INTRODUCTION

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The function of the swallowing reflex can serve as a protective reflex for the upper respiratory tract (1). It has been suggested that the precise coordination of breathing and swallowing might be an important mechanism to prevent pulmonary aspiration (2). Factors that alter the breathing pattern and ventilation may influence the precise coordination of breathing and swallowing. Issa and Porostocky (5) studied the effect of continuous swallowing on breathing pattern and ventilation in adult subjects and demonstrated that although repetitive swallowing influences the rate and depth of breathing, the level of ventilation is maintained constant during continuous swallowing. They also showed that continuous swallowing did not change the slope of the CO₂ response curve assessed by a rebreathing technique and that repetitive swallowing did not result in a single incidence of aspiration or coughing during progressive hypercapnia (6). Their results suggest that coordinating mechanisms integrating breathing and swallowing allow repetitive swallowing to occur without compromising ventilation. However, since their experiments were performed with the CO₂ rebreathing technique, which exposed the subjects to a relatively short period of a hypercapnic condition, it remains unknown whether or not a prolonged exposure to hypercapnia would lead to the same results. In the course of experiments studying the effect of prolonged hypercapnia on the swallowing reflex in normal adult subjects, we noticed that during continuous infusion of water into the pharyngeal cavity, signs of aspiration were more frequently observed with hypercapnia than with eucapnia. In order to test the hypothesis that the coordination of swallowing and respiration may be compromised during hypercapnia, in the present study we examined the coordination of swallowing and respiration at four different levels of end-tidal PCO₂ in normal subjects.

	METHODS

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Eleven healthy volunteer subjects (seven men and four women) 25 to 50 yr of age were studied. None of the subjects had clinical evidence of respiratory, cardiovascular, rhinolaryngologic, or neuromuscular disorders. The research was carried out in accordance with the Declaration of Helsinki (1989) of the World Medical Association, and the protocol was approved by the Ethics Committee of Chiba University School of Medicine. Each subject gave informed written consent. None was familiar with the hypothesis being tested.

Prior to the start of the experiment, a thin polyethylene catheter having a diameter of 1.35 mm was inserted through the naris so that the tip of the catheter lay in the epipharynx. Each subject was seated during the experiment and breathed through a tightly fitting face mask connected to a pneumotachograph (CP-100; Allied Health Care Products Inc., St. Louis, MO) and then to a T-piece system. The total instrumental dead space was about 150 ml. Ventilatory airflow was measured with the pneumotachograph, and tidal volume (VT) was obtained by electrical integration of the inspired flow signal. Mask pressure was measured with a pressure transducer (Transpac IV; Abbott Critical Care Systems, Chicago, IL). End-tidal PCO₂ (PET_CO2) was monitored with an infrared CO₂ analyzer (Aika MEL RAS-41; Aika, Tokyo, Japan) through a port in the face mask.

The swallowing reflex was induced by continuous infusion of distilled water through the nasal catheter at the rate of 2.5 ml/min and was identified by a submental electromyogram (EMG), a transient interruption of respiratory airflow, and visual observation of the characteristic laryngeal swallowing movements. During the experiments, hyperoxia was maintained by passing 100% oxygen, with a total gas flow of 15 to 20 L/min, through the T-piece. Before the start of the main study, a preliminary experiment was performed to familiarize the subject with the apparatus and the continuous infusion of water into the pharynx.

The main study was conducted 10 min after the preliminary study. The usual protocol of the study is as follows.

The subject breathed through the face mask and pneumotachograph for at least 5 min. When all of the respiratory variables were stable, breathing was recorded during a 3- to 4-min period (eucapnic baseline recording). The last 2 min were used for data analysis. Subsequently, instillation of water was started to induce the swallowing reflex. The instillation of water was continued for at least 4 min to ensure that the swallowing response to continuous infusion of water was stable. The last 2 min of the above condition were used for data analysis.

