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J Appl Physiol 91: 2289-2297, 2001;
8750-7587/01 $5.00
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Vol. 91, Issue 5, 2289-2297, November 2001

Negative pressure effects on mechanically opposing pharyngeal muscles in awake and sleeping goats

Thom R. Feroah1,2, H. V. Forster1, L. Pan3, N. E. Schlick1, Paul Martino1, and Tom Rice2

Departments of 1 Physiology and 2 Pediatrics, Medical College of Wisconsin and Zablocki Veterans Affairs, Milwaukee 53226; and 3 Department of Physical Therapy, Marquette University, Milwaukee, Wisconsin 53201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our aim was to investigate the effects of the negative pressure reflex on mechanically opposing pharyngeal muscles during wakefulness, slow-wave sleep (SWS), and rapid eye movement (REM) sleep. In four goats with isolated upper airways, we measured tracheal airflow and electrical activity of the thyropharyngeus (TP; constricting), the stylopharyngeus (SP; dilating), and the diaphragm (Dia). In the wakefulness state in response to negative pressure tests, TP decreased (65%), SP increased (198%), and tidal volume (VT) (66%) and rate of rise of Dia (Diaslope, 69%) decreased (P < 0.02). Similarly, during SWS, the negative pressure response of TP (31%), VT (61%), and Diaslope (60%) decreased, whereas SP (113%) increased, relative to SWS control (P < 0.02). In REM sleep, the negative pressure response by TP and SP were small, whereas both VT (38%) and Diaslope (24%) were greatly decreased (P < 0.02) compared with REM control. Inspiratory duration remained unchanged in response to negative pressure tests in all states. These data provide evidence that mechanically opposing inspiratory and expiratory pharyngeal muscles are reciprocally controlled and their response to negative pressure are state dependent.

slow-wave sleep; rapid eye movement sleep; electromyography; sleep apnea; upper airways; thyropharyngeus; stylopharyngeus


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE STRUCTURAL COMPLEXITY of the upper airway (UAW) is derived from its functional diversity in respiration, phonation, olfaction, air-conditioning, and deglutition. The operation of this multiprocessing phenomenon depends on the proper orchestration and coordination of 28 pairs of UAW muscles with different functional pattern generators and numerous UAW reflexes. In response to the presence of a negative intraluminal pressure, the laryngeal and nasal mechanoreceptors have been found to mediate a patency-maintaining response in the UAW by selectively activating upper airway-dilating muscles (e.g., genioglossus and elevator tensor palatini) and by decreasing inspiratory drive (10, 11, 16-20, 33-35, 38). Both actions in concert are proposed to increase the UAW-dilating forces and limit the effect of collapsing forces with negative intraluminal pressures during inspiration. Few studies have investigated this reflex in unanesthetized animals while examining the drive and timing to the diaphragm (Dia) and the activation of UAW muscles. Instead, the previous investigations have focused primarily on the activation UAW dilator muscles.

We recently have shown the control and activation of two mechanically opposing pharyngeal constrictor [thyropharyngeus (TP)] and dilator [stylopharyngeus (SP)] muscles, which are reciprocally activated during eupnea and the opposing extremes of hypercapnic and hypocapnic stimulation (3). In addition, we provided evidence that the TP and the SP muscles, depending on their level of activation, functionally control the position of the lateral and posterior wall of the oropharynx in goats. We also have shown that the level of activity in these mechanically opposing muscles during eupneic breathing and in response to mechanically induced apneas progressively decreased from wakefulness to slow-wave sleep (SWS) and were the lowest in rapid eye movement (REM) sleep (3a). In the subsequent recovery from the induced apnea during wakefulness and SWS, we also found an elevation in constrictor muscle activity relative to dilator muscle activity that persisted beyond the return of Dia activity. Taken together, these data suggest that an imbalance in neuromuscular forces may exist in certain states and conditions and, therefore, contribute to an increase in collapsing forces in the UAW, resulting in airflow limitation and obstructive events.

