Coupling of dyspnea perception and tachypneic breathing during hypercapnia

Respiratory Physiology & Neurobiology 179 (2011) 276–286

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Coupling of dyspnea perception and tachypneic breathing during hypercapnia Masahiko Izumizaki ∗ , Yuri Masaoka, Ikuo Homma Department of Physiology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

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Article history: Accepted 8 September 2011 Keywords: Dyspnea Hypercapnia Respiratory frequency

a b s t r a c t Respiratory rhythm is susceptible to behavioral influences including emotions. Since laboratory dyspnea induces negative emotions, we examined whether tachypneic breathing occurs in relation to perception of dyspnea during CO2 rebreathing (n = 21). Dyspnea intensity scored by a visual analog scale and respiratory frequency started to increase rapidly once the intensity of the stimuli exceeded a threshold for the end-tidal CO2 fraction. The thresholds for dyspnea and respiratory frequency were similar (7.5 ± 0.1% and 7.6 ± 0.2% of the end-tidal CO2 fraction, respectively), while the threshold for tidal volume (8.0 ± 0.2%), when the tidal volume had stabilized, was significantly higher than the thresholds for dyspnea (p < 0.01) and respiratory frequency (p < 0.05). A positive correlation was found between the thresholds for dyspnea and respiratory frequency (r = 0.81, p < 0.001), and these thresholds showed good agreement on a Bland–Altman plot. These findings suggest that the start of tachypneic breathing is coupled with the threshold for dyspnea. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Respiratory output is primarily regulated by an autonomic metabolic control system in the brainstem that maintains blood gas homeostasis through reflex feedback pathways from chemoreceptors. In addition, there is a behavioral control system that relies on inputs from the higher centers and alters the respiratory output in response to changes in the internal and external environments (Shea, 1996). The behavioral influences on breathing include situations involving voluntary activities such as singing and speech, and involuntary activities such as arousal state and emotions (Shea, 1996). We have designated this type of breathing as “behavioral breathing”, and it includes “emotional breathing” (Homma and Masaoka, 2008). Behavioral breathing is thought to be controlled by a different mechanism from metabolic breathing (Homma and Masaoka, 2008). Presumably, metabolic and behavioral breathing are coordinated to produce the final respiratory output from the spinal motoneurons during normal daily activities. In the laboratory, the ventilatory responses to experimental challenges such as hypercapnia and exercise have been investigated to evaluate the ability of metabolic breathing to cope with an increased demand for pulmonary gas exchange. The ventilatory response to experimental stimuli comprises an initial increase in the tidal volume (VT ) and a subsequent rapid increase in the respiratory frequency (fR ) at the VT plateau (Clark et al., 1983; Duffin et al., 2000; Hey et al., 1966). This tachypneic breathing occurs once

∗ Corresponding author. Tel.: +81 3 3784 8113; fax: +81 3 3784 0200. E-mail address: [email protected] (M. Izumizaki). 1569-9048/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2011.09.007

the intensity of the stimuli exceeds a threshold level. Duffin et al. (2000) referred to this as the breathing patterning threshold, above which the contribution of fR to the ventilatory response exceeds the contribution of VT . The possible mechanisms underlying tachypneic breathing include reflexes mediated by pulmonary stretch receptors and chest wall receptors, in addition to the mechanical properties of the respiratory system. However, the exact mechanisms are still unclear, particularly in humans. Physical stimuli such as hypercapnia and exercise increase ventilation, being accompanied by differing levels of dyspnea. The intensity of dyspnea increases in a threshold-like manner with the pulmonary O2 uptake (VO2 ) during exercise (O’Donnell et al., 1997) and with the end-tidal CO2 fraction (FETCO2 ) during CO2 rebreathing (Wan et al., 2008), similar to fR . Banzett et al. (2008) reported that negative emotions are associated with laboratory-induced dyspnea. We previously showed that the breathing patterns regulated by the brainstem can be influenced by input from the higher centers during rest periods (Homma and Masaoka, 2008). In particular, the respiratory rhythm is susceptible to the influence of behavioral breathing. With negative emotions, behavioral breathing increases ventilation with a marked increase in fR during rest periods (Bechbache et al., 1979; Masaoka and Homma, 2001; Masaoka et al., 2005). Nevertheless, the role of behavioral breathing in the ventilatory response to physical stimuli, which was traditionally thought to be driven by metabolic breathing, is poorly understood. It is possible that the ventilatory response to experimental stimuli is also achieved by an integration of metabolic and behavioral breathing driven by their respective demands. Since dyspnea causes negative emotions (Banzett et al., 2008), the perception

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of dyspnea is potentially associated with an increased respiratory rhythm (i.e., tachypneic breathing) during exposure to laboratory dyspnea stimuli. In addition, both fR and dyspnea increase in a threshold-like manner with the intensity of respiratory stimuli. Therefore, in the present study, we tested the hypothesis that tachypneic breathing occurs in relation to the perception of dyspnea during exposure to CO2 rebreathing. Accordingly, we measured the ventilatory and dyspnea responses to hypercapnia, and compared the thresholds for dyspnea perception and fR .

breath holding, you may have felt an uncomfortable sensation with the urge to breath. This sensation is generally called air hunger. During breathing with resistive loading, your breathing requires more work/effort than usual. Can you see the difference in dyspnea descriptors between air hunger and respiratory work/effort? (The difference was discussed if necessary.) For the subsequent formal testing (i.e., CO2 rebreathing), we want you to score the intensity of dyspnea similar to that experienced during breath-holding and not to score the level of respiratory work/effort.”