After completion of the above studies, a telescopic plastic tube with an internal diameter of approximately 5 cm was connected between the pneumotachograph and the T-piece. This telescopic tube has three sections that slide one within the other and can produce any volume of dead space ranging from 400 to 1,800 ml by simply lengthening it. By adjusting the amount of dead space, PET_CO2 was elevated by 3 to 4 mm Hg steps to obtain three consecutively elevated steady-state PET_CO2 levels. These three elevated levels of PET_CO2 were designated as Hypercapnia 1 (PET_CO2: baseline + 3 to 4 mm Hg), Hypercapnia 2 (Hypercapnia 1 + 3 to 4 mm Hg), and Hypercapnia 3 (Hypercapnia 2 + 3 to 4 mm Hg), respectively. After a change in PET_CO2 with an addition of external dead space, 8 to 10 min was allowed for establishment of a new steady-state pattern. The instillation of water was then started and continued for at least 4 min while the same amount of dead space was maintained. As in the previous trials, recording of respiratory variables was performed during the steady-state breathing for a 3- to 4-min period, and the last 2 min of each trial was used for data analysis.

Submental EMG, airflow, VT, Pmask, and PET_CO2 all were recorded on a thermal array recorder (Omniace RT3424; NEC, Tokyo, Japan).

For quantitative analysis of the effects of swallowing on respiratory pattern, breath-by-breath analysis of VT, inspiratory time (TI), expiratory time (TE), respiratory frequency (f), and PET_CO2 was performed during the steady-state conditions before and during the instillation of water. Minute ventilation (I) was also calculated as the product of VT and f. In addition, frequency of swallows, average duration of interrupted flow (Tac) during swallowing, and the timing of swallows in relation to the phases of the respiratory cycle were determined. Swallows preceded by and followed by inspiratory flow were marked as inspiratory swallows, whereas swallows preceded by and followed by expiratory flow were marked as expiratory swallows. Swallows occurring at transition between inspiration and expiration were designated "inspiratory-expiratory (I-E) transition swallows" and swallows occurring at the transition between expiration and the inspiratory phase of the next breath were designated "expiratory- inspiratory (E-I) transition swallows."

The CO₂ ventilatory response was obtained by plotting points of ventilation for varying PET_CO2 levels. The CO₂ response curves before and during the continuous infusion of water were obtained by linear regression analysis. The CO₂ response curves were analyzed in terms of two variables: the slope and the intercept of the curve on the PET_CO2 axis when extrapolated to a theoretical zero ventilation.

Incidents of laryngeal irritation and/or pending aspiration were defined as a single cough, a series of coughs, or a short period of apnea.

Statistical analysis was performed by the use of two-way ANOVA followed by Scheffe's test, Friedman's rank test, chi-square test, and paired t test, when appropriate; p < 0.05 was considered significant.

	RESULTS

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Ten of 11 subjects tolerated the continuous infusion of water at all the elevated levels of PET_CO2 and completed the experimental protocol. However, one subject could not tolerate the 4 min of continuous infusion of water at the highest level of PET_CO2 (Hypercapnia 3) because of the repetitive occurrence of coughing. In this subject, the data at the highest level of PET_CO2 were obtained from the first 1 min after the start of continuous infusion of water.

The respiratory pattern of a single subject before and during continuous infusion of water with and without an additional dead space is shown in Figure 1. Before the infusion of water at eucapnic level of PET_CO2 (Figure 1A), the pattern of respiration was regular. Infusion of water during the eucapnia baseline breathing caused repetitive swallows and the pattern of breathing became somewhat irregular, but the level of PET_CO2 was maintained constant (Figure 1A). With the addition of dead space (Figure 1B), there were considerable increases in PET_CO2 and respiration. During the infusion of water at this elevated level of PET_CO2 (Hypercapnia 2), repetitive swallows similar to those observed at eucapnia occurred, although the frequency of swallows was much less than that observed at the eucapnic level of PET_CO2. Despite considerable changes in breathing pattern, the level of PET_CO2 was almost identical to that observed before the continuous infusion of water (Figure 1B).

View larger version (26K): [in this window] [in a new window]   Figure 1.   Experimental records illustrating the respiratory pattern before and during infusion of water without (A) and with (B) an added external dead space. VT = tidal volume; Pmask = mask pressure; PETCO2 = end-tidal PCO2.