Our primary aim was to investigate the effects of the negative pressure reflex in our model of mechanically opposing TP and SP muscles during conditions of wakefulness, SWS, and REM sleep. Our hypothesis is that, in the awake state, SP activity would increase and TP activity would reciprocally decrease during a negative pressure (Neg) test. Furthermore, because the respiratory-related activity of the UAW muscles progressively decrease from wakefulness to SWS and REM, we hypothesize that the reflex response of the TP and SP muscles would also decrease. We further investigated the negative pressure reflex on the drive to the main respiratory pump muscle, the Dia, and on respiratory timing.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Four adult goats of various breeds were studied. The study protocol was approved by the Institutional Animal Care Committee of the Medical College of Wisconsin.

Surgical preparation. Two surgeries were performed ~6-8 wk apart. The first surgery was performed to implant chronic electromyography (EMG) electrodes in the muscles of the Dia (EMGDia), TP (EMGTP), and the SP (EMGSP). The second surgery was performed to isolate the UAW by creating a tracheostoma at the level of the eighth tracheal ring and to implant electroencephalographic (EEG) and electrooculographic (EOG) electrodes. Before surgery, the animals received an intravenous injection of ketamine (Ketaset) and xylazine (12:1, 15 mg/kg) for induction of anesthesia before intubation. Anesthesia was maintained with 1.5% halothane (in oxygen).

EMG electrode placement has been described previously (3). Briefly, Teflon-coated 32-gauge stainless steel bipolar microelectrodes were inserted into the muscles defined above (no. AS637, Cooner Wire). For implants into airway muscles, a midline incision was made on the ventral surface of the neck from the hyoid bone to 4 cm below the thyroid cartilage to expose the lateral aspect of the pharynx, the TP and SP muscles. The EMGTP electrode was sewn into the TP muscle midway between the posterior midline of the pharynx and the insertion on the thyroid cartilage 0.5 cm below the cranial laryngeal nerve. The SP muscle was located at the cranial tip of the parotid gland, just caudal to the insertion of the styloglossus muscle, and followed to where it descends below the hypopharyngeus muscle. The electrode was sewn into the SP muscle close to its disappearance under the hypopharyngeus. The wires were all looped in the subcutaneous layer of connective tissue and exited on the lateral ventral surface of the neck. For the EMGDia, a lateral thorocotomy was performed between the ninth and tenth ribs midway between the sternum and spine. The Dia electrodes were implanted in the costal portion of the Dia and exteriorized next to the incision.

EEG and EOG electrodes were constructed of the same wire as above and soldered to a screw (0.25-in. no. 6 metal screw) and insulated with dental cement. For placement of the EEG electrodes, a midline incision was made on the dorsal surface of the skull between the base of the horns (cornual process) caudally to the insertion of the nuchal ligament on the occipital bone to expose the skull. Three electrodes were screwed into the skull equal distances along the midline and covered with dental cement to electrically isolate the electrodes from muscle activity. The wires were then exteriorized next to the incision. After an incision above each eye, EOG electrodes were placed into the frontal bone midway between the superior orbit of the eye and the cornual process. The EOG electrodes were then covered with dental cement, and the wires were exteriorized.

The animals received daily intramuscular antibiotics (ceftiofur sodium, 2 mg/kg) throughout the period of time they were studied. The rectal temperature, eating habits, and behavior were monitored throughout the study to evaluate the overall health and fitness of the animals.

Measurements. For measurements of airflow, an 8-Fr cuffed tracheostomy tube was inserted into the trachea, and the cuff was inflated and connected to a one-way breathing circuit. The breathing circuit consisted of a standard home ventilation circuit (B & F Medical Products) connected to a Bear-2 ventilator. A pneumotachograph was connected in-line on the inspiratory side of both ventilatory circuits and connected to a differential pressure transducer to measure inspiratory airflow.

For the measurement of UAW pressure and the application of a negative pressure to the isolated upper airway, a second cuffed tracheostomy tube (6 Fr) was inserted cranially into the trachea until the tip was just below the cricoid cartilage, and the cuff was inflated. The upper airway tracheostomy tube was connected to a negative pressure system consisting of a balloon valve attached to a 0.2-liter compliant hose that was then attached to a variable negative pressure blower. With this system, the negative pressure in the UAW could be set at any pressure between 0 and -30 cmH2O. All goats wore a tight-fitting muzzle mask to seal the nasal and oral passages. Negative pressure in the upper airway was measured by a tracheal cannula connected to a calibrated pressure transducer (model P45, Validyne). The average response time of the negative pressure system from atmospheric pressure to -10 cmH2O was 84 ± 8 ms. A negative pressure of -10 cmH2O was used in all goats because it was found to be just beyond the minimum pressure (-8 cmH2O) to evoke a consistent maximal response in the TP and SP muscles. The negative pressure was reduced in the UAW during expiration after postinspiratory activity of the Dia. To assess whether the negative pressure reflex was activated as traditionally found in previous studies, short negative pressure pulses (-10 cmH2O, 0.5 s) were also performed during inspiration only (7, 10, 35).

