Inhibition of inspiratory motor output by high-frequency lowpressure oscillations in the upper airway of sleeping dogs

Transcription

1 8738 Journal of Physiology (1999), 517.1, pp Inhibition of inspiratory motor output by high-frequency lowpressure oscillations in the upper airway of sleeping dogs Peter R. Eastwood, Makoto Satoh, Aidan K. Curran, Maria T. Zayas, Curtis A. Smith and Jerome A. Dempsey John Rankin Laboratory of Pulmonary Medicine, Department of Preventive Medicine, University of Wisconsin, Madison, WI 53705, USA (Received 15 September 1998; accepted after revision 12 February 1999) 1. We utilized a chronically tracheostomized, unanaesthetized dog model to study the reflex effects on inspiratory motor output of low-amplitude, high-frequency pressure oscillations (HFPOs) applied to the isolated upper airway (UA) during stable non-rapid eye movement (NREM) sleep. 2. HFPOs (30 Hz and ±2 to ±4 cmhµo) were applied via a piston pump during eupnoea, inspiratory resistive loading and tracheal occlusion. 3. When applied to the patent UA during expiration, and especially during late expiration, HFPOs prolonged expiratory time (TE) and tonically activated the genioglossus muscle EMG. When applied to the patent UA during inspiration, HFPOs caused tonic activation of the genioglossus muscle EMG and inhibition of inspiratory motor output by either: (a) a shortening of inspiratory time (TI), as inspiration was terminated coincident with the onset of HFPOs; or (b) a prolonged TI accompanied by a decreased rate of rise of diaphragm EMG and rate of fall of tracheal pressure. These effects of HFPOs were observed during eupnoea and inspiratory resistive loading, but were maximal during tracheal occlusion where the additional inhibitory effects of lung inflation reflexes were minimized. 4. During eupnoea, topical anaesthesia of the UA abolished the HFPO-induced prolongation of TE, suggesting that the response was mediated primarily by mechanoreceptors close to the mucosal surface; whereas the TE-prolonging effects of a sustained square wave of negative pressure (range, 4 0 to 14 9 cmhµo) sufficient to close the airway were preserved following anaesthesia. 5. These results demonstrate that high-frequency, low-amplitude oscillatory pressure waves in the UA, similar to those found in snoring, produce reflex inhibition of inspiratory motor output. This reflex may help maintain UA patency by decreasing the collapsing pressure generated by the inspiratory pump muscles and transmitted to the UA. Snoring is characterized by high-frequency oscillations of the soft palate, pharyngeal walls, epiglottis and tongue (Liistro et al. 1991). Previous investigators, applying highfrequency pressure oscillations to the upper airway (UA) at a similar frequency (30 Hz) as seen during snoring (Robin, 1968; Liistro et al. 1991), have demonstrated reflex activation of muscles in the UA in anaesthetized, waking and sleeping dogs, and in sleeping humans with and without obstructive sleep apnoea (Plowman et al. 1990b; Henke & Sullivan, 1993; Brancatisano et al. 1996). The results from these studies have led to the hypothesis that snorers may be able to resist complete UA obstruction, despite the generation of substantial negative pressures in the UA, because the pressure oscillations trigger reflex activation of dilator muscles in the UA which serve to stiffen it (Plowman et al. 1990b; Henke & Sullivan, 1993; Brancatisano et al. 1996). Low-amplitude, high-frequency pressure oscillations (HFPOs) in the UA have also been shown to elicit reflex changes in breathing pattern and in the magnitude of respiratory motor output; however, the results are variable. HFPOs applied to the UA of normal sleeping subjects and patients with obstructive sleep apnoea during a single inspiration cause increased diaphragm EMG activity, implying a reflex excitation of inspiratory motor output (Henke & Sullivan, 1993). However, when applied to the UA over successive inspirations in sleeping dogs, HFPOs cause a decreased minute-averaged inspiratory time (TI), implying an inhibitory effect on inspiratory motor output (Plowman et al. 1990b). When applied during expiration HFPOs result in a prolonged, shortened or unchanged expiratory time (TE) (Plowman et al. 1990b; Henke & Sullivan, 1993).

