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1Biomedical Engineering Department, Boston University, Boston, Massachusetts; 2Pfizer Inc., Groton, Connecticut; and 3School of Veterinary Medicine, University of California, Davis, California
Submitted 15 September 2004 ; accepted in final form 7 November 2005
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ABSTRACT |
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airway resistance; methacholine; histamine; Ascaris suum; augmented breaths
There have been a very limited number of studies examining the change in deep-breath frequency during increased resistive loads in humans or animals. One such study, by Orehek et. al. (36), has shown that deep-breath frequency increases in asthmatic subjects with carbachol-induced bronchoconstriction and returns to baseline (BL) levels following administration of a bronchodilator.
It is generally accepted that deep breaths are regulated by vagal afferents from lung rapidly adapting receptors (RARs) (3, 8–10, 16, 25, 31, 46, 47). However, the mode of stimulation of the RARs is unclear and seems to be a somewhat ambiguous combination of mechanical (21–23, 25, 42, 43, 49) and chemical (31, 42, 44, 52) stimuli.
The hypothesis tested in this study was that deep-breath frequency increases following experimentally induced bronchoconstriction, and the magnitude of the increase is dependent on the mechanism used to induce the constriction. To this end, cynomolgus monkeys were challenged with methacholine (MCh), Ascaris suum (AS), histamine, or an added external resistance, and the deep-breath frequencies, observed during bronchoconstriction, were compared with BL levels. In analyzing the change in deep-breath frequency as well as the simultaneous changes in the respiratory and ventilatory parameters, we hope to elucidate a relationship between the mechanical characteristics and neural regulation of the respiratory system.
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METHODS |
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Nine male cynomolgus monkeys (Macaca fascicularis) with a mean weight of 7.3 kg (SD 1.24) (4.9–8.5 kg) were studied. All monkeys demonstrated AS sensitivity evaluated by both skin test and airway hyperreactivity. The animals were located and the studies were performed at Pfizer, Groton, CT. The facility is accredited by the American Association for the Accreditation of Laboratory Animal Care, and all animals were maintained, according to the standards specified in the Institute of Laboratory Animal Resources (18, 19). The Internal Animal Care and Use Committee at Pfizer approved all experimental protocols.
Experimental Protocol
The animals were anesthetized with xylazine (1 mg/kg) and ketamine (10 mg/kg), intubated with a cuffed endotracheal tube, and seated upright in a head-out body plethysmograph box during experimentation.
Each of the nine monkeys was tested on four different occasions. On 1 day, a 60-min trial was performed to determine the deep-breath frequency during BL conditions. On the other 3 days, BL measurements were made (2, 5, or 15 min) to determine mean Raw, tissue compliance (Cti), and ventilatory parameters. The BL measurements were followed by a dose-response determination, involving a specific aerosolized agent, MCh, AS, or histamine, to determine their effect on deep-breath frequency, Raw, Cti, and the ventilatory parameters. On these challenge days, measurements were made for 15 min following each dose, but only the data at the dose that most closely doubled the mean BL Raw are reported. On 1 of the challenge days, following BL measurements and before the dose-response determination, measurements were made (15 min) with the external resistance.
Bronchoconstrictor Challenge
The pharmacological challenges (MCh, AS, histamine) were delivered in an aerosolized phosphate-buffered saline solution via an ultrasonic nebulizer (97% of particles are 1–3 µm) connected to the endotracheal tube through a mechanical ventilator (Harvard Apparatus), whose tidal volume (VT) was adjusted according to the monkey's body weight. The agents were delivered for 2 min at increasing concentrations, until the total BL respiratory resistance was approximately doubled. The actual concentrations that elicited a doubling of the respiratory resistance were different for each monkey and challenge. For the external resistance challenge, an external mechanical resistor was attached, in series, to the endotracheal tube. The resistor was standard for all trials and consisted of multiple mesh screens designed to provide a resistance equivalent to the mean of the BL Raw of the monkeys. This would, on average, double the monkey's effective Raw.
Transfer Impedance Measurements
Transfer impedance (Ztr) data were obtained by measuring pressure oscillations imposed on the monkey's chest wall and the resulting flow at the airway opening, as described in detail by Madwed and Jackson (27), with one modification. The flow signal was split into two analog channels. The first analog channel was band-pass filtered (2 > frequency > 256 Hz) and represented the oscillatory flow that resulted from the oscillatory pressure imposed on the chest wall. This signal was used in the calculation of Ztr. The second analog channel was low-pass filtered (frequency > 2 Hz) and used as a measure of the tidal flow since the superimposed oscillatory flow was removed. Integration of this signal resulted in changes in volume, from which VT values were computed. This flow channel was not calibrated, and thus absolute values of VT were not calculated. Instead, VT for each monkey, during each challenged condition, was normalized by the mean of their BL VT measured on that same day.