The proximal ends of the EMG, EEG, and EOG wires were connected via microclips to a Grass recorder for signal processing and recording on paper. The EMG signals were filtered at a band pass of 3-500 Hz. The band pass was set at 1-70 Hz for the EEG signals, and the high-pass filtering was set at 10 Hz for EOG signals. The raw EMG, EEG, EOG, and flow signals were sent to a CODAS computer data acquisition system at a sampling rate of 250 Hz for display, digital recording, and analysis.

Experimental design. Before surgery and at least 2 days after, the goats were acclimated to experimental protocols during wakefulness and sleep. All goats were studied in the prone, recumbent position. The goats underwent 48 h of sleep restriction before the first sleep study to promote consolidation of sleep. During sleep restriction, the goats were prevented from lying down except for the same time of day and period of time (7-8 h) during which they were allowed to sleep. This restriction protocol was maintained throughout the study. While the goats were in the quiet awake state, SWS, and REM sleep, eupneic breathing and EMG activity and their response to Neg tests in the isolated UAW were acquired. At least three sleep studies (3 days) were performed to obtain artifact-free data sets during the Neg tests for wakefulness, SWS, and REM sleep.

Sleep staging. Sleep was assessed via EEG and EOG standard criteria and/or behavioral criteria (24, 28). The awake state was defined as low-voltage, mixed-frequency EEG with concurrent behavior of head holding, alerting response to random ambient noises, and eye blinks. Head holding was define as a behavior in which the animal was able to maintain the usual head-erect position and is associated with the presence of neuromuscular tone to the neck muscles. SWS was defined as a synchronized low-frequency EEG (2 Hz or less) with an amplitude two to three times greater than found during awake and a concurrent absence of rapid eye movements. REM sleep was defined as a low-voltage, mixed-frequency desynchronization of the EEG with frequent rapid eye movements in the EOG channels and postural muscle atonia indicated by an inability of head holding. REM sleep was also associated with frequent twitching movements of the nose, ears, and lips. Rapid eye movements were distinguished from eye blinks because they were defined as sharp waves with an amplitude >30 µV from baseline in the EOG tracing and regularly confirmed visually with twitching movements. Only measurements taken during unequivocal awake sleep, SWS, and REM sleep were analyzed.

Topical anesthesia. The role of mechanoreceptors in the laryngeal and nasal airway in mediating the response to Neg tests was assessed by the application of a topical anesthesia to the isolated UAW. First, a benzocaine spray (20%) was applied in the larynx via the UAW tracheostomy tube and into each naris. After 4 min, 2 ml of viscous lidocaine (2%) were injected into the UAW via a multilumen nasal catheter. Successful application of topical anesthetic was assessed by the absence of swallowing during movement of the nasal catheter and injection of 1 ml of water into UAW. The goats were tested in the awake state before and 10 min after the successful application of the anesthesia.

Data analysis. Respiratory airflow, UAW pressure, and EMG, EEG, and EOG signals were processed and analyzed (WinDaq, DATAQ Instruments). Raw EMG data were full-wave rectified and passed through a moving time averager (time constant of 0.1 s) to obtain an integrated EMG signal. The integrated EMG signals for TP, SP, and Dia were analyzed to obtain the breath-by-breath phasic activity of each muscle. The mean activity of EMGTP during eupnea was calculated from the peak of EMGDia to the subsequent peak of EMGDia. The mean EMGSP activity was obtained from the start of EMGDia to the peak of EMGDia activity. Breath-by-breath calculations for tidal volume (VT), the duration of inspiration (TI), and the duration of expiration (TE) were made from the airflow signal. The total duration of a breath was calculated as the sum of TI and TE. The slope of the EMGDia (Diaslope) was used as a measure of ventilatory drive to the Dia and was calculated as the rate of rise in EMGDia from onset of Dia to peak activity.