2. Materials and methods

2.5. CO2 rebreathing

2.1. Subjects

The intensity of dyspnea and the breathing pattern were measured while the subjects rebreathed from a 6-L plastic bag containing a gas mixture of 5% CO2 and 95% O2 . The bag was connected to the facemask once the breathing had stabilized. The subject rebreathed into the bag until the FETCO2 exceeded 9% or the VAS score exceeded 80 mm. The subject was asked to score the dyspnea VAS at 10-s intervals. The breath-by-breath fR , VT , and VE were plotted against the FETCO2 . The individual VAS scores were plotted against the FETCO2 at the corresponding time, which was predicted by a linear regression analysis between time and FETCO2 . The baseline dyspnea VAS was plotted against the mean FETCO2 before CO2 inhalation. The CO2 rebreathing procedure was repeated twice with a 15-min interval. The first procedure was performed to familiarize the subjects with the measurements and experimental setting. Thus, the second response was used for analysis.

A total of 21 young adult males (mean age, 22.1 years) participated in this study. All of the subjects provided written informed consent before participating in the experiments, which were approved by the Showa University Committee for Human Experimentation. The study conformed to the principles of the Declaration of Helsinki. 2.2. Measurement of ventilation The subjects wore a facemask during all trials. Ventilation was measured with a respiratory monitor (Aeromonitor AE280; Minato Medical Science, Osaka, Japan). This monitor measured the flow rate at the mouth using a hot-wire flowmeter and continuously calculated the ventilation (VE , L/min), VT (mL), fR (breaths/min), inspiratory time (TI , s), and expiratory time (TE , s). The monitor was calibrated before each test. 2.3. Measurement of dyspnea The intensity of dyspnea was measured using a 100-mm horizontal visual analog scale (VAS) with the maximum feeling of dyspnea on the right side of the scale and the feeling of no dyspnea on the left side of the scale. A piece of paper displaying the VAS was presented to the subjects at 10-s intervals. The subjects marked the scale using a pen and were not allowed to see their previous scores during each test. 2.4. Prior experience of dyspnea Before the experiments, the subjects experienced both (1) voluntary breath-holding at the end-expiratory position and (2) breathing under a resistive load with a 100-mm long 8-mm ID acrylic tube. These were performed to give the subjects experience with two distinct types of dyspnea (air hunger and work/effort) before CO2 rebreathing. During hypercapnia, the most common descriptors for dyspnea reported by spontaneously breathing subjects are an uncomfortable urge to breath (air hunger) and work/effort (Banzett et al., 1996). It was previously reported that breath-holding elicits air hunger and that resistive loading adds respiratory work/effort (Simon et al., 1989). Therefore, this prior experience was designed to help the subjects to distinguish between air hunger and respiratory work/effort during CO2 rebreathing. During breath-holding, which was repeated three times with rest periods in between, the subjects were asked to score the intensity of dyspnea using the VAS at 10-s intervals until the VAS score exceeded 80 mm. After three breath-holding trials, the acrylic tube was connected to the facemask. The subject was directed to breathe under the resistive load for 30 s, which was performed once in each subject. Finally, we provided an explanation to each subject about the differences in the quality of dyspnea between air hunger and respiratory work/effort. This was explained in Japanese, as “During

2.6. Thresholds for dyspnea, respiratory frequency, and tidal volume Commercial software was used for the analyses (SigmaPlot 11; Systat Software Inc., San Jose, CA, USA). We used a piecewise regression model in the regression library of SigmaPlot. Each plot was fitted with a segmented linear regression model consisting of two segments connected by a breakpoint to predict the thresholds for dyspnea VAS and fR (Duffin et al., 2000; Mohan et al., 1999; Wan et al., 2008). The dyspnea VAS and fR began to increase rapidly at the breakpoint. The threshold for VT was also determined separately where the regression slope started to decrease. The thresholds were taken as the FETCO2 value at the breakpoint. The piecewise regression technique has been described elsewhere (Toms and Lesperance, 2003). In one subject, the threshold for VT was obtained by plotting breath-by-breath VT against time as performed by Wan et al. (2008). For fR and VT , we excluded data collected before starting CO2 inhalation from the final analysis. Although a three-segment regression model could be used if these data were included, our preliminary analyses using three-segment regression models did not always provide threshold values, consistent with the report by Duffin et al. (2000). Thus, we used a two-segment regression model with the values of fR and VT recorded after starting CO2 inhalation to estimate the thresholds for fR and VT . 2.7. Thresholds for duty cycle and mean inspiratory flow The thresholds for TI , TE , and duty cycle (TI /TTOT ), given by dividing TI by the length of respiratory cycle (TTOT ), were determined by fitting the segmented linear regression model. For this purpose, breath-by-breath data of TI , TE , and TI /TTOT were smoothed using a three-breath moving average. For TI , TE , and TI /TTOT , we excluded data collected before starting CO2 inhalation. Then, the ratio of VT to TI was defined as mean inspiratory flow (VT /TI ). Breath-by-breath VT /TI was plotted against FETCO2 , and the threshold for VT /TI was determined using the segmented linear regression model.