The mean values of respiratory variables before and during continuous infusion of water in response to increasing levels of PET_CO2 obtained in all 11 subjects are shown in Table 1. Increases in PET_CO2 progressively increased I mainly because of increases in VT both before and during infusion of water. Infusion of water caused a significant decrease in f because of prolongation of TE only at the eucapnic level of PET_CO2. However, f during infusion of water progressively increased during hypercapnia because of the shortening of TE. A statistically significant decrease in TE was observed only between the values at eucapnia and those at the highest level of PET_CO2 (Hypercapnia 3). There was no change in the duration of interrupted flow (Tac) during swallowing at different levels of PET_CO2 (Table ).

The slopes and intercepts of the CO₂ ventilatory response curves obtained from all 11 subjects before and during continuous infusion of water are summarized in Table 2. There was no significant difference between the mean values of the slopes and intercepts of CO₂ response curves measured before and during continuous infusion of water.

The continuous infusion of water invariably caused repetitive swallows in all subjects, although the frequency of swallows varied from subject to subject. A one-to-one or one-to-two rhythmic coupling of swallowing and respiration was observed in six of 11 subjects at the eucapnic level of PET_CO2, whereas irregularly repeated swallows were observed in the other five subjects. The relationship between the frequency of swallows and PET_CO2 is shown in Figure 2. Although changes in the frequency of swallows in response to increasing PET_CO2 varied from subject to subject, there was a significant decrease in the frequency of swallows during hypercapnia.

View larger version (15K): [in this window] [in a new window]   Figure 2.   Changes in the frequency of swallows with increasing PETCO2 during continuous infusion of water. Closed circles represent the mean values of the frequency of swallows measured at four different levels of PETCO2. Values are mean ± SEM. Open asterisks indicate p < 0.01, compared with the baseline eucapnic values. Open squares represent the individual data points obtained from all 11 subjects. The regression line obtained from all the data is also shown.

The phase relationship between continuous swallows during continuous infusion of water and respiration is summarized in Figure 3. Although there was a wide variation in the timing of swallows in relation to the phase of the respiratory cycle, during baseline eucapnia the majority of swallows were expiratory and I-E transition swallows. Inspiratory and I-E transition swallows were very rare. Increases in PET_CO2 considerably modified the types of swallows. Expiratory swallows progressively decreased, whereas inspiratory and E-I transition swallows progressively increased with increasing levels of PET_CO2.

View larger version (53K): [in this window] [in a new window]   Figure 3.   Timing of swallows in relation to the phase of the respiratory cycle during continuous infusion of water. Percentage of swallows coinciding with each phase of the respiratory cycle was calculated for individual subjects; the values shown are mean ± SEM of these percentages for each type of swallow.

Data regarding the incidence of laryngeal irritation during continuous infusion of water are shown in Figure 4. In seven of 11 subjects, signs of laryngeal irritation were observed during hypercapnia, whereas such signs were never observed during eucapnia. The incidence of laryngeal irritation was higher, the higher the PET_CO2 (Figure 4). Although the signs of laryngeal irritation such as a series of coughs and a short period of apnea (Figure 5) were occasionally observed, the most frequently observed sign of laryngeal irritation was a single cough (Figure 6).

View larger version (14K): [in this window] [in a new window]   Figure 4.   Incidence of laryngeal irritation during continuous infusion of water at different levels of PETCO2. (A) Occurrence of laryngeal irritation during continuous infusion of water (% of subjects). (B) Data for individual subjects on the frequency of laryngeal irritations. Individual subjects are represented by different symbols.

View larger version (30K): [in this window] [in a new window]   Figure 5.   Experimental records illustrating signs of laryngeal irritation during continuous infusion of water. (A) A series of coughs observed in a subject who did not show any change in swallowing frequency during hypercapnia. (B) A short apnea observed in a subject different from the one shown in A.

