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Muscle relaxation attenuates the reflex response to laryngeal negative pressure
Respiration Physiology 107 (1997) 219 – 230
Muscle relaxation attenuates the reflex response to laryngeal negative pressure Wiktor A. Janczewski * Department of Neurophysiology, Medical Research Centre, Polish Academy of Sciences, 3 Dworkowa Str. 00 -784 Warsaw, Poland Accepted 15 November 1996
Abstract Negative pressure (NP) within the upper airway (UA) excites the activity of the genioglossus muscle and other UA dilators. For unknown reasons this reflex response is attenuated during sleep. Sleep reduces skeletal muscle tone, therefore we hypothesized that the magnitude of the pressure reflex is modulated by muscle tension. We have compared the threshold and the magnitude of the reflex response to laryngeal NP before and during muscle relaxation in 16 rabbits. The threshold increased from 79 2 (mean9S.D.) to 17 9 4 cm H2O. Peak amplitude during NP pulse dropped from 167 to 139 arbitrary units (a.u.) for the hypoglossal nerve (n.XII) and from 134 to 114 a.u. for the facial nerve (n.VII). Independently of the pressure reflex muscle relaxation reduced the phasic n.XII activity by 18% and tonic activity by 20%. We conclude that muscle atonia attenuates n.XII activity and the response to UA pressure change. Our findings when extrapolated to state of sleep suggest that sleep-induced muscle relaxation may be an independent factor suppressing pressure reflex. © 1997 Elsevier Science B.V. Keywords: Mammals; Rabbit; Muscle; Upper airway dilators; Reflex; Laryngeal; Sleep; Upper airway patency; Upper airways; Patency; Negative pressure reflex
1. Introduction During each inspiration the upper airway (UA) is subjected to negative pressure (NP) of 2 – 3 cm * Corresponding author. Present address: Laboratory of Clinical Pharmacology, Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Str, 02-106 Warsaw, Poland. Tel.: +48 22 6086523; fax: +48 22 6685416; e-mail: [email protected]
H2O generated by contraction of the diaphragm and other thoracic respiratory muscles. In normal subjects, UA negative pressure increases during sleep due to increase of the UA resistance. Such an increase is more pronounced in habitual snorers (Pack, 1994). In obstructive sleep apnea (OSA) patients apneic episodes are characterized by progressive (Kimoff et al., 1994) increase in intrathoracic and laryngeal negative pressure during efforts against the occluded airway. Maximal
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value of transdiaphragmatic pressure is typically reached on the last (83% of all apneas) obstructed effort and usually amounts ca. −56 cm H2O (Kimoff et al., 1994). Upper airway pressure more negative than few cm H2O indicates pathological narrowing of the upper airway, therefore it seems reasonable to assume that the amount of negative pressure is somehow sensed and used as a feedback information for the systems controlling excitation of the UA dilating muscles. This was found to be correct by Mathew et al. (1982a). They have shown that in rabbits NP pulse applied to UA markedly activates the hypoglossal nerve (n.XII) and at the same time depresses phrenic nerve (n.Ph) activity (Mathew et al., 1982a). Laryngopharyngeal compartment of the upper airway was found to be the main reflexogenic zone responding to NP, nosopharyngeal compartment played subsidiary role, whereas oropharyngeal compartment was of a minor importance (Mathew et al., 1982b). The Internal branches of the superior laryngeal nerves, the nasal trigeminal nerves and the glossopharyngeal nerves were mediating the responses from these compartments, respectively (Mathew et al., 1982b). Augmentation of UA dilator muscles by negative intraluminal pressure have been demonstrated also in man (Aronson et al., 1989; Horner et al., 1991, 1994; Wheatley et al., 1993). Negative pressure reflex activates not only hypoglossal muscle but other muscles dilating and stiffening UA, as well (Wheatley et al., 1993). Remmers et al. (1978) have proposed that the collapse of the upper airspace occurs when the negative pressures generated during inspiration are inadequately opposed by activation of the upper airway dilator muscles. Therefore, the pressure reflex, which selectively increases activity of the UA dilators and at the same time decreases the amount of negative pressure by suppressing the activity of the diaphragm should be able to prevent UA collapse. This theoretically potent reflex fails to prevent episodes of prolonged obstructive apneas during sleep in some individuals. Likely, because during sleep the magnitude of the reflex UA muscle activation is lower. Aronson et al. (1989) reported
an immediate increase in electromyographic genioglossal muscle activity in response to nasal continuous negative airway pressure of − 2.5 to − 7.5 cm H2O in normal awake humans. The response disappeared during NREM and REM sleep. Wheatley et al. (1993) have found that an increase of the tensor palatini electromyogram was attenuated during non-REM sleep in comparison to that during wakefulness. Horner et al. (1994) have shown that non-REM sleep attenuates reflex genioglossus muscle activation by stimuli of negative airway pressure. Although studies of Wheatley et al. (1993) and Horner et al. (1994) do not present such data it is likely that the response to UA negative pressure is further reduced during REM sleep. Impairment of the pressure reflex during sleep in humans has been discussed previously as an example of neural disfacilitation of reflex motor systems during sleep (Wheatley et al., 1993) or as a state dependent central effect (Horner et al., 1994). It seems also likely that the magnitude of the reflex response is affected by sleep related withdrawal of an excitatory drive to hypoglossal motorneurons (Kubin et al., 1996; Pack, 1994). We think that other mechanism may contribute to attenuation of the pressure reflex during sleep. We propose that the primary reason for the reduction of the pressure reflex during sleep is not the state of sleep but muscle relaxation. We think that reduction of the skeletal muscle tone may modify afferent information from UA receptors in such a way that the response to negative pressure will be reduced. If our concept is correct it should be possible to modify reflex response in a similar way as described during sleep only by relaxing skeletal muscles. Therefore, our aim was to determine if the reflex hypoglossal and facial nerve activation observed in response to stimuli of negative pressure in non-paralyzed rabbits is altered in magnitude during muscle relaxation. To answer this question we have compared responses to the same stimuli before and during muscle relaxation. Our working hypothesis was that the magnitude of the reflex response to negative pressure depends on the muscle tension.
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Since, this is the first study to record UA nerve activity continuously while muscles are being relaxed we have utilized this opportunity to determine whether the magnitude of hypoglossal and facial nerve activities are affected by changes in skeletal muscle tone. A preliminary account has been published (Janczewski, 1993; Janczewski and Janczewski, 1995).
2. Methods Nine decerebrate and seven halothane (1.5 vol%) anaesthetized rabbits (2.9 – 3.5 kg) were used. Decerebration was performed under halothane (3.0 vol%) anesthesia. In decerebrate rabbits anesthesia was discontinued more than 30 min before recordings. Animals were placed supine on a heating blanket to maintain body temperature in the range of 38 – 38.5°C. Femoral artery and vein were cannulated. Arterial cannula was connected to a transducer (Viggo-Spectramed DTX/Plus) and Neurolog 108 amplifier in order to measure arterial pressure. Vagus nerves and sympathetic trunks were bilaterally cut in the neck caudal to the origin of the superior laryngeal nerves. Animals were artificially ventilated at the constant level with air supplemented with oxygen to maintain arterial oxygen tension (PaO2) above 200 mmHg and PaCO2 in the range of 39–45 mmHg. If, despite sectioning of both phrenic nerves the animal still ‘struggled’ with the respirator, thoracotomy was performed to ensure that the spontaneous respiratory movements were ventilatory inefficient. End-tidal CO2 and O2 were monitored (Nec San-El IH 206). Arterial blood samples (0.4 ml) were drown anaerobically into heparinized syringes and immediately analyzed (Coming 238) in order to determine pH, PaO2, PaCO2 before and after muscle relaxation and before and after application of negative pressure. The corrections for temperature differences between the rabbit and blood gas analyzer were introduced by the Coming system. If the PaCO2 tension after muscle paralysis was more then 1 mmHg lower then before muscle paralysis the results were not included in the statistical analysis.
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Arterial bicarbonate concentration and base excess were also measured. If necessary, sodium bicarbonate was administered (i.v.) to maintain the arterial bicarbonate concentration above 20 mEq/L.
2.1. Laryngeal pressure changes To apply pulses of negative pressure we modified the protocol of Mathew et al. (1982a). Tracheostomy was performed below the cricoid cartilage. Two cannulas were inserted through the tracheostomy. The upper cannula was inserted in the direction of larynx and secured with its tip just below the larynx. The upper cannula was connected to the source of the negative pressure. The lower tracheal cannula was inserted in the direction of the lungs and connected to a constant volume mechanical ventilator (Ugo Basile 6025 ventilator). We mimicked the situation of the upper airway obstruction by inflating a small rubber balloon placed just rostral to larynx between tongue and posterior pharyngeal wall. This arrangements ensured that the negative pressure stimulated receptors only in the larynx and a part of pharynx close to epiglottis (i.e. epipharynx). Since mouth and the nares of the rabbits were opened, the pressure in the nosopharynx was equal to atmospheric.