2 260 P. R. Eastwood and others J. Physiol Therefore the purpose of this study was to investigate the reflex effects of HFPOs on timing, drive and UA muscle EMG activity in the sleeping dog when HFPOs were applied during inspiration and expiration, together or separately, on backgrounds of eupnoea, increased resistance and airway occlusion. We also used mucosal blockade to examine the role of surface receptors in mediating the reflex responses. METHODS General Studies were performed on each of three unanaesthetized female mixed-breed dogs (20 25 kg) during non-rapid eye movement (NREM) sleep. The dogs were trained to sleep in an air-conditioned sound-attenuated chamber. Throughout all experiments the dogs behaviour was monitored by an investigator seated within the chamber, and also by closed-circuit television. The protocol for this study was approved by the Animal Care and Use Committee of the University of Wisconsin. Animal preparation Sterile surgical techniques were used to create a permanent tracheostomy, and to implant electrodes for recording of inspiratory and UA muscle activities, and permit staging of sleep state. The dogs were premedicated with acepromazine (0 5 mg kg s.c.), induced with sodium thiopental (20 mg kg i.v.) and maintained on a mechanical ventilator with 1 % halothane in oxygen. A mid-line cervical incision and removal of the ventral aspect of four to five cartilagenous rings was used to create the chronic tracheostomy. Bipolar Teflon-coated multistrand stainless steel wire electrodes were sewn into the crural diaphragm and genioglossus muscles for measurement of EMG activity. The raw EMGs were filtered ( Hz), amplified, rectified and moving-time averagedwithatimeconstantof100ms. Five wire electrodes were implanted subcutaneously, consisting of an EEG, two electrooculograms (EOGs), a common reference and a ground electrode. The EEG and EOGs were amplified (BMA_831, CWE Inc., PA, USA) and filtered at 1 50 Hz. These methods have been described in detail previously (Chow et al. 1994). All electrodes were tunnelled subcutaneously to the cephalad portion of the dog s back, where they were exteriorized. Analgesics (butorphanol, 0 3 mg kg s.c.) and antibiotics (enrofloxacin, 2 2 mg kg per os (p. o.) or trimethoprim sulfa, 24 mg kg p. o.) were administered postoperatively as required. At least 2 months were allowed for recovery from surgery before any experiments were performed. At the end of these experiments, each dog was killed with methods consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. Experimental set-up and measurements The dogs breathed via a cuffed tracheostomy tube (o.d mm) inserted into the permanent tracheostomy. A heated (37 C) pneumotachograph system (model 3700, Hans Rudolph, Kansas City, MO, USA; model MP_45_14_871, Validyne, Northbridge, CA, USA) connected to the tracheostomy tube was used to measure airflow and was calibrated prior to each study with five known flows. A low-resistance two-way valve (model 1400, Hans Rudolph) was attached to the pneumotachograph. The inspiratory port of the two-way valve was connected to a circuit which, by inflating or deflating different combinations of balloon-valves, could allow inspiration to proceed without external load, apply an inspiratory flow-resistive load (80 cmhµo l s at 0 2 l s ), or occlude inspiration (Eastwood et al. 1998). The dead space of this system wasapproximately40ml. The UA was isolated by sealing both around a snout mask and below the larynx (Fig. 1). A light-weight polyethylene mask with polymer gel inserts (Silipos, Niagara Falls, NY, USA) was placed over the dog s snout to form an airtight seal around the mouth and nose. An additional cuffed endotracheal tube (o.d. 6 0 mm) was inserted rostrally into the tracheostomy above the tracheostomy tube to lie just caudal to the larynx. Inflation of this cuff produced an isolated UA. Airway pressure changes were simultaneously measured at three sites: mask pressure (PM) was measured with a catheter passed through the mask near the nares; sub-laryngeal pressure (PSl) was measured via the laryngeal endotracheal tube; tracheal pressure (PTr) was measured from a catheter passed into the tracheostomy tube. Each catheter was connected to a pressure transducer (model MP_45_14_871, Validyne) and was calibrated prior to each study by applying eight known pressures. Oscillating pressure waves of 30 Hz and between ±2 and ±4 cmhµo were generated from a mechanical piston pump (Metrex Instruments, Toronto, Canada) and applied to the dog s isolated UA via the snout mask or the laryngeal endotracheal tube. All signals were collected on a 12-channel polygraph (Gould ES 2000, Rolling Meadows, IL, USA), and were passed via an analogto-digital converter and stored on the hard disk of a microcomputer for subsequent analysis. Experimental protocol Over separate days, multiple trials were performed on each dog in stable NREM sleep. Trials consisted of the application of HFPOs to the isolated UA for a single respiratory effort, during inspiration or expiration, while the tracheostomy tube remained unloaded, during inspiratory resistive loading or during occlusion. Time of application (inspiratory or expiratory) and mode of breathing (unloaded or loaded) were randomized. Any trials in which there was EEG evidence of arousal were discarded. Application of HFPOs during expiration and inspiration. At various times during expiration HFPOs were initiated and then maintained throughout the following inspiration, which was either unloaded (eupnoea), or against a resistance or occlusion. HFPOs were terminated at the beginning of the subsequent expiration. Application of HFPOs during inspiration alone. At various times during unloaded inspiration (eupnoea) or during inspiratory efforts against a resistance or occlusion, HFPOs were initiated and then maintained until the beginning of the subsequent expiration. Upper airway anaesthesia. To anaesthetize the surface mucosa of the UA, lidocaine aerosol (4 %) was introduced into the UA of dogs 2 and 3 using a high-output extended aerosol nebulizer (B&B Medical Technologies Inc., Orangevale, CA, USA) which delivered high density particles (2 3 ìm) at approximately 50 ml h. The aerosol was delivered via the laryngeal endotracheal tube and vented through the dog s unmasked nose and mouth. Before and immediately after 1 h of anaesthetic application the awake dog s responses to probing the larynx with small polyethylene tubing and to ammonium hydroxide passed near the nares were tested. Once the UA was anaesthetized the mask was immediately positioned on

