Phasic mechanoreceptor stimuli can induce phasic activation of upper airway muscles in humans

doi:10.1111/j.1469-7793.2001.0677h.x

Journal List > J Physiol > v.531(Pt 3); Mar 15, 2001

J Physiol. 2001 March 15; 531(Pt 3): 677–691.

doi: 10.1111/j.1469-7793.2001.0677h.x.

PMCID: PMC2278497

Phasic mechanoreceptor stimuli can induce phasic activation of upper airway muscles in humans

Toshiki Akahoshi, David P White, Jill K Edwards, Josee Beauregard, and Steven A Shea

Harvard Medical School and Division of Sleep Medicine, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, MA 02115, USA

Corresponding author S. A. Shea: Circadian, Neuroendocrine and Sleep Disorders Section, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, MA 02115, USA. Email: steven_shea/at/hms.harvard.edu

Received August 28, 2000; Accepted October 31, 2000.

This article has been cited by other articles in PMC.

Abstract

Upper airway dilator muscles are phasically activated throughout breathing by respiratory pattern generator neurons. Studies have shown that non-physiological upper airway mechanoreceptive stimuli (e.g. rapidly imposed pulses of negative pressure) also activate these muscles. Such reflexes may become activated during conditions that alter airway resistance in order to stabilise airway patency.
To determine the contribution of ongoing mechanoreceptive reflexes to phasic activity of airway dilators, we assessed genioglossal electromyogram (GG EMG: rectified with moving time average of 100 ms) during slow (physiological) oscillations in negative pressure generated spontaneously and passively (negative pressure ventilator).
Nineteen healthy adults were studied while awake, during passive mechanical ventilation across normal physiological ranges of breathing rates (13–19 breaths min⁻¹) and volumes (0.5–1.0 l) and during spontaneous breathing across the physiological range of end-tidal carbon dioxide (P_ET,CO₂; 32–45 mmHg).
Within-breath phasic changes in airway mechanoreceptor stimuli (negative pressure or flow) were highly correlated with within-breath phasic genioglossal activation, probably representing a robust mechanoreceptive reflex. These reflex relationships were largely unchanged by alterations in central drive to respiratory pump muscles or the rate of mechanical ventilation within the ranges studied. A multivariate model revealed that tonic GG EMG, P_ET,CO₂ and breath duration provided no significant independent information in the prediction of inspiratory peak GG EMG beyond that provided by epiglottic pressure, which alone explained 93 % of the variation in peak GG EMG across all conditions. The overall relationship was: Peak GG EMG = 79.7 − (11.3 x Peak epiglottic pressure), where GG EMG is measured as percentage of baseline, and epiglottic pressure is in cmH₂O.
These data provide strong evidence that upper airway dilator muscles can be activated throughout inspiration via ongoing mechanoreceptor reflexes. Such a feedback mechanism is likely to be active on a within-breath basis to protect upper airway patency in awake humans. This mechanism could mediate the increased genioglossal activity observed in patients with obstructive sleep apnoea (i.e. reflex compensation for an anatomically smaller airway).

Obstructive sleep apnoea (OSA) is a common disease characterised by repeated pharyngeal collapse during sleep. Upper airway patency depends on the balance between the dilating force of pharyngeal muscles and the collapsing force of intra-luminal negative pressure (generated during inspiration by respiratory ‘pump’ muscles) (Remmers et al. 1978). Many pharyngeal muscles are phasically active with breathing, yet we are uncertain whether the increased dilator muscle activation throughout inspiration spontaneously emanates from the respiratory pattern generator or is primarily a product of ongoing upper airway mechanoreceptor reflexes. If ongoing upper airway mechanoreceptor reflexes predominate, this would provide a feedback mechanism whereby local changes in the mechanical conditions of the upper airway could directly affect dilator muscle activity and protect upper airway patency within each breath.

Numerous studies have demonstrated that imposing large, rapid pulses of negative pressure can activate pharyngeal dilator muscles (Horner et al. 1991a, b; Wheatley et al. 1993). The principal aim of this research was to determine whether or not this reflex is active during normal breathing. Our three specific aims were to determine: (i) the effects on pharyngeal muscle activation of upper airway pressure changes that are slow, phasic and within the physiological range (i.e. at normal breathing frequencies and tidal volumes), (ii) whether such physiological mechanoreceptive stimuli can induce phasic activation of upper airway muscles when the spontaneous central respiratory drive to the diaphragm is relatively quiescent, and (iii) whether increased spontaneous central respiratory drive affects the relationship between upper airway mechanoreceptor stimuli and pharyngeal muscle activation. For aim (i), a negative pressure ventilator was used to apply slow, physiological mechanoreceptor stimuli at a range of breathing frequencies. For aim (ii), to lessen the effects of central respiratory drive, subjects were studied during hypocapnia while being mechanically ventilated, as hypocapnia is associated with a reduction or cessation of spontaneous breathing (Shea, 1996). For aim (iii), hypercapnia was induced to increase spontaneous central respiratory drive. We chose to study the genioglossus (GG), as this is one of the principal upper airway dilator muscles (Remmers et al. 1978; Kobayashi et al. 1996). If upper airway mechanoreceptor reflexes are primarily responsible for phasic GG activation, we would expect phasic GG activation to persist throughout passive mechanical ventilation, that the magnitude of this phasic GG activity would be related to the level of passive mechanical ventilation, and that the relationship between upper airway mechanoreceptor stimuli and GG activity would be similar during passive mechanical ventilation and spontaneous breathing. On the other hand, if the spontaneous central respiratory drive to the diaphragm is primarily responsible for phasic GG activity, we would expect little phasic GG activation during passive mechanical ventilation and increased phasic GG activity when spontaneous breathing becomes stimulated during hypercapnia.