2.2. Electroneurograms and electromyogram In order to record genioglossus muscle (m.GG) electromyogram (EMG) bipolar fine wire electrodes (Biomed Wire AS 631, Cooner Wire) were inserted 4–6 mm deep through one mylohyoid muscle midway between chin and hyoid bone. Phrenic nerves (C5-C6 roots) were bilaterally cut and prepared for recording. Both n.XIIs were placed on bipolar electrodes and covered with petroleum jelly to prevent drying. Hypoglossal nerves were not cut, phrenic nerves were bilaterally cut to prevent movements of diaphragm while animals were not paralyzed. In ten animals one bucco-labial branch of the facial nerve (n.VII) was cut distally and placed on a bipolar electrode. All signals were amplified, filtered, rectified, and integrated by means of RC integrator with time
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constant of 100 msec. Neural signals together with arterial pressure, laryngeal pressure and end-tidal PaCO2 were recorded on Honeywell Omnilight recorder and after analog to digital conversion on a hard disk of IBM-486 PC.
2.3. Muscle relaxation Data were obtained from 16 rabbits. In order to produce a state of muscle atonia a short acting neuromuscular blocking agent Norcuron (Organon Teknika), i.e. vecuronium bromide 0.05 mg/kg i.v. have been administered to 11 animals. The remaining five rabbits were treated with a longer acting neuromuscular blocking agent Tubocurarin (Orion), i.e. D-Tubocurarinum chloratum i.v. 0.6 mg/kg. Muscle relaxants were dissolved in physiological solution and administered in a 0.5 ml bolus to the femoral artery. Vercuronium bromide is widely used in humans, however we were not aware of any data defining the rabbit dose. Therefore in the preliminary experiments we have tried several doses. We have found that the dose of 0.04 mg/ kg of vecuronium bromide was the smallest dose effectively relaxing UA muscles. Total elimination of the genioglossus muscle EMG activity was considered to be a reliable marker of muscle relaxation. Our experimental arrangements ensured constant ventilation throughout the experiment, however in two rabbits after administration of vecuronium bromide blood gas test revealed 2 mmHg decrease of PaCO2. It was not clear whether those two animals really developed hypercapnia or sampling or measurements were not accurate. Nevertheless, these data were excluded from further analysis. More than 1 h later vecuronium bromide was administered again in all rabbits that received short acting agent before. For statistical analysis we have only used data from the first successful trial. This means from the first trial in 14 animals and from the second trial in those two, which did not have stable PaCO2 during the first attempt.
2.4. Measurements of ner6e acti6ity The changes of n.XVI n.XII, n.VII and n.Ph nerves activity and m.GG EMG activity were evaluated from integrated signals. The level of 0 arbitrary unit (a.u.) for total activity was defined as the level of nerve/EMG activity during expiration. The level of 100 a.u. for total activity was defined as an average of peak activity during ten successive respiratory cycles just before experimental intervention. Such a procedure is simple and commonly used to evaluate the total UA and other nerve activity (Kubin et al., 1996). However, for evaluation of the decrease of UA nerve activity induced by administration of muscle relaxant, we have separately measured decrease of tonic and phasic activity. The reason was that total UA neural or m.GG muscle activity is a sum of tonic (expiratory) activity and phasic inspiratory activity. In a case when an experimental intervention reduces total nerve/EMG activity it may happen that the tonic activity is totally eliminated and the phasic activity is reduced so much that it drops below the zero-line (see m.GG EMG signal in Fig. 2). In such a case peak activity would be negative. The 0 and 100 a.u. for phasic activity were as described previously. The 100 a.u. for tonic activity was the expiratory level of activity (i.e. zero-line for phasic activity). To find the zero-line for tonic activity we had hyperventilated the animals until the PaCO2 was 20 mmHg. Signal from electrodes at this PaCO2 partial pressure was our 0 a.u. level for tonic activity. Such a procedure was justified by our observation that n.XII/ m.GG activity was totally eliminated when PaCO2 was lower than 24 mmHg and n.VII activity disappeared when PaCO2 was below 22 mmHg. To estimate control inspiratory activity the mean9 S.D. have been calculated for ten respiratory cycles just preceding NP pulse. By definition we treated the mean value as 100 a.u. S.D. value for UA nerves was usually close to 3 a.u.
2.5. E6aluation of the threshold of the reflex response To determine the threshold for the reflex response we have applied a series of NP pulses.
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Fig. 1. Excitation of the UA nerves in response to 2.5 s duration pressure stimuli of -30 cm H2O. Traces from bottom to top: n.Ph, n.XII, m.GG EMG, n.VII, laryngeal pressure and end-tidal CO2. NP pulse was applied during the second half of inspiration. This resulted in a rapid increase of the UA muscle and nerve activities and depression of n.Ph activity. Note that UA activity remain increased for more than seven respiratory cycles after termination of the NP pulse.
Pulses were rectangular and started at the end of expiration and lasted for three respiratory cycles. The response was classified as positive if the mean peak n.XII activity increased by 3 S.D. a.u. (typically ca. 10 a.u.) during the pulse.
2.6. Characteristic of the negati6e pressure pulse Once the threshold have been determined we could decide what value of negative pressure will be used during the experiment. We used pressure equal to 4 times the threshold. The mean value was − 28 cm H2O. Our source of negative pressure was efficient enough to produce nearly rectangular pulses. The pressure pulse was re-
stricted to the larynx and epipharynx.
2.7. Estimation of the magnitude of the reflex response To determine the magnitude of the reflex response average of peak integrated activities observed during five consecutive NP pulses was calculated. Therefore, one animal provided a single number representing the amount of activation of particular nerve activity or EMG activity. The mean for all 16 animals was calculated and presented in the form of mean9 S.D. For the purpose of comparison before vs. during muscle relaxation the same procedure was repeated after
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Fig. 2. Attenuation of the n.XII nerve activity and attenuation of the response to NP pulse induced by i.v. administration of muscle relaxant. Traces from bottom to top: integrated activity of the n.Ph( − ), integrated activity of the n.XII, integrated EMG activity of the m.GG, intralaryngeal pressure. Note decrease of both tonic expiratory n.XII activity and n.XII amplitude concomitant with disappearance of m.GG EMG. Before muscle relaxation, pulse of −30 cm H2O increased n.XII peak inspiratory activity to the value of 350 a.u. When muscles were relaxed NP pulse increased peak n.XII activity to the value of 165 a.u., which was less then half of the stimulation before muscle relaxation. The relative increase was calculated according to formula: the amplitude during NP pulse divided by the amplitude just before NP pulse. It amounted to 165/78 a.u. =212% during MR vs. 350/100 a.u. =350% before muscle relaxation. Post-stimulus potentiation of the n.XII and GG EMG activity may be observed both before and during muscle relaxation.
muscle relaxation. Therefore, each one of 16 animals provide one pair of numbers, i.e. average peak response before vs. during muscle relaxation. The values were compared by means of a paired t-test.
TI. Control values for TI and TE were obtained by averaging the values for ten breaths immediately preceding each trial.
2.8. Measurements of respiratory rhythm
At the end of each experiment we interrupted the afferent pathway of the reflex by sectioning of the internal branch of the SLN. When it was difficult to separate the internal branch from the external branch we sectioned bilaterally both branches of the SLN. This happened in seven rabbits. After deafferentation of the larynx NP pulses of −40 to − 70 cm H2O were applied.
Duration of the inspiratory and expiratory phases were calculated from the raw diaphragmatic neurogram. TI was defined as a duration of a burst of phrenic activity. The silent period between burst was defined as TE. Slope of n.Ph was obtained by dividing the peak n.Ph amplitude by
2.9. Sectioning of the superior laryngeal ner6es
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3. Results
3.1. Respiratory response to upper airway negati6e pressure before muscle relaxation We have found that the threshold for the reflex response to the negative laryngeal pressure was 792 cm H2O (mean 9 S.D.) in vagotomized anaesthetized rabbits (Section 2.5 and Fig. 3). We measured the response of n.XII, n.VII and n.Ph nerves together with an m.GG EMG to subatmospheric pressure equal to four times threshold pressure. The pressure pulse was restricted to larynx and epipharynx. In response to NP the amplitude of the hypoglossal nerve phasic activity increased to 1679 50% of the control value. Amplitude of the genioglossus muscle phasic activity increase to 220990%. Facial nerve phasic activity increased to 1349 27% and phrenic nerve decreased to 989 2% of the control value. Increase of UA nerves was significant (P B0.01). Tonic expiratory activity increased to 112, 109, 103% of their initial value for m.GG EMG, n.XII, n.VII, respectively. Slope of the phrenic nerve activity decreased to 859 12% of its control value. Inspiratory time increased to 116914% and expiratory time increased to 109 9 10% of the control value. Simultaneous recordings of the m.GG EMG and n.XII
Fig. 3. Decrease of n.XII activity in response to muscle relaxation. Left hand side graph presents integrated activity of the hypoglossal nerve just before and during muscle relaxation. The n.XII exhibits two types of activity, tonic and phasic. During expiration only tonic activity is present. During inspiration total activity is a sum of phasic and tonic activity. Muscle relaxation results in a decrease of both tonic and phasic component. Bar graph represents mean phasic hypoglossal nerve activity in 16 animals after muscle relaxation. Suppression of the phasic and tonic n.XII activity was significant with a P value of less than 0.01.
