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J Appl Physiol 88: 1915-1923, 2000;
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Vol. 88, Issue 5, 1915-1923, May 2000

HIGHLIGHTED TOPICS
Consequences of periodic augmented breaths on tongue muscle activities in hypoxic rats

Patrick L. Janssen, James S. Williams, and Ralph F. Fregosi

Department of Physiology, University of Arizona, Tucson, Arizona 85721


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was designed to investigate the influence of hypoxia-evoked augmented breaths (ABs) on respiratory-related tongue protrudor and retractor muscle activities and inspiratory pump muscle output. Genioglossus (GG) and hyoglossus (HG) electromyogram (EMG) activities and respiratory-related tongue movements were compared with peak esophageal pressure (Pes; negative change in pressure during inspiration) and minute Pes (Pes × respiratory frequency = Pes/min) before and after ABs evoked by sustained poikilocapnic, isocapnic, and hypercapnic hypoxia in spontaneously breathing, anesthetized rats. ABs evoked by poikilocapnic and isocapnic hypoxia triggered long-lasting (duration at least 10 respiratory cycles) reductions in GG and HG EMG activities and tongue movements relative to pre-AB levels, but Pes was reduced transiently (duration of <10 respiratory cycles) after ABs. Adding 7% CO2 to the hypoxic inspirate had no effect on the frequency of evoked ABs, but this prevented long-term declines in tongue muscle activities. Bilateral vagotomy abolished hypoxia-induced ABs and stabilized drive to the tongue muscles during each hypoxic condition. We conclude that, in the rat, hypoxia-evoked ABs 1) elicit long-lasting reductions in protrudor and retractor tongue muscle activities, 2) produce short-term declines in inspiratory pump muscle output, and 3) are mediated by vagal afferents. The more prolonged reductions in pharyngeal airway vs. pump muscle activities may lead to upper airway narrowing or collapse after spontaneous ABs.

sighs; vagal mechanisms; genioglossus; hyoglossus


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BREATHS THAT ARE LARGER than the usual tidal volume punctuate normal breathing in mammals. Periodic augmented breaths (ABs), or sighs, are caused by an inspiration-augmentation reflex mediated by vagal afferents (10, 17). Chemoreceptor stimulation increases the frequency of ABs (1, 5, 10). The proposed physiological role for periodic ABs is in the prevention of atelectasis (2, 17).

Mechanisms responsible for ABs have been previously shown to act similarly on upper airway muscles and respiratory pump muscles (14, 24). During an AB, inspiratory motor output is markedly increased to the diaphragm, intercostal, and genioglossus (GG) muscles (4, 5, 10, 24). After an AB, breathing has been shown to be rapid and shallow for a brief period of time (5, 6, 18, 21). Accordingly, phrenic and hypoglossal nerve activities, and diaphragm and GG electromyogram (EMG) activities, have been shown to be depressed for one to five breaths following an AB before returning to baseline levels (4, 5, 24).

This study, however, demonstrates for the first time in rats that pharyngeal airway muscles and inspiratory pump muscles behave differently after ABs evoked by hypoxia. EMG activities of two muscles that control tongue position and respiratory-related tongue movements were compared with inspiratory pump muscle output [esophageal pressure (Pes)] before and after ABs in hypoxic, spontaneously breathing anesthetized rats. On the basis of the known protrusive action of the GG muscle, it is generally thought that GG contraction and forward tongue displacement help to maintain pharyngeal airway patency by preventing prolapse of the tongue against the posterior pharyngeal wall during inspiration (16). In the present study, both protrudor (GG) and retractor [hyoglossus (HG)] muscles of the tongue were examined. Recent experiments in our laboratory showed that in the rat the GG and HG are coactivated by hypoxia and hypercapnia (7) and that simultaneous stimulation of both muscles improves pharyngeal airflow mechanics more than the independent activation of either muscle (8).