Data Analysis
Analysis of Ztr data. The DuBois six-element model (13) was fit to the Ztr data to determine the values of Raw and Cti (27). Each Ztr spectrum was fit with the model, generating a Raw and Cti estimate per s, thus providing a representation of the time dependence of these parameters. The analysis was performed for BL, and each challenge and the percent changes in Raw and Cti were calculated.
Analysis of ventilatory parameters.
Changes in lung volume were obtained by numerical integration of the tidal flow signal. The VT was obtained in arbitrary units for each breath using a peak-and-valley detection method. The breath-by-breath respiratory rate (RR) was obtained by calculating the inverse of the period of each breath. The challenge VT and RR levels were normalized by the mean BL VT and RR, respectively. Minute ventilation (
E) was calculated from the product of VT and RR for each respiratory cycle. The mean challenge VT was normalized to the mean BL VT. The mean challenge RR and E were normalized to their mean BL values.
Deep-breath analysis. The deep-breath frequency was determined by calculating the number of deep breaths divided by the trial time. To distinguish between deep breaths and normal breaths for each trial, the histogram of the VT values for the specific trial was qualitatively considered. The population with the vast majority of breaths was considered to be the non-deep breaths, and the outliers that were larger were considered to be the deep breaths. Deep breaths were then separated into augmented and nonaugmented breaths by considering the derivative of flow (volume acceleration). Deep breaths were classified as nonaugmented breaths, if the slope of the flow curve (i.e., flow acceleration), during the inspiratory phase of the deep breath, was never negative (Fig. 1A). Those breaths in which flow acceleration was negative for some portion of the inspiratory phase were considered to be augmented breaths (Fig. 1B) (7, 16, 47, 50, 51).
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Statistical Analysis
For each modality, the mean and SD of the changes, from BL, in the deep-breath frequency, as well as the respiratory and ventilatory parameters, were calculated for the entire sample population. ANOVA tests were performed to determine whether the different modalities produced significant differences in one or more of the parameters. Additionally, paired t-tests were performed to elucidate significant differences produced by specific modalities within each parameter. Statistical significance was determined at P < 0.05.
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RESULTS |
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The mean deep-breath frequencies for BL, MCh, AS, histamine, and external resistance challenges were 0.061 (SD 0.046), 0.156 (SD 0.173), 0.370 (SD 0.186), 0.467 (SD 0.216), and 0.117 breaths/min (SD 0.082), respectively. The deep-breath frequencies during AS and histamine challenge were significantly higher than during BL conditions, but MCh and external resistance deep-breath frequencies were not different from BL. There were no significant differences in the mean deep-breath frequency when comparing AS and histamine challenge.
Deep-Breath Volumes and Characteristics
Mean BL deep-breath volume [2.31 (SD 0.65)] was not significantly different from the postchallenge deep-breath volumes for MCh, histamine, and the external resistance [1.90 (SD 0.29), 1.90 (SD 0.49), and 1.95 (SD 0.62), respectively] but was significantly greater than the deep-breath volumes during AS challenge [1.37 (SD 0.59)]. Comparing the deep-breath volumes among the modalities, there was a significant difference only between AS and the external resistance.
The percentages of deep breaths that were augmented breaths for BL conditions and during MCh, AS, histamine, and external resistance challenges were 97.5 (SD 7), 64.3 (SD 50.1), 92.5 (SD 12.8), 68.6 (SD 36.3), and 90.6% (SD 26.5), respectively. The only significant differences occurred between BL and histamine, as well as between histamine and external resistance.
Comparing the characteristics of the nonaugmented and augmented breaths indicated that there were significant differences between VT [2.12 (SD 0.46) and 1.72 (SD 0.66), respectively] and VT/TI [1.05 (SD 0.30) and 0.77 (SD 0.29), respectively], but there was no significant difference between TI [2.16 (SD 0.43) and 2.42 s (SD 0.89), respectively] or expiratory time [2.57 (SD 0.65) and 2.20 s (SD 0.79), respectively]. Because there was no difference in TI, the difference in the VT/TI is due to the difference in VT. A comparison of the histograms of these parameters for nonaugmented and augmented breaths did not indicate the presence of two separate populations.