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activities enabled us to compare changes of these two signals. Representative recording is presented on Fig. 1. In this rabbit peak m.GG EMG activity increased ca. three times that of the peak n.XII activity both during and after the NP where the first volleys were still greater than control levels. Example on Fig. 1 illustrated the general trend. In all animals (n= 16) mean increase of n.XII activity was found to be 679 50%, which was significantly less (PB 0.01) than mean increase of m.GG EMG activity (120 9 90%). Augmentation of the first volley after NP pulse was also significantly (PB 0.01) different and amounted to 249 22 vs. 409 38% for n.XII and m.GG EMG volleys, respectively.
3.2. Changes of the respiratory and physiological parameters in response to muscle relaxation In 11 rabbits, which received vercuronium bromide the first symptoms of muscle relaxation (i.e. decrease m.GG EMG) were observed not earlier than 1996 sec after drug administration. Then EMG gradually decreased to disappear 70952 sec after drug administration. Interestingly, in some animals for the next 70–160 sec it was still possible to elicit GG EMG volleys by negative pressure (see Fig. 2). Decrease and disappearance of the m.GG EMG was associated with gradual decrease of hypoglossal nerve activity (Fig. 2 and 3). This effect was observed in all 16 animals. Two minutes after administration of muscle relaxant the amplitude of the hypoglossal nerve volleys (i.e. the difference between peak inspiratory and expiratory activity) was 189 16% lower than just before muscle relaxation (Fig. 3). At the same time n.XII tonic activity decreased by 20 9 14%. These changes were significant (PB 0.01). Changes of the facial nerve peak activity were smaller than 10% and n.Ph peak activity remained unchanged. Muscle atonia did not introduce any changes to respiratory rhythm. Inspiratory time remained to be 0.5 90.1 s and expiratory time was 0.99 0.2 sec. Mean respiratory frequency 60/ (TI + TE) was 43.89 8.9 per min. Mean control heart rate was 243.29 33.6 strokes/min and also remained unchanged. Arterial blood pressure was unaffected and remained constant at the level of
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Fig. 4. Effect of muscle relaxation on the threshold for response to negative pressure within the larynx. Note marked differences of the threshold value between subjects.
15.59 4.5/10.29 3.1 23.3 mmHg).
kPa
cation of muscle relaxant. For both UA nerves the difference was significant (PB 0.001). Attenuation of the peak n.XII activity during NP pulse had two causes. The first, that the initial (i.e. just before NP) UA nerve amplitude was smaller than before muscle relaxation. The second that in all but six rabbits the relative increase (i.e. the amplitude during NP pulse/the amplitude just before NP pulse) was smaller as well. The responses to a pulse of upper airway negative pressure before and during muscle relaxation are presented on Fig. 5. At the end of each experiment we interrupted the afferent pathway of the reflex by bilateral sectioning of the internal branch of the SLN. This totally abolished reflex response to negative pressure.
(116.5 933.8/76.79
3.3. Comparison of the response to upper airway negati6e pressure before and during muscle relaxation We have determined the threshold level for the reflex response during muscle relaxation. Three minutes after administration of muscle relaxant the threshold value increased more then twice and amounted 16.99 3.9 cm H2O in 16 rabbits. Threshold level varied considerably and ranged from 5 to 25 cm H2O (see Fig. 4). Typically pulses less negative than − 10 cmH2O where not able to initiate any response. Threshold levels and their changes in response to muscle relaxation are presented on Fig. 4. In none of the 16 animals decrease of the threshold during muscle atonia was observed. There were marked individual differences in the threshold value. In every one of 16 rabbits the peak n.XII and n.VII activity in response to the negative pressure pulse was lower during than before muscle relaxation. Consequently, the mean for all animals was lower during muscle relaxation. In response to NP pulse mean peak n.XII activity reached the value of 139 a.u. during vs. 167 a.u. before muscle relaxation. Mean peak n.VII activity was 114 u.a. during muscle relaxation vs. 134 a.u. before appli-
4. Discussion We studied the effect of muscle relaxation on the response of upper airway muscles to negative pressure. We found that the threshold for the reflex increased and the magnitude of the response decreased after muscle relaxation. We have also
Fig. 5. Effect of applying negative pressure to larynx on peak m.GG EMG, n.XII, n.VII and n.Ph inspiratory activity. Large values of S.D. indicate that there is a variability between animals in the magnitude of response. Nevertheless, mean changes of UA nerve activity are significant (PB 0.01) both before and during muscle relaxation. Changes of n.Ph activity are significant with PB 0.05. For n.XII and n.VII comparison of the response before and during muscle relaxation (paired t-test) shows significant (P B0.001) differences. Responses of the n.Ph are not significantly affected by muscle relaxation.
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shown that reduction of skeletal muscle tone attenuated the activity of the hypoglossal nerve even when laryngeal pressure was kept at the atmospheric level. Our interpretation of these data is that muscle relaxation changes the sensory feedback from the larynx and other segments of the upper airway. This in turn reduces both the gain of the pressure reflex and central respiratory drive activating UA dilators. Muscle tone throughout the body decreases during sleep, with relaxation of the UA dilating muscles. Therefore we believe that our findings may at least in part explain impairment of the pressure reflex during sleep (Aronson et al., 1989; Wheatley et al., 1993; Horner et al., 1994). We may only speculate about the mechanism by which muscle relaxation influences pressure reflex. We propose that when motor activity to the muscles increases, the sensory feedback from the muscles also increases and further stimulates motor activity. Consequently, such a positive feedback reduces motor activity when feedback signal decreases. Two mechanisms by which muscle tension may influence the magnitude of the pressure reflex may be considered. The first, that muscle relaxation specifically changes the pattern and magnitude of the SLN pressure sensitive endings. The second that muscle relaxation generally modifies feedback from tongue and other UA mechanoreceptors, while information about the pressure remains basically unchanged. The second concept is based on the assumption that muscle atonia modifies microenvironment of mechanoreceptors within the muscle and in tendon organs, therefore it may change feedback from muscle spindles in the tongue and other UA muscle receptors. This in turn could modify excitability of n.XII motorneurons. Muscle spindles are present in the tongue musculature of primates (Bowman, 1971) and increased afferent n.XII activity has been recorded in response to tongue stretch in the anterior-posterior direction (Lowe, 1984). The concept of direct influence of muscle relaxation on SLN afferent activity may be supported by findings of Mathew et al. (1984). They have found that mean discharge frequency of negative pressure receptors at atmospheric pressure was 10.8 impulse/sec when laryngeal muscles were not
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paralysed. During paralysis of laryngeal muscles induced by cooling of the recurrent laryngeal nerves or by i.v. gallamine mean discharged frequency was only 6.4 impulse/sec. In the other study (Mathew et al., 1988) discharge frequency of 65 receptors of different types which was 15.4 impulse/sec before laryngeal paralysis dropped to 6.6 impulse/s during paralysis of laryngeal muscles. However, there is also some data against the concept that laryngeal muscle paralysis may change excitability of receptors responsible for pressure response. Mathew et al. (1988) have shown that selective paralysis of laryngeal muscles obtained by cooling of the recurrent laryngeal nerve do not introduce any significant change in the peak activity of the cricothyroid muscle during NP pulse. Similarly, there was no significant difference between the occluded TI before and during laryngeal paralysis. These findings of Mathew et al. (1988) may suggest that relaxation of other UA muscles than laryngeal muscles is necessary to obtain results observed in our study. Alternatively, cricothyroid muscle activity similarly to n.VII activity is much less sensitive to the reflex then n.XII activity. We have not enough data to choose between presented options. However, even if activation of laryngeal receptors by negative pressure is significantly modified during muscle relaxation, changes in feedback information from other UA receptors seem to be also important. This can be supported by our finding that muscle relaxation decreased tonic n.XII activity by 20% and phasic activity by 18% when larynx and other segments of the UA were not subjected to negative pressure. This study is the first to analyze the pressure reflex both before and after muscle relaxation. All previous studies, but that of Hwang et al. (1984) examined non-paralyzed spontaneously breathing animals or human subjects and measured only EMG signals. Threshold for the reflex activation of the n.XII by negative pressure has not been previously determined. Some comparisons are however possible. Aronson et al. (1989) found that the reflex response to NP could be triggered by negative pressures of −7.5 cm H2O in awake but not