ABs evoked by isocapnic and poikilocapnic hypoxia triggered sustained reductions in GG and HG EMG activities and respiratory-related tongue movements, but peak Pes was only transiently reduced after ABs. Consequently, the response to sustained hypoxia was characterized by reduced tongue motor activities during periods of increased inspiratory pump muscle activity. Adding 7% CO2 to the inspired gas mixture during hypoxia had no effect on the frequency of evoked ABs, but it prevented long-term reductions in GG and HG EMG activities and tongue movements following ABs. On the basis of our preliminary findings and information from previous studies showing that spontaneous ABs are vagally mediated in rats (1), we hypothesized that bilateral vagotomy would abolish hypoxia-induced ABs and stabilize drive to the tongue muscles. Our hypothesis was confirmed, and the significance of the present findings in the maintenance of pharyngeal airway patency in hypoxia is discussed.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Surgical Procedures

Eighteen male Sprague-Dawley rats [358.0 ± 9.3 (SE) g] were used for the experimental procedures. All procedures adhered to the guidelines established by the Institutional Animal Care and Use Committee at the University of Arizona. The animals were anesthetized with a series of intraperitoneal injections of urethane to achieve a dose of 1.3 g/kg. The animals were considered to be in a state of surgical anesthesia if they were not responsive to deep pressure application to the paws. Body temperature was monitored with a rectal thermistor (Yellow Springs Instruments) and maintained at 37°C with the use of a servo-controlled heating lamp. Polyethylene catheters (PE-50 tubing) were placed in a femoral vein for administration of fluids and in a carotid artery for arterial blood sampling. Arterial blood samples were withdrawn in 0.2- to 0.4-ml aliquots over a period of ~10 s and analyzed <1 min after sampling for arterial blood gases and pH. Blood-gas and pH values for 6 of 18 rats were analyzed with a Cameron Instruments (model BGM) analyzer; values for the remaining animals were studied with an Instrumentation Laboratories (model 1640) analyzer. If a base deficit existed, it was corrected by intravenous infusion of sodium bicarbonate. Donor blood was obtained from a urethane-anesthetized littermate; donor blood was administered intravenously to replace the volume of blood extracted during arterial sampling. The trachea was cannulated for delivery of inspired gases. Mixtures of O2, N2, and CO2 were delivered to the spontaneously breathing animal by connecting the outflow port of a rotameter to the tracheal cannula with a "t-tube" system. The concentrations of O2 and CO2 in the inspired air were monitored with rapidly responding O2 (Applied Electrochemistry, model S-3A) and CO2 (Beckman, model LB-2) analyzers. End-tidal CO2 was monitored with a very low dead space analyzer (model 1265, Novametrix) placed in series between the tracheal cannula and the t tube. At the end of the experiment, the animals were euthanized with an overdose of pentobarbital sodium.

Measurement of Inspiratory Pump Muscle Function

Pes was measured by a saline-filled catheter (PE-160) connected to a pressure transducer (Gould, model P23XL). The tip of the catheter was advanced to the level of the heart. Peak Pes (negative change in pressure during inspiration) was used as an index of tidal volume. The product, Pes × respiratory frequency (minute Pes), was used to estimate total ventilatory drive.

Preliminary experiments in our laboratory in urethane-anesthetized rats showed that changes in peak Pes correlated with both peak inspiratory intercostal and diaphragm EMG activities during a response to a 3-min hypercapnic challenge with 10% inspired CO2 (Fig. 1). On the basis of these data, we used Pes as an index of inspiratory pump muscle output rather than either inspiratory intercostal or diaphragm EMG activities. Our rationale for using Pes instead of EMG activities was the following. By not opening the abdomen to access the diaphragm or dissecting the muscles overlying the inspiratory intercostal muscles, we were able to preserve chest wall mechanics. However, chest wall mechanics may not have been completely conserved in this model; it is likely that anesthesia had depressive effects on the muscles of the chest wall.


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  		Fig. 1.   Line plots showing correlations between peak esophageal pressure (Pes) and peak inspiratory intercostal (A) and diaphragm (B) integrated electromyogram (iEMG) activities. Values are expressed as a percentage of maximal responses during a 3-min hypercapnic challenge (10% inspired CO2). Data were collected at 20-s intervals during the first 2 min of the progressive response to maximum levels and at the plateau of the response during each trial. n = No. of rats.