View larger version (41K): [in this window] [in a new window]   Figure 6.   Experimental records illustrating laryngeal irritation, i.e., single coughs during continuous swallowing caused by continuous infusion of water. (A) A single cough after an expiration-inspiration transition swallow (arrow indicates E-I swallow). (B) A single cough after a swallow coinciding with inspiration (swallow is indicated by an arrow). The subject is the same as in A, but the level of hypercapnia is different (A: Hypercapnia 3; B: Hypercapnia 2). Note also that the frequency of swallows transiently increases after the single coughs in A and B. (C ) An incident of laryngeal irritation (single cough) without preceding swallows. The subject is different from the one shown in A and B.

When a total of 36 laryngeal irritations consisting of single coughs in seven subjects were analyzed, 20 incidents occurred after E-I transition swallows (Figure 6A), nine incidents occurred after inspiratory swallows (Figure 6B), and seven incidents occurred without preceding swallows (Figure 6C). No signs of laryngeal irritation after expiratory swallows or I-E transition swallows were ever observed.

	DISCUSSION

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In this study we have shown that during continuous infusion of water into the pharynx (1) the frequency of swallowing decreases with increasing PET_CO2, (2) the timing of swallows in the phase of the respiratory cycle changes with increasing PET_CO2, and (3) the incidence of laryngeal irritations increases with increasing PET_CO2.

The major finding of this study is that the incidence of laryngeal irritation during continuous infusion of water into the pharynx increases with increasing PET_CO2. This finding is in sharp contrast to the study of Issa and Porostocky (6) who reported that infusion of water into the oral cavity did not cause a single incidence of coughing or aspiration during progressive hypercapnia. There is no obvious explanation for this difference. However, the difference between our observation and that of Issa and Porostocky (6) may be related to the difference in methodology. For instance, in the study by Issa and Porostocky (6), the observation was made under progressively increasing hypercapnia induced by the CO₂ rebreathing technique, whereas in our study the observation was made under steady-state hypercapnic conditions induced by addition of an external dead space. In our study, subjects were hypercapnic for a longer period of time. Presumably, the prolonged hypercapnia with the use of the dead space may cause more remarkable increases in the tonic and phasic contraction of upper airway muscles than those produced by the CO₂ rebreathing, which may have a stronger impact on the coordination of breathing and swallowing.

Despite the different methods (rebreathing technique versus steady-state technique), in this study we confirmed the results of Issa and Porostocky (6) that CO₂ chemosensitivity is not changed by repeated swallows during continuous infusion of water. The fact that we reached the same conclusion indicates that the method of assessing CO₂ responsiveness is not crucial for determining the effects of continuous swallows on CO₂ ventilatory chemosensitivity. These results are also compatible with the results reported previously by Smith and colleagues (3) and Issa and Porostocky (5) who showed that the level of ventilation is maintained during eating and drinking.

In our study we also observed that the frequency of swallows during continuous infusion of water decreases during hypercapnia. This observation agrees with the results of Issa and Porostocky (6) but is in contrast to the observation made in anesthetized, vagotomized, paralyzed, and artificially ventilated cats that graded hypercapnia had no effect on the swallowing reflex (7). The difference in swallowing responses to CO₂ between humans and cats may be ascribed to the species difference, but it is also possible that the difference may be due to different methods and experimental conditions. The finding that the frequency of swallows decreases during hypercapnia is analogous to the observations made in anesthetized humans that airway protective reflexes are attenuated during hypercapnia (8). Thus, it may be possible that the attenuation of reflex activities during hypercapnia is a common feature of airway protective reflexes, including the swallowing reflex. It seems to be reasonable to speculate that the automatic respiratory control system prevails over the swallowing reflexes when the maintenance of ventilation is particularly important in a condition of loaded breathing since the time available for breathing could be significantly reduced during repeated swallowing. The attenuation of the swallowing reflex during hypercapnia is compatible with the finding that hypercapnia enhances the chance of laryngeal irritation during continuous infusion of water. One might argue that the decrease in the frequency of swallows does not reflect the depression of the swallowing reflex since subjects can clear larger volumes of water per swallow while decreasing the number of swallows (6). In fact, signs of laryngeal irritation were repeatedly observed in a subject who did not show any decrease in the frequency of swallows during hypercapnia (Figure 5A). Furthermore, there was a great variability in the decrease in frequency of swallows among subjects with increasing PET_CO2. Therefore, the incidence of laryngeal irritation during hypercapnia increased regardless of a change in the frequency of swallows.