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sleeping (non-REM or REM sleep) healthy individuals. Horner et al. (1991) observed significant m.GG EMG activation with pressures of at least −5 cm H2O in awake subjects. During non-REM sleep Wheatley et al. (1993) was able to trigger the reflex with a NP of −11 to −14 cm H2O. Horner et al. (1994) with pressure of − 25 cm H2O. These results indicate that the threshold in awake humans is lower then − 7.5 cm H2O and increases to a value of ca. −11 to − 14 cm H2O during sleep. Both in human and animal studies quantitative data were obtained while pressure changes were induced in the entire upper airway respiratory tract. Laryngopharyngeal compartment of the upper airway seems to be the main reflexogenic zone responding to NP, nosopharyngeal compartment plays subsidiary role whereas oropharyngeal compartment is of a minor importance (Mathew et al., 1982b; Horner et al., 1991). The internal branches of the superior laryngeal nerves, the nasal trigeminal nerves and the glossopharyngeal nerves are mediating the responses from these compartments, respectively (Mathew et al., 1982b). Wide receptor area could be one of the reasons of an enormous variability (presented later) of reported quantitative results. In our study NP pulses were restricted to the laryngopharyngeal compartment. This was verified by the observation that the response disappeared after sectioning of the internal branches of the superior laryngeal nerves, which are known (Gray, 1985) to supply sensory endings to the larynx from the epiglottis to the level of vocal cords. Restricted receptive area may explain rather high threshold value and lower, than reported previously, magnitude of the response determined in our study. The other reason may be lower chemical respiratory drive than in the previous studies. Our animals were hyperoxic (Section 2) and were ventilated at constant level, while in all but one previous studies animals were breathing spontaneously with air. Reduced respiratory rate and n.Ph amplitude must have increased chemical drive during application of NP in the previous studies. Van Lunteren (1987) and Gauda et al. (1994) found that increased chemical drive enhances the response to UA negative pressure. The
above mentioned factors may have increased the magnitude of the response in previous reports as compared to our study. We observed excitation by 120, 67 and 34% of the peak m.GG EMG, n.XII and n.VII activity, respectively. For comparison Mathew and Faber (1983) (Table 1 and Fig. 4 of Mathew and Faber, 1983) reported a 1200% increase of the m.GG EMG in response to mean NP pulse of 10.2 cm H2O in spontaneously breathing rabbits with intact vagi. Woodall et al. (1989) reported a 765% increase of m.GG EMG in response to NP pulses of 200 msec duration, which were applied within the first 200 msec of inspiration in anesthetized rabbits with intact vagi. In similar preparation (rabbits with intact vagi) Zhang and Mathew (1992) (Fig. 3) observed only ca. 92% increase event though their NP pulses were longer (− 20 cm H2O for 300 msec). In the same study during partial inactivation of the pulmonary stretch receptors an increase of m.GG EMG was ca. 236% and an increase of alae nasi muscle EMG was ca. 132% (Zhang and Mathew, 1992). Gauda et al. (1994) stated that negative pressure of − 25 cm H2O applied to the isolated upper airway during spontaneous breathing did not elicit reflex activation of the genioglossus muscle in anaesthetized cats with intact vagi. In awake humans m.GG EMG signal increases by 82 and 88.6% in response to NP pulses of − 25 and − 35 cm H2O, respectively (Horner et al., 1991). In the study of Aronson et al. (1989) the EMG increase was 24% in response to negative pressure equal to − 7.5 cm H2O in awake human subjects. All above quoted studies utilized not paralyzed preparations and recorded m.GG EMG. The only previous study on paralyzed preparation was that of Hwang et al. (1984). They reported hypoglossal nerve excitation of 83% in response to NP pulses of − 21 cm H2O applied to the whole UA in paralyzed, ventilated, bivagotomized cats. The excitation determined by Hwang et al. (1984) was much smaller than other animal results quoted above. Our results in rabbits are comparable with those reported in humans by Horner et al. (1991) and only a little smaller than those of Hwang et al. (1984). Generally, these major quantitative differences reported by different investigators may
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suggest that the magnitude of the reflex response is influenced by yet unrecognized factors. Our hypothesis assumed that skeletal muscle tone is such a factor. Although, it is speculative we think that Hwang et al. (1984) obtained markedly lower amount of n.XII excitation because they used paralyzed preparation. In our study we observed that in response to NP pulse genioglossus muscle EMG signal increased by 120% (i.e. to 220% of its control value), while at the some time hypoglossal nerve activity increased only by 67%. The difference was significant. We are not aware of any other report of simultaneous recordings of the hypoglossal nerve activity and genioglossus muscle EMG and our data is insufficient to explain this result. Significant disproportion between changes of the GG EMG amplitude in comparison to n.XII amplitude may have physiological meaning or it may be purely methodological. Hypoglossal nerve innervates four intrinsic and four extrinsic lingual muscles. If we assume that all fibres exhibit phasic respiratory activity but only those which supply genioglossus muscles contribute to the response to negative pressure the relative increase of the whole n.XII activity should be smaller than m.GG EMG response. Another explanation would be that during reflex excitation of the m.GG more distant motor units start to contribute to the potential of the EMG electrode. If this explanation is correct the ratio between the increase of the n.XII and m.GG EMG activity would depend on the physical properties of the EMG electrode. This study is one more attempt to determine factors modulating the reflex response to negative pressure. Previously, such factors were recognised as: (1) hypercapnia (Van Lunteren, 1987; Gauda et al., 1994); (2) release from influence of the pulmonary stretch receptors (Zhang and Mathew, 1992; Gauda et al., 1994); and (3) sleep (Aronson et al., 1989; Wheatley et al., 1993; Horner et al., 1994). We have added a new factor to this list-muscle relaxation. Presumably, those factors are not independent. Attenuation of the pressure reflex during sleep may be primary due to sleep induced biochemical changes within the brainstem struc-
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tures exciting the hypoglossal nerve motor nuclei (Kubin et al., 1996) or they may be primarily the consequence of sleep induced muscle atonia. Also, magnification of the reflex response during hypercapnia may be only secondary to an increased chemical drive but primary due to hypercapnia induced increase of UA muscle tension. It may be also speculated that other factors diminishing muscle tone like administration of anesthetics, benzodiazepines, barbiturates and ethanol may influence the magnitude of the pressure reflex and in this way, predispose to snoring or obstructive apneas (Krol et al., 1984). Functional importance of the factors influencing the magnitude of the pressure reflex remain to be elucidated.
Acknowledgements The author thank Miss K. Goldsztajn for skillful technical assistance. This study was supported by KBN Grant 6 P20702805.
References Aronson, R.M., E. Onal, D.W. Carley and M. Lopata (1989). Upper airway and respiratory muscle response to continuous negative airway pressure. J. Appl. Physiol. 66: 1373 – 1382. Bowman, J.P. (1971). The Muscle Spindle and Neural Control of the Tongue, edited by Charles C. Thomas. Springfield, IL, USA. Gauda, E.B., T.P. Carroll, A.R. Schwartz, P.L. Smith and R.S. Fitzgerald (1994). Mechano- and chemoreceptor modulation of respiratory muscles in response to upper airway negative pressure. J. Appl. Physiol. 76: 2656 – 2662. Gray Anatomy (1985). 30th American edition, edited by C.D. Clemente. Philadelphia, PA: Lea and Febiger. Horner, J.A., J.A. Innes, K. Murphy and A. Guz. (1991). Evidence for reflex upper airway dilator muscle activation by sudden negative airway pressure in man. J. Physiol. (London) 436: 15 – 29. Horner, J.A., J.A Innes, M.J. Morrell, S.A. Shea and A. Guz. (1994). The effect of sleep on reflex genioglossus muscle activation by stimuli of negative airway pressure in humans. J. Physiol. (London) 476: 141 – 151. Hwang, J.C., W.M. St. John and D. Bartlett, Jr. (1984). Afferent pathways for hypoglossal and phrenic responses to changes in upper airway pressure. Respir. Physiol. 55: 341 – 354.
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Janczewski, W.A (1993). Relaxation of upper airway muscles reduces activity of the hypoglossal nerve-positive feedback leading to UA obstruction. Eur. Respir. J. 6 (Suppl. 17): 1641. Janczewski, W.A. and P.H. Janczewski (1995). Relaxation of skeletal muscles reduces the magnitude of the reflex response to negative upper airway pressure-mechanism leading to upper airway obstruction. Sleep Res. 24A: 329. Kimoff, R.J., T.H. Cheong, A.E. Olha, M. Charbonneau, R.D. Levy, M.G. Cosio and S.B. Gottfried (1994). Mechanisms of apnea termination in obstructive sleep apnea. Am. J. Respir. Crit. Care Med. 149: 707–714. Krol, R.C., S.L. Knut and D. Bartlett (1984). Selective reduction of genioglossus muscle activity by alcohol in normal subjects. Am. Rev. Respir. Dis. 129: 247–250. Kubin, L., H. Tojima, C. Reignier, A.I. Pack and R.O. Davis (1996). Interaction of serotonergic excitatory drive to hypoglossal motoneurons with carbachol-induced, REM sleep-like atonia. Sleep 19: 187–195. Lowe, A.A. (1984). Tongue movements-brainstem mechanisms and clinical postulates. Brain Behav. Evol. 25: 128–137. Mathew, O.P., Y.K. Abu-Osba and B.T. Thach (1982a). Influence of upper airway pressure changes on genioglossus muscle respiratory activity. J. Appl. Physiol. 52: 438 – 444. Mathew, O.P., Y.K. Abu-Osba and B.T. Thach (1982b). Genioglossus muscle responses to upper airway pressure changes: afferent pathways. J. Appl. Physiol. 52: 445– 450.