GG and HG EMG Recordings

EMG activities of the GG and HG were recorded by inserting two fine wire (diameter of 0.125 mm; Formvar, California Fine Wire) electrodes into the belly of each muscle. The wires were insulated except for the terminal 2 mm. Correct electrode placement was confirmed before each experiment by stimulating the muscles with supramaximal shocks through the wires to ensure that the tongue retracted with HG stimulation and protruded with GG stimulation (7). EMG recordings were amplified, filtered (30- to 3,000-Hz bandwidth) with AC-coupled differential amplifiers (Grass Instruments, model 79511K), rectified, and moving-time averaged with a time constant of 100 ms (Coulbourn Instruments, model S76-01). The processed EMG signal will be referred to as the integrated EMG (iEMG).

Measurement of Respiratory-Related Tongue Movement

The methods for measurement of respiratory-related tongue movement in the anesthetized rat have been described previously in detail (7). Briefly, the animal was placed in a custom-designed head frame, and a silk suture was connected from the tip of the rat's tongue to an isometric force transducer (Grass Instruments, model FT03). The transducer was mounted on a micromanipulator that allowed for precise alteration of muscle length. With this system, tongue retraction pulls, or loads, the transducer, and tongue protrusion releases, or unloads, the transducer. To record both protrusion and retraction of the tongue, tension was placed on the line connecting the tongue to the force transducer. The passive line tension was standardized by determining the optimal length of both GG and HG muscles. This was done by stimulating the GG and HG individually through the electrodes with single supramaximal shocks at different line tensions and recording the resultant twitch force. The line tension was increased by moving the transducer away from the animal in 1-mm increments. The peak muscle twitch force at each increment of passive line tension was recorded for both retractor and protrudor tongue muscles. If the optimal line tensions for the GG and HG twitch forces were different, the tension was set at the average of the two values and used for the entire protocol.

Experimental Protocol

The protocol consisted of 6-min exposures to three levels of hypoxia: poikilocapnic, isocapnic, and hypercapnic hypoxia. Each trial was separated by 15-30 min of recovery. The rats breathed 100% O2 during control and recovery periods. The order of administration of each level of hypoxia was randomized for each experiment.

Approximately 1 min before the start of the first stimulation period, an arterial blood sample was drawn to obtain control levels of blood gases and pH. Poikilocapnic hypoxia was produced by adjusting the inspired O2 concentration to between 12 and 15%, with the balance gas being N2. Isocapnic hypoxia was produced by adjusting the inspired O2 and CO2 concentrations to 12-15% and 3%, respectively. For hypercapnic hypoxia, the inspired gas concentration was adjusted to 12-15% O2 and 7% CO2. Arterial blood was sampled for blood gas and pH analysis at 1 and 6 min after the start of each stimulation period. In recovery periods after each hypoxic trial, an equal volume of donor blood was administered intravenously to replace the amount of blood extracted for blood-gas analysis. The maximum number of blood samples extracted from an individual rat ranged from 9 to 15. This number included samples that were repeated in control conditions, for example, after correction of the animal's acid-base status with intravenous injections of sodium bicarbonate.

Approximately 30 min after the end of the final hypoxic bout, the influence of hyperoxic hypercapnia was studied by adding progressively greater amounts of CO2 to the inspired gas mixture. The level of inspired CO2 was raised until GG and HG iEMG, tongue force, and Pes responses reached a plateau. The purpose of this part of the experiment was to obtain maximal levels for each experimental parameter to normalize steady-state data on a percentage of maximal basis (see Analysis of steady-state responses to hypoxia below). However, maximal levels of tongue muscle or pump muscle responses often occurred during one of the hypoxic trials. Therefore, steady-state data were expressed as a percentage of maximal responses recorded at any time during the experimental protocol.

The protocol was completed in 10 rats with intact vagus nerves. A separate group of rats (n = 8) was vagotomized bilaterally at the midcervical level during preparatory surgery, and the protocol was repeated.