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Fig. 1. Changes in the intensity of dyspnea (dyspnea VAS), respiratory frequency (fR ), tidal volume (VT ), and ventilation (VE ) during CO2 rebreathing in one subject (A) and in 14 subjects (B). The variables are plotted against the end-tidal CO2 fraction (FETCO2 ). Each plot is fitted with a segmental linear regression model consisting of two segments connected by a breakpoint to predict the threshold FETCO2 value. Dyspnea VAS and fR show similar rapid increases once FETCO2 exceeds the threshold. VT and VE start to increase earlier. The thresholds for fR and dyspnea VAS are similar. Values are means ± SEM.

2.8. Bland–Altman plot We used Bland–Altman plots to measure the agreement between the thresholds for dyspnea VAS and fR and between the thresholds for dyspnea VAS and VT . The Bland–Altman plot is typically used to assess the level of agreement between measurements derived from two different methods (Bland and Altman, 1986). In this plot, the within-subject differences between the two methods (Y-axis) are plotted against the mean of both methods (X-axis). This plot provides “95% limits of agreement” as the mean value for the within-subject difference ±2 standard deviations. The 95% limits of agreement indicate graphically how well the two methods agree. The 95% confidence interval (CI) of the mean within-subject

difference is calculated to detect systematic bias. Linear regression analysis between the within-subject differences and the mean of both methods is used to show proportional bias. A study exploring the relationship between the threshold for dyspnea and the ventilatory threshold during exercise used this plot and showed similarities between these thresholds (Amiard et al., 2007).

2.9. Hypercapnic ventilatory response (HCVR) The HCVR was determined by the piecewise regression technique in all subjects. VE plot was fitted with a segmented linear regression model composed of two segments connected by a

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Fig. 2. Typical examples of changes in dyspnea VAS and fR that differed from the most common response. (A) fR does not increase with FETCO2 . The VE response is caused by the VT response. (B) fR increases at the beginning. VT starts to increase when fR becomes stabilized. In both subjects, dyspnea VAS increases in a threshold-like manner. However, rapid increases in dyspnea VAS after the breakpoint are not accompanied by increases in fR .

breakpoint where VE began to increase. The slope of the second segment was considered to represent HCVR (L/min/% CO2 ).

regression analysis was carried out to detect proportional bias. For all analyses, the level of significance was set at p < 0.05. All values are presented as means ± standard error of the mean (SEM).

2.10. Statistical analysis 3. Results The data were statistically analyzed using SigmaPlot 11. The thresholds for dyspnea VAS, fR , and VT were compared by oneway repeated-measures analysis of variance (ANOVA) with Tukey’s multiple comparison post hoc tests. The paired t-test was used if appropriate. Correlation analysis was used to explore the relationship between the thresholds for dyspnea VAS and fR and between the thresholds for dyspnea VAS and VT . We performed post hoc power analyses for correlations with an ˛ level of 0.05 when no significant differences were found. In the Bland–Altman plot, linear

3.1. Thresholds for dyspnea, respiratory frequency, and tidal volume First, we confirmed that the changes in the intensity of dyspnea expressed as VAS scores during hyperoxic hypercapnia consisted of two linear segments (Fig. 1). In the first segment, the dyspnea increased gradually or sometimes minimally as FETCO2 increased. Subsequently, the intensity of dyspnea started to increase rapidly