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Mathew, O.P. and J.P. Faber (1983). Effect of upper airway negative pressure on respiratory timing. Respir. Physiol. 54: 259 – 268. Mathew, O.P., G. Sant’Ambrogio, J.T. Fisher and F.B. Sant’Ambrogio (1984). Laryngeal pressure receptors. Respir. Physiol. 57: 113 – 122. Mathew, O.P., F.B. Sant’Ambrogio and O. Sant’Ambrogio (1988). Laryngeal paralysis on receptor and reflex responses to negative pressure in the upper airway. Respir. Physiol. 74: 25 – 34. Pack, A.I. (1994). Obstructive sleep apnea. Adv. Intern. Med. 39: 517 – 567. Remmers, J.E., W.J. de Oroot, E.K. Sauerland and A.M. Anch (1978). Pathogenesis of upper airway occlusion during sleep. J. Appl. Physiol. 44: 931 – 938. Woodall D.L., J.A. Hakanson and O.P. Mathew (1989). Time of application of negative pressure pulses and upper airway muscle activity. J. Appl. Physiol. 67: 366 – 370. Wheatley J.R., D.J. Tangel, W.S. Mezzanotte and D.P. White (1993). Influence of sleep on response to negative airway pressure of tensor palatini muscle and retropalatal airway. J. Appl. Physiol. 75: 2117 – 2124. Van Lunteren, E. (1987). Interactive effects of CO2 and upper airway negative pressure on breathing pattern. J. Appl. Physiol. 63: 229 – 237. Zhang S. and O.P. Mathew (1992). Decrease in lung volumerelated feedback enhances laryngeal reflexes to negative pressure. J. Appl. Physiol. 73: 832 – 836.
Muscle relaxation attenuates the reflex response to laryngeal negative pressure Wiktor A. Janczewski * Department of Neurophysiology, Medical Research Centre, Polish Academy of Sciences, 3 Dworkowa Str. 00 -784 Warsaw, Poland Accepted 15 November 1996
Abstract Negative pressure (NP) within the upper airway (UA) excites the activity of the genioglossus muscle and other UA dilators. For unknown reasons this reflex response is attenuated during sleep. Sleep reduces skeletal muscle tone, therefore we hypothesized that the magnitude of the pressure reflex is modulated by muscle tension. We have compared the threshold and the magnitude of the reflex response to laryngeal NP before and during muscle relaxation in 16 rabbits. The threshold increased from 79 2 (mean9S.D.) to 17 9 4 cm H2O. Peak amplitude during NP pulse dropped from 167 to 139 arbitrary units (a.u.) for the hypoglossal nerve (n.XII) and from 134 to 114 a.u. for the facial nerve (n.VII). Independently of the pressure reflex muscle relaxation reduced the phasic n.XII activity by 18% and tonic activity by 20%. We conclude that muscle atonia attenuates n.XII activity and the response to UA pressure change. Our findings when extrapolated to state of sleep suggest that sleep-induced muscle relaxation may be an independent factor suppressing pressure reflex. © 1997 Elsevier Science B.V. Keywords: Mammals; Rabbit; Muscle; Upper airway dilators; Reflex; Laryngeal; Sleep; Upper airway patency; Upper airways; Patency; Negative pressure reflex
1. Introduction During each inspiration the upper airway (UA) is subjected to negative pressure (NP) of 2 – 3 cm * Corresponding author. Present address: Laboratory of Clinical Pharmacology, Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Str, 02-106 Warsaw, Poland. Tel.: +48 22 6086523; fax: +48 22 6685416; e-mail: [email protected]
H2O generated by contraction of the diaphragm and other thoracic respiratory muscles. In normal subjects, UA negative pressure increases during sleep due to increase of the UA resistance. Such an increase is more pronounced in habitual snorers (Pack, 1994). In obstructive sleep apnea (OSA) patients apneic episodes are characterized by progressive (Kimoff et al., 1994) increase in intrathoracic and laryngeal negative pressure during efforts against the occluded airway. Maximal
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value of transdiaphragmatic pressure is typically reached on the last (83% of all apneas) obstructed effort and usually amounts ca. −56 cm H2O (Kimoff et al., 1994). Upper airway pressure more negative than few cm H2O indicates pathological narrowing of the upper airway, therefore it seems reasonable to assume that the amount of negative pressure is somehow sensed and used as a feedback information for the systems controlling excitation of the UA dilating muscles. This was found to be correct by Mathew et al. (1982a). They have shown that in rabbits NP pulse applied to UA markedly activates the hypoglossal nerve (n.XII) and at the same time depresses phrenic nerve (n.Ph) activity (Mathew et al., 1982a). Laryngopharyngeal compartment of the upper airway was found to be the main reflexogenic zone responding to NP, nosopharyngeal compartment played subsidiary role, whereas oropharyngeal compartment was of a minor importance (Mathew et al., 1982b). The Internal branches of the superior laryngeal nerves, the nasal trigeminal nerves and the glossopharyngeal nerves were mediating the responses from these compartments, respectively (Mathew et al., 1982b). Augmentation of UA dilator muscles by negative intraluminal pressure have been demonstrated also in man (Aronson et al., 1989; Horner et al., 1991, 1994; Wheatley et al., 1993). Negative pressure reflex activates not only hypoglossal muscle but other muscles dilating and stiffening UA, as well (Wheatley et al., 1993). Remmers et al. (1978) have proposed that the collapse of the upper airspace occurs when the negative pressures generated during inspiration are inadequately opposed by activation of the upper airway dilator muscles. Therefore, the pressure reflex, which selectively increases activity of the UA dilators and at the same time decreases the amount of negative pressure by suppressing the activity of the diaphragm should be able to prevent UA collapse. This theoretically potent reflex fails to prevent episodes of prolonged obstructive apneas during sleep in some individuals. Likely, because during sleep the magnitude of the reflex UA muscle activation is lower. Aronson et al. (1989) reported
an immediate increase in electromyographic genioglossal muscle activity in response to nasal continuous negative airway pressure of − 2.5 to − 7.5 cm H2O in normal awake humans. The response disappeared during NREM and REM sleep. Wheatley et al. (1993) have found that an increase of the tensor palatini electromyogram was attenuated during non-REM sleep in comparison to that during wakefulness. Horner et al. (1994) have shown that non-REM sleep attenuates reflex genioglossus muscle activation by stimuli of negative airway pressure. Although studies of Wheatley et al. (1993) and Horner et al. (1994) do not present such data it is likely that the response to UA negative pressure is further reduced during REM sleep. Impairment of the pressure reflex during sleep in humans has been discussed previously as an example of neural disfacilitation of reflex motor systems during sleep (Wheatley et al., 1993) or as a state dependent central effect (Horner et al., 1994). It seems also likely that the magnitude of the reflex response is affected by sleep related withdrawal of an excitatory drive to hypoglossal motorneurons (Kubin et al., 1996; Pack, 1994). We think that other mechanism may contribute to attenuation of the pressure reflex during sleep. We propose that the primary reason for the reduction of the pressure reflex during sleep is not the state of sleep but muscle relaxation. We think that reduction of the skeletal muscle tone may modify afferent information from UA receptors in such a way that the response to negative pressure will be reduced. If our concept is correct it should be possible to modify reflex response in a similar way as described during sleep only by relaxing skeletal muscles. Therefore, our aim was to determine if the reflex hypoglossal and facial nerve activation observed in response to stimuli of negative pressure in non-paralyzed rabbits is altered in magnitude during muscle relaxation. To answer this question we have compared responses to the same stimuli before and during muscle relaxation. Our working hypothesis was that the magnitude of the reflex response to negative pressure depends on the muscle tension.
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Since, this is the first study to record UA nerve activity continuously while muscles are being relaxed we have utilized this opportunity to determine whether the magnitude of hypoglossal and facial nerve activities are affected by changes in skeletal muscle tone. A preliminary account has been published (Janczewski, 1993; Janczewski and Janczewski, 1995).