3 J. Physiol Upper airway pressure oscillations during sleep 261 the dog and multiple HFPO trials and square-wave negative pressure trials (see Results, Responses to square-wave negative pressures ) were performed during eupnoea in NREM sleep. Generally, the dogs required less than 5 min to attain stable NREM sleep following repositioning of the snout mask. Data were collected for approximately 30 min, immediately following which the dog was woken and the UA retested to confirm the maintenance of anaesthesia. Data selection and statistical analyses For any given trial, occluded or resistive-loaded efforts with or without HFPOs and eupnoeic trials with HFPOs, were compared with control values, which were obtained by averaging the values for the preceding three breaths. Inspiratory effects of HFPOs were compared for each of the three conditions (eupnoea, resistance or occlusion). Expiratory effects of HFPOs were analysed using the combined data from all trials, regardless of the condition, because the subsequent inspiration had no effect on the preceding expiratory responses. During eupnoea, paired t tests were used to compare control breaths and HFPO breaths, and unpaired t tests to compare the responses when HFPOs were applied during inspiration alone or maintained during inspiration and expiration. During efforts against an inspiratory resistance and inspiratory occlusion, ANOVA was used to compare the responses when HFPOs were not applied, applied during inspiration only, or applied during both inspiration and expiration. While the data in Tables 1 4 are presented as a percentage change from eupnoeic control values, statistical analyses for each dog were performed on the raw data. A Student Newman Keuls post hoc test was applied to correct for multiple comparisons. Statistical significance was inferred when P < RESULTS Representative polygraph examples of the responses to HFPOs during single efforts against an inspiratory resistance are shown in Fig. 2A D. The mean changes in timing, volume, pressure and EMG activity for each of the three conditions (eupnoea, inspiratory resistance and inspiratory occlusion) are shown for each dog in Tables 1 3. Route of application of HFPOs To assess the potential effect of the route of application of HFPOs, in dogs 1 and 2 the responses to HFPOs applied from the mask and sub-laryngeal catheters were compared. We found no significant differences between TI, TE, tidal volume (VT), diaphragm EMG activity (EMGDi) or PTr changes in response to HFPOs between the two routes of application. Because of this lack of effect, measurements in dog 3 were obtained from the mask only, and mask and sublaryngeal data were pooled in each of dogs 1 and 2. Effects of HFPOs applied during expiration on expiratory time During all trials in which HFPOs were applied during expiration, expiratory time was identified by ete, in order to distinguish these changes from the expiratory time following an inspiration, which was identified by TE. Application of HFPOs during expiration caused ete to be prolonged by 151 ± 55, 129 ± 26 and 128 ± 18 % Figure 1. Schematic representation of the isolated upper airway (UA) preparation The preparation allowed high-frequency pressure oscillations (HFPOs) to be applied to the isolated UA via the mask or sub-laryngeal catheters. During application of HFPOs continuous measurements were made of mask pressure (PM), sub-laryngeal pressure (PSl) and tracheal pressure (PTr). Sleep state was assessed from electro-oculograms (EOG), EEG and genioglossus EMG (EMGGg).

4 262 P. R. Eastwood and others J. Physiol Figure 2. Polygraph examples of the effects of HFPOs applied to the isolated UA Each panel shows the changes in EEG, integrated and raw diaphragm EMG (EMGDi), raw genioglossus muscle activity (EMGGg), respired flow and volume, and mask, sub-laryngeal and tracheal pressure changes (PM, PSl, PTr) during NREM sleep. In each example two unloaded control breaths are followed by a single inspiratory effort against an inspiratory resistance (80 cmhµo l s ) without (A) and with (B, C and D) application of HFPOs to the isolated UA. Application of HFPOs during expiration (B) resulted in a

5 J. Physiol Upper airway pressure oscillations during sleep 263 prolongation of expiratory time (ete) and, in this example, activation of genioglossus muscle EMG activity. Application of HFPOs during inspiration resulted in a prolongation of inspiratory time and depression of EMGDi activity, particularly at the onset of HFPOs (C), or early termination of inspiration coincident with the onset of HFPOs (D). It was notable that the UA remained patent throughout all trials, as similar changes were observed in PM and PSl.

6 264 P. R. Eastwood and others J. Physiol Table 1. Effects of HFPOs during eupnoea Dog 1 Dog 2 Dog 3 Absolute values during eupnoea No. of trials TI (s) 1 6 ± ± ± 0 1 TE (s) 3 0 ± ± ± 0 3 VT (ml) 380 ± ± ± 35 PTrÏTI (cmhµo s ) 0 9 ± ± ± 0 1 HFPOs applied during inspiration only No. of trials TI (%) 114±16* 107±9 121±13* TE(%) 112±46 122±26 98±5 VT(%) 106±14 100±7 103±11 PTrÏTI (%) 81±19* 90±12 62±24* EMGDiÏTI (%) 91±17* 88±12* 79±13* HFPOs applied during expiration and inspiration No. of trials TI (%) 133 ± 23 * 116 ± 14 * 147 ± 20 * TE (%) 148±59* 133±27* 128±18* VT(%) 120 ± 22 * 110 ± 17 * 110 ± 19 * PTrÏTI (%) 70±24* 84±18* 60±21* EMGDiÏTI (%) 83±23* 91±15* 70±10* Values are means ± s.d. of multiple trials on each dog, expressed as absolute values or as a percentage of eupnoeic control values. TI, inspiratory time (from the inspiratory period); TE, expiratory time (from the expiratory period following this inspiratory period); ete, expiratory time (from the expiratory period prior to the inspiratory period); VT, tidal volume; PTrÏTI, rate of fall of tracheal pressure; EMGDiÏTI, rateofrise of diaphragm EMG. * Raw values significantly different from eupnoeic control breaths, P < Raw values significantly different from HFPOs applied during inspiration only, P < (means ± s.d.) of control values