Respiratory Parameters
Raw. The mean postchallenge percent increases in Raw were statistically significant compared with BL for MCh, AS, histamine, and an external resistance [98.9 (SD 51.1), 98.0 (SD 37.8), 108.0 (SD 59.5), and 165.3% (SD 85.7), respectively]. An ANOVA test suggested that there were no significant differences in the percent changes in Raw among the four modalities. This characteristic was confirmed through paired t-tests, which indicated that there were no significant differences in the percent changes in Raw when comparing any two modalities.
Cti. The mean postchallenge percent decreases in Cti were statistically significant compared with BL for MCh, AS, histamine, and external resistance challenges [–48.0 (SD 21.0), –53.8 (SD 19.1), –37.0 (SD 20.7), and –19.0% (SD 9.6), respectively]. An ANOVA test indicated that there were significant differences in the Cti percent changes among the four modalities. Indeed, paired t-tests confirmed that the percent decreases in Cti were similar for MCh, AS, and histamine challenges, and these were significantly greater than the percent decrease due to the external resistance.
Ventilatory Parameters
VT. An ANOVA test indicated that there were significant differences in the postchallenge non-deep-breath VT values, normalized to their BL VT, among the four modalities. Furthermore, paired t-tests confirmed that non-deep-breath VT values were significantly smaller for AS and histamine challenges [0.67 (SD 0.13) and 0.79 (SD 0.15), respectively] compared with BL, but they were not significantly different for the MCh or external resistance [1.04 (SD 0.32) and 1.07 (SD 0.14), respectively]. There was a significant difference between non-deep-breath VT for AS and histamine, but there was no significant difference between MCh and external resistance.
RR. Mean RRs for MCh, AS, and histamine [1.39 (SD 0.35), 1.75 (SD 0.45), and 1.32 (SD 0.24), respectively] were significantly increased compared with BL. The mean RR for the external resistor [1.03 (SD 0.09)] was not significantly different from BL.
E.
The mean normalized E were not statistically significant compared with BL for MCh, AS, histamine challenges, or the external resistance [1.14 (SD 0.47), 1.00 (SD 0.28), 1.05 (SD 0.17), and 1.03 (SD 0.19), respectively]. An ANOVA test indicated that there were no significant differences in the E responses among the four modalities, and this characteristic was confirmed through paired t-tests.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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All modalities increased mean deep-breath frequency, with the increase due to histamine and AS being significantly different from BL, whereas the increases due to MCh and the external resistance were not. Given that each modality increased deep-breath frequency and the differences in the effects of each modality, our data suggest that, while alterations in Raw and Cti may contribute, the primary factors that elevate deep-breath frequency are increased vascular permeability (AS and histamine) and/or direct activation of RARs (histamine).
Most studies that have reported a relationship between deep breaths and altered mechanical loads have measured either the occurrence of a deep breath with decreased capacitive loads or changes in compliance resulting from deep breaths (3, 4, 14, 29, 46, 50). Mead and Collier (29) showed that, during periods of normal tidal breathing, compliance decreases, but, without exception, these compliance changes were reversed following a deep breath. Ferris and Pollard (14) demonstrated that a deep breath elicited an increase in compliance, and, during a period of normal tidal breathing, following the deep breath, compliance decreased back toward pre-deep-breath values. Both of these investigators (14, 29) attributed the decrease in lung compliance to an increase in surface tension at the liquid-gas interface, resulting from maldistribution or reabsorption of surfactant. Thus it has become widely accepted that deep breaths act as a regulatory mechanism to increase compliance by the redistribution of surfactant (3, 4, 14, 29, 46, 50). While there have been numerous studies that have investigated the relationship between altered elastic loads and deep breaths, we were able to find only one study in the literature that reported deep-breath frequency during increased resistive loads (36). This study reported that carbachol-induced bronchoconstriction in asthmatic humans elicits an increase in deep-breath frequency.
Although the intended goal of the study was to increase Raw, all of the modalities also resulted in a significant decrease in dynamic Cti. The concomitant decrease in Cti with the increase in Raw could be caused by two different mechanisms in the three pharmacological modalities (MCh, AS, and histamine). First, the challenge agent could directly alter the inherent lung tissue properties, resulting in decreased Cti. The second mechanism could be decreased effective, dynamic compliance due to increased parallel Raw heterogeneity with little or no direct effect on surface tension or Cti. Unfortunately, it is not possible to separate the changes in deep-breath frequency due to increased Raw from those due to decreased effective dynamic compliance. The increased deep-breath frequency observed following the pharmacological modalities could be due to increased Raw, decreased Cti, or a combination of both. However, with the added external resistance, increased heterogeneity would not be expected, and the decrease in compliance could only be attributed to altered tissue properties, presumably because the animals breathed at a higher or lower lung volume.