2. Methods Nine decerebrate and seven halothane (1.5 vol%) anaesthetized rabbits (2.9 – 3.5 kg) were used. Decerebration was performed under halothane (3.0 vol%) anesthesia. In decerebrate rabbits anesthesia was discontinued more than 30 min before recordings. Animals were placed supine on a heating blanket to maintain body temperature in the range of 38 – 38.5°C. Femoral artery and vein were cannulated. Arterial cannula was connected to a transducer (Viggo-Spectramed DTX/Plus) and Neurolog 108 amplifier in order to measure arterial pressure. Vagus nerves and sympathetic trunks were bilaterally cut in the neck caudal to the origin of the superior laryngeal nerves. Animals were artificially ventilated at the constant level with air supplemented with oxygen to maintain arterial oxygen tension (PaO2) above 200 mmHg and PaCO2 in the range of 39–45 mmHg. If, despite sectioning of both phrenic nerves the animal still ‘struggled’ with the respirator, thoracotomy was performed to ensure that the spontaneous respiratory movements were ventilatory inefficient. End-tidal CO2 and O2 were monitored (Nec San-El IH 206). Arterial blood samples (0.4 ml) were drown anaerobically into heparinized syringes and immediately analyzed (Coming 238) in order to determine pH, PaO2, PaCO2 before and after muscle relaxation and before and after application of negative pressure. The corrections for temperature differences between the rabbit and blood gas analyzer were introduced by the Coming system. If the PaCO2 tension after muscle paralysis was more then 1 mmHg lower then before muscle paralysis the results were not included in the statistical analysis.
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Arterial bicarbonate concentration and base excess were also measured. If necessary, sodium bicarbonate was administered (i.v.) to maintain the arterial bicarbonate concentration above 20 mEq/L.
2.1. Laryngeal pressure changes To apply pulses of negative pressure we modified the protocol of Mathew et al. (1982a). Tracheostomy was performed below the cricoid cartilage. Two cannulas were inserted through the tracheostomy. The upper cannula was inserted in the direction of larynx and secured with its tip just below the larynx. The upper cannula was connected to the source of the negative pressure. The lower tracheal cannula was inserted in the direction of the lungs and connected to a constant volume mechanical ventilator (Ugo Basile 6025 ventilator). We mimicked the situation of the upper airway obstruction by inflating a small rubber balloon placed just rostral to larynx between tongue and posterior pharyngeal wall. This arrangements ensured that the negative pressure stimulated receptors only in the larynx and a part of pharynx close to epiglottis (i.e. epipharynx). Since mouth and the nares of the rabbits were opened, the pressure in the nosopharynx was equal to atmospheric.
2.2. Electroneurograms and electromyogram In order to record genioglossus muscle (m.GG) electromyogram (EMG) bipolar fine wire electrodes (Biomed Wire AS 631, Cooner Wire) were inserted 4–6 mm deep through one mylohyoid muscle midway between chin and hyoid bone. Phrenic nerves (C5-C6 roots) were bilaterally cut and prepared for recording. Both n.XIIs were placed on bipolar electrodes and covered with petroleum jelly to prevent drying. Hypoglossal nerves were not cut, phrenic nerves were bilaterally cut to prevent movements of diaphragm while animals were not paralyzed. In ten animals one bucco-labial branch of the facial nerve (n.VII) was cut distally and placed on a bipolar electrode. All signals were amplified, filtered, rectified, and integrated by means of RC integrator with time
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constant of 100 msec. Neural signals together with arterial pressure, laryngeal pressure and end-tidal PaCO2 were recorded on Honeywell Omnilight recorder and after analog to digital conversion on a hard disk of IBM-486 PC.
2.3. Muscle relaxation Data were obtained from 16 rabbits. In order to produce a state of muscle atonia a short acting neuromuscular blocking agent Norcuron (Organon Teknika), i.e. vecuronium bromide 0.05 mg/kg i.v. have been administered to 11 animals. The remaining five rabbits were treated with a longer acting neuromuscular blocking agent Tubocurarin (Orion), i.e. D-Tubocurarinum chloratum i.v. 0.6 mg/kg. Muscle relaxants were dissolved in physiological solution and administered in a 0.5 ml bolus to the femoral artery. Vercuronium bromide is widely used in humans, however we were not aware of any data defining the rabbit dose. Therefore in the preliminary experiments we have tried several doses. We have found that the dose of 0.04 mg/ kg of vecuronium bromide was the smallest dose effectively relaxing UA muscles. Total elimination of the genioglossus muscle EMG activity was considered to be a reliable marker of muscle relaxation. Our experimental arrangements ensured constant ventilation throughout the experiment, however in two rabbits after administration of vecuronium bromide blood gas test revealed 2 mmHg decrease of PaCO2. It was not clear whether those two animals really developed hypercapnia or sampling or measurements were not accurate. Nevertheless, these data were excluded from further analysis. More than 1 h later vecuronium bromide was administered again in all rabbits that received short acting agent before. For statistical analysis we have only used data from the first successful trial. This means from the first trial in 14 animals and from the second trial in those two, which did not have stable PaCO2 during the first attempt.
2.4. Measurements of ner6e acti6ity The changes of n.XVI n.XII, n.VII and n.Ph nerves activity and m.GG EMG activity were evaluated from integrated signals. The level of 0 arbitrary unit (a.u.) for total activity was defined as the level of nerve/EMG activity during expiration. The level of 100 a.u. for total activity was defined as an average of peak activity during ten successive respiratory cycles just before experimental intervention. Such a procedure is simple and commonly used to evaluate the total UA and other nerve activity (Kubin et al., 1996). However, for evaluation of the decrease of UA nerve activity induced by administration of muscle relaxant, we have separately measured decrease of tonic and phasic activity. The reason was that total UA neural or m.GG muscle activity is a sum of tonic (expiratory) activity and phasic inspiratory activity. In a case when an experimental intervention reduces total nerve/EMG activity it may happen that the tonic activity is totally eliminated and the phasic activity is reduced so much that it drops below the zero-line (see m.GG EMG signal in Fig. 2). In such a case peak activity would be negative. The 0 and 100 a.u. for phasic activity were as described previously. The 100 a.u. for tonic activity was the expiratory level of activity (i.e. zero-line for phasic activity). To find the zero-line for tonic activity we had hyperventilated the animals until the PaCO2 was 20 mmHg. Signal from electrodes at this PaCO2 partial pressure was our 0 a.u. level for tonic activity. Such a procedure was justified by our observation that n.XII/ m.GG activity was totally eliminated when PaCO2 was lower than 24 mmHg and n.VII activity disappeared when PaCO2 was below 22 mmHg. To estimate control inspiratory activity the mean9 S.D. have been calculated for ten respiratory cycles just preceding NP pulse. By definition we treated the mean value as 100 a.u. S.D. value for UA nerves was usually close to 3 a.u.
2.5. E6aluation of the threshold of the reflex response To determine the threshold for the reflex response we have applied a series of NP pulses.
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Fig. 1. Excitation of the UA nerves in response to 2.5 s duration pressure stimuli of -30 cm H2O. Traces from bottom to top: n.Ph, n.XII, m.GG EMG, n.VII, laryngeal pressure and end-tidal CO2. NP pulse was applied during the second half of inspiration. This resulted in a rapid increase of the UA muscle and nerve activities and depression of n.Ph activity. Note that UA activity remain increased for more than seven respiratory cycles after termination of the NP pulse.
Pulses were rectangular and started at the end of expiration and lasted for three respiratory cycles. The response was classified as positive if the mean peak n.XII activity increased by 3 S.D. a.u. (typically ca. 10 a.u.) during the pulse.
2.6. Characteristic of the negati6e pressure pulse Once the threshold have been determined we could decide what value of negative pressure will be used during the experiment. We used pressure equal to 4 times the threshold. The mean value was − 28 cm H2O. Our source of negative pressure was efficient enough to produce nearly rectangular pulses. The pressure pulse was re-
stricted to the larynx and epipharynx.
2.7. Estimation of the magnitude of the reflex response To determine the magnitude of the reflex response average of peak integrated activities observed during five consecutive NP pulses was calculated. Therefore, one animal provided a single number representing the amount of activation of particular nerve activity or EMG activity. The mean for all 16 animals was calculated and presented in the form of mean9 S.D. For the purpose of comparison before vs. during muscle relaxation the same procedure was repeated after
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Fig. 2. Attenuation of the n.XII nerve activity and attenuation of the response to NP pulse induced by i.v. administration of muscle relaxant. Traces from bottom to top: integrated activity of the n.Ph( − ), integrated activity of the n.XII, integrated EMG activity of the m.GG, intralaryngeal pressure. Note decrease of both tonic expiratory n.XII activity and n.XII amplitude concomitant with disappearance of m.GG EMG. Before muscle relaxation, pulse of −30 cm H2O increased n.XII peak inspiratory activity to the value of 350 a.u. When muscles were relaxed NP pulse increased peak n.XII activity to the value of 165 a.u., which was less then half of the stimulation before muscle relaxation. The relative increase was calculated according to formula: the amplitude during NP pulse divided by the amplitude just before NP pulse. It amounted to 165/78 a.u. =212% during MR vs. 350/100 a.u. =350% before muscle relaxation. Post-stimulus potentiation of the n.XII and GG EMG activity may be observed both before and during muscle relaxation.
muscle relaxation. Therefore, each one of 16 animals provide one pair of numbers, i.e. average peak response before vs. during muscle relaxation. The values were compared by means of a paired t-test.
