INTRODUCTION

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The sensation of discomfort associated with the act of breathing may be modulated by afferent information from a variety of sources, including the upper airways, chest wall, lower airways, and lungs (1). It is a commonplace experience that patients who suffer from dyspnea can occasionally obtain subjective relief when they sit near an open window or in front of a fan. Also, it has been reported that, in patients with chronic obstructive pulmonary disease (COPD), breathing cold air reduces the sensation of dyspnea and improves exercise performance (2).

There is much evidence (3) to suggest the presence of a specific type of upper airway receptors responding to inspiratory airflow both in animals and humans. These receptors, which operate as thermoreceptors responsive to small decreases in luminal temperature of the upper airways, are now called "cold receptors" (5). Cold receptors in the upper airway may be one source of information on inspiratory flow rate and inspired volume, and therefore, stimulation of cold receptors may modulate the respiratory sensation.

Although breathing cold air has been shown to alter both the pattern of breathing and the response to CO₂ (7, 8), little information is available as to the effect of stimulation of cold receptors in the upper airway on respiratory sensation. Furthermore, a decrease in upper airway temperature not only stimulates specific cold receptors but also inhibits mechanoreceptors in the upper airway (9), and the contribution of the latter cannot be disregarded when evaluating the changes in respiratory sensation during cold air inhalation.

Schwartzstein and colleagues (10) demonstrated that a flow of cold air against the face reduces the sensation of breathlessness associated with loaded breathing in normal subjects. However, their subjects breathed gases maintained at room temperature without inspiring the cold air directed against the face, and therefore, stimulation of cold receptors in the upper airway as a cause of the respiratory relief is unlikely.

l-menthol is known as a specific stimulant of cold receptors and the potential use of l-menthol as a tool for the respiratory study has been suggested (11). In the present study we investigated the effect of nasal inhalation of l-menthol on changes in perception of respiratory discomfort induced by addition of external loads in normal subjects. We postulated that inhalation of l-menthol would reduce the sensation of respiratory discomfort associated with loaded breathing.

	METHODS

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We studied 11 volunteers (10 males, 1 female) who ranged in age from 23 to 39 yr. None had clinical evidence of cardiovascular, respiratory, neuromuscular, and rhinolaryngological disorders. All subjects were nonsmokers. The protocol was approved by the institutional ethics committee, and each subject gave informed consent. None was familiar with the hypothesis being tested.

Each subject was seated during the experiment and breathed through an experimental apparatus containing a nasal mask, a two-way valve, a pneumotachograph (CP-100, Allied Health Care Product Inc., St. Louis, MO), and a bypass circuit (Figure 1). The experimental apparatus had a resistance of 5.5 cm H₂O/L/s at a flow rate of 0.5 L/s with or without the use of the bypass circuit. The bypass circuit had a total respiratory space of approximately 250 ml in which 300 mg of l-menthol crystal was deposited. The choice of this dose of l-menthol was based on the results of several preliminary studies in which the subjects inhaled through the bypass circuit containing different doses of l-menthol, and the dose that produced the most comfortable cooling sensation was assessed.

View larger version (18K): [in this window] [in a new window]   Figure 1.   Schematic illustration of the experimental apparatus.

In order to induce the sensation of respiratory discomfort in subjects, two different types of external loads, i.e., a flow-resistive load and an elastic load, were added to the experimental apparatus. To apply the flow-resistive loading, an easily exchangeable flow resistor (2.5 mm in diameter and 10 cm in length) was placed in the distal inspiratory limb of the experimental apparatus. The total inspiratory resistance of the respiratory circuit with the resistor in place was 180 cm H₂O/L/s at a flow of 0.5 L/s with or without the use of the bypass circuit. To apply the elastic loading, a 12-L rigid glass bottle with a vent valve was connected to the distal inspiratory limb of the experimental apparatus. The vent valve was closed throughout each inspiration and opened promptly during expiration in order to replenish the load with fresh air and maintain the load constant from breath to breath. Closing and opening of the valve was effected by a solenoid triggered by the flow signal of the pneumotachograph. The magnitude of the elastic load was 75.5 cm H₂O/L without the use of the bypass circuit and 74.0 cm H₂O/L when the bypass circuit was used.