METHODS

The primary variables of interest were upper airway pressure and flow (the stimuli), and the level of phasic GG activity (the response). To avoid subject discomfort, is was not possible to collect data in all conditions in one experimental session. Therefore, we performed two experiments. Experiment A was designed to establish the relationship between upper airway mechanoreceptor stimuli and GG activity during passive mechanical ventilation at two controlled levels of upper airway pressure and at two breathing frequencies. Experiment B was designed to determine whether increased central respiratory drive (hypercapnia) affected the relationship between upper airway mechanoreceptor stimuli and GG activity found during passive mechanical ventilation.

Subjects

The protocol was approved by the local Human Subjects Committee, and was performed in accordance with the Declaration of Helsinki. Subjects provided written informed consent. None of the subjects had symptoms of neurological, cardiovascular, pulmonary or sleep disorders or a history of snoring, hypersomnolence or smoking. Women were studied in the follicular phase of their menstrual cycle. No subject had taken alcohol or medication on the day of the study. We studied 22 healthy adults. Three of these subjects did not complete the experiment because they could not meet the criteria for establishing passive mechanical ventilation. Of the 19 remaining subjects, 12 were men and 7 were women. Their ages ranged from 23 to 44 years and their body mass indices were normal (ranging from 18.0 to 24.5). We did not perform an overnight screening polysomnogram to exclude OSA syndrome, as OSA is very unlikely to occur in this population based on medical history, age, body habitus and questionnaire results.

Physiological monitoring

Subjects were instrumented with two channels of electro-encephalography and one channel of electro-oculography to document wakefulness. The genioglossal electromyogram (GG EMG) was measured using a pair of intramuscular electrodes referenced to a single earth. Following mild topical anaesthesia with aerosolised 4 % lidocaine (lignocaine) on the floor of the mouth, two 25 gauge needles, each containing a 36 gauge stainless steel wire, were inserted 15-20 mm into the body of the GG muscle bilaterally, 3-5 mm lateral to the frenulum and anterior to the lingular salivary duct. The needles were then removed leaving the recording wires in place. Diaphragmatic EMG (DIA EMG) was obtained from electrodes placed at the right sixth to eighth intercostal spaces adjacent to the costal margin (assessed only in experiment A). Airway pressures were measured using catheters with pressure transducers in their tip (MPC-500, Millar Instruments, Houston, TX, USA) placed at the choanae and the epiglottic regions of the airway, following nasal decongestion (2-3 inhalations of oxymetazoline HCl, 0.05 %) and light topical anaesthesia (< 1 ml of 4 % lidocaine aerosol). Subjects were instructed to breathe exclusively through a nasal mask connected to one-way inspiratory and expiratory valves. Pressure in the mask was also determined using a differential pressure transducer. Inspiratory airflow was measured with a pneumotachometer and a differential pressure transducer. Inspiratory tidal volume was determined from the integrated flow signal. End-tidal partial pressure of carbon dioxide (P_ET,CO₂) was measured from expired air sampled within the mask. All measurements were recorded on a chart recorder, and selected signals were digitised (12 bit resolution: 4096 levels per channel) at 200 Hz and stored on a microcomputer for off-line analysis.

Experimental protocol

Subjects reported to the laboratory after fasting for at least 3 h. Following an acclimatisation period of at least 10 min with all equipment in place, the basal eucapnic level for each subject was estimated as the mean P_ET,CO₂ over a period of 5-10 min while the subject was relaxed, awake and supine. Subjects were asked to remain awake with eyes open for all experimental conditions.

Non-invasive negative pressure ventilator (‘iron lung’) Subjects were studied while supine with the head outside and body within a negative pressure ventilator (Iron Lung; Series J; Emerson, MA, USA). The ventilator was switched on only for specific parts of the experiment. This device was lightly sealed around the neck with a flexible twisted nylon sheet while an external piston created negative pressure that gently sucked on the chest wall thus assisting each breath. The iron lung could be adjusted to achieve the desired upper airway pressure and breathing frequency. To control the level of P_ET,CO₂, the inspired fraction of CO₂ was adjusted. All subjects required some initial coaching to enable passive mechanical ventilation. This involved asking the subjects to remain completely relaxed and providing feedback on a breath-by-breath basis to achieve consistent timing and shapes of the pressure and flow traces. Recordings were stopped when there was departure from this passive pattern until adequate passivity could be achieved, or the experiment was terminated.

Experiment A (11 subjects) Measurements were made in seven steady-state conditions (Table 1). The conditions included spontaneous breathing and passive mechanical ventilation at two controlled levels of peak epiglottic pressure (peak P_epi) (i.e. medium (-6 cmH₂O) and high (~-10 cmH₂O)), at two breathing frequencies (rapid (breath duration ~ 3 s) and slow (breath duration ~ 5 s)), and at two levels of P_ET,CO₂ (eucapnia and hypocapnia (8 mmHg below eucapnia)).

Table 1

Protocols for experiments A and B

Experiment B (8 subjects) Measurements were made in four steady-state conditions (Table 1). The conditions included spontaneous breathing at eucapnia, spontaneous breathing during mild hypercapnia (4 mmHg above eucapnia), mechanical ventilation at eucapnia, and mechanical ventilation during mild hypercapnia (4 mmHg above eucapnia).

Within each experiment, once a steady state was achieved in each condition (5 min), data were recorded for approximately 25-40 consecutive breaths. The order of presentation of the conditions was randomised among subjects.