in each of dogs 1, 2 and 3, respectively (Tables 1 3, Fig. 2B). Linear regression analyses of these data demonstrated a significant positive correlation between the time of application of HFPOs and the magnitude of ete prolongation for each dog: regression coefficient 0 485, and 0 556, respectively (P < 0 05); slope of the regression line 1 015, and 0 427, respectively (P < 0 05). Thus the magnitude of ete prolongation was dependent on when during expiration HFPOs were applied, such that the greatest prolongation was observed when HFPOs were initiated during late expiration (Fig. 3). Effects of HFPOs on inspiratory timing and motor output Application of HFPOs during expiration and continuing through the subsequent inspiration invariably prolonged that inspiration. Application of HFPOs during inspiration alone resulted in two distinct responses, represented by either (i) a prolonged TI or, (ii) a shortened TI as a consequence of the termination of inspiratory effort coincident with the onset of HFPOs. Trials in which TI was prolonged During eupnoea. Relative to eupnoeic control values, initiation of HFPOs during inspiration increased TI (by 14 ± 7 %, significant in 2 dogs only), decreased the rate of rise of EMGDi (by 14 ± 6 %), and decreased the rate of fall of PTr (by 22±14%) (means±s.d. from the 3 dogs, Table 1). Similar changes were observed when HFPOs were initiated in expiration and maintained throughout the subsequent inspiration. That is, relative to eupnoeic control values TI increased (by 32 ± 15 %), VT increased (by 13 ± 6 %), rate of rise of EMGDi decreased (by 19 ± 11 %) andrateoffallofptr decreased (by 29 ± 12 %) (means ± s.d. from 3 dogs, Table 1). In 2 of the 3 dogs the magnitude of increase of TI was significantly greater when HFPOs were initiated in expiration than inspiration, while the changes in VT, rateof rise of EMGDi andrateoffallofptr were generally similar whether HFPOs were initiated in expiration or inspiration. During efforts against an inspiratory resistance. Relative to eupnoeic control values, application of an inspiratory resistance increased TI (127 ± 10 %), decreased VT (50 ± 6 %), decreased the rate of rise of EMGDi (88 ± 2 %) and increased the rate of fall of PTr (732 ± 171 %) (means ± s.d. from3dogs,p< 0 05 for each dog; Table 2 and Fig. 2A). Initiation of HFPOs during inspiration caused termination of inspiration in all trials performed in dog 1 (see below, Trials in which TI was shortened ). In dogs 2 and 3, relative

7 J. Physiol Upper airway pressure oscillations during sleep 265 Table 2. Effects of HFPOs during efforts against an inspiratory resistance Dog 1 Dog 2 Dog 3 Without HFPOs No. of trials TI (%) 120±11 138±16 123±13 TE(%) 106±27 111±18 106±16 VT (%) 47±7 57±7 45±8 PTrÏTI (%) 574 ± ± ± 244 EMGDiÏTI (%) 87±12 87±16 90±16 HFPOs applied during inspiration only No. of trials TI (%) 165±30* 155±12* TE(%) 103±11 109±13 VT(%) 67 ± 7 * 55 ± 10 * PTrÏTI (%0 564 ± 181 * 621 ± 129 * EMGDiÏTI (%) 70 ± 18 * 72 ± 8 * HFPOs applied during expiration and inspiration No. of trials TI (%) 158±32* 156±24* 157±16* ete(%) 158±32* 121±19 129±19* VT(%) 58±13* 71±19* 59±8* PTrÏTI (%) 426 ± 152 * 728 ± ± 116 * EMGDiÏTI (%) 73±21* 87±24 81±12 Values are means ± s.d. of multiple trials on each dog, expressed as a percentage of eupnoeic control values. TI, inspiratory time (from the inspiratory period); TE, expiratory time (from the expiratory period following this inspiratory period); ete, expiratory time (from the expiratory period prior to the inspiratory period); VT, tidal volume; PTrÏTI, rate of fall of tracheal pressure; EMGDiÏTI, rate of rise of diaphragm EMG. For dog 1, HFPOs applied during inspiration only caused termination of inspiratory efforts (see Results). * Raw values significantly different from efforts without HPFOs, P < Raw values significantly different from HFPOs applied during inspiration only, P < to the control responses against a resistance (without HFPOs), initiation of HFPOs during inspiration increased TI (by 25 ± 11 %), increased VT (by 31 ± 4 %), decreased therateofriseofemgdi (19 ± 1 %) and decreased the rate of fall of PTr (by 24±13%) (means±s.d. from 2 dogs, Table 2 and Fig. 2C). It was possible to obtain data from all dogs when HFPOs were initiated in expiration. Relative to the control responses against a resistance, HFPOs increased TI (24 ± 14 %), increased VT (by 32 ± 7 %), decreased the rate of rise of EMGDi (8 ± 8 %, significant in dog 1 only) and decreased the rate of fall of PTr (by 14±18%, significant in dogs 1 and 3) (means ± s.d. from 3 dogs; Table 2 and Fig. 2B). Generally, the magnitudes of increase of TI and VT and decrease of rate of rise of EMGDi andrateoffallofptr were similar whether HFPOs were applied in expiration or inspiration. During efforts against an inspiratory occlusion. Relative to eupnoeic control values, inspiratory occlusion increased TI (133 ± 15 %), decreased the rate of rise of EMGDi (90 ± 10 %, significant in 2 dogs only) and increased the rate of fall of PTr (1398 ± 465 %) (means ± s.d. of 3 dogs, P < 0 05 for each dog; Table 3). Initiation of HFPOs during inspiration caused arousal in dog 1, and these data were discarded. In dogs 2 and 3, relative to the control values against an occluded airway (without HFPOs), initiation of HFPOs during inspiration increased TI (by 62 ± 37 %), decreased the rate of rise of EMGDi (by 38 ± 21 %) and decreased the rate of fall of PTr (28±14%)(means±s.d. from 2 dogs, Table 3). Data could be obtained from all dogs when HFPOs were initiated in expiration. Relative to control responses against an occlusion, HFPOs increased TI (by 40 ± 7 %), decreased the rate of rise of EMGDi (by 20 ± 10 %) and decreased the rate of fall of PTr (by24±12%) (means±s.d. from 3 dogs, Table 3). Generally, the magnitudes of increase of TI and VT and decrease of rate of rise of EMGDi andrateoffallofptr were similar whether HFPOs were applied in expiration or inspiration. Therefore, relative to their respective controls, HFPOs increased TI during eupnoea, resistance and occlusion trials. Analysis of the group data showed that the magnitude of the increase in TI with HFPOs was greater during inspiratory efforts against occlusion than during resistive loading and eupnoea, which were similar (P < 0 05, n = 3, 2-way ANOVA).