Acquisition and Analysis of Data

Experimental parameters were monitored on a digital storage oscilloscope and/or Grass polygraph. The signals were recorded simultaneously on videocassette recorder tape using a pulse code modulation system (Vetter) for subsequent off-line analysis.

Analysis of ABs. The frequency of ABs was quantified during each hypoxic trial. In addition, tongue muscle activities and pump muscle output were quantified on a breath-by-breath basis before and after each AB evoked by hypoxia. Peak GG and HG iEMG amplitudes, tongue force, and Pes were measured during the breath preceding each AB and during the first, fifth, and tenth breaths following each AB. The data collected during the breath before the appearance of the first sigh in each hypoxic trial were considered baseline levels for the remaining breaths analyzed within that trial. All subsequent breaths included in the AB analysis within each trial were expressed as fractions of the baseline response to hypoxia. Tongue muscle and pump muscle parameters were not quantified during ABs for the following reasons: 1) we could not, with confidence, separate contributions from neural drive and movement artifact in the raw or iEMG signals, and 2) this was not the focus of the present study.

Analysis of steady-state responses to hypoxia. To determine the influence of vagal mechanisms on steady-state tongue muscle and pump muscle responses to sustained hypoxia, the time course of changes in GG and HG iEMG activities, tongue force, and ventilatory drive (minute Pes) was examined in both intact and bilaterally vagotomized rats during 6 min of poikilocapnic, isocapnic, and hypercapnic hypoxia. Five-breath bins of data were measured and averaged during periods of stable breathing in control conditions and at the end of each minute of poikilocapnic, isocapnic, and hypercapnic hypoxia. ABs, and the first 10 breaths after each AB, were excluded from the time-course analysis. GG and HG iEMG activities, tongue force, and minute Pes responses to hypoxia were expressed as a percentage of the maximal response.

Statistical analysis. The time courses of changes in GG and HG iEMG amplitudes, tongue force, and minute Pes during sustained hypoxia were analyzed by two-way repeated-measures ANOVA. Similarly, a two-way ANOVA was used to determine whether ABs had a significant influence on post-AB tongue muscle and Pes responses to hypoxia; t-tests were used to determine whether respiratory frequency was different from baseline after individual ABs. A P value of 0.05 or lower was considered significant for all tests.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Vagally Intact Rats

Figure 2 shows GG and HG iEMG activities, tongue force, and Pes from a vagally intact rat in control conditions and during poikilocapnic, isocapnic, and hypercapnic hypoxia. Note that the GG and HG muscles are phasically active during inspiration in each condition and that respiratory-related tongue movements result in tongue retraction (negative tongue force deflections). This record shows the first three ABs (denoted as single-breath augmentations in negative Pes) evoked during each hypoxic trial. The first AB occurred ~60 s after the onset of hypoxia in each trial. Note that, during poikilocapnic and isocapnic hypoxia, GG and HG iEMG activities and tongue force are diminished stepwise after each AB, whereas peak Pes is reduced for only one to two breaths after ABs. During hypercapnic hypoxia, the extent and duration of inhibition of GG and HG iEMG amplitudes and tongue force after ABs are markedly reduced relative to the other trials. The average frequency of periodic ABs was not different during poikilocapnic, isocapnic, and hypercapnic hypoxia (1.1 ± 0.3, 1.1 ± 0.3, 1.9 ± 0.2 ABs/min, respectively). ABs were not observed in control conditions (100% O2).


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  		Fig. 2.   Record of genioglossus (GG) and hyoglossus (HG) iEMG activities, tongue force, and Pes obtained from a vagally intact rat before (control) and during poikilocapnic, isocapnic, and hypercapnic hypoxia. Gap of time between the control period and the start of the recordings in hypoxia is ~1 min. * Augmented breaths (ABs) in each condition.