Data analysis All unusual breaths, such as swallows and coughs, were excluded from the analysis. In addition, during the iron lung conditions, only those breaths where there were consistent airway pressure and flow profiles synchronised with the ventilator were analysed. The signals from each breath were averaged using the start of inspiration time as zero, yielding an average breath waveform for each variable, individual and condition (Sigavg software, Cambridge Electronic Design, Cambridge, UK).

Quantification of phasic EMG Electrocardiographic (EKG) artifacts were removed from the raw DIA EMG signal using an EKG ‘blanker’ (CWE: SB-1, Ardmore, PA, USA). Thereafter, the raw GG and DIA EMG signals were amplified, rectified, filtered (30-1000 Hz bandpass) and integrated using a time constant of 100 ms (CWE: BMA-400), yielding moving time average signals. To normalise the GG EMG, the moving time average was scaled between 0 % (electrical zero, i.e. earthed electrodes) and 100 % (maximal activation encountered during swallowing, maximal inspiratory effort or maximal tongue pressure (White et al. 1998)). GG EMG measurements included tonic activity (minimum during expiration) and phasic activity (peak during inspiration). DIA EMG was normalised in the same fashion with 100 % being assigned to the greatest DIA EMG occurring during maximal voluntary inspiratory efforts against an occluded airway. The magnitude of the phasic DIA EMG was determined as the mean level during inspiration minus the mean during expiration.

Other respiratory measurements The mean P_ET,CO₂, tidal volume and breath duration were calculated for each condition. Airflow resistance was calculated at a constant inspiratory flow (0.2 l s⁻¹) for two segments of the airway: pharyngeal resistance (from choanae to epiglottis: (P_choaP_epi)/flow: experiment A) and supraglottic resistance (from nosemask to epiglottis: (P_maskP_epi)/flow: experiment B).

Statistical comparisons

For each variable, the following planned comparisons were performed: (i) spontaneous breathing vs. negative pressure ventilation (at equivalent P_ET,CO₂; experiments A and B); (ii) medium vs. high P_epi (at equivalent P_ET,CO₂ and breath duration; experiment A); (iii) short vs. long breath duration (at equivalent P_ET,CO₂ and P_epi; experiment A); (iv) eucapnia vs. hypocapnia (at equivalent P_epi and breath duration; experiment A); and (v) eucapnia vs. hypercapnia (experiment B). Comparisons between conditions were performed using one-way repeated measures analysis of variance (SigmaStat v 2.0, Jandel Scientific, San Rafael, CA, USA). These were parametric analyses when data satisfied tests for normality and sphericity, and non-parametric analyses (Friedman repeated measures analyses of variance on ranks) when data were not normally distributed. Comparisons between specific pairs of conditions were performed using Student-Neuman-Keuls post hoc tests for multiple comparisons. A probability of less than 0.05 was considered to be significant for all statistical analysis. Data in the Results section are reported as means ±s.e.m. In addition, multivariate models (both forward and backward stepwise linear regression) were used to test which variables provided independent information in the prediction of peak GG EMG. Finally, for each condition, analysis included within-breath linear regressions and correlations between mechanoreceptor stimuli (pressure, flow and resistance) and GG muscle response.

RESULTS

Extent of passivity during negative pressure ventilation

Three of the twenty-two subjects did not complete the protocol because they were unable to achieve adequate passivity based upon the criteria of uniform airflow and airway pressure profiles synchronised with the ventilator cycle. All remaining subjects reported that they were relaxed and not voluntarily controlling breathing or voluntarily holding their breath during the mechanical ventilation conditions. Additional assessment of passivity was based upon recordings of DIA EMG (experiment A). Detectable phasic DIA EMG occurred during baseline spontaneous breathing in only 5 of 11 subjects due to the insensitivity of surface electrode EMG recordings. Each of these subjects had a substantial decrease in inspiratory phasic DIA EMG during the conditions of negative pressure ventilation (Fig. 1). A further example in one subject is shown in Fig. 2, which demonstrates that the inspiratory phasic DIA EMG can be abolished by negative pressure ventilation (at various pressures, breath durations and P_ET,CO₂ levels) at a time when phasic activity of the GG increases.

Figure 1

Negative pressure ventilation reduces inspiratory diaphragmatic activity

Figure 2

Phasic inspiratory genioglossus activation persists during passive negative pressure ventilation

During spontaneous breathing, central respiratory pattern generator neurons activate GG immediately before inspiratory airflow on each breath (i.e. ‘pre-activation’, Strohl et al. 1980). In the current experiment, evidence of dissociation of central respiratory drive to the diaphragm from the timing of the ventilator cycle, which provides indirect evidence of passivity, comes from the disappearance of the usual pre-activation of the GG before the onset of airflow during negative pressure ventilator breaths. During all conditions of spontaneous breathing throughout both experiments, the group mean time between GG activation and onset of airflow was -91 ms, indicating clear pre-activation. In contrast, there was usually a lack of pre-activation in the conditions of mechanical ventilation, as indicated by GG activation occurring after the onset of airflow (range of group averages among conditions of mechanical ventilation was 0 to +111 ms throughout both experiments). The differences between spontaneous breathing and mechanical ventilation were statistically significant in each experiment.