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after reaching the threshold for FETCO2 until the end of CO2 inhalation. Data from a representative subject were fitted using a segmented linear regression model consisting of two segments connected by a breakpoint (Fig. 1A, upper panel). After the breakpoint, the dyspnea intensity increased rapidly. The threshold for dyspnea VAS in this subject was estimated to be a FETCO2 of 7.27%. The changes in the breathing patterns in response to hyperoxic hypercapnia varied among the subjects. The typical breathing pattern presented by most subjects is shown in Fig. 1A. The pattern of changes in fR was similar to that for dyspnea in most subjects, consisting of a small gradual increase and a subsequent rapid increase. The threshold for fR was 7.50% in this subject. In contrast, the VT responses differed from those of dyspnea VAS and fR , and showed a rapid increase starting immediately after the onset of CO2 inhalation, followed by a plateau above the FETCO2 threshold. In this subject, the estimated threshold was 7.47%. The VE , representing VT multiplied by fR , started to increase after the onset of CO2 inhalation and the breakpoint was visible. The threshold for VE was 6.25% in this subject. Therefore, for the analysis of VE , the breakpoints derived from the two-segment regression model represent the ventilatory recruitment threshold (Jensen et al., 2005) or chemoreflex drive threshold (Duffin et al., 2000). In other words, the VE response consisted of an initial increase in VT followed by an increase in fR , thereby showing a linear increase with FETCO2 . A total of 21 subjects performed the experiment. We excluded five subjects from the statistical analyses because their fR responses differed from the typical pattern described above. In three

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Fig. 3. The mean threshold values of 14 subjects differ significantly among the three parameters. The threshold for VT (8.0 ± 0.2%) is significantly higher than those for dyspnea VAS (**p < 0.01) and fR (*p < 0.05), while the thresholds for dyspnea VAS (7.5 ± 0.1%) and fR (7.6 ± 0.2%) are similar (one-way ANOVA with Tukey’s multiple comparison post hoc tests). Values are means ± SEM.

subjects, fR did not increase with FETCO2 (Fig. 2A). In two subjects, fR started to increase at the beginning and then reached a plateau (Fig. 2B). Therefore, the statistical analyses included 16 subjects showing typical fR responses. In the five excluded subjects, the dyspnea VAS responses were similar to the others. However, rapid increases in dyspnea VAS after the breakpoint were not accompanied by increases in fR (Fig. 2A and B).

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Fig. 4. Correlation and agreement between the thresholds for dyspnea VAS and fR and between the thresholds for dyspnea VAS and VT . (A) The thresholds for dyspnea VAS and fR are positively correlated (n = 16, r = 0.81, p < 0.001), while the thresholds for dyspnea VAS and VT are not significantly correlated (n = 14, r = 0.40). (B) Bland–Altman plots for the difference between the thresholds for fR and dyspnea VAS (fR − VAS) against the mean of the thresholds for fR and dyspnea VAS [(fR + VAS)/2] in 16 subjects (left) and the difference between the thresholds for VT and dyspnea VAS (VT − VAS) against the mean of the thresholds for VT and dyspnea VAS [(VT + VAS)/2] in 14 subjects (right). The widths of the 95% limits of agreement are 1.6% for the thresholds for fR and dyspnea VAS and 2.5% for the thresholds for VT and dyspnea VAS (dashed lines). The 95% CI of the mean difference between the thresholds for VT and dyspnea VAS does not include zero (dotted lines, right panel), while that between the thresholds for fR and dyspnea VAS does include zero (dotted lines, left panel). The slopes of the regression analyses do not differ significantly from zero (thin solid lines).

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Fig. 5. Changes in inspiratory time (TI ), expiratory time (TE ), and the duty cycle (TI /TTOT ), given by dividing TI by the length of respiratory cycle (TTOT ), during CO2 rebreathing. (A) The mean regression lines and breakpoints of TI and TE for 16 subjects, who showed the typical fR response. (B) The TI /TTOT response to FETCO2 of the subject, presented in Fig. 1A, is shown in the left panel. TI /TTOT shows a slight downward inflection at 8.12% in this subject. The mean regression lines and breakpoint of TI /TTOT for the 16 subjects are shown in the right panel. (C) The TI /TTOT responses of the two subjects presented in Fig. 2. The left and right panels represent the subjects in Fig. 2A and B, respectively. Values are means ± SEM.

The mean regression lines and breakpoints for 14 subjects are shown in Fig. 1B. The trends were similar to those of the typical subject shown in Fig. 1A. We excluded two of the 16 subjects from the one-way repeated-measures ANOVA because they showed VT increases that were proportional to FETCO2 until the end, even though we successfully determined the thresholds for dyspnea VAS and fR in both patients. The mean dyspnea VAS scores at the threshold for dyspnea VAS were 10.7 ± 2.0 mm for the 14 subjects (Fig. 1B, top panel) and 10.1 ± 1.7 mm for the 16 subjects. The mean threshold values of the 14 subjects are shown in Fig. 3 and differed significantly among the three thresholds (p < 0.01, one-way repeated-measures ANOVA). Tukey’s multiple comparison post hoc tests showed that the threshold for VT (8.0 ± 0.2%) was significantly

higher than those for dyspnea VAS (p < 0.01) and fR (p < 0.05). The thresholds for dyspnea VAS (7.5 ± 0.1%) and fR (7.6 ± 0.2%) were similar (p = 0.732). The mean FETCO2 of the 14 subjects before CO2 inhalation was 5.6 ± 0.1%. The mean difference in FETCO2 between before CO2 inhalation and at the threshold for dyspnea VAS was 1.9 ± 0.1%. 3.2. Correlation between the thresholds for dyspnea VAS and ventilatory variables A positive correlation was found between the thresholds for dyspnea VAS and fR (Fig. 4A). Correlation analysis showed a significant relationship between the thresholds for dyspnea VAS and fR (n = 16,