For the effect of sleep on eupneic ventilatory data, TP, SP, and Dia activity, the average EMG values for each variable was computed for a minimum of 2- to 5-min periods before a series of Neg tests for awake, SWS, and REM states. During Neg tests, the average values of TP, SP, and Dia activity were computed differently from above. The average TP activity was calculated from the onset of the Neg test to the beginning of Dia activity and from the end of peak Dia activity to the beginning of the next breath or until the UAW pressure was returned to atmospheric pressure. The average value for SP and Dia activity was computed from their signal from the onset of the Dia activity to its end. The mean activities of Dia, TP, and SP during the Neg test were expressed as a percentage of their control period during the awake state, SWS, and REM sleep for each goat.

Statistical analysis. The TI and VT values during control and in response to Neg tests during awake, SWS, and REM states were compared using a two-way repeated-measures analysis of variance (P < 0.02). The posttest analysis was performed using the Bonferroni test (awake vs. SWS, awake vs. REM, and SWS vs. REM; P < 0.02). The average percent change in TP, SP, Dia activity, and Diaslope during the Neg tests awake and in SWS and REM sleep were analyzed for statistical significance using a Student's paired t-test (P < 0.02, adjusted for multiple comparisons).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of state on TP, SP, and Dia activity. Figure 1A presents a representative tracing in the awake goat during a Neg test. Concurrent with the decrease in UAW pressure, there is an immediate decrease in TP activity and an increase in SP tonic expiratory activity. During the subsequent inspiration, phasic inspiratory SP activity significantly increased, whereas Peak Dia activity decreased. After inspiration, except for a small burst in TP activity seen just after the peak of Dia activity, TP activity remained decreased until the UAW pressure was raised. An elevated tonic SP expiratory activity is not seen after inspiration during the Neg test. In the awake state, the presence of a burst of TP activity after peak inspiration varied within and between animals during the awake state. In addition, a reciprocal increase in tonic SP activity with a decrease in TP activity was not a consistent finding before and after phasic inspiratory SP activity. Figure 1B illustrates the effect seen in each goat when a short negative pressure pulse was given during inspiration in the awake state. During this test, SP activity was increased during the duration of the negative pressure pulse, Dia activity was reduced and airflow decreased. As a consequence of the negative pressure, TI was lengthened.


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  		Fig. 1.   Polygraphs of an awake goat during eupneic breathing with a negative pressure (Neg) test during expiration (A) and a short Neg test during inspiration (B). EEG, electroencephalogram; EOG, electrooculogram; V, inspiratory airflow; TP, thyropharyngeus electromyogram; TPMTA, moving time average of the TP; SP, stylopharyngeus electromyogram; SPMTA, moving time average of the SP; Dia, diaphragm electromyogram; DiaMTA, moving time average of the Dia; PUAW, pressure in the upper airway.

During SWS (Fig. 2A), although eupneic TP and SP activity was reduced, a similar response to a Neg test was still present. In this example, UAW pressure was reduced at the beginning of Dia activity and maintained for two inspiratory efforts. During both inspirations, there was a significant increase in phasic SP with a small decrease in peak Dia activity in the first breath. During both subsequent expirations, TP activity was greatly decreased until the return of the UAW pressure to atmospheric pressure. At this time, a rapid return of both the TP and SP signals to eupneic activity was seen. The lack of a decrease in peak Dia in the second breath compared with the first was a common observation in both wakefulness and SWS when the UAW pressure was held down for two inspiratory efforts.


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  		Fig. 2.   Polygraphs of a goat during slow-wave sleep (SWS; A) and rapid eye movement (REM) sleep (B) with a Neg test during eupneic breathing.

During REM sleep, the response to a reduction in UAW pressure by TP and SP was variable. Figure 2B presents an example during REM sleep in which both eupneic TP and SP activity are at their lowest levels. During the Neg test, a reduction in TP activity during expiration is seen with no increase in SP observed. However, a response was still observed while UAW pressure was reduced as TI increased and peak Dia decreased in the first breath but not in the second breath. In other cases of REM sleep, when phasic inspiratory activity was seen in the preceding breaths, a slight increase in phasic SP activity could be observed.