Figure 3 shows average breath-by-breath GG and HG iEMG, tongue force, and Pes responses to poikilocapnic, isocapnic, and hypercapnic hypoxia before and after three consecutive ABs. The breath before the first AB is also shown in Fig. 3 and is considered the baseline response to hypoxia within each condition. These data suggest that ABs evoked long-lasting reductions in GG and HG iEMG activities and tongue movements relative to pre-AB values during poikilocapnic and isocapnic hypoxia. Diminished GG and HG iEMG activities and tongue force typically persisted until the onset of the next AB, after which further reductions in tongue muscle parameters ensued (especially obvious in poikilocapnic hypoxia). However, tongue muscle activities were not completely abolished after ABs: residual tongue muscle activities were consistently recorded after successive ABs in each hypoxic condition. Pes was reduced transiently and recovered to baseline levels within five breaths after each AB in poikilocapnic and isocapnic hypoxia. In hypercapnic hypoxia, GG and HG iEMG, tongue force, and Pes responses recovered within 10 breaths after each AB.


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  		Fig. 3.   Breath-by-breath changes in peak Pes, GG and HG iEMG activities, and tongue force before and after 3 consecutive ABs evoked by poikilocapnic (A), isocapnic (B), and hypercapnic (C) hypoxia. Leftmost data point in each panel represents the breath before first AB and is considered the baseline response to hypoxia in each condition (1.0; dashed line). All subsequent data shown are means ± SE, expressed as fractions of the baseline response to hypoxia within each trial. P: breath before each AB; 1, 5, and 10: 1st, 5th, and 10th breath, respectively, after each AB. Gap of time between successive ABs was ~45 s. * Values within brackets are significantly less than their respective baseline levels (P < 0.05).

Effect of ABs on Respiratory Rate

Respiratory frequency did not change significantly after individual ABs in any of the three hypoxic conditions. The changes in frequency that occurred immediately after each of the first three ABs in a given trial were averaged for each condition. These data show that breathing frequency increased slightly after ABs evoked by hypercapnic and isocapnic hypoxia [change in frequency = 2.6 ± 1.2 and 2.2 ± 0.8 (SE) breaths/min, respectively] and decreased by 0.7 ± 1.2 breaths/min after ABs in poikilocapnic hypoxia.

Figure 4 shows the time course of changes in GG and HG iEMG activities and tongue force during 6 min of poikilocapnic, isocapnic, and hypercapnic hypoxia in vagally intact rats. Phasic inspiratory GG and HG EMG activities and retractive tongue movements were consistently recorded in hyperoxic control conditions. Hypercapnic hypoxia caused sustained increases in phasic GG and HG iEMG amplitudes and tongue retraction force. In contrast, steady-state tongue muscle responses were reduced relative to control levels during poikilocapnic hypoxia and were not different from control during isocapnic hypoxia.


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  		Fig. 4.   Line plots showing peak GG and HG iEMG activities, tongue force, and minute Pes before, during (solid lines), and after 6 min of poikilocapnic (A), isocapnic (B), and hypercapnic (C) hypoxia in vagally intact rats. Data are mean ± SE (n = 10), expressed as % of maximal responses. Note that iEMG and tongue force data are expressed on a per breath basis, and Pes is expressed on a per minute basis. * Minute Pes values are significantly different from corresponding prehypoxia levels (P < 0.05). and  GG and HG iEMG and tongue force values are significantly different from corresponding prehypoxia levels (P < 0.05).

Each hypoxic condition caused an increase in minute Pes, due to increases in Pes and respiratory frequency (Table 1), which was maintained above control levels throughout the trial (Fig. 4). Peak changes in respiratory frequency and Pes consistently occurred 1 min after the start of hypoxia (Table 1). The greatest change in respiratory frequency (change in frequency = peak - control) occurred during poikilocapnic hypoxia (change in frequency = 28.4 breaths/min), followed by isocapnic hypoxia (change in frequency = 20.1 breaths/min) and hypercapnic hypoxia (change in frequency = 8.3 breaths/min). By the last minute of hypoxia (6-min time point), respiratory frequency had rolled off from peak levels in each condition (Table 1).