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Fig. 6. Changes in the mean inspiratory flow (VT /TI ), given by dividing VT by TI during CO2 rebreathing. (A) A typical example, which is taken from the subject presented in Fig. 1A. (B) The VT /TI response of the subject presented in Fig. 2A. (C) The response of the subject presented in Fig. 2B. (D) The mean regression lines and breakpoint of VT /TI for all 21 subjects. Values are means ± SEM.

r = 0.81, p < 0.001) (Fig. 4A, left panel), but not between the thresholds for dyspnea VAS and VT (n = 14, r = 0.40, p = 0.162) (Fig. 4A, right panel). In the post hoc power analysis of the correlation between the thresholds for dyspnea VAS and VT , the statistical power was 28.3%.

3.3. Agreement between the thresholds for dyspnea VAS and ventilatory variables We assessed the level of agreement between the thresholds for dyspnea VAS and fR (Fig. 4B, left panel) and between the thresholds for dyspnea VAS and VT (Fig. 4B, right panel) using Bland–Altman plots, which suggested that the thresholds for dyspnea VAS and fR showed good agreement based on the narrow width of the 95% limits of agreement. The widths of the 95% limits of agreement were 1.6% of FETCO2 for the thresholds for dyspnea VAS and fR , and 2.5% for the thresholds for dyspnea VAS and VT . This suggests better agreement between the thresholds for dyspnea VAS and fR than between the thresholds for dyspnea VAS and VT . The 95% CI for the mean difference between the thresholds for dyspnea VAS and VT did not include zero (Fig. 4B, right panel), suggestive of systematic bias and differences between the thresholds for dyspnea VAS and VT , as suggested by the one-way ANOVA with Tukey’s multiple comparison post hoc tests. However, the 95% CI for the thresholds for dyspnea VAS and fR did include zero (Fig. 4B, left panel). This supports the similarities between the thresholds for dyspnea VAS and fR . Finally, regression analysis showed that the slope did not differ significantly from zero in either Bland–Altman plot (Fig. 4B), suggesting no proportional bias in either plot.

3.4. Thresholds for duty cycle and mean inspiratory flow We determined the thresholds for TI , TE , TI /TTOT (duty cycle), and VT /TI (mean inspiratory flow). The mean regression lines and breakpoints of TI and TE for 16 subjects who showed typical fR responses are shown in Fig. 5A. Both thresholds for TI and TE , where they started to decrease, were 7.6 ± 0.2%, and were similar to the threshold for fR for these 16 subjects (7.5 ± 0.2%). Fig. 5B and C shows changes in TI /TTOT with FETCO2 . The left panel in Fig. 5B shows TI /TTOT for the subject presented in Fig. 1A. In this subject, TI /TTOT shows a slight downward inflection at 8.12%. The mean regression lines and breakpoint of TI /TTOT for the 16 subjects are shown in the right panel of Fig. 5B. This suggests that the duty cycle, TI /TTOT , was broadly constant during CO2 rebreathing. The left and right panels of Fig. 5C show the TI /TTOT response to increasing FETCO2 in the two subjects presented in Fig. 2A and B, respectively. Again, the TI /TTOT value was broadly constant in both subjects who did not show the most common response. To summarize these findings, the tachypneic pattern was due to shortening of TI and TE with constant TI /TTOT . Breath-by-breath VT /TI was plotted against FETCO2 . All 21 subjects showed similar VT /TI responses to increasing FETCO2 . Fig. 6A represents a typical example, which is taken from the subject presented in Fig. 1A. The threshold for VT /TI , at which VT /TI starts to increase, was 6.23% in this subject. The threshold for VT /TI (6.23%) was lower than the threshold for fR (7.50%), and it was similar to the threshold for VE (6.25%). Fig. 6B and C shows the VT /TI response in the two subjects presented in Fig. 2. Fig. 6D shows the mean regression lines and breakpoint of VT /TI for all 21 subjects. In the 16 subjects who showed typical fR responses, the threshold for

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4.1. Changes in breathing pattern and dyspnea intensity

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Fig. 7. Hypercapnic ventilatory responses (HCVR) of all subjects (n = 21). The VE plot was fitted with a segmented linear regression model consisting of two segments connected by a breakpoint where VE began to increase. The slope of the second segment was considered to represent HCVR (L/min/% CO2 ). The subjects were classified into four groups on the basis of breathing pattern responses to CO2 rebreathing: (1) those who showed the most common pattern (common, n = 14), (2) those with no threshold for VT (no VT threshold, n = 2), (3) those in who fR started to increase at the beginning and then reached a plateau (early fR , n = 2), and (4) those with no fR response (no fR response, n = 3).