TI. Control values for TI and TE were obtained by averaging the values for ten breaths immediately preceding each trial.
2.8. Measurements of respiratory rhythm
At the end of each experiment we interrupted the afferent pathway of the reflex by sectioning of the internal branch of the SLN. When it was difficult to separate the internal branch from the external branch we sectioned bilaterally both branches of the SLN. This happened in seven rabbits. After deafferentation of the larynx NP pulses of −40 to − 70 cm H2O were applied.
Duration of the inspiratory and expiratory phases were calculated from the raw diaphragmatic neurogram. TI was defined as a duration of a burst of phrenic activity. The silent period between burst was defined as TE. Slope of n.Ph was obtained by dividing the peak n.Ph amplitude by
2.9. Sectioning of the superior laryngeal ner6es
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3. Results
3.1. Respiratory response to upper airway negati6e pressure before muscle relaxation We have found that the threshold for the reflex response to the negative laryngeal pressure was 792 cm H2O (mean 9 S.D.) in vagotomized anaesthetized rabbits (Section 2.5 and Fig. 3). We measured the response of n.XII, n.VII and n.Ph nerves together with an m.GG EMG to subatmospheric pressure equal to four times threshold pressure. The pressure pulse was restricted to larynx and epipharynx. In response to NP the amplitude of the hypoglossal nerve phasic activity increased to 1679 50% of the control value. Amplitude of the genioglossus muscle phasic activity increase to 220990%. Facial nerve phasic activity increased to 1349 27% and phrenic nerve decreased to 989 2% of the control value. Increase of UA nerves was significant (P B0.01). Tonic expiratory activity increased to 112, 109, 103% of their initial value for m.GG EMG, n.XII, n.VII, respectively. Slope of the phrenic nerve activity decreased to 859 12% of its control value. Inspiratory time increased to 116914% and expiratory time increased to 109 9 10% of the control value. Simultaneous recordings of the m.GG EMG and n.XII
Fig. 3. Decrease of n.XII activity in response to muscle relaxation. Left hand side graph presents integrated activity of the hypoglossal nerve just before and during muscle relaxation. The n.XII exhibits two types of activity, tonic and phasic. During expiration only tonic activity is present. During inspiration total activity is a sum of phasic and tonic activity. Muscle relaxation results in a decrease of both tonic and phasic component. Bar graph represents mean phasic hypoglossal nerve activity in 16 animals after muscle relaxation. Suppression of the phasic and tonic n.XII activity was significant with a P value of less than 0.01.
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activities enabled us to compare changes of these two signals. Representative recording is presented on Fig. 1. In this rabbit peak m.GG EMG activity increased ca. three times that of the peak n.XII activity both during and after the NP where the first volleys were still greater than control levels. Example on Fig. 1 illustrated the general trend. In all animals (n= 16) mean increase of n.XII activity was found to be 679 50%, which was significantly less (PB 0.01) than mean increase of m.GG EMG activity (120 9 90%). Augmentation of the first volley after NP pulse was also significantly (PB 0.01) different and amounted to 249 22 vs. 409 38% for n.XII and m.GG EMG volleys, respectively.
3.2. Changes of the respiratory and physiological parameters in response to muscle relaxation In 11 rabbits, which received vercuronium bromide the first symptoms of muscle relaxation (i.e. decrease m.GG EMG) were observed not earlier than 1996 sec after drug administration. Then EMG gradually decreased to disappear 70952 sec after drug administration. Interestingly, in some animals for the next 70–160 sec it was still possible to elicit GG EMG volleys by negative pressure (see Fig. 2). Decrease and disappearance of the m.GG EMG was associated with gradual decrease of hypoglossal nerve activity (Fig. 2 and 3). This effect was observed in all 16 animals. Two minutes after administration of muscle relaxant the amplitude of the hypoglossal nerve volleys (i.e. the difference between peak inspiratory and expiratory activity) was 189 16% lower than just before muscle relaxation (Fig. 3). At the same time n.XII tonic activity decreased by 20 9 14%. These changes were significant (PB 0.01). Changes of the facial nerve peak activity were smaller than 10% and n.Ph peak activity remained unchanged. Muscle atonia did not introduce any changes to respiratory rhythm. Inspiratory time remained to be 0.5 90.1 s and expiratory time was 0.99 0.2 sec. Mean respiratory frequency 60/ (TI + TE) was 43.89 8.9 per min. Mean control heart rate was 243.29 33.6 strokes/min and also remained unchanged. Arterial blood pressure was unaffected and remained constant at the level of
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Fig. 4. Effect of muscle relaxation on the threshold for response to negative pressure within the larynx. Note marked differences of the threshold value between subjects.
15.59 4.5/10.29 3.1 23.3 mmHg).
kPa
cation of muscle relaxant. For both UA nerves the difference was significant (PB 0.001). Attenuation of the peak n.XII activity during NP pulse had two causes. The first, that the initial (i.e. just before NP) UA nerve amplitude was smaller than before muscle relaxation. The second that in all but six rabbits the relative increase (i.e. the amplitude during NP pulse/the amplitude just before NP pulse) was smaller as well. The responses to a pulse of upper airway negative pressure before and during muscle relaxation are presented on Fig. 5. At the end of each experiment we interrupted the afferent pathway of the reflex by bilateral sectioning of the internal branch of the SLN. This totally abolished reflex response to negative pressure.
(116.5 933.8/76.79
3.3. Comparison of the response to upper airway negati6e pressure before and during muscle relaxation We have determined the threshold level for the reflex response during muscle relaxation. Three minutes after administration of muscle relaxant the threshold value increased more then twice and amounted 16.99 3.9 cm H2O in 16 rabbits. Threshold level varied considerably and ranged from 5 to 25 cm H2O (see Fig. 4). Typically pulses less negative than − 10 cmH2O where not able to initiate any response. Threshold levels and their changes in response to muscle relaxation are presented on Fig. 4. In none of the 16 animals decrease of the threshold during muscle atonia was observed. There were marked individual differences in the threshold value. In every one of 16 rabbits the peak n.XII and n.VII activity in response to the negative pressure pulse was lower during than before muscle relaxation. Consequently, the mean for all animals was lower during muscle relaxation. In response to NP pulse mean peak n.XII activity reached the value of 139 a.u. during vs. 167 a.u. before muscle relaxation. Mean peak n.VII activity was 114 u.a. during muscle relaxation vs. 134 a.u. before appli-
4. Discussion We studied the effect of muscle relaxation on the response of upper airway muscles to negative pressure. We found that the threshold for the reflex increased and the magnitude of the response decreased after muscle relaxation. We have also
Fig. 5. Effect of applying negative pressure to larynx on peak m.GG EMG, n.XII, n.VII and n.Ph inspiratory activity. Large values of S.D. indicate that there is a variability between animals in the magnitude of response. Nevertheless, mean changes of UA nerve activity are significant (PB 0.01) both before and during muscle relaxation. Changes of n.Ph activity are significant with PB 0.05. For n.XII and n.VII comparison of the response before and during muscle relaxation (paired t-test) shows significant (P B0.001) differences. Responses of the n.Ph are not significantly affected by muscle relaxation.