Comparisons of mean respiratory data between all conditions

The results of comparisons of mean respiratory data between all conditions in each experiment are presented in Table 2. For experiment A, it can be seen that all six iron lung conditions were associated with an increased airflow and more negative epiglottic pressure when compared to spontaneous breathing (as dictated by the protocol), and that there were concomitant increases in peak phasic GG (significantly different from spontaneous breathing in those four comparisons where n = 11 subjects, while a trend toward significance occurred in the other two comparisons involving fewer subjects). Thus, increased stimuli (negative pressure and flow) resulted in increased responses (phasic GG activity). There was little or no change in pharyngeal resistance or tonic GG EMG in these iron lung conditions when compared to spontaneous breathing (no significant differences from spontaneous breathing in 11 of 12 comparisons for these two variables). When comparing iron lung conditions at similar levels of P_ET,CO₂, phasic GG activity was directly proportional to the peak P_epi (i.e. conditions 2 vs. 4, and 3 vs. 5). In contrast, there was no effect upon GG activity of altering the breath duration (i.e. rate of iron lung ventilation; conditions 2 vs. 6, and 4 vs. 7) or the P_ET,CO₂ (i.e. conditions 2 vs. 3, and 4 vs. 5).

Table 2

Groupmean results inrandomised steady-stateconditions

For experiment B, comparisons of spontaneous breathing and negative pressure ventilation at equivalent levels of P_ET,CO₂ (i.e. conditions 8 vs. 10, and 9 vs. 11) revealed, as occurred in experiment A, that iron lung conditions with increased airflow and epiglottic negative pressure resulted in significantly increased peak phasic GG EMG, without changes in tonic GG EMG or supraglottic resistance. (Note, choanal pressure was not measured in experiment B, and thus supraglottic resistance is provided.) Again, as occurred in experiment A, there were no changes in peak phasic GG EMG associated with alterations in P_ET,CO₂ (during similar modes of ventilation, i.e. conditions 8 vs. 9, and 10 vs. 11).

Prediction of peak inspiratory genioglossus activity

We used a multivariate model to determine which of the variables provided independent information in the prediction of peak GG EMG by using both forward and backward stepwise linear regression. We used a number of models including:

A mathematical equation, expression, or formula that is to be displayed as a block (callout) within the narrative flow. The name of referred object is tjp0531-0677-mu1.jpg

and,

For these models, as experiment A and experiment B were performed on different subjects, it was necessary to normalise peak GG EMG to the percentage of the peak GG EMG obtained during the baseline condition within each experiment. The results of each of these multivariate analyses were identical and conclusive (and consistent with the results of the repeated measures analyses of variance noted above). The multivariate analysis showed that: tonic GG EMG, P_ET,CO₂ and breath duration (within the ranges studied) provided no significant independent information in the prediction of peak GG EMG beyond that provided by epiglottic pressure alone, which explained 93 % of the variation in peak GG EMG across all 11 conditions. The overall relationship was:

where GG EMG was measured as a percentage of baseline and epiglottic pressure in cmH₂O.

In addition to the pattern for the group mean data, within most subjects there was a significant linear relationship between peak inspiratory negative P_epi and peak inspiratory GG activity across all conditions (spontaneous breathing; medium and high level negative pressure ventilation; slow and rapid breathing frequencies; hypocapnia, eucapnia and hypercapnia). In experiment A, the mean correlation coefficient (r) among subjects was 0.844 (range 0.49-0.99). In experiment B, the mean correlation coefficient among subjects was 0.855 (range 0.46–0.98). The individuals’ data are shown in Fig. 3 (experiment A only). This robust relationship between peak negative P_epi and peak GG activity across conditions also occurred when the data were averaged for the group (experiment A: r = 0.97, P = 0.0002; experiment B: r = 0.96, P = 0.04). These group relationships are shown in Fig. 4.

Figure 3

Peak intra-pharyngeal negative pressure predicts peak genioglossus activity in all subjects (experiment A; individual results)

Figure 4

Peak negative intra-pharyngeal pressure predicts peak genioglossus activity in all conditions

Genioglossus activity is correlated with airway negative pressure and flow throughout spontaneous inspiration and passive inflation

In addition to the robust relationship between peak negative airway pressure and peak inspiratory phasic GG activity shown above, within-breath analysis revealed highly significant relationships between negative pressure (or flow) and genioglossus activity across inspiration in all conditions.

Individual subjects The shapes and amplitudes of the average waveforms in Fig. 2 (inset) demonstrate that there is an inverse relationship between P_epi and GG EMG. These correlations were significant across the mean breath profiles in every subject in almost every condition in both experiments A and B (64 out of 65 correlations were significant in A, and 28 out of 30 were significant in B). The mean results of these relationships are shown in Table 3. Thus negative P_epi and flow were equally good predictors of GG EMG during spontaneous breathing in each subject at various levels of P_ET,CO₂, and during passive mechanical ventilation at various tidal volumes, breathing frequencies and levels of P_ET,CO₂. Furthermore, these relationships were very similar across conditions within each experiment. There were no significant differences between conditions in the slopes of the relationships between flow and GG EMG or between negative P_epi and GG EMG, with the single exception being during eucapnia in experiment B where the slope of negative P_epivs. GG EMG was higher during spontaneous breathing than during medium negative pressure ventilation (Table 3). There were no significant differences in r between conditions in each experiment.