VT /TI (6.8 ± 0.2%) was significantly different to that for fR (7.5 ± 0.2%, p < 0.001, paired t-test). The threshold for VT /TI was significantly lower than the threshold for fR .

3.5. HCVR The HCVR values for all subjects (n = 21) are shown in Fig. 7. The subjects were classified into four groups based on their breathing pattern responses to CO2 rebreathing: (1) those who showed the most common pattern (common, n = 14), (2) those with no threshold for VT (no VT threshold, n = 2), (3) those in who fR started to increase at the beginning and then reached a plateau (early fR , n = 2), and (4) those with no fR response (no fR response, n = 3). Although we did not perform statistical comparisons because of the small numbers of subjects in the latter three groups, the HCVR values were in the lower range in subjects with no fR response.

4. Discussion In this study, we found that the thresholds for dyspnea VAS and fR were similar during CO2 rebreathing. In the initial phase of CO2 rebreathing, VT contributed to the ventilatory response more than fR . However, a tachypneic breathing pattern started when the respiratory stimulus reached the threshold for CO2 , with a trend toward leveling off of VT . Interestingly, the intensity of dyspnea also exhibited a threshold-like behavior at a similar level. The thresholds for fR and dyspnea VAS were significantly correlated and showed good agreement. In other words, the dyspnea intensity and fR both showed rapid increases that were virtually simultaneous. Our findings suggest that the start of tachypneic breathing is coupled with the threshold for dyspnea VAS. Our explanation for this is that changes in emotional states with dyspnea perception play a causative role in tachypneic breathing during hypercapnia. However, we did not identify emotions in the present study. This explanation awaits further testing with a direct measure of emotions in future studies.

The present results confirm the previously reported changes in breathing patterns during CO2 rebreathing. Changes in breathing patterns during hypercapnia have been reported by a number of investigators over the past century. Hey et al. (1966) reported the effects of various respiratory stimuli on the rate and depth of breathing. In their study, the combinations of the rate and depth of breathing were similar for hypercapnia at rest and during moderate exercise. Both stimuli increased VT first, and a tachypneic breathing pattern started with a plateau of VT once ventilation reached a threshold. This change in breathing pattern is consistent with our findings during CO2 rebreathing. Nevertheless, there are different types of changes in breathing patterns (Bechbache et al., 1979). Duffin et al. (2000) reported that, in some subjects, fR and VT contributed equally during the initial phase of hypercapnia, but fR and VT presented the most common response during hypercapnia, which is similar to that reported by Hey et al. (1966). In the present study, we excluded five subjects whose responses differed from the most common response to determine the common relationship between the thresholds for dyspnea VAS and fR . Nevertheless, it is worth noting that subjects with no fR response tended to have lower HCVR values. The lack of increases in fR may have resulted in the lower HCVR, but it is also possible that CO2 sensitivity is associated with the breathing pattern formation. This finding provides a basis for future work on this topic. 4.2. Relationship between thresholds for ventilatory variables and dyspnea intensity We found that changes in the subjective measure of dyspnea nearly coincided with those in the objective measure of respiration, particularly the onset of tachypneic breathing. The dyspnea intensity (i.e., VAS scores) increased gradually before reaching the threshold for FETCO2 , and then increased more rapidly thereafter. This pattern of response was similar to the changes in fR . Furthermore, the thresholds for fR and dyspnea VAS were significantly correlated and substantially agreeable. In other words, the inflection points for dyspnea VAS and fR were very close to one another. In addition, the VAS score for the intensity of dyspnea at the time when tachypneic breathing started was about 10 mm. In a study by Wan et al. (2008), the estimated FETCO2 values for the fR threshold ranged from 6.94% to 7.97% during CO2 rebreathing, depending on the level of anxiety and how many times the subject performed the test. They simultaneously measured the intensity of “hunger” for air and determined the threshold for air hunger as the perception of air hunger with an intensity of 5 on a 0–100 scale. However, the thresholds for fR and air hunger differed in their study, probably because they used a different method to determine the threshold for air hunger compared with that used to determine dyspnea in our study. Nevertheless, fR and air hunger started to rise simultaneously at an air hunger rate of about 10 on a 0–100 scale in the representative subject shown in Fig. 1 of their paper (Wan et al., 2008). Their findings seem to be consistent with our observations. The findings below suggest the following explanation for the onset of tachypneic breathing: unpleasantness accompanied by dyspnea reaches a level that induces emotional respiratory reactions to stimulate a tachypneic breathing pattern. The similar thresholds for dyspnea VAS and fR could explain the tachypneic breathing pattern that emerged with a strong respiratory stimulus in terms of emotional respiratory behavior. A prominent type of dyspnea experienced by healthy subjects during CO2 rebreathing is air hunger (Banzett et al., 1996; Moosavi et al., 2003; O’Donnell et al., 1997). In the present study, we directed the subjects to rate the intensity of dyspnea similar to that perceived