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shown that reduction of skeletal muscle tone attenuated the activity of the hypoglossal nerve even when laryngeal pressure was kept at the atmospheric level. Our interpretation of these data is that muscle relaxation changes the sensory feedback from the larynx and other segments of the upper airway. This in turn reduces both the gain of the pressure reflex and central respiratory drive activating UA dilators. Muscle tone throughout the body decreases during sleep, with relaxation of the UA dilating muscles. Therefore we believe that our findings may at least in part explain impairment of the pressure reflex during sleep (Aronson et al., 1989; Wheatley et al., 1993; Horner et al., 1994). We may only speculate about the mechanism by which muscle relaxation influences pressure reflex. We propose that when motor activity to the muscles increases, the sensory feedback from the muscles also increases and further stimulates motor activity. Consequently, such a positive feedback reduces motor activity when feedback signal decreases. Two mechanisms by which muscle tension may influence the magnitude of the pressure reflex may be considered. The first, that muscle relaxation specifically changes the pattern and magnitude of the SLN pressure sensitive endings. The second that muscle relaxation generally modifies feedback from tongue and other UA mechanoreceptors, while information about the pressure remains basically unchanged. The second concept is based on the assumption that muscle atonia modifies microenvironment of mechanoreceptors within the muscle and in tendon organs, therefore it may change feedback from muscle spindles in the tongue and other UA muscle receptors. This in turn could modify excitability of n.XII motorneurons. Muscle spindles are present in the tongue musculature of primates (Bowman, 1971) and increased afferent n.XII activity has been recorded in response to tongue stretch in the anterior-posterior direction (Lowe, 1984). The concept of direct influence of muscle relaxation on SLN afferent activity may be supported by findings of Mathew et al. (1984). They have found that mean discharge frequency of negative pressure receptors at atmospheric pressure was 10.8 impulse/sec when laryngeal muscles were not
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paralysed. During paralysis of laryngeal muscles induced by cooling of the recurrent laryngeal nerves or by i.v. gallamine mean discharged frequency was only 6.4 impulse/sec. In the other study (Mathew et al., 1988) discharge frequency of 65 receptors of different types which was 15.4 impulse/sec before laryngeal paralysis dropped to 6.6 impulse/s during paralysis of laryngeal muscles. However, there is also some data against the concept that laryngeal muscle paralysis may change excitability of receptors responsible for pressure response. Mathew et al. (1988) have shown that selective paralysis of laryngeal muscles obtained by cooling of the recurrent laryngeal nerve do not introduce any significant change in the peak activity of the cricothyroid muscle during NP pulse. Similarly, there was no significant difference between the occluded TI before and during laryngeal paralysis. These findings of Mathew et al. (1988) may suggest that relaxation of other UA muscles than laryngeal muscles is necessary to obtain results observed in our study. Alternatively, cricothyroid muscle activity similarly to n.VII activity is much less sensitive to the reflex then n.XII activity. We have not enough data to choose between presented options. However, even if activation of laryngeal receptors by negative pressure is significantly modified during muscle relaxation, changes in feedback information from other UA receptors seem to be also important. This can be supported by our finding that muscle relaxation decreased tonic n.XII activity by 20% and phasic activity by 18% when larynx and other segments of the UA were not subjected to negative pressure. This study is the first to analyze the pressure reflex both before and after muscle relaxation. All previous studies, but that of Hwang et al. (1984) examined non-paralyzed spontaneously breathing animals or human subjects and measured only EMG signals. Threshold for the reflex activation of the n.XII by negative pressure has not been previously determined. Some comparisons are however possible. Aronson et al. (1989) found that the reflex response to NP could be triggered by negative pressures of −7.5 cm H2O in awake but not
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sleeping (non-REM or REM sleep) healthy individuals. Horner et al. (1991) observed significant m.GG EMG activation with pressures of at least −5 cm H2O in awake subjects. During non-REM sleep Wheatley et al. (1993) was able to trigger the reflex with a NP of −11 to −14 cm H2O. Horner et al. (1994) with pressure of − 25 cm H2O. These results indicate that the threshold in awake humans is lower then − 7.5 cm H2O and increases to a value of ca. −11 to − 14 cm H2O during sleep. Both in human and animal studies quantitative data were obtained while pressure changes were induced in the entire upper airway respiratory tract. Laryngopharyngeal compartment of the upper airway seems to be the main reflexogenic zone responding to NP, nosopharyngeal compartment plays subsidiary role whereas oropharyngeal compartment is of a minor importance (Mathew et al., 1982b; Horner et al., 1991). The internal branches of the superior laryngeal nerves, the nasal trigeminal nerves and the glossopharyngeal nerves are mediating the responses from these compartments, respectively (Mathew et al., 1982b). Wide receptor area could be one of the reasons of an enormous variability (presented later) of reported quantitative results. In our study NP pulses were restricted to the laryngopharyngeal compartment. This was verified by the observation that the response disappeared after sectioning of the internal branches of the superior laryngeal nerves, which are known (Gray, 1985) to supply sensory endings to the larynx from the epiglottis to the level of vocal cords. Restricted receptive area may explain rather high threshold value and lower, than reported previously, magnitude of the response determined in our study. The other reason may be lower chemical respiratory drive than in the previous studies. Our animals were hyperoxic (Section 2) and were ventilated at constant level, while in all but one previous studies animals were breathing spontaneously with air. Reduced respiratory rate and n.Ph amplitude must have increased chemical drive during application of NP in the previous studies. Van Lunteren (1987) and Gauda et al. (1994) found that increased chemical drive enhances the response to UA negative pressure. The
above mentioned factors may have increased the magnitude of the response in previous reports as compared to our study. We observed excitation by 120, 67 and 34% of the peak m.GG EMG, n.XII and n.VII activity, respectively. For comparison Mathew and Faber (1983) (Table 1 and Fig. 4 of Mathew and Faber, 1983) reported a 1200% increase of the m.GG EMG in response to mean NP pulse of 10.2 cm H2O in spontaneously breathing rabbits with intact vagi. Woodall et al. (1989) reported a 765% increase of m.GG EMG in response to NP pulses of 200 msec duration, which were applied within the first 200 msec of inspiration in anesthetized rabbits with intact vagi. In similar preparation (rabbits with intact vagi) Zhang and Mathew (1992) (Fig. 3) observed only ca. 92% increase event though their NP pulses were longer (− 20 cm H2O for 300 msec). In the same study during partial inactivation of the pulmonary stretch receptors an increase of m.GG EMG was ca. 236% and an increase of alae nasi muscle EMG was ca. 132% (Zhang and Mathew, 1992). Gauda et al. (1994) stated that negative pressure of − 25 cm H2O applied to the isolated upper airway during spontaneous breathing did not elicit reflex activation of the genioglossus muscle in anaesthetized cats with intact vagi. In awake humans m.GG EMG signal increases by 82 and 88.6% in response to NP pulses of − 25 and − 35 cm H2O, respectively (Horner et al., 1991). In the study of Aronson et al. (1989) the EMG increase was 24% in response to negative pressure equal to − 7.5 cm H2O in awake human subjects. All above quoted studies utilized not paralyzed preparations and recorded m.GG EMG. The only previous study on paralyzed preparation was that of Hwang et al. (1984). They reported hypoglossal nerve excitation of 83% in response to NP pulses of − 21 cm H2O applied to the whole UA in paralyzed, ventilated, bivagotomized cats. The excitation determined by Hwang et al. (1984) was much smaller than other animal results quoted above. Our results in rabbits are comparable with those reported in humans by Horner et al. (1991) and only a little smaller than those of Hwang et al. (1984). Generally, these major quantitative differences reported by different investigators may
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suggest that the magnitude of the reflex response is influenced by yet unrecognized factors. Our hypothesis assumed that skeletal muscle tone is such a factor. Although, it is speculative we think that Hwang et al. (1984) obtained markedly lower amount of n.XII excitation because they used paralyzed preparation. In our study we observed that in response to NP pulse genioglossus muscle EMG signal increased by 120% (i.e. to 220% of its control value), while at the some time hypoglossal nerve activity increased only by 67%. The difference was significant. We are not aware of any other report of simultaneous recordings of the hypoglossal nerve activity and genioglossus muscle EMG and our data is insufficient to explain this result. Significant disproportion between changes of the GG EMG amplitude in comparison to n.XII amplitude may have physiological meaning or it may be purely methodological. Hypoglossal nerve innervates four intrinsic and four extrinsic lingual muscles. If we assume that all fibres exhibit phasic respiratory activity but only those which supply genioglossus muscles contribute to the response to negative pressure the relative increase of the whole n.XII activity should be smaller than m.GG EMG response. Another explanation would be that during reflex excitation of the m.GG more distant motor units start to contribute to the potential of the EMG electrode. If this explanation is correct the ratio between the increase of the n.XII and m.GG EMG activity would depend on the physical properties of the EMG electrode. This study is one more attempt to determine factors modulating the reflex response to negative pressure. Previously, such factors were recognised as: (1) hypercapnia (Van Lunteren, 1987; Gauda et al., 1994); (2) release from influence of the pulmonary stretch receptors (Zhang and Mathew, 1992; Gauda et al., 1994); and (3) sleep (Aronson et al., 1989; Wheatley et al., 1993; Horner et al., 1994). We have added a new factor to this list-muscle relaxation. Presumably, those factors are not independent. Attenuation of the pressure reflex during sleep may be primary due to sleep induced biochemical changes within the brainstem struc-
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tures exciting the hypoglossal nerve motor nuclei (Kubin et al., 1996) or they may be primarily the consequence of sleep induced muscle atonia. Also, magnification of the reflex response during hypercapnia may be only secondary to an increased chemical drive but primary due to hypercapnia induced increase of UA muscle tension. It may be also speculated that other factors diminishing muscle tone like administration of anesthetics, benzodiazepines, barbiturates and ethanol may influence the magnitude of the pressure reflex and in this way, predispose to snoring or obstructive apneas (Krol et al., 1984). Functional importance of the factors influencing the magnitude of the pressure reflex remain to be elucidated.
Acknowledgements The author thank Miss K. Goldsztajn for skillful technical assistance. This study was supported by KBN Grant 6 P20702805.
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