Ventilatory airflow was measured through the pneumotachograph while the subject was breathing only through the nose. 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; Tokyo, Japan).

During the experiment, the subject was asked to rate continuously the intensity of the sensation of respiratory discomfort using a visual analog scale (VAS). The analog scale, ranging from 0 to 100, was displayed on an oscilloscope screen, and the subjects measured the VAS score by adjusting the knob of a potentiometer, thus altering the display on the oscilloscope anywhere on the scale from 0 to 100 arbitrary units. The numerical value of 0 was given for the sensation of "not at all unpleasant" and 100 for the sensation of "intolerable." Respiratory discomfort was defined as "an unpleasant urge to breathe." No further clarification or definitions were given, and the subjects were not asked to distinguish different qualities or dimensions of the respiratory sensation. Mask pressure, VT, PET_CO2, and VAS score all were recorded on a four-channel recorder (Graphtec WR-3701; Tokyo, Japan). Before the start of the main study, a preliminary experiment was performed to familiarize the subject with the apparatus, the protocol, the sensation of breathing against the added loads, and the use of VAS.

The main study was conducted 10 min after the preliminary study, and the usual protocol is described in the following paragraph.

When the subject breathed through the experimental apparatus without the load in place and the breathing pattern was stable (unloaded breathing), the subject rated the magnitude of respiratory discomfort, and values of ventilation were calculated from measurements of 30-s periods. Then either the resistive load or the elastic load was applied and maintained throughout inspirations continuously for at least 11 min. This time was required to evaluate the effects of l-menthol on the sensation of respiratory discomfort under a near steady respiratory state in the presence of added loads. After the application of the respiratory load, when breathing pattern and VAS values had remained nearly steady for at least 2 min, the subject started to breathe through the bypass circuit, thus inhaling the air containing l-menthol for 4 min, after which time the bypass circuit was closed and the inhalation of l-menthol was discontinued while the measurements of ventilatory variables and VAS score were continued for another 3 min, and then the load was removed.

The order of application of the resistive load and the elastic load was randomized, but each subject received two trials of both the elastic and resistive loads with an interval of 5 min between each trial. The results obtained from two trials for one type of load on each subject were averaged. On different days, in 8 of 11 subjects the same experiment as in the above protocol was repeated. However, in this series of experiments a strawberry essence was deposited in the bypass circuit so that the subjects inhaled strawberry-flavored air instead of l-menthol during loaded breathing.

The effects of inhalation of l-menthol or strawberry-flavored air on ventilation and respiratory sensation were evaluated by comparing the steady-state values of ventilatory variables and VAS scores obtained from measurements of at least five consecutive breaths at 1 min before the opening of the bypass circuit (baseline period), at 3 min after the opening of the bypass circuit (test condition), and at 3 min after the closing of the bypass circuit (recovery period). Data are expressed as mean ± SD.

Statistical analysis was performed by using two-way analysis of variance (ANOVA) followed by Scheffe's test, paired t test with Bonferroni correction, where appropriate.

	RESULTS

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All subjects tolerated both flow-resistive and elastic loads and completed the experimental protocol.

Immediately after the addition of an external load, regardless of the type of loading, there were changes in VT and respiratory frequency (f) with a concomitant increase in VAS score. These changes gradually stabilized within 2 min, and thereafter, breathing patterns as well as VAS scores remained nearly steady. Although there was an intersubject variability, in general the breathing pattern with the flow-resistive load during the steady-state was characterized by a slow breathing with a marked prolongation of inspiratory time (TI) while the breathing pattern with the elastic load was characterized by a rapid, shallow breathing with a shortening of expiratory time (TE) (Table 1).