Previous studies (2, 9, 10) have demonstrated that in awake human adults, the majority of swallows occur during the expiratory phase, and respiratory movement resumes after a swallow in the same expiratory phase that had been interrupted. In this study, swallowing/respiration patterns constantly changed during continuous swallowing. Furthermore, the swallowing/respiratory patterns varied widely among subjects during continuous swallowing. Thus, irregularly repeated swallows occurred with no relation to respiratory phase in some subjects, whereas a one-to-one coupling of swallowing and respiration was observed in other subjects. Nevertheless, expiratory swallows were observed most frequently during baseline eucapnia. Our results are quite different from the results of Issa and Porostocky (5) who reported that the majority of swallows coincided with inspiration during continuous swallowing. Although the difference may be due to the different definition of swallowing type, it is also possible that the difference may be due to the different methods for elicitation of reflex swallowing. For example, Issa and Porostocky (5) induced the repetitive swallows by infusion of water into the oral cavity with an infusion rate that was 16 to 40 times higher than the rate used in our study. Instillation of such a large amount of water into the oral cavity may cause voluntary adjustment of swallowing timing, whereas the swallowing act produced by infusion of a small amount of water into the pharynx may be more reflex in nature.

Another possibility comes from the consideration of physiologic responses that the methodologic difference might elicit. The recent study of Anderson and colleagues (11) showed that swallowing occurs in sleeping cats by injecting water through a nasopharyngeal tube, whereas Issa (12) demonstrated that water infusion through a feeding tube into the oropharynx fails to induce swallowing in sleeping dogs. These findings suggest that the different responses can be obtained depending on the site of stimulation. In addition, large fluid volume in the oral cavity may cause changes in the pharyngeal and tongue contours, which in turn modulates proprioceptors in the oropharyngeal cavity and thereby affects the timing of swallowing. Thus, it is likely that the differences in site and volume of fluid delivery may considerably change the timing of swallowing.

We demonstrated in this study that increases in PET_CO2 considerably change the timing of swallows in relation to the phase of the respiratory cycle (Figure 3). A progressive decrease in occurrence of expiratory swallows with concomitant increases in occurrence of E-I and inspiratory swallows in response to increasing PET_CO2 suggests that the timing of swallows coinciding with expiration may shift towards the inspiratory phase of the next breath with increasing PET_CO2. This finding is compatible with the result that TE during infusion of water progressively shortened with increasing PET_CO2.

Analysis of the phase relationship between swallowing and respiration revealed that the majority of laryngeal irritations occurred either when the swallows were initiated at the E-I transition or when the swallows coincided with inspiration. Therefore, the timing of swallows may be another important factor that determines the occurrence of aspiration, particularly during hypercapnia when an increased negative pressure of inspiration can enhance the chance of aspiration. In this regard, Paydarfar and colleagues (13), who measured the time between inspiration and a laryngeal exposure to a barium bolus using videofluoroscopic techniques, showed that the time from the departure of a laryngeal bolus to the onset of the next inspiration was shortest for late expiratory swallows, suggesting that the phase of E-I transition is the most vulnerable for aspiration. The results of our study are compatible with their suggestion.

In conclusion, we have shown that during continuous infusion of water in the pharynx, CO₂ ventilatory response in humans is not influenced by continuous swallowing and that the frequency of swallows decreases during steady-state hypercapnia. Although swallows coinciding with expiration were observed most frequently during eucapnia, the occurrence of these expiratory swallows progressively decreased, whereas the occurrence of swallows during inspiration and the expiration-inspiration transitional period progressively increased with increasing levels of PCO₂.

Signs of laryngeal irritation and/or pending aspiration such as coughing or a short apnea were never observed during continuous infusion of water at eucapnia, but these responses were frequently observed during hypercapnia in more than half of the subjects, and the incidence of laryngeal irritation was higher the greater the PET_CO2. These results suggest that the automatic respiratory control system is not influenced by continuous swallowing but that the coordination of swallowing and respiration may be compromised during hypercapnia.