The average response for all goats to Neg test is presented in Fig. 3. In the awake state, TP activity was significantly reduced to 65 ± 15.5% (P < 0.0 2) of control, whereas SP activity increased to 198 ± 14.7% (P < 0.02) relative to control. In SWS, whereas control TP (65.08 ± 14.0%) and SP (57.1 ± 10.1%) activity were reduced (P < 0.02) compared with awake values, a comparable reduction in response to Neg test in TP activity (53.1 ± 9.6% of SWS control; P < 0.02) and an increase in SP activity (196 ± 22.7% of SWS control; P < 0.02) were still observed. REM sleep was characterized by a significant decrease in control TP (31.1 ± 14.5%; P < 0.02) and SP (23.9 ± 13.6%; P < 0.02) activity compared with awake controls. In response to Neg test, TP activity was decreased further (13.8 ± 3% of REM control; P < 0.02), whereas SP activity rose only slightly (174.2 ± 63.9% of REM control; P > 0.02). This later results not only shows a decrease in the basal level of TP and SP activity but also a decreased amplitude of the Neg test response compared with SWS.


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  		Fig. 3.   Effect of Neg tests on TP and SP electromyograph activity during wakefulness (AWK), SWS, and REM sleep. Values are means ± SD. dagger  TP SWS or REM control means were significantly different from AWK control means, or TP means during Neg tests were significantly different from their same-state controls, P < 0.02. Dagger  SP SWS or REM control means were significantly different from AWK control means, or SP means during Neg tests were significantly different from their same-state controls, P < 0.02.

Effect of state and negative pressure on ventilatory drive and timing. In the awake state (Table 1), a consistent decrease in VT (66 ± 16.2% compared with control; P < 0.02) was observed in response to Neg tests compared with control values. Although three of the four goats demonstrated increases in TI, no significant changes in TI (103.9 ± 10.1%) were found. During SWS (Table 2), mean control VT was reduced (90.1 ± 7.6%; P < 0.02), whereas TI was not significantly different (P > 0.02), from awake control values. In response to Neg tests during SWS, VT further decreased (61.3 ± 13.9% of SWS control; P < 0.02) and, similar to the awake response, TI remained unchanged (98.9 ± 5.6% of SWS control). In REM sleep (Table 3), control values for VT were also significantly reduced (85.3 ± 13.5%; P < 0.02) compared with awake values but not with SWS values. A considerable within-animal variation occurred for control TI values in REM, and, although the mean control TI was less than awake and SWS control values, it was not significantly different from either (P > 0.02). In response to Neg tests in REM sleep, VT was again reduced (38.9 ± 8.4% compared with REM control; P < 0.02), and, although TI increased (108.5 ± 9.1%), it was not significantly different from control (P > 0.02).

                              
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  		Table 1.   Awake control and Neg test ventilatory values


                              
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  		Table 2.   SWS control and Neg test ventilatory values


                              
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  		Table 3.   REM sleep control and Neg test ventilatory values

Similar to changes in VT, we consistently observed a decrease in Diaslope during eupneic breathing during SWS (84.2 ± 5.4%; P < 0.02) and REM sleep (78 ± 12.3%; P < 0.02) compared with awake controls (Fig. 4), but they were not significantly different from each other. In response to Neg tests in the awake state, Diaslope decreased (P < 0.02) to 68 ± 18.1%. A comparable decrease in Diaslope was found in SWS (71.6 ± 5.9% of SWS control; P < 0.02) in response to Neg tests. In REM sleep, a large decrease in Diaslope was observed in response to Neg tests compared with the REM control (23.5 ± 17.1% of awake controls; P < 0.02). However, no apneas were observed (lasting longer than 8.5 s) after the decrease in UAW pressure in any state or goat. The state-related changes in Diaslope are in contrast to the lack of changes in TI as discussed above and are shown for comparison in Fig. 4.


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  		Fig. 4.   Effect of Neg tests on the rate of rise of the integrated Dia electromyograph (Diaslope) and duration of inspiration (TI) during AWK, SWS, and REM sleep compared with awake controls. Values are means ± SD. dagger  Diaslope SWS or REM control means were significantly different from AWK control means, or Diaslope means during Neg tests were significantly different from their same-state controls, P < 0.02.