Table 3

Mean relationships between P_epi and GG, and flow and GG in individual subjects

Group results Since negative P_epi and flow had identical relationships with GG EMG, all further analysis will address only the relationship between negative P_epi and GG EMG. The correlations between negative P_epi and GG EMG are shown for the group data in Fig. 5 for both experiments. In each condition there was a clear relationship between negative P_epi and GG EMG throughout the breath. This can be seen both in the plot of these signals against time (upper traces) and in the x-y plots (lower plots). As with the individual subjects’ results (Table 3), the slopes of the relationships between negative P_epi and GG EMG were very similar among the conditions within each experiment (Fig. 5). It should be noted that there was usually some ‘hysteresis’ in the relationship whereby GG EMG changed relatively more for a given change in pressure (or flow) early in the inspiration when compared to late in the inspiration. This hysteresis is represented as a ‘loop’ in the x-y plots in Fig. 5. Within each experiment there was no difference between conditions in the degree of hysteresis (Student's paired t tests among conditions on correlation coefficient r between negative P_epi and GG EMG). Such hysteresis was also observed in individual subjects’ data (not shown). Because of the existence of this hysteresis, in a post hoc analysis we assessed the slope of the relationship and correlation coefficient specifically for this early portion of inspiration (from flow onset until peak GG EMG). In all instances the correlation between epiglottic negative pressure and GG EMG improved and the early inspiratory slope increased (which is predictable from observation of the loops). However, comparisons of slopes and correlation coefficients among conditions revealed almost identical results for analysis of the entire loop (Table 3) and analysis of the early portion of the loop alone. The only difference was that analysis of the whole loop had revealed a significant difference between conditions 8 and 10 for epiglottic negative pressure vs. GG EMG, whereas this failed to reach significance when considering the initial part of the loop alone. The possible explanation for this hysteresis is discussed further below. The mean within-breath relationships between epiglottic negative pressure and GG EMG for this early portion of the breath were virtually identical across all eleven experimental conditions (Fig. 6).

Figure 5

Relationship between negative airway pressure and genioglossus activity across inspiration

Figure 6

Robust relationship between airway negative pressure and genioglossus activity across early portion of inspiration during all 11 experimental conditions

DISCUSSION

This study has led to a number of new findings in awake humans: (i) relatively physiological (i.e. slow and phasic) changes in upper airway mechanoreceptor stimuli throughout inspiration result in phasic activation of an upper airway dilator muscle; (ii) throughout inspiration the magnitude of intra-pharyngeal negative pressure and inspiratory airflow changes were both highly correlated with the degree of GG activation; and (iii) the slopes of the relationships between intra-pharyngeal negative pressure and GG activity and between flow and GG activity were unchanged across virtually all conditions, both within a breath and between breaths. Thus, there was a robust relationship between upper airway mechanoreceptor stimuli and pharyngeal muscle activation despite greatly altered degrees of central drive to the respiratory pump muscles (i.e. diminished or absent drive during passive mechanical ventilation and hypocapnia, and increased drive during hypercapnia and spontaneous breathing). These data provide strong evidence that upper airway dilator muscles can be activated throughout inspiration via ongoing upper airway mechanoreceptor reflexes. Such a feedback mechanism may naturally protect upper airway patency within each breath in awake humans.

Evidence that mechanoreceptive reflexes contribute to upper airway dilator muscle control during spontaneous breathing

Several studies both in humans and animals have demonstrated the existence of an upper airway reflex in response to the application of relatively non-physiological, sudden onset, large negative airway pressure pulses (Horner et al. 1991a, b; Wheatley et al. 1993). We found that a mechanoreceptive reflex is still present at normal breathing rates, and that the timing and magnitude of this reflex is adequate to account for the GG activity that occurs throughout normal breathing, stimulated breathing and all conditions of mechanical ventilation used. Several additional studies suggest that this mechanoreceptive reflex may be active during spontaneous breathing. First, breathing through a tracheotomy (absence of upper airway stimuli) immediately reduces upper airway dilator muscle activity in patients with obstructive sleep apnoea (Malhotra et al. 2000) and in vagotomised rabbits (Mathew et al. 1982a). Second, nasopharyngeal anaesthesia reduces spontaneous GG activation during inspiration both in healthy subjects (White et al. 1998) and in patients with OSA (Fogel et al. 2000). These observations, together with the results of the current experiment, strongly suggest that ongoing mechanoreceptive reflexes contribute significantly to the phasic activation of upper airway dilator muscles throughout spontaneous breathing in awake humans.

The specific airway mechanoreceptive stimulus to genioglossus activation

The precise airway mechanoreceptive stimulus that is responsible for activation of the upper airway muscles is unknown. Numerous receptors exist in the upper airway, including superficial receptors responsive to pressure, flow (or temperature) and mechanical deformation, and deeper receptors responsive to muscle stretch and motion (Widdicombe et al. 1988). Any number of these receptors could contribute to the mechanoreceptor reflex control of upper airway muscles. Studies in humans using topical nasopharyngeal anaesthesia and local anaesthetic block of internal branches of the superior laryngeal nerves (SLN) (Horner et al. 1991b), and denervation studies in anaesthetized rabbits (Mathew et al. 1982b) and unanaesthetized dogs (Issa et al. 1988; Curran et al. 1997) have demonstrated that mucosal and/or submucosal laryngeal receptors mediate an important component of this reflex response to upper airway pressure. However, some proportion of the reflex response remains after SLN blockade or section (Horner et al. 1991b; Curran et al. 1997), suggesting that other nerve afferents are involved to some degree (e.g. trigeminal or glossopharyngeal afferents). Our results demonstrate equally robust relationships between negative P_epi and GG EMG, and between airflow and GG EMG in individual within-breath analyses and across all conditions. While we could not distinguish the actions of pressure and flow receptors in our study, pressure receptors may be the more likely candidates for producing the mechanoreceptive reflexes for several reasons (Widdicombe et al. 1988). Pressure receptors constitute the largest proportion of the mechanosensitive endings in the larynx (Sant'Ambrogio et al. 1983), and a correlation exists between peak phasic GG EMG and oesophageal pressure swings generated by the respiratory pump muscles during obstructive apnoeas when there is no airflow (Berry et al. 1997).