                              
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  		Table 1.   Blood-gas, pH, and ventilatory data in control conditions (100% O2) and in the 1st and 6th min of hypoxia in vagally intact rats

Vagotomized Rats

Phasic inspiratory GG and HG EMG activities and retractive tongue movements were consistently recorded in hyperoxic control conditions in vagotomized rats. Respiratory frequency was 43% lower in vagotomized animals compared with vagally intact rats during hyperoxia (intact: 89.0 ± 2.1 breaths/min, vagotomized: 51.0 ± 1.5 breaths/min; P < 0.05). PCO2 in arterial blood was 6.5 Torr higher in vagotomized animals during hyperoxia (intact: 41.3 ± 1.0 Torr, vagotomized: 47.8 ± 1.5 Torr; P < 0.05). Blood-gas, pH, and ventilatory data for vagotomized rats are shown in Table 2.

                              
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  		Table 2.   Blood-gas, pH, and ventilatory data in control conditions (100% O2) and in the 1st and 6th min of hypoxia in vagotomized rats

Vagotomized rats showed increases in GG and HG iEMG activities, tongue retraction force, and minute Pes in each condition (Fig. 5). ABs did not appear during hypoxic stimulation in vagotomized animals. In contrast to the animals with intact vagi, GG and HG iEMG activities, tongue force, and minute Pes changed in parallel during sustained poikilocapnic, isocapnic, and hypercapnic hypoxia in vagotomized rats (Fig. 5).


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  		Fig. 5.   Line plots showing peak GG and HG iEMG activities, tongue force, and minute Pes before, during (solid lines), and after 6 min of poikilocapnic (A), isocapnic (B), and hypercapnic (C) hypoxia in vagotomized rats. Data are mean ± SE (n = 8), expressed as percentage of maximal responses. Note that iEMG and tongue force data are expressed on a per breath basis, and Pes is expressed on a per minute basis. * Minute Pes values are significantly different from corresponding prehypoxia levels (P < 0.05). and  GG and HG iEMG and tongue force values are significantly different from corresponding prehypoxia levels (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present experiments were designed to investigate in anesthetized rats the influence of hypoxia-evoked ABs on respiratory-related tongue muscle behavior and inspiratory pump muscle output. The main finding was that ABs evoked by poikilocapnic and isocapnic hypoxia triggered prolonged reductions in peak GG and HG iEMG amplitudes and tongue retraction force relative to pre-AB levels. In contrast, inspiratory pump muscle output (Pes) was only transiently reduced following ABs in these conditions. Consequently, the overall response to sustained hypoxia was characterized by reduced tongue motor activities but elevated drive to inspiratory pump muscles. We also tested the hypothesis that bilateral vagotomy would abolish hypoxia-evoked ABs and would stabilize drive to the tongue muscles. This hypothesis was confirmed. Finally, we verified previous findings in our laboratory (7), showing that tongue protrudor and retractor muscles are coactive during inspiration in the rat and respond in parallel to chemoreceptor stimulation and that coactivation is associated with retractive tongue movements.

Critique of Methods

Peak Pes was used to estimate tidal volume in the present experiments. Although it is reasonable to assume that tidal volume was increasing as Pes increased during chemoreceptor stimulation in these tracheostomized animals, we acknowledge that changes in pulmonary resistance may alter this relationship. Preliminary experiments in our laboratory using the present model show that Pes is correlated with both peak inspiratory intercostal and peak diaphragm EMG activities (Fig. 1). Thus we feel that Pes is a reliable index of inspiratory pump muscle output in the rat.

Minute Pes was used to represent total ventilatory drive in this study. The patterns of change in minute Pes during sustained poikilocapnic, isocapnic, and hypercapnic hypoxia in the present study were qualitatively similar to changes in minute ventilation that were reported during sustained chemoreceptor stimulation in humans and other mammals (25, 26). The present data suggest that minute Pes is a useful method for representing total ventilatory drive in the rat.

Respiratory-related tongue movements were measured by attaching the tongue to a force transducer via a suture. This method has been shown to accurately record protrusive and retractive movements of the tongue (7, 9). However, despite the primary protrusive action of the GG and retractive action of the HG, both muscles are known to depress the tongue. Thus one limitation of the present model is that tongue depression cannot be quantified. In addition, because there are other extrinsic tongue muscles that retract the tongue (e.g., styloglossus), we cannot attribute all retractive forces measured to the HG muscle. Previous experiments in our laboratory (7) and the present data show that tongue muscle EMG activities consistently change in parallel with tongue force measurements. Therefore, despite its limitations, the present methods offer a reasonable representation of the coupling between neural drive and mechanical output of the extrinsic tongue muscles during breathing.