8 266 P. R. Eastwood and others J. Physiol Table 3. Effects of HFPOs during efforts against an inspiratory occlusion Dog 1 Dog 2 Dog 3 Without HFPOs No. of trials TI (%) 124±11 150±12 124±12 TE(%) 99 ± ± ± 22 PTrÏTI (%) 965 ± ± ± 437 EMGDiÏTI (%) 86±17 83±11 102±18 HFPOs applied during inspiration only No. of trials 11 3 TI (%) 204±22* 213±18* TE(%) 99 ± 13 * 76 ± 16 PTrÏTI (%) 1053 ± 284 * 1110 ± 247 * EMGDiÏTI (%) 64 ± 15 * 48 ± 3 * HFPOs applied during expiration and inspiration No. of trials TI (%) 186±27* 211±38* 186±18* ete(%) 174±40* 132±32 127±12* PTrÏTI (%) 628 ± 131 * 1052 ± 325 * 1167 ± 625 * EMGDiÏTI (%) 73±21* 75±30* 66±12* Values are means ± s.d. of multiple trials on each dog, expressed as a percentage of eupnoeic control values. TI, inspiratory time (from the inspiratory period); TE, expiratory time (from the expiratory period following this inspiratory period); ete, expiratory time (from the expiratory period prior to the inspiratory period); VT, tidal volume; PTrÏTI, rate of fall of tracheal pressure; EMGDiÏTI, rate of rise of diaphragm EMG. For dog 1, HFPOs appplied during inspiration only caused arousal in all trials (see Results). * Raw values significantly different from efforts without HPFOs, P < Raw values significantly different from HFPOs applied during inspiration only, P < Table 4. Effects of UA anaesthesia on responses to HFPOs during eupnoea Dog 2 Dog 3 HFPOs applied during inspiration only No. of trials 6 8 TI (%) 100±9 104±9 TE(%) 100±13 103±10 VT(%) 112 ± ± 14 PTrÏTI (%) 122 ± ± 11 EMGDiÏTI (%) 100 ± 8 98 ± 13 HFPOs applied during expiration and inspiration No. of trials TI (%) 106±14 108±12 ete(%) 93 ± ± 7 VT (%) 120±17 105±20 PTrÏTI (%) 148 ± ± 34 EMGDiÏTI (%) 113 ± ± 19 Values are means ± s.d. of multiple trials on each dog, expressed as a percentage of eupnoeic control values. TI, inspiratory time (from the inspiratory period); TE, expiratory time (from the expiratory period following this inspiratory period); ete, expiratory time (from the expiratory period prior to the inspiratory period); VT, tidal volume; PTrÏTI, rate of fall of tracheal pressure; EMGDiÏTI, rate of rise of diaphragm EMG. * Raw values significantly different from eupnoeic control breaths, P < Raw values significantly different from HFPOs applied during inspiration only, P < 0 05.