We note that, as well as upper airway receptors, pulmonary and chest wall mechanoreceptors (muscle spindles, Golgi tendon organs and body surface receptors) are stimulated in synchrony with ventilatory movements and could have affected GG activity in the current study. However, we doubt that pulmonary and chest wall mechanoreceptors explain the robust relationship between upper airway mechanoreceptor stimuli and GG activation that we observed because pulmonary stretch receptors activated during inspiration are more likely to inhibit rather than facilitate GG activity (Gauda et al. 1994), and the pattern of activation of muscle spindles and body surface receptors would differ between spontaneous and mechanical ventilation.

The mild degree of hysteresis in the relationship between flow and GG and between negative intra-pharyngeal pressure and GG activity (Fig. 5A and B) suggest that there may be non-linearities in this reflex pathway. This hysteresis could have resulted from the summation of inputs to the GG motoneurons from various mechanoreceptors. For instance, airflow and negative intra-pharyngeal pressure receptors in the upper airway may facilitate GG while ongoing pulmonary stretch receptor activity throughout inspiration may inhibit GG. Furthermore, such receptors could have different thresholds for activation and may undergo different degrees of adaptation throughout a breath (Bartlett et al. 1976). Also, pharyngeal wall surface tension may differ across inspiration and affect the relationship between receptor activation and muscle activity. In addition, these mechanoreceptive inputs will summate with any central respiratory drive to the diaphragm related to ongoing activity of the central respiratory pattern generator. Despite the multiple potential sources of such non-linearities, we were most impressed by the relatively stable mean linear relationship between mechanoreceptive stimuli and GG activation. Moreover, negative pressure and flow always changed together, and we found no significant difference in airway resistance across conditions (Table 2), and no correlation between airway resistance and GG activation (data not shown). Thus, it seems reasonable to hypothesise that the integration of receptor information concerning pressure differences across regions of the airway could result in the active regulation of airway resistance. While the airway sensors are evidently in place, the central integration of such a mechanism is hypothetical at this stage.

Central respiratory drive versus mechanoreceptive reflexes as the principal stimulus to phasic muscle activation

Although our data suggest that upper airway dilator muscles can be activated throughout inspiration via ongoing upper airway mechanoreceptor reflexes, a large body of evidence in humans and animals demonstrates that chemoreceptive stimuli can also drive these muscles. Two studies in humans (Redline et al. 1987; Innes et al. 1995) have demonstrated that laryngectomised patients who can only breathe through a tracheotomy retain some inspiratory phasic GG activity during spontaneous breathing, and that this phasic activity increases during imposed hypercapnia and while breathing against a resistive load (despite an absence of intra-pharyngeal negative pressure swings). In the present study we demonstrated that the linear relationship between peak negative P_epi and peak phasic GG EMG was largely unaffected by alterations in central ventilatory drive across the normal physiological range (during mild hypocapnia, eucapnia and mild hypercapnia). These new data are consistent with the results of a separate study (Shea et al. 2000) in which spontaneous ventilation was stimulated across wider ranges of blood gases including combined hypercapnia and hypoxia (arterial oxygen saturation was 87 % combined with +4 mmHg hypercapnia, resulting in spontaneous ventilation of 23 l min⁻¹). Interestingly, the GG response in those more stimulated conditions could also be fully explained based upon the mechanoreceptive stimuli alone. These observations suggest that local mechanoreceptive reflexes are more potent than chemoreceptive mechanisms in mediating upper airway dilator muscle activation across a wide physiological range. Thus, in the current experiment it appears likely that much of the relationship between central respiratory drive and phasic GG activation occurs indirectly via mechanoreceptor reflex mechanisms.

Integration of spontaneous respiratory rhythm and mechanoreceptive reflex effects on genioglossus activity

We remain uncertain of the specific pathways involved in the integration of spontaneous respiratory rhythm and mechanoreceptive reflex effects on GG activation. Spontaneous respiratory phasic activity can be recorded from central pattern generator neurons in the brainstem that activate the pump muscles (e.g. pre-Botzinger complex) and from hypoglossal motor nuclei which activate the genioglossus muscle (Bianchi et al. 1995). However, the functional link between these nuclei has not been well studied. In particular, we do not know whether the premotor neurons that activate respiratory pump muscles are the same as those premotor neurons that activate upper airway muscles, how differences in these pathways result in the pre-activation of upper airway muscles relative to pump muscles, and where in this pathway the mechanoreceptive stimuli exert their effect (i.e. via central pattern generator neurons or directly onto hypoglossal motoneurons). While chemoreceptive stimuli concurrently activate both pump and airway dilator muscles, specific mechanoreceptive stimuli have been shown to have differential effects on these muscles. For instance, airway deformation can activate airway dilator muscles and simultaneously inhibit respiratory pump muscles (Harms et al. 1996). Nonetheless, in the current study we found the same relationship between mechanoreceptive stimuli and upper airway dilator muscle activity during spontaneous breathing and during passive mechanical ventilation when the spontaneous respiratory rhythm output was diminished or absent. This dissociation between pump muscle and airway muscle activation suggests that phasic mechanoreceptive stimuli exerted their influence on upper airway dilator muscles by directly stimulating hypoglossal motoneurons (rather than by facilitating the central respiratory drive to the diaphragm which is absent or diminished during passive ventilation). A possible caveat to this last interpretation is that subjects may have volitionally suppressed their pump muscles during mechanical ventilation, or even voluntarily activated their GG muscle. Although we do not have any direct evidence to this effect, we note that our subjects were instructed to remain completely relaxed (i.e. to not actively suppress breathing), and they reported that they were not voluntarily controlling breathing. Whether or not spontaneous breathing can be suppressed in awake humans during mechanical ventilation is the subject of controversy (e.g. Leevers et al. 1993; Morrell et al. 1993; reviewed by Shea, 1996). New evidence from a functional magnetic imaging study shows that there are motor cortical regions activated during volitional inspiration which are relatively inactive during mechanical ventilation in awake humans, suggesting that the usual voluntary respiratory motor control regions need not be activated during mechanical ventilation (Evans et al. 1999). Overall, we do not consider the possibility of volitional control of pump muscles to greatly affect our interpretation because this eventuality would add even one more condition in which the robust relationship between airway mechanoreceptor activity and genioglossus activity is stable, i.e. during basal spontaneous breathing, stimulated spontaneous breathing, passive mechanical ventilation (if present), volitional activation of pump muscles (if present), and volitional suppression of pump muscles (if present). Since we used slow stimuli during mechanical ventilation, it remains possible that these stimuli could result in voluntary activation of GG. However, this seems unlikely as it would require precise moment-to-moment voluntary activation of GG during spontaneous breathing and mechanical ventilation to yield a robust relationship between mechanoreceptor stimuli and GG activity in all conditions.