Effect of topical anesthesia on negative pressure response. Before topical anesthesia, movement of the nasal catheter and drips of water into the UAW produced coughing and gagging in all goats, and awake responses to Neg tests were present. After topical anesthesia in the awake state, Neg tests produced no significant changes (P > 0.02) in TP, SP, Dia, Diaslope, and VT compared with preanesthesia tests. Approximately 45 min after the application of anesthesia, the response to Neg tests had returned in TP, SP, Dia, Diaslope, and VT to preanesthesia levels.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our data demonstrate that the negative pressure reflex originating in the UAW has a selective and reciprocal effect on the electrical activity of mechanically opposing TP and SP muscles in the pharyngeal airway, which is attenuated in SWS and REM sleep compared with wakefulness. Our data also show that the reciprocal responses in the TP and SP muscles are coordinated with the decrease in VT and drive to the Dia. In contrast to the effect of sleep on UAW muscles, the effect of Neg tests on VT and Diaslope was greatest in REM sleep compared with awake and SWS states. Furthermore, our data support the concept that mechanoreceptors in the UAW mediate these responses. These data provide further evidence in the control and activation of mechanically opposing pharyngeal muscles that may lead to an imbalance between the opening and closing forces in the UAW, further leading to problems maintaining patency during sleep.

Effect of negative pressure on pharyngeal muscles in the awake state. Although no studies have shown the effect of Neg tests on TP and SP activity, many studies have focused on the UAW dilator muscles response to negative pressure in anesthetized animal models. An increase in genioglossus activity in response to a Neg test in the UAW has been observed in anesthetized rabbits (16-19), anesthetized (38) and unanesthetized dogs (7, 20), and anesthetized cats (4). Similarly, the activities of the sternothyroid, sternohyoid, and the posterior cricoarytenoid muscles have also been shown to increase after Neg tests in isolated UAW animal preparations (18, 38). In a study using anesthetized goats, Sekizawa et al. (31) observed a significant increase in the posterior cricoarytenoid muscle. Our results extend these observations by demonstrating that SP dilator muscle is selectively activated as a result of Neg tests in the isolated UAW. Conversely, our data also show that the mechanically opposing TP constrictor muscle is selectively deactivated in response to this reflex. In addition, the immediate response by SP and TP activity to decrease and then the return of UAW pressure to atmospheric affirm the short latency of this reflex as shown by Horner et al. (10, 11) and by Wheatley et al. (35) in humans. Our results, from short pulses in negative pressure during inspiration and from topical anesthesia, further suggest that superficial mechanoreceptors in the UAW are mediating the observed responses.

Harms et al. (7) found a persistent increase in the genioglossus activity after the application of negative pressure to the isolated UAW during expiration. In response to mechanically induced apneas, we also found a persistent reciprocal increase in TP activity and a decrease in SP activity compared with Dia activity that continued in the recovery from the apnea (3). The subsequent hypothesis by Harms et al. was that a reciprocal excitatory/inhibitory "memory" effect persisted on the UAW respiratory motoneurons far beyond the effect on the central output to the Dia. However, we did not see a long-lasting increase in SP or a decrease in TP activity after Neg tests. One possible explanation for the observed differences found between Harms et al. in dogs and the present data concerning Dia activity and TE prolongation may in part be due to the greater negative pressures they applied the Neg tests during expiration.

Negative pressure effect on drive and timing in the awake state. Although our primary focus was on the effects of negative pressure on TP and SP activity, our data also demonstrated a decrease in mean Dia activity, Diaslope, and VT with Neg tests, whereas TI was unchanged. Previous studies examining the effect of Neg tests on the isolated UAW preparation have shown mixed effects on Dia activity, drive to the Dia, and airflow. In anesthetized rabbits, Mathew and Farber (19) showed a decrease in peak Dia activity and slope of the integrated Dia activity with Neg tests. Van Lunteren et al. (37) reported in anesthetized dogs an insignificant decrease in peak Dia activity, whereas Diaslope significantly decreased. In anesthetized cats, Gauda et al. (4) did not find changes in Dia activity in response to Neg tests. In a study with a similar preparation and method to ours, Thach et al. (33), observed a decrease in flow rate and VT in unanesthetized tracheotomized children with Neg tests, which coincided with the results of this study.