9 J. Physiol Upper airway pressure oscillations during sleep 267 Trials in which TI was shortened Figure 2D shows an example of a trial in which HFPOs initiated during inspiration caused early termination of inspiration, coincident with the onset of HFPOs. This was observed in 50 ± 24 % of trials in eupnoea (means ± s.d. from 3 dogs), 57 ± 44 % of trials during resistive loading (3 dogs) and 63 ± 26 % of trials during occlusion (2 dogs), otherwise TI was prolonged (see above). During eupnoea and breaths against a resistance and occlusion, TI was decreased to 69 ± 5, 70 ± 13 and 83 ± 6 % of eupnoeic control values, respectively. The time of application of HFPOs during inspiration ranged between 42 and 99 % of TI, andboreno relationship to whether TI was terminated or prolonged. Shortening of TI wasneverseenandtiprolongation was always observed when HFPOs were initiated in expiration and maintained throughout the subsequent inspiration. previously reported by Harms et al. (1996) in the sleeping dog when the upper airway was not anaesthetized. DISCUSSION The main finding of this study was that low-amplitude HFPOs applied to the isolated UA of sleeping dogs caused inhibition of inspiratory motor output. The findings are summarized as follows. (1) TE was prolonged when HFPOs were applied during expiration, and the magnitude of TE UA muscle activation Augmentation of tonic genioglossus muscle EMG activity was observed in 70 ± 21 % of all trials when HFPOs were initiated in expiration (mean ± s.d. from 3 dogs). When HFPOs were initiated in inspiration, augmentation of tonic genioglossus muscle EMG activity was observed in 30 ± 52 % of trials in eupnoea (3 dogs), 64 ± 47 % of trials during resistive loading (3 dogs) and 83 ± 24 % of trials during occlusion (2 dogs). Phasic genioglossus activity was not observed in this experimental model. Topical anaesthesia Responses to HFPOs The eupnoeic responses to HFPOs after topical anaesthesia oftheuawereexaminedindogs2and3(table 4). HFPOs were initiated at various times during expiration (60 ± 14 % of TE) and inspiration (48 ± 14 % of TI). Relative to the responses seen in the unanaesthetized UA (Table 1), topical anaesthesia abolished the prolongation of ete (Table 4 and Fig. 3), and decreased or abolished the prolongation or shortening of TI, the decrease of the rate of rise of EMGDi and the decrease in the rate of fall of PTr (Table 4). Abolition of these responses occurred regardless of when during inspiration or expiration HFPOs were initiated. Responses to square-wave negative pressures While the UA was still anaesthetized, square waves of negative pressure of sufficient magnitude to cause UA collapse (a divergence of PSl from PM) were randomly applied from the sub-laryngeal catheter. Pressures were initiated during expiration and held until the beginning of the next inspiratory effort. Five trials were performed in each of two dogs. Square waves of 8 ± 4 and 12 ± 3 cmhµo applied to the isolated UA of dog 2 and dog 3 resulted in an increase in TE to 159 ± 29 and 168 ± 25 % of eupnoeic TE values, respectively. These effects of square-wave negative pressure on TE in the dog with upper airway anaesthesia are similar to those Figure 3. The effect of time of application of HFPOs on expiratory time (ete) In each of the three dogs, during NREM sleep, the magnitudeofeteprolongationdependedonwhenduring expiration HFPOs were initiated, with the greatest prolongation seen when HFPOs were initiated during late expiration (0). Topical anaesthesia of the upper airway (1) abolished the prolongation of ete.

10 268 P. R. Eastwood and others J. Physiol prolongation was greatest when HFPOs were initiated in late expiration. (2) When HFPOs were applied during inspiration the response was either a prolongation of TI, or a shortening of TI. The decreased TI was a consequence of termination of inspiration at a time coincident with the onset of HFPOs. (3) The prolongation of TI by HFPOs was observed during eupnoea and inspiratory resistive loading, but was maximal during tracheal occlusion where the additional inhibitory effects of lung inflation reflexes were minimized. (4) Tonic activation of the genioglossus muscle was observed in most trials in response to HFPOs. (5) Unlike the effects of square-wave negative pressure, UA collapse was not required for HFPOs to inhibit respiratory motor output when applied during inspiration or expiration: the UA remained patent throughout all trials, as would be expected with airway pressures between ±2 and ±4 cmhµo. (6) Topical anaesthesia of the UA abolished the effects of HFPOs on breath timing but not the effects of square-wave negative pressure, suggesting that the response to HFPOs was mediated primarily by mechanoreceptors close to the mucosal surface. HFPO-induced prolongation of expiration Application of HFPOs to the isolated UA of sleeping dogs caused a central apnoea, the length of which was dependent on the time of initiation of HFPOs during expiration. The greatest TE prolongation was observed when HFPOs were applied in late expiration. Similar effects on prolonging TE were demonstrated when square waves (< 5 cmhµo) and pulses (< 20 cmhµo) of negative pressure of sufficient magnitude to cause UA collapse were applied to the isolated UA of the sleeping dog (Harms et al. 1996) and the anaesthetized rabbit (Mathew & Farber, 1983). In the present study the airway remained patent and only small pressure oscillations (±2 to ±4 cmhµo) were required to prolong TE. Thus it would appear that the UA is more sensitive to oscillating than to static pressure changes. This conclusion is consistent with the observation by Zhang & Mathew (1992) that some respiratory-modulated receptors in the UA of the anaesthetized dog are activated in response to small oscillating pressures (±2 5 cmhµo) but not to maintained pressure changes of greater magnitude (±10 cmhµo). In contrast to our finding of inhibition of inspiratory motor output by HFPOs, Henke & Sullivan (1992) demonstrated in sleeping humans that application throughout inspiration of HFPOs at the mouth to an intact UA during eupnoeic breathing and during a spontaneous obstructive apnoea caused activation of diaphragm EMG. Reasons for the disparate findings are not clear; however, contributing factors may include the presence of occult arousal, the effect of stimulating the entire tracheobronchial tree in addition to the UA, differences between the state of the central respiratory oscillator during an apnoea and a eupnoeic expiratory pause, or differences in the background