F., T.Y., S.K., and T.O. conception and design of research; K.H.U.K. performed experiments; K.H.U.K., C.K., Y.F., M.H., T.I., K.F., T.Y., S.K., and T.O. analyzed data; K.H.U.K., C.K., Y.F., M.H., T.I., K.F., T.Y., S.K., and T.O. interpreted results of experiments; K.H.U.K. prepared figures; K.H.U.K. drafted manuscript; K.H.U.K., C.K., S.K., and T.O. edited and revised manuscript; S.K. and T.O. approved final version of manuscript. REFERENCES 1. Bailey EF, Fregosi RF. Coordination of intrinsic and extrinsic tongue muscles during spontaneous breathing in the rat. J Appl Physiol 96: 440 –449, 2004. 2. Bailey EF, Jones CL, Reeder JC, Fuller DD, Fregosi RF. Effect of pulmonary stretch receptor feedback and CO2 on upper airway and respiratory pump muscle activity in the rat. J Physiol 532: 525–534, 2001. 3. Bailey EF, Huang YH, Fregosi RF. Anatomic consequences of intrinsic tongue muscle activation. J Appl Physiol 101: 1377–1385, 2006. 4. Brouillette RT, Thach BT. Control of genioglossus muscle inspiratory activity. J Appl Physiol 49: 801–808, 1980. 5. Burke RE, Levine DN, Tsairis P, Zajac FE. Physiological types and histochemical profiles in motor units of the cat gastrocnemius. J Physiol 234: 723–748, 1973. 6. Chamberlin NL, Eikermann M, Fassbender P, White DP, Malhotra A. Genioglossus premotoneurons and the negative pressure reflex in rats. J Physiol 579: 515–526, 2007. 7. Connor NP, Ota F, Nagai H, Russell JA, Leverson G. Differences in age-related alterations in muscle contraction properties in rat tongue and hindlimb. J Speech Lang Hear Res 51: 818 –827, 2008. 8. Doran GA, Bagget H. The genioglossus muscle: a reassessment of its anatomy in some mammals, including man. Acta Anat (Basel) 81: 403– 410, 1972. 9. Dutra EH, Maruo H, Vianna-Lara MS. Electromyographic activity evaluation and comparison of the orbicularis oris (lower fascicle) and mentalis muscles in predominantly nose- or mouth-breathing subjects. Am J Orthod Dentofacial Orthop 129: 722-e1–722-e9, 2006. 10. Fregosi RF, Fuller DD. Respiratory-related control of extrinsic tongue muscle activity. Respir Physiol 110: 295–306, 1997. 11. Fuller DD, Fregosi RF. Co-activation of the tongue protrudor and retractor muscles during chemoreceptor stimulation in the rat. J Physiol 507: 265–276, 1998.

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