A hyperoxic inspirate was used for control and recovery periods in the present experiments. This was done to limit the appearance of ABs to the hypoxic trials. Preliminary experiments showed that spontaneous ABs often occur during room-air breathing and cause unstable tongue muscle activities. ABs were not observed when animals breathed 100% inspired O2.

Effects of ABs on Respiratory-Related Tongue Muscle and Ventilatory Pump Muscle Responses to Hypoxia

Regularly occurring ABs evoked by poikilocapnic and isocapnic hypoxia in anesthetized rats triggered prolonged reductions in GG and HG iEMG amplitudes and tongue retraction force relative to pre-AB levels, whereas peak Pes was only transiently diminished after ABs in these conditions. To our knowledge, this is the first study to show that pharyngeal airway muscles and inspiratory pump muscles behave differently following spontaneous ABs. Previous studies in anesthetized dogs (24) and rabbits (4) have shown that inspiratory motor output is markedly increased to the diaphragm and GG muscles during an AB and that the activities of both muscles are depressed for one breath after the AB. In both of these studies, ABs were induced by increased CO2, either by the CO2 rebreathing method (4) or by exposure to hyperoxic hypercapnia (24). It is possible that hypercapnia masked the secondary depressive effects of ABs on GG activity in those studies. Indeed, in the present experiments, long-term post-AB reductions in GG and HG iEMG activities and tongue force were prevented when 7% CO2 was added to the hypoxic inspirate (see Fig. 3C). Other studies have shown that ABs elicit a period of rapid and shallow breathing, which is restored to control levels in three to six breaths (5). In agreement with those findings, our present results show that Pes was depressed for <10 breaths after ABs in each hypoxic condition. However, respiratory frequency did not change significantly after ABs in the present experiments.

Effect of Vagotomy on ABs and Steady-State Responses to Hypoxia

In the present experiments, bilateral vagotomy slowed respiratory frequency and abolished hypoxia-evoked ABs. These findings are in agreement with a previous report in urethan-anesthetized rats, which showed a slowing of breathing frequency and ablation of spontaneous deep breaths after vagotomy (1). In pentobarbital sodium-anesthetized cats, ABs have been shown to be temporarily abolished after vagotomy but reappear after 1-2 h (5). Perhaps the difference in species or anesthesia used in these experiments could account for the contradictory results.

In contrast to results from animals with intact vagi, the drive to tongue and pump muscles changed in parallel during sustained hypoxia in vagotomized rats. These data suggest that bilateral vagotomy stabilized the drive to the tongue muscles during hypoxia by eliminating perturbations due to periodic ABs. This idea is not supported by findings from an earlier study in chloralose-anesthetized cats with intact vagus nerves, which showed that the time course of changes in GG and diaphragm EMG activities was similar during sustained poikilocapnic hypoxia (22). However, ABs were not reported to have occurred during hypoxia in those experiments. Similar to the present findings, results from one study in awake humans showed that GG muscle activity declined to a greater extent than inspiratory intercostal muscle activity during sustained isocapnic hypoxia (15). Unfortunately, it is impossible to know whether spontaneous ABs contributed to this response because only steady-state data were presented and no ABs were reported to have occurred during hypoxia.

Potential Mechanisms and Physiological Significance

Given the findings of our present experiments, it is possible that a long-lasting inhibitory vagal memory could have been activated by periodic stimulation of pulmonary stretch receptors to differentially suppress tongue muscle activities after ABs. Previous reports suggest that a long-term memory of inhibitory vagal feedback reduces short-term potentiation of ventilation after isocapnic hypoxia exposure (27) and diminishes long-term facilitation of upper airway muscle activities after repeated carotid sinus nerve stimulation (12). The more potent inhibitory effect on GG and HG iEMG amplitudes compared with peak Pes after ABs could be explained by the fact that the lung inflation reflex reduces drive to upper airway muscles more than inspiratory pump muscles (11, 23). The inhibitory effects after ABs were most dramatic during poikilocapnic hypoxia. It is likely that hyperventilation-induced hypocapnia and AB-induced vagal inhibition interacted to markedly depress tongue muscle activities in these conditions.