Figure 2 shows experimental records illustrating changes in breathing pattern and VAS score in response to inhalation of l-menthol during the flow-resistive loading (Figure 2A) and the elastic loading (Figure 2B). During the flow-resistive loading, shortly after the start of inhalation of l-menthol there was a slight decrease in VT with a concomitant increase in PET_CO2, but soon these changes returned to the pre-inhalation baseline level that remained nearly steady for the remainder of inhalation of l-menthol. The VAS score also decreased shortly after inhalation of l-menthol and reached its nadir within 60 s after the start of inhalation of l-menthol. Thereafter, there was a slight attenuation of the decrease in VAS score, but the VAS score remained low throughout the period of inhalation of l-menthol. Discontinuation of inhalation of l-menthol caused a prompt recovery of the VAS score to the baseline level.

View larger version (27K): [in this window] [in a new window]   Figure 2.   Experimental records illustrating changes in breathing pattern and VAS score in response to inhalation of l-menthol during the flow-resistive (A) and elastic loading (B). Arrows indicate the start and end of inhalatian of l-menthol. VAS = visual analog scale; Pmask = mask pressure; VT = tidal volume; PETCO2 = end-tidal PCO2.

Unlike the changes observed during the flow-resistive loading, inhalation of l-menthol during the elastic loading caused almost no change in breathing pattern. However, the changes in VAS score were qualitatively similar to those observed during the flow-resistive loading, and thus, the VAS score decreased immediately after the start of inhalation of l-menthol and returned to the baseline level shortly after the discontinuation of inhalation of l-menthol. Similar changes in breathing patterns and VAS scores in response to inhalation of l-menthol were observed in the majority of subjects, although in 2 of 11 subjects l-menthol inhalation did not cause any change in VAS score during the elastic loading. Table 1 summarizes changes in breathing patterns in response to inhalation of l-menthol during loaded breathing in 11 subjects. Both during the flow-resistive loading and the elastic loading, the inhalation of l-menthol caused neither changes in breathing pattern nor changes in mean inspiratory flow rate (VT/TI).

Figure 3 shows changes in VAS scores in response to inhalation of l-menthol. Before the start of inhalation of l-menthol, the values of the VAS score during the flow-resistive loading and the elastic loading were 62 ± 14 and 68 ± 13, respectively. There was no significant difference between the two values. The inhalation of l-menthol caused significant decreases in VAS scores both during the flow-resistive loading (36 ± 16, p < 0.01) and during the elastic loading (55 ± 17, p < 0.01), but the values of VAS scores during the elastic loading were significantly higher (p < 0.01) than those during the flow-resistive loading. After the discontinuation of inhalation of l-menthol, the values of VAS scores during the flow-resistive loading and the elastic loading were 63 ± 14 and 68 ± 13, respectively. These values were almost identical to those obtained before the start of inhalation of l-menthol.

View larger version (19K): [in this window] [in a new window]   Figure 3.   Changes in VAS scores in response to inhalation of l-menthol during the loaded breathing. Both individual data (upper panel  ) and averaged data (lower panel  ) are shown. p value < 0.01, significantly different from the baseline values; p < 0.01, compared with the corresponding values of the elastic loading.

The results of inhalation of strawberry-flavored air are summarized in Table 2. These results show that neither breathing patterns nor VAS scores changed in response to inhalation of the strawberry-flavored air both during the flow-resistive loading and the elastic loading.

	DISCUSSION

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In this study we have demonstrated that nasal inhalation of l-menthol can considerably reduce the sensation of respiratory discomfort produced by addition of external respiratory loads. Although underlying mechanisms of this reduction in the sensation of respiratory discomfort are not entirely clear, cold receptors in the upper airway seem to play an important role since l-menthol is known as a specific stimulant of cold receptors in the upper airway (11).