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during breath holding, and not to rate respiratory work/effort. It is accepted that the type of dyspnea felt during breath holding is air hunger (O’Donnell et al., 2007). Therefore, we believe our subjects primarily rated the intensity of air hunger. Banzett et al. (2008) reported that dyspnea causes negative emotions, and that air hunger is more unpleasant in relation to the intensity than respiratory work/effort from the affective dimension. A review article by Boiten et al. (1994) discusses the relationships between emotion and respiration, and describes many studies that have shown the effects of emotions on the rate and depth of respiration. As an extension of these earlier studies, Masaoka et al. (2005) demonstrated that unpleasantness at rest does not affect the metabolic rate, but does increase fR with a slight decrease in VT . Human neuroimaging studies have demonstrated connections between dyspnea and limbic structures (Nishino, 2011). Corfield et al. (1995) presented evidence for limbic system activation during CO2 breathing. Evans et al. (2002) showed anterior insular activation with air hunger. The contribution of pulmonary vagal reflexes to tachypneic breathing needs to be considered. We analyzed respiratory volume–time relationships to discern between vagal reflex control and behavioral influences in tachypneic breathing during hypercapnia. Clark and von Euler (1972) proposed that the depth and rate of breathing are determined by (1) the central inspiratory drive, represented by VT /TI , and (2) the inspiratory off-switch mechanism. Increased VT /TI accelerates filling of the lung, leading to an early termination of inspiration, specifically in the range above 1.5–2 times the eupneic VT values in humans. In the present study, VT /TI increased linearly with the levels of CO2 above the threshold for VT /TI . However, this threshold was significantly lower than the threshold for fR . This suggests that increases in VT /TI do not solely explain the start of tachypneic breathing at the threshold for fR . From the perspective of Clark and von Euler (1972), the early termination of inspiration, which was observed above the threshold for TI , may be due to increased excitability of the inspiratory off-switch mechanism. For behavioral influences, we suggest that dyspnea and negative emotions are associated with the decreased TE , rather than TI . Clark and von Euler (1972) further proposed that the duration of TE depends on the duration of the preceding TI . In the present study, both TI and TE started to decrease almost simultaneously at the threshold for fR , and TI /TTOT was broadly constant during CO2 rebreathing. These findings are consistent with Clark and von Euler’s proposal. However, recent studies suggest that a central pattern generator for respiration, which includes rhythm generators, determines the start of inspiration (Onimaru et al., 2009), and that respiratory rhythm generation is influenced by various inputs from higher centers (Homma and Masaoka, 2008). Masaoka and Homma (1999) showed that the magnitude of reduction in TE is correlated with individual anxiety levels during physical load and noxious audio stimulation. These earlier observations suggest that the duration of TE can change independently from the duration of TI . We believe that the decreased TE was due to modification of respiratory rhythm generation primarily by behavioral influences during CO2 rebreathing, while vagal reflexes were associated with the decreased TI . The possibility that peripheral chemoreceptors are involved in tachypneic breathing needs to be considered. Tachypneic breathing also occurs during exercise at the VT plateau (Hey et al., 1966), which is consistent with our results during CO2 rebreathing. Martin and Weil (1979) showed that fR and VT inflected simultaneously at the ventilatory (anaerobic) threshold, where fR starts to increase rapidly and VT stabilizes. Amiard et al. (2007) demonstrated that the threshold for dyspnea and the ventilatory threshold occur concomitantly. Increased H+ stimulation of the carotid chemoreceptors is the most prevalent explanation for the hyperventilation during heavy exercise in humans (Dempsey et al., 1995). However,

during hyperoxic hypercapnia, as used in our study, the carotid chemoreceptors are likely to be silent because hyperoxia markedly diminishes carotid body activity (Lahiri and DeLaney, 1975). Mechanisms determining the level of total ventilation required for the metabolic demand may differ between exercise and CO2 rebreathing. However, assuming that the same mechanism determines the breathing patterning threshold, our results indicate that behavioral pathways are likely to be responsible for tachypneic breathing.