In previous studies, the effect of Neg tests had a more consistent effect on respiratory timing than what was observed in this study. In anesthetized rabbits (19, 36) and in dogs (38), TI increased with Neg tests to the isolated UAW. In unanesthetized dogs (20), TI increased when negative pressure was applied across several breaths. In anesthetized goats (31), Neg tests applied to the UAW had no effect of respiratory timing. The basis for the difference of our findings in drive and timing compared with previous studies may have several causes. First, a species difference may exist in response to Neg tests applied to the isolated UAW as suggested by Sekizawa et al. (31) and Sant'Ambrogio et al. (27). Second, the negative pressure used in this study (-10 cmH2O) was the minimum pressure to produce a consistent UAW effect while minimizing swallowing as determined during a preliminary study. This same pressure in awake dogs had a minimum effect on TI while providing an ~20% increase in genioglossus activity (20). Similar results have been found for a threshold effect for Neg test response for UAW muscles (between -4 to -8 cmH2O) in anesthetized rabbits (20) and in dogs (37). In addition, negative pressures of -10 to -30 cmH2O had a larger effect on increasing TI and decreasing peak Dia activity than negative pressures between 0 and -10 cmH2O (20). Therefore, it is possible that the -10 cmH2O used in this study may be at a minimum threshold for producing an effect on TI.

Another important finding in this study was the lack of induced central apneas with the application of negative pressure during expiration in our isolated UAW preparation. Mathew and Farber (19) found induced central apneas in anesthetized rabbits in 15% of the trials. In anesthetized dogs, Van Lunteren et al. (38) observed induced apneas in some animals at more negative pressures (less than -10 cmH2O). In unanesthetized dogs, McNamara et al. (20) did not find any induced apneas in Neg tests in the pressure range of -10 to -30 cmH2O. Harms et al. (7), in anesthetized dogs, observed a delay in the onset of Dia activity (induced apnea) with negative pulses applied during expiration that correlated to the increases in the magnitude and duration of the Neg tests. At approximately -15 cmH2O pressure during wakefulness, the applied negative pressure was found to transiently close the UAW and thus contribute to the induced apnea observed. The lack of induced apneas observed in this study, therefore, may be due to the pressures used during the Neg tests, the lack of occlusion of the UAW during the test, or a species difference.

The objective of applying a topical anesthetic to the UAW during wakefulness was to test whether the responses to the Neg tests were mediated via mucosa pressure receptors in the UAW, which are carried primarily by superior laryngeal and trigeminal nerves (16, 17, 38). Results from Mathew et al. (17) and Harms et al. (7) suggest that submucosal mechanoreceptors in the UAW and muscle spindle stretch reflex in UAW muscles can respond to deformation of the UAW with pressures less than -5 cmH2O. The entire isolated UAW was anesthetized to abolish the response of the superficial receptors while not affecting the joint receptors and muscles spindles. The results from this anesthetic intervention support the concept that mucosa pressure receptors in the UAW mediate the negative pressure reflex at the pressure used in this study. However, the protocol used could not exclude that greater negative pressures, which may cause more UAW deformation and, thereby, stimulating muscle spindle reflex, would produce changes in the Neg test responses during UAW anesthesia in goats. Therefore, the role of muscle spindle reflex stimulation during Neg tests in goats is unclear.

Effect of SWS and REM sleep on the response to negative pressure. SWS has been shown to have little effect on resting minute ventilation, with a modest effect on Dia activity (25, 26). In REM sleep, minute ventilation and Dia activity may even increase (25). In contrast, SWS has been shown to produce a decrease in UAW muscle activity with further decreases in REM sleep (8, 14, 15, 24, 34, 35). Our data during control breathing confirm previous findings by us (unpublished observations) showing an ~30% decrease in TP and SP activity during SWS and a 70% reduction in both during REM sleep and findings by others in TP activity (5, 29, 32). However, as our data reaffirm, the Dia is relatively spared from the reduction of neuromuscular activity during eupneic breathing in SWS and REM sleep.