Relevance to OSA

We chose to study healthy awake subjects rather than patients with OSA as there may be differences in the mechanoreceptive reflex pathways brought on by the disease or the sleep-wake state. For instance, it seems clear from several studies that the pharyngeal airway is smaller in OSA patients compared to normal subjects during wakefulness (Horner et al. 1989; Schwab et al. 1995). Also, numerous studies indicate that during wakefulness OSA patients have an abnormally high pharyngeal dilator muscle activity (e.g. Mezzanotte et al. 1992). Since nasal continuous positive airway pressure diminishes upper airway dilator muscle activity during wakefulness in OSA, it has been proposed that their abnormally high muscle activity is driven by a reflex response to intra-luminal negative pressure (Mezzanotte et al. 1992). Results from the current study are consistent with these hypotheses. However, the precise stimulus to the neuromuscular compensation in OSA patients during wakefulness is not fully understood, with the greatest conundrum being that augmented pharyngeal muscle activity occurs in the face of relatively normal intra-luminal pressures while awake. This suggests that there may have been adaptation in the mechanoreceptive reflex pathway in OSA patients.

Conclusion

The current study demonstrates that in awake humans relatively physiological (i.e. slow and phasic) changes in upper airway mechanoreceptor stimuli throughout inspiration result in phasic activation of an upper airway dilator muscle. Intra-pharyngeal negative pressure and airflow were equally good predictors of genioglossus activity during spontaneous breathing at various levels of ventilation, and during passive mechanical ventilation at various tidal volumes, breathing frequencies and levels of P_ET,CO₂. This mechanoreceptive reflex appeared to be much stronger than any chemoreceptive influence on upper airway muscle activation, at least within the physiological range that we studied (from mild hypocapnia to mild hypercapnia). These data collected across the normal physiological ranges of breathing frequency, volume and end-tidal carbon dioxide provide strong evidence that upper airway dilator muscles can be activated throughout inspiration via ongoing upper airway mechanoreceptor reflexes. Such a feedback mechanism may naturally protect upper airway patency within each breath in awake humans. This mechanism may be important in the neuromuscular compensation to upper airway dilator muscles present in the awake patient with obstructive apnoea (Mezzanotte et al. 1992). Loss of this reflex response during sleep could lead to obstructive sleep apnoea (Wheatley et al. 1993; Horner et al. 1994; Shea et al. 1999).

Acknowledgments

This research project was supported by NHLBI grants HL48531 and HL60292, and in part by NIH grant NCRR GCRC M01 RR02635 to the Brigham and Women's Hospital, General Clinical Research Center.