MCh, AS, and histamine challenges resulted in rapid, shallow breathing compared with BL conditions, which was consistent with what has been reported in the literature (e.g., Refs. 24, 30, 31, 35, 37). The data indicate that, among the modalities, there were no significant differences in VT, RR, or E that could account for changes in deep-breath frequency, and there is nothing in the literature to suggest that this happens.
Each modality would be expected to change the discharge pattern of one or more of the known lung sensory nerves: slowly adapting receptors, RARs, and/or lung C fibers. However, it is widely accepted that stimulation of lung RARs is responsible for deep breaths (3, 8–10, 16, 25, 31, 46, 47). Our data show that AS and histamine challenges resulted in a similar, significantly elevated deep-breath rate compared with BL, whereas MCh challenge and an added external resistance produced no significant change in deep-breath rate compared with BL. Thus we can infer that AS and histamine initiated RAR stimulation, whereas MCh and an added external resistance did not elevate RAR activity from BL levels.
RARs have been shown to increase their activity in response to numerous mechanical stimuli, including forced inflations and deflations of the lung (25, 31), decreases in compliance (21, 42), or increased mucosal interstitial hydrostatic pressure (22, 23, 43). RARs have also been shown to be affected by chemical stimuli, including aerosol or intravenous administering of histamine (11, 31, 44, 52) or acetylcholine (11), as well as inhalation of substances such as allergen (5), phenyl diguanide (31), ammonia gas (31, 44), cigarette smoke (42, 44), carbon dust (42), alcohol, acetone, or ether (44). The degree that any of these substances act by way of specific receptors on RARs or indirectly by one of the other identified mechanisms is uncertain.
In this study, Raw and Cti during bronchoconstriction were significantly different compared with BL values. Furthermore, Raw was not significantly different among the challenges, and Cti with the external resistance was significantly different compared with MCh, AS, and histamine. If the RARs were stimulated solely by altered lung mechanics, then their activity should be similar for each modality and the corresponding deep-breath rates should also be similar, which was not the case.
Histamine and AS may work indirectly to stimulate RAR activity because of the increased interstitial fluid resulting from the increased permeability of the capillaries and bronchial venules upon exposure to histamine. This is consistent with reported evidence that increased left atrial pressure and increased lung parenchymal extravascular fluid volume increase RAR activity (22, 23, 43), which is thought to be the result of anastomoses between the bronchial and pulmonary veins (32).
One possible discrepancy occurs between the reported location of the RARs and the site of action of AS and histamine. It has been reported that the total lung resistance increases that occur following AS challenge or histamine challenge primarily affect the peripheral airways (12, 37). Thus we can infer that the site of action of AS and histamine is in the smooth muscles of the bronchioles rather than the trachea and cartilagenous bronchi, where the RARs are reported to be located (16). However, allergen and histamine challenges have been shown to increase vascular permeability in the large airways (6, 34). It could be that there is histamine release in central and peripheral airways, resulting in RAR activation by extravascular fluid centrally and smooth muscle constriction peripherally.
The vast majority (79%) of the deep breaths that we observed were augmented breaths. It would be of interest to determine whether the augmented breaths and nonaugmented deep breaths are maneuvers controlled by two different mechanisms. If this was the case, one might expect some characteristic, or characteristics, related to their shapes to be represented by two separate populations. There were differences in VT and VT/TI for the two groups, which suggest the possibility of different mechanisms. However, histograms of these parameters for nonaugmented and augmented deep breaths do not indicate that they are represented by two distinctly separate populations. A more detailed analysis, or separate study, might provide deeper insight into this question, but this is beyond the scope of the present study.
In conclusion, our data show that deep-breath frequency is increased, and non-deep-breath VT is decreased, compared with BL, during bronchoprovocation with AS and histamine. However, deep-breath frequency is not significantly changed from BL during administration of MCh or with an added external resistance. Because all modalities resulted in similar changes in Raw and Cti, these data suggest that the Raw and/or Cti changes are not the sole mechanisms responsible for RAR activity. Instead, the increased vascular permeability that accompanies AS and histamine challenge likely acts through one or more mechanisms to increase RAR activity and deep-breath frequency.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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