level of respiratory motor output (Eldridge et al. 1989). Species differences most probably do not contribute to the difference in the findings as many studies have now shown similar reflex effects of pressure stimuli in the UA, regardless of species: static negative pressures applied during expiration cause TE prolongation and genioglossus muscle activation in humans and dogs (Horner et al. 1991; Harms et al. 1996) and HFPOs have been shown to elicit reflex UA muscle activation in humans and dogs (Plowman et al. 1990b; Henke & Sullivan, 1992; Brancatisano et al. 1996). In this regard the finding of increased activation of the human diaphragm in response to HFPOs is an anomaly, requiring further investigation. We found that the time of application of HFPOs was an important determinant of the duration of the apnoea, such that the greatest magnitude of TE prolongation occurred when HFPOs were initiated in late expiration. A similar phase dependency has been reported in sleeping dogs by Harms et al. (1996), who described TE prolongation when brief negative pressure pulses were applied during late expiration, but not during early expiration. Prolongation of TE has also been reported to occur in adult cats by activation of pulmonary afferents via lung inflation or moderate electrical stimulation of the vagus nerve (Feldman & Gautier, 1976). However, in this study lung inflation or vagal stimulation prolonged TE only when the stimulus was applied during early expiration: late-expiratory stimulation had no effect on TE (Feldman & Gautier, 1976). Thus, the prolongation of TE seen with UA HFPOs occurs independently of vagal influences, as lung inflation was absent during the expiratory pause. The differences in responses to UA and vagal stimuli indicate distinctly different pathways for mechanoreceptor afferent modulation of respiratory rhythm. HFPO-induced effects on inspiratory motor output HFPO-induced lengthening of TI was observed during eupnoea and breathing against a resistance, but the effects were greatest when inspiration occurred against an occluded airway. Under this circumstance the inhibitory effects of lung inflation reflexes, which in the dog appear to mask any inhibitory influences originating from the UA, are abolished. In each condition, the prolonged TI was accompanied by a decreased rate of rise of diaphragm EMG activity, which was paralleled by a decrease in the rate of fall of PTr. This depression of the rate at which the UA is exposed to negative intrathoracic pressure may be beneficial for airway patency by reducing the collapsing pressure transmitted to it (Remmers et al. 1978). The prolongation of TI had the effect of offsetting the decreased rate of rise of inspiratory neural activity, thereby increasing VT when breathing against an inspiratory resistance. Henke & Sullivan (1992) also observed an increased VT in 46 % of all trials performed in individuals with obstructive sleep apnoea and narrow UAs in response to HFPOs, a response these authors attributed to the dilatation of the UA by reflex activation of UA muscles. In the present study the changes in VT were independent of changes in UA calibre as the dog was breathing via the trachea and the UA was bypassed. Thus

11 J. Physiol Upper airway pressure oscillations during sleep 269 the reflex effects of HFPOs on prolonging TI can also contribute to an augmented VT. Application of HFPOs during inspiration also resulted in a shortening of TI, with inspiratory termination always coinciding with the onset of HFPOs. A decreased minuteaveraged TI has previously been noted by Plowman et al. (1990b) when inspiratory-timed HFPOs were applied to the isolated UA of sleeping dogs over a 1 min interval. The reduction in TI observed in this study was a consequence of rapid termination of inspiration by HFPOs. A similar effect was also noted by Harms et al. (1996) when pulses of negative pressure of sufficient magnitude to collapse the UA caused early termination of inspiration when applied at various times during inspiration. In the present study UA collapse was not required for early termination of TI to occur when low-amplitude HFPOs were applied, as the UA remained patent throughout. Furthermore, whether TI was lengthened or shortened was independent of when during inspiration HFPOs were applied. Why application of HFPOs of identical magnitudes and durations caused lengthening of TI in some trials and early termination of TI in others is unclear. The time of application of HFPOs had no effect on whether TI was lengthened or shortened, and nor did changes in sleep state, amplitude of HFPOs, or magnitude of background inspiratory resistance, whichwereconstantinallofthe reported trials. We speculate that, while both responses reflect inhibition of inspiratory motor output, whether TI was shortened or prolonged was a consequence of differing magnitudes of inhibitory stimuli reaching the central pattern generator. A characteristic of breaths in which TI was prolonged was a momentary abolition or fractionation of diaphragm activity at the onset of HFPOs, following which inspiratory diaphragm EMG activity would proceed. These sudden decreases in diaphragm EMG activity were accompanied by a momentary positive deflection in PTr (see Fig. 2C). Another characteristic of the effect of HFPOs applied during inspiration was that when HFPOs were initiated during expiration and maintained throughout the next inspiration, TI was never shortened, although depression of inspiratory motor output was still evident. It is possible that the increase in chemical stimuli as a consequence of the prolonged TE prevented the inhibitory effect of HFPOs on TI seen when HFPOs were initiated during inspiration (Eldridge et al. 1989). However, the observation in sleeping dogs by Plowman et al. (1990b) that early termination of inspiration occurred during all eupnoeic breaths over a 1 min period, suggests that, to the extent that change in breathing pattern resulted in hypoventilation over this period, the HFPO-induced shortening of TI will persist, even in a background of high chemical drive. Alternatively, it may be that the largest pulse of afferent stimuli occurs at the onset of HFPOs, and beyond this point sensory adaptation occurs. Consistent with this latter hypothesis is the finding from fibre-optic visualization of the UA during HFPOs of a transient vibration of tissues in the UA followed by an indefinitely sustained period of greatly reduced tissue movement (Plowman et al. 1990a). Similar variable effects on TI were observed by Mathew & Farber (1983) when static negative pressures of 10 2 ± 2 9 cmhµo were applied to the isolated UA of the anaesthetized rabbit. In their study negative pressures applied in expiration and maintained throughout the next inspiration usually produced prolongation of TI, whereas initiation of the negative pressure during inspiration produced a shortened, prolonged or unchanged TI. These authors hypothesized a role for rapidly and slowly adapting receptors in mediating this response: application of negative pressure during expiration would decrease rapidly adapting receptor activity and maintain the activity of slowly adapting receptors by the next inspiration, whereas both rapidly and slowly adapting receptors would probably be excited when pressure changes were initiated during inspiration. The marked similarities between the effects on TI of static negative pressure (Mathew & Farber, 1983) and HFPOs suggest a potential role for these receptors in these timing changes. Afferent pathways Our data using topical anaesthesia of the UA suggest that surface, not deeper, receptors are primarily responsible for mediating the reflex inhibition of inspiratory motor output. Supporting this conclusion is the finding that UA anaesthesia abolished the effects of HFPOs but not of square-wave negative pressures of sufficient magnitude to collapse the UA. It would seem reasonable to assume that the degree of airway wall distortion associated with UA collapse would provoke activation of deeper mechanoreceptors than lowpressure oscillations of the patent UA. The importance of surface receptors in mediating this response is supported by a study by Sampson & Eyzaguirre (1964) which demonstrated that vibratory stimuli applied to the laryngeal mucosa readily excited touch receptors, but had no effect on deeper mechanoreceptors. However this finding is in contrast to that of Zhang & Mathew (1992) who demonstrated that HFPOs were capable of activating essentially all respiratorymodulated laryngeal mechanoreceptors. Application of square-wave negative pressures more negative than 5 cmhµo (i.e. sufficient to cause collapse of the isolated UA) are required before a prolongation of TE is evident (Harms et al. 1996), whereas HFPOs of smaller amplitudes produce significant TE prolongation. We have previously shown in the sleeping dog that TI increases by 164 % of control when the UA is exposed to graded negative pressures spontaneously generated in response to an occluded airway (Eastwood et al. 1998). In the present study HFPOs of much smaller magnitudes, when applied during inspiratory occlusion, caused an increase in TI of approximately 200 % of control. It appears, therefore, that UA mechanoreceptors are more sensitive to HFPOs than to static or graded negative pressures (Zhang & Mathew, 1992). These are most probably neural receptors that are

12 270 P. R. Eastwood and others J. Physiol particularly sensitive to rate of change and not static pressure (Davis & Nail, 1987), or rapidly adapting laryngeal mechanoreceptors (Sant Ambrogio et al. 1983, 1985; Mathew et al. 1984). Implications The findings from this study have implications for understanding the mechanical behaviour of the UA in snorers. Snoring is characterized by high-frequency oscillations of the soft palate, pharyngeal walls, epiglottis and tongue (Liistro et al. 1991) at a similar frequency (30 Hz) to the HFPOs artificially applied to the UA in the present study (Robin, 1968; Liistro et al. 1991). Previous investigators have hypothesized that a reason snorers can resist complete UA obstruction, despite the generation of substantial negative pressures in the UA (Lugaresi et al. 1975) is that the pressure oscillations cause reflex activation of the genioglossus and other UA muscles (Plowman et al. 1990b; Henke & Sullivan, 1993; Brancatisano et al. 1996). Our data suggest an additional mechanism for the maintenance of airway patency during snoring, that of a depression of the rate at which the UA is exposed to negative intrathoracic collapsing pressures. Of the three conditions used in the present study, HFPOs applied during inspiratory efforts against a high resistive load is the most analogous to snoring. Breathing against a resistance will increase TI as a consequence of a depression of inhibitory lung inflation reflexes. Application of HFPOs further inhibited inspiratory motor output, as indicated by an even greater increase in TI and a further depression of the rate of rise of inspiratory neural activity and the rate of fall of PTr. VT was maintained despite this depression of inspiratory motor output, because of the compensatory effect of an increased TI offsetting the depressed rate of rise of inspiratory activity. The data from the present study also have implications for ventilatory control during high-frequency ventilation (HFV), whereby small tidal volumes are delivered at high frequency. The typical response of anaesthetized, intubated animals to HFV is apnoea or expiratory prolongation (Thompson et al. 1981; Banzett et al. 1983). This inhibition has been shown to depend on chest wall as well as vagal afferent input (Thompson et al. 1981; England et al. 1984), with the magnitude of the response modified by factors including lung volume (Banzett et al. 1983), depth of general anaesthesia (Banzett et al. 1983) and sleep state (England et al. 1985). In conscious humans, the response to HFV at Hz is a prolongation of TI ataconstantvtïti, an increase in VT and a fall in respiratory frequency (DeWeese et al. 1985). These changes are blunted by airway anaesthesia and to a lesser extent by pharyngeal anaesthesia, suggesting that receptors in the larynx and lower airways are of more importance than pharyngeal receptors in mediating the response (DeWeese et al. 1985). In the present study the greatest prolongation of TI was seen when HFPOs were applied during efforts against an occluded airway, under which circumstance the contribution of inhibitory lung inflation reflexes was minimal. It appears therefore, that the dominance of inhibitory reflexes originating in the lung and chest wall, relative to those originating in the UA, may lessen as lung volume decreases. This may be particularly relevant when UA resistance increases, such as occurs in individuals with a narrow UA or during sleep. Banzett, R., Lehr, J. & Geffroy, B. (1983). High-frequency ventilation lengthens expiration in the anesthetized dog. Journal of Applied Physiology 55, Brancatisano, A., Van der Touw, T., O Neill, N. & Amis, T. C. 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