The bulk of the research on the response of UAW muscles to Neg tests have shown an increase in UAW dilator activity, but only a few studies have shown the response during sleep. Issa et al. (14) demonstrated a reduced genioglossal response to negative pressure in SWS compared with wakefulness with further reductions in REM. In humans, Wheatley et al. (34) and Horner et al. (10) demonstrated that the genioglossus response to negative pressure was reduced in non-REM sleep. Harms et al. (7) found a comparable increased in genioglossus activity to Neg tests between SWS and wakefulness, but the latency to genioglossus activation was increased in SWS. In REM sleep, they observed that the genioglossus activity was absent throughout the control period as well as during the negative pressure application. Our data extend these findings for UAW muscles with previously unexamined reciprocally controlled pharyngeal muscles in sleep demonstrating both a reduction in the control levels and the response to Neg tests. The reduction in the control levels of TP and SP activity from wakefulness to SWS corresponds to their decrease in activity associated with a disfacilitation from the raphe serotonergic neurons during SWS [reviewed in Horner (9)]. However, the magnitude of the responses (change from same-state control levels) to Neg tests was not different in SWS compared with wakefulness, suggesting that reflex loop is operating at the same gain as wakefulness even though the total activity is reduced. In contrast, the almost compete absence of TP and SP muscle activity and of the response to Neg tests during REM sleep supports the concept that an active inhibition of the respiratory motoneurons occurs in REM sleep (22, 25).

Only a handful of studies have investigated the effects of sleep on ventilatory drive and timing in response to Neg tests. Eastwood et al. (2) found no significant changes in the mean Dia activity during wakefulness, SWS, and REM sleep, but they observed a comparable (50%) reduction in Diaslope in dogs with intact UAWs that were exposed to negative tracheal pressures during tracheal occlusions. The decrease in Diaslope coincided with increases in TI in all states, although wakefulness increases were slightly greater than those in SWS and REM sleep. In the preparation by Eastwood et al., tracheal occlusions would result in no increases in VT and, therefore, a loss of the pulmonary stretch receptors inhibition (Hering-Breuer reflex). In contrast in the isolated UAW preparation used in this study, the effects of the Hering-Breuer reflex are maintained with unobstructed increases in VT. Therefore, we would anticipate a greater inspiratory inhibition with our isolated UAW preparation during Neg test compared with the findings of Eastwood et al. Our 30% decrease in Diaslope in response to Neg tests during SWS is less than that found by Eastwood et al. and could be explained by the presence of Hering-Breuer reflex in our results, even with a decrease in VT compared with wakefulness. However, the 76% decrease in Diaslope during REM sleep was considerably greater in our study compared with SWS and the changes observed by Eastwood et al., although both of our REM control levels were quite similar. We were careful not to use Neg tests with concurrent eye saccades or inspiration with swallows. Thus we are confident that our observations were not in part due to pontogeniculooccipital-related eye movements that may effect Dia activity (13) or to other reflexes in the UAW (27). The neurophysiological basis for the effect of Neg test on VT and Diaslope in REM is presently unknown.

Implications of negative pressure reflex on airway patency. From our results and others, it is clear that negative pressure in the UAW will cause a dual reflex consisting of 1) decrease in drive to the Dia and an increased duration of Dia and inspiratory flow and 2) a selective increase in UAW dilator activity and a corresponding decrease in constrictor activity. As a result, this dual reflex is thought to mediate several patency-maintaining responses in the UAW. We have demonstrated two mechanisms at work in counteracting the collapsing effect of negative pressure: an increased activation of the UAW dilating muscles and a reciprocal decrease in mechanically opposing constrictors. The resultant effect would be an enlargement and stiffening of the UAW to resist the collapsing forces of a negative intraluminal pressure during inspiration. The second effect is to reduce inspiratory drive and activity of the Dia, therefore reducing the negative pressure transmitted to the UAW during inspiration. Although both drive to the Dia and to the UAW muscles are decreased during SWS, the response to Neg test is still present. However, in REM sleep, neuromuscular activity to the genioglossus (9, 34, 35) and to the SP and TP in this study clearly shows a loss of neuromuscular tone to the UAW in response to negative pressure with maintenance of Dia activity. Therefore, the response of the upper airway muscles becomes less effective with sleep.

Conclusions. These data provide further evidence that mechanically opposing inspiratory and expiratory pharyngeal muscles are reciprocally controlled by negative pressure reflexes originating in the UAW. In addition, the reciprocal response by the UAW muscles and Dia to this protective reflexes are state dependent.


    ACKNOWLEDGEMENTS

The authors thank Julie Wenninger, Matt Hodges, and Dr. Alex Serra for technical assistance and Donna Dale for help in preparation of the manuscript.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant HL-25739 and the Veterans Affairs.

Address for reprint requests and other correspondence: H. V. Forster, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.

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.

Received 5 September 2000; accepted in final form 26 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 91(5):2289-2297
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