4.3. Study limitations Several mechanisms may explain the coupling between dyspnea perception and tachypneic breathing. Indeed, we found that the intensity of dyspnea started to rise near the threshold for fR rather than the threshold for VT . However, we must acknowledge that a cause-and-effect relationship between dyspnea perception and respiratory rhythm remains to be proven. An alternative behavioral explanation for tachypneic breathing is that voluntary control of breathing increases fR because the act of breathing relieves respiratory discomfort (Chonan et al., 1987; Harty et al., 1996). Why did fR not increase when the dyspnea VAS increased rapidly at the breakpoint in five subjects? If the tachypneic breathing pattern is mainly caused by emotional changes, the levels of emotional reaction to dyspnea perception would be different in these subjects compared with those showing the most common response. We should have simultaneously measured the emotional reactions to confirm this possibility. Alternatively, the contribution of behavioral breathing to the ventilatory response is likely to small in subjects who show an uncoupling between dyspnea perception and respiratory rhythm. We also need to consider the possibility that these subjects primarily rated respiratory work/effort, which causes fewer emotional responses (Banzett et al., 2008). The simulation we used to induce air hunger may receive some criticism. Dyspnea-related negative emotions are experienced when subjects perceive air hunger induced by hypercapnia at 6.1–7.7 mmHg of the end-tidal CO2 pressure above baseline combined with restricted ventilation (Banzett et al., 2008). However, ventilation in our subjects was not specifically restricted. It was reported that the act of breathing reduces the level of air hunger during hypercapnia (Banzett et al., 1996). This raises the question of whether the level of air hunger was sufficient to cause negative emotions in our subjects. However, in the present study, the mean difference in FETCO2 between baseline and the threshold for dyspnea VAS was 1.9 ± 0.1%, equivalent to approximately 13.5 mmHg. A rise in the end-tidal CO2 pressure increases the level of air hunger even if ventilation is not restricted (Banzett et al., 1996). Although neither we nor Banzett et al. identified emotions associated with dyspnea during hypercapnic free breathing, we believe air hunger caused the negative emotions in our subjects. The repeated exposure to hypercapnia possibly affected the relationship between the thresholds for dyspnea VAS and breathing pattern components. Our subjects were exposed to hypercapnia twice with a 15-min interval, and we analyzed data from the second exposure. Wan et al. (2008) demonstrated that the threshold for fR during CO2 rebreathing increases across trials, whereas the threshold for air hunger does not change. However, they used a different method to determine the threshold for air hunger compared with our method. They measured the ‘just noticeable’ level of air hunger. Therefore, their results do not necessary imply that the thresholds for dyspnea VAS, which we measured in the present study, are constant. Instead, the similarity between the thresholds for fR and dyspnea VAS during the second hypercapnic exposure suggests that the threshold for dyspnea VAS, where dyspnea starts to increase rapidly, may increase across trials along with an increase in the threshold for fR .

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We did not include female subjects in the present study because the menstrual cycle affects HCVR (Dutton et al., 1989). Jensen et al. (2005) reported gender differences in the VT response to hypercapnia. Thus, our findings may only be applicable to a male population. However, the exclusion of female subjects eliminates any confounding factors associated with the menstrual cycle and gender differences, which is particularly important considering the possible effects of testosterone on peripheral chemoreflex sensitivity (Mateika et al., 2004). Nevertheless, this is unlikely to be an issue in the present study because the carotid chemoreceptors were likely silent under hyperoxic conditions. We found a significant correlation between the thresholds for dyspnea VAS and fR (r = 0.81), but not between the thresholds for dyspnea VAS and VT (r = 0.40). The latter may be due to the low statistical power because of the small number of subjects. However, the correlation between the thresholds for dyspnea VAS and VT was weak. 4.4. Clinical relevance Dyspnea is a very important symptom of chronic obstructive pulmonary disease (COPD). Lung hyperinflation is a physiologic abnormality in patients with COPD. It progresses dynamically during exercise because of limited expiratory flow, contributing to the occurrence of dyspnea and limits the exercise capacity in these patients (O’Donnell et al., 2007). Based on our findings, the resultant dyspnea potentially worsens hyperinflation in COPD because tachypneic breathing patterns likely decrease the duration of TE and lead to worsening of air trapping. Cooper (2006) has already described a similar dyspnea-related vicious circle in terms of patient-centered outcomes of COPD. The qualitative aspects of breathlessness were recently described by Smith et al. (2009), who concluded that air hunger is the dominant sensation during exercise in patients with pulmonary disorders, including COPD. Furthermore, affective components are particularly important at rest in such patients. Therefore, it is worth investigating whether a vicious circle of worsening hyperinflation caused by dyspnea perception is associated with further impairments in quality of life outcomes in COPD. 5. Conclusions The thresholds for dyspnea VAS and fR during hypercapnia were significantly correlated and showed good agreement. Our results suggest that the start of tachypneic breathing is coupled with the threshold for dyspnea. Metabolic and behavioral breathing seem to be coordinated to produce the final respiratory output not only during normal daily activities but also during exposure to physical stimuli. Thus, we are planning future experiments to establish the cause-and-effect relationship between dyspnea perception and respiratory rhythm during physical stimuli to provide new insights into the mechanisms underlying breathing control in conscious humans. Future directions of this work should include exploring whether dyspnea perception contributes to tachypneic breathing patterns in pulmonary diseases and particularly whether it leads to further progression of dynamic hyperinflation in patients with COPD. Acknowledgement This work was partially supported by KAKENHI (21500491). References Amiard, V., Jullien, H., Nassif, D., Maingourd, Y., Ahmaidi, S., 2007. Relationship between dyspnea increase and ventilatory gas exchange thresholds during

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