In the present study, it appeared that post-AB responses were influenced by the underlying rate of respiration associated with each hypoxic condition. The greatest reductions in GG and HG EMG activities and tongue force occurred when respiratory frequency was greatest, that is, during the initial response to poikilocapnic hypoxia (see Table 1 and Fig. 4). Accordingly, the post-AB responses were less pronounced in isocapnic hypoxia, and lowest in hypercapnic hypoxia, during which the smallest change in respiratory frequency occurred. Moreover, as the frequency response declined over the course of the poikilocapnic hypoxia trial, tongue muscle activities returned toward baseline levels. Thus post-AB reductions in tongue muscle activities may be the result of an interaction between 1) prolonged inhibitory vagal memory evoked by ABs and 2) the underlying respiratory timing associated with chemoreceptor stimulation. Our results also indicate that respiratory frequency did not change significantly after periodic ABs. This suggests that post-AB reductions in tongue muscle activities may be mediated by changes in hypoglossal motoneuron output rather than by changes in central pattern generation. Future studies are required to address these hypotheses in a systematic manner.

The consequence of reduced tongue protrudor and retractor muscle activities during heightened ventilatory drive could be an increase in pharyngeal airway collapsibility. Indeed, stimulation of the whole hypoglossal nerve, which innervates both protrudor and retractor tongue muscles, has been shown to preserve pharyngeal airway patency when airflow is artificially enhanced in the cat isolated upper airway (20). Similarly, recent experiments in our laboratory using a rat isolated upper airway model show that simultaneous electrical stimulation of tongue protrudor and retractor muscles improves pharyngeal airflow mechanics more than independent stimulation of either muscle, despite retractive tongue movements (8). It was suggested that coactivation "stiffens, retracts, and depresses the tongue as the antagonistic muscles work against one another, resulting in a less collapsible retroglossal airway" (see Ref. 8). Unfortunately, the tracheotomized rat model, as used in the present study, offers no information about changes in pharyngeal flow mechanics following ABs. Future experiments should be designed to investigate the relationship between changes in protrudor and retractor tongue muscle activities and pharyngeal airway resistance in animals with an intact upper airway.

Our present findings could be of clinical and physiological significance. From a clinical standpoint, reduced upper airway muscle activities in the face of sustained inspiratory negative pressure are thought to be central to the pathogenesis of obstructive sleep apnea (16). From a physiological standpoint, results from the poikilocapnic hypoxia trials could provide insights into the mechanism of periodic breathing during sleep at high altitude, which is characterized by periods of ventilatory stimulation and inhibition, alternating with periods of apnea (3). However, we concede that the physiological changes that occur during sleep are quite different from those evoked by anesthesia. In addition, the present model does not account for the function of upper airway receptors, which may evoke excitation of hypoglossal motoneurons in response to changes in intraluminal pressure or flow (13, 19).


    ACKNOWLEDGEMENTS

We thank J. Reeder for excellent technical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-51056 and HL-56876.

Present address of P. L. Janssen: Dept. of Animal Biology, School of Veterinary Medicine, Univ. of Pennsylvania, Philadelphia, PA 19104.

Present address of J. S. Williams: 229 Turner Center, Division of Exercise Science, Univ. of Mississippi, University, MS 38677.

Original submission in response to a special call for papers on "Hypoxia Influence on Gene Expression."

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. F. Fregosi, Dept. of Physiology, Gittings Building, Univ. of Arizona, Tucson, AZ 85721-0093 (E-mail: fregosi{at}u.arizona.edu).

Received 9 July 1999; accepted in final form 14 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 88(5):1915-1923
8570-7587/00 $5.00 Copyright © 2000 the American Physiological Society


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