There is some evidence to suggest that cooling of the upper airway or stimulation of cold receptors in the upper airway depresses respiratory activity both in humans and animals (7, 8, 12). It has been also proposed that an increase in inspiratory motor output or respiratory drive causes an increased sense of effort and dyspnea (13). Thus, one mechanism by which l-menthol reduces the sensation of respiratory discomfort might be through a reduction in respiratory drive. In fact, in our study there was a slight decrease in ventilation with a concomitant decrease in the sensation of respiratory discomfort immediately after the start of inhalation of l-menthol during the flow-resistive loading. However, such decrease in ventilation was transient and was never observed during the elastic loading. Furthermore, during both the flow-resistive loading and the elastic loading the breathing patterns and ventilation at 3 min after the start of inhalation of l-menthol did not differ from those during the baseline period, despite that the improvement of respiratory discomfort continued throughout the inhalation of l-menthol. We did not measure occlusion pressure at the steady-state, but the lack of changes in mean inspiratory flow rate and breathing patterns suggests that there is little, if any, change in respiratory drive during inhalation of l-menthol. Therefore, the reduction in respiratory discomfort following inhalation of l-menthol cannot be explained solely by the reduction in respiratory drive.

Another possible mechanism by which inhalation of l-menthol reduces the respiratory discomfort might be through an altered perception of the respiratory discomfort. Afferent information from the skin, the airway, or chest wall, which is projected to the sensory cortex, is known to alter respiratory sensation (10, 14). Indeed, Schwartzstein and colleagues (10) showed that a flow of cold air directed to the face reduces the intensity of breathlessness associated with loaded breathing without causing a significant reduction in ventilation. They hypothesized that the reduction in breathlessness is due to stimulation of trigeminal nerve afferents, the information from which may be processed in the central nervous system to alter the perception of breathlessness. Also, it has been shown that chest wall vibration reduces breathlessness in normal subjects (17) and in patients with chronic lung disease (18), presumably through stimulation of receptors in the chest wall. Thus, it is likely that increased information from cold receptors in the upper airway may decrease the sensation of respiratory discomfort independently of changes in ventilation and pattern of breathing. This notion is compatible with the work of Simon and coworkers (19), who showed that, in patients with COPD, stimulation of oral mucosa flow receptors reduces the intensity of breathlessness induced by hypercapnia and an inspiratory resistive load, independently of changes in ventilation or pattern of breathing.

The possibility exists that the observed reduction in sensation of respiratory discomfort might not depend on specific receptor mechanisms but rather on nonspecific effects of l-menthol, including distraction. In this connection, it is worthy to note that l-menthol is not only a specific stimulant of cold receptors but also a strong olfactory stimulus. Our finding that inhalation of strawberry-flavored air did not cause significant changes in breathing patterns and VAS scores suggests that the distraction associated with olfaction may not improve the respiratory discomfort during loaded breathing. However, it is in practice difficult to dissociate the effects of l-menthol on cold receptors from that of olfaction (11). Therefore, we cannot exclude the possibility that the cooling sensation following l-menthol inhalation might have provided the potential distraction from the respiratory discomfort during loaded breathing.

The difference in the relief of respiratory discomfort between the flow-resistive loading and the elastic loading may be related to the difference in breathing patterns between the two different types of loading. For example, the breathing patterns during flow-resistive loading are characterized by a slow breathing, whereas those during elastic loading are characterized by a rapid, shallow breathing. Thus, it is likely that the relatively prolonged stimulation of cold receptors due to the marked prolongation of TI during the flow-resistive loading may cause the greater relief of respiratory discomfort than during the elastic loading.

During continued inhalation of l-menthol, in the majority of subjects there was a slight attenuation of the reduction in sensation of respiratory discomfort. This attenuation of relief of respiratory discomfort is consistent with either an adaptation of cold receptors to l-menthol or central adaptation, or both. Considering the nature of cold receptors that adapt rapidly to a constant airflow (5), it is possible that the adaptation of cold receptors to l-menthol may in part explain the slight attenuation of relief of respiratory discomfort observed during continued inhalation of l-menthol. The central adaptation is another important feature of sensory mechanisms. Indeed, the sensory magnitude of any stimulus declines following prolonged periods of stimulation for all sensory modalities, and the respiratory sensation is not an exception (20). Thus, we cannot deny the possibility that the attenuation of relief of respiratory discomfort during continued inhalation of l-menthol may be due to the central adaptation mechanism.

In conclusion, our results support the hypothesis that stimulation of cold receptors in the upper airway alleviates the sensation of respiratory discomfort.