References

Bartlett, D; Sant'Ambrogio, G; Wise, JCM. Transduction properties of tracheal stretch receptors. Journal of Physiology. 1976;258:421–432. [PubMed]
Berry, RB; McNellis, MI; Kouchi, K; Light, RW. Upper airway anesthesia reduces phasic genioglossus activity during sleep apnea. American Journal of Respiratory and Critical Care Medicine. 1997;156:127–132. [PubMed]
Bianchi, AL; Denavit-Saubie, M; Champagnat, J. Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters. Physiological Reviews. 1995;75:1–31. [PubMed]
Curran, AK; Eastwood, PR; Harms, CA; Smith, CA; Dempsey, JA. Superior laryngeal nerve section alters responses to upper airway distortion in sleeping dogs. Journal of Applied Physiology. 1997;83:768–775. [PubMed]
Evans, KC; Shea, SA; Saykin, AJ. Functional MRI localisation of central nervous system regions associated with volitional inspiration in humans. Journal of Physiology. 1999;520:383–392. [PubMed]
Fogel, R; Malhotra, A; Edwards, JK; Shea, SA; White, DP. Reduced genioglossal activity with upper airway anesthesia in awake patients with obstructive sleep apnea. Journal of Applied Physiology. 2000;88:1346–1354. [PubMed]
Gauda, EB; Carroll, TP; Schwartz, AR; Smith, PL; Fitzgerald, RS. Mechano and chemoreceptor modulation of respiratory muscles in response to upper airway negative pressure. Journal of Applied Physiology. 1994;76:2656–2662. [PubMed]
Harms, CA; Zeng, YJ; Smith, CA; Vidruk, EH; Dempsey, JA. Negative pressure-induced deformation of the upper airway causes central apnea in awake and sleeping dogs. Journal of Applied Physiology. 1996;80:528–539.
Horner, RL; Innes, JA; Holden, HB; Guz, A. Evidence for reflex upper airway dilator muscle activation by sudden negative airway pressure in man. Journal of Physiology. 1991a;436:15–29. [PubMed]
Horner, RL; Innes, JA; Holden, HB; Guz, A. Afferent pathway(s) for pharyngeal dilator reflex to negative pressure in man: a study using upper airway anaesthesia. Journal of Physiology. 1991b;436:31–44. [PubMed]
Horner, RL; Innes, JA; Morrell, MJ; Shea, SA; Guz, A. The effect of sleep on reflex genioglossus muscle activation by stimuli of negative airway pressure in humans. Journal of Physiology. 1994;476:141–151. [PubMed]
Horner, RL; Shea, SA; McIvor, J; Guz, A. Pharyngeal size and shape during wakefulness and sleep in patients with obstructive sleep apnoea. Quarterly Journal of Medicine. 1989;72:719–735. [PubMed]
Innes, JA; Morrell, MA; Kobayashi, I; Hamilton, RD; Guz, A. Central and reflex neural control of genioglossus in subjects who underwent laryngectomy. Journal of Applied Physiology. 1995;78:2180–2186. [PubMed]
Issa, FG; Edwards, P; Szeto, E; Lauff, D; Sullivan, C. Genioglossus and breathing responses to airway occlusion: effect of sleep and route of occlusion. Journal of Applied Physiology. 1988;64:543–549. [PubMed]
Kobayashi, I; Perry, A; Rhymer, J; Wuyam, B; Hughes, P; Murphy, K; Innes, JA; McIvor, J; Cheesman, AD; Guz, A. Inspiratory coactivation of the genioglossus enlarges retroglossal space in laryngectomized humans. Journal of Applied Physiology. 1996;80:1595–1604. [PubMed]
Leevers, AM; Simon, PM; Xi, L; Dempsey, JA. Apnoea following normocapnic mechanical ventilation in awake mammals: a demonstration of control system inertia. Journal of Physiology. 1993;472:749–768. [PubMed]
Malhotra, A; Fogel, RB; Edwards, JK; Shea, SA; White, DP. Local mechanisms drive genioglossus muscle activation in obstructive sleep apnea. American Journal of Respiratory and Critical Care Medicine. 2000;161:1746–1749. [PubMed]
Mathew, OP; Abu-Osba, YK; Thach, BT. Influence of upper airway pressure changes on genioglossus muscle respiratory activity. Journal of Applied Physiology. 1982a;52:438–444. [PubMed]
Mathew, OP; Abu-Osba, YK; Thach, BT. Genioglossus muscle responses to upper airway pressure changes: afferent pathways. Journal of Applied Physiology. 1982b;52:445–450. [PubMed]
Mezzanotte, WS; Tangel, DJ; White, DP. Waking genioglossal electromyogram in sleep apnea patients versus normal controls (a neuromuscular compensatory mechanism). Journal of Clinical Investigation. 1992;89:1571–1579. [PubMed]
Morrell, MJ; Shea, SA; Adams, L; Guz, A. Effects of inspiratory support upon breathing in humans during wakefulness and sleep. Respiration Physiology. 1993;93:57–70. [PubMed]
Redline, S; Strohl, KP. Influence of upper airway sensory receptors on respiratory muscle activation in humans. Journal of Applied Physiology. 1987;63:368–374. [PubMed]
Remmers, JE; de Groot, WJ; Sauerland, EK; Anch, AM. Pathogenesis of upper airway occlusion during sleep. Journal of Applied Physiology. 1978;44:931–938. [PubMed]
Sant'Ambrogio, G; Mathew, OP; Fisher, JT; Sant'Ambrogio, FB. Laryngeal receptors responding to transmural pressure, airflow and local muscle activity. Respiration Physiology. 1983;54:317–330. [PubMed]
Schwab, RJ; Gupta, KB; Gefter, WB; Metzger, LJ; Hoffman, EA; Pack, AI. Upper airway and soft tissue anatomy in normal subjects and patients with sleep-disordered breathing. Significance of the lateral pharyngeal walls. Journal of Applied Physiology. 1995;152:1673–1689.
Shea, SA. Behavioural and arousal-related influences on breathing in humans. Experimental Physiology. 1996;81:1–26. [PubMed]
Shea, SA; Akahoshi, T; Edwards, JK; White, DP. Influence of chemoreceptor stimuli on genioglossal response to negative pressure in humans. American Journal of Respiratory and Critical Care Medicine. 2000;162:559–565. [PubMed]
Shea, SA; Edwards, JK; White, DP. Effect of wake-sleep transitions and REM sleep on pharyngeal muscle response to negative pressure in humans. Journal of Physiology. 1999;520:897–908. [PubMed]
Strohl, KP; Hensley, MJ; Hallett, M; Saunders, NA; Ingram, RH., Jr Activation of upper airway muscles before onset of inspiration in normal humans. Journal of Applied Physiology. 1980;49:638–642. [PubMed]
Wheatley, JR; Mezzanotte, WS; Tangel, DJ; White, DP. Influence of sleep on genioglossus muscle activation by negative pressure in normal men. American Review of Respiratory Diseases. 1993;148:597–605. [PubMed]
White, DP; Edwards, JK; Shea, SA. Local reflex mechanisms: influence on basal genioglossal muscle activation in normal subjects. Sleep. 1998;21:719–728. [PubMed]
Widdicombe, JG; Santo'Ambrogio, G; Mathew, OP. Nerve receptors of the upper airway. In: Mathew OP, Santo'Ambrogio G. , editors. Respiratory Function of the Upper Airway. New York: Dekker; 1